Interaction between Hyaluronic Acid and Its Receptors (CD44, RHAMM) Regulates the Activity of Inflammation and Cancer -

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INTERACTION BETWEEN HYALURONIC ACID AND ITS RECEPTORS (CD44, RHAMM) REGULATES THE ACTIVITY OF INFLAMMATION AND CANCER EDITED BY : David Naor PUBLISHED IN : Frontiers in Immunology Frontiers Copyright Statement About Frontiers © Copyright 2007-2016 Frontiers Media SA. All rights reserved. Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering All content included on this site, such as text, graphics, logos, button approach to the world of academia, radically improving the way scholarly research icons, images, video/audio clips, is managed. The grand vision of Frontiers is a world where all people have an equal downloads, data compilations and software, is the property of or is opportunity to seek, share and generate knowledge. Frontiers provides immediate and licensed to Frontiers Media SA permanent online open access to all its publications, but this alone is not enough to (“Frontiers”) or its licensees and/or subcontractors. 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Find out more on how to host your own Frontiers ISSN 1664-8714 Research Topic or contribute to one as an author by contacting the Frontiers Editorial ISBN 978-2-88919-913-6 DOI 10.3389/978-2-88919-913-6 Office: [email protected] Frontiers in Immunology 1 July 2016 | Interaction Between Hyaluronic Acid and Its Receptors INTERACTION BETWEEN HYALURONIC ACID AND ITS RECEPTORS (CD44, RHAMM) REGULATES THE ACTIVITY OF INFLAMMATION AND CANCER Topic Editor: David Naor, Hebrew University of Jerusalem, Israel The biological outcome of Hyaluronan (also hyaluronic acid, abbreviated HA) interaction with its CD44 or RHAMM receptors recently attracted much attention within the scientific community owing to a Nature article by Tian X et al. (Nature 2013; 499:346-9). The article described a life span exceeding 30 years in naked mole rats, whereas the maximal lifespan of mice, to which the naked mole rat is related, is only 4 years. This observation is accompanied by the finding that the naked mole rat, in contrast to the mouse, does not develop spontaneous tumors during this exceptional longevity. The article provides evidence that interaction of long tissue HA (6000-12,000 kDa) of the naked mole rat with cell surface CD44, in contrast to the interaction of short tissue HA (less than 3000 kDa) with the mouse CD44, makes the difference. More specifically, this communication shows that the interaction of short HA with fibroblasts’ CD44 imposes on them susceptibility for malignant transformation, whereas the corresponding interaction with long HA imposes on the fibroblasts a resistance to malignant transformation. The article does not explain the mechanism that underlines these findings. However, the articles, that will be published in the proposed Research Topic in the Inflammation section of Frontiers in Immunology, can bridge not only this gap, but also may explain why interaction between short HA and cell surface CD44 (or RHAMM, an additional HA receptor) enhances the development of inflammatory and malignant diseases. Furthermore, the articles included in the proposed Frontiers Research Topic will show that cancer cells and inflammatory cells share several properties related to the interaction between short HA and cell surface CD44 and/ or RHAMM. These shared properties include: 1. Support of cell migration, which allows tumor metastasis and accumulation of inflammatory cells at the inflammation site; 2. Delivery of intracellular signaling, which leads to cell survival of either cancer cells or inflammatory cells; 3. Delivery of intracellular signaling, which activates cell replication and population expansion of either cancer cells or inflammatory cells; and 4. Binding of growth factors to cell surface CD44 Frontiers in Immunology 2 July 2016 | Interaction Between Hyaluronic Acid and Its Receptors of cancer cells or inflammatory cells (i.e., the growth factors) and their presentation to cells with cognate receptors (endothelial cells, fibroblasts), leading to pro-malignant or pro-inflammatory activities. Going back to the naked mole rat story, we may conclude from the proposed articles of this Frontiers Research Topic that the long HA, which displays anti- malignant effect, interferes with the above described pro-malignant potential of the short HA (perhaps by competing on the same CD44 receptor). Extrapolating this concept to Inflammation, the same mechanism (competition?) may be valid for inflammatory (and autoimmune) activities. If this is the case, long HA may be used for therapy of both malignant and inflammatory diseases. Moreover, targeting the interaction between short Hyaluronic acid localization on pancreatic β cells. Section HA and CD44 (e.g. by anti-CD44 blocking from pancreatic islets derived from diabetic NOD mice were subjected to double fluorescence staining with anti-insulin antibodies) may display also a therapeutic (green) and biotinylated hyaluronic acid binding protein effect on both malignant and inflammatory (HABP ;red). DAPI staining was used to detect cell nuclei. diseases, an issue that encourages not only Analysis by confocal microscopy revealed that hyaluronic fruitful exchange of views, but also practical acid (HA;red) are localized on β cell membrane (green). experimental collaboration. It was suggested by Nathalie Assayag-Asherie from Naor’s laboratory that the Interaction of the cell bound HA with the Citation: Naor, D., ed. (2016). Interaction β cell surface CD44 imposes apoptosis on these cells resulting Between Hyaluronic Acid and Its Receptors in type 1 diabetes (Nathalie Assayag-Asherie et.al., Can CD44 (CD44, RHAMM) Regulates the Activity of be a mediator of cell destruction ? the challenge of type 1 Inflammation and Cancer. Lausanne: Frontiers diabetes. PLoS One. 2015; 10(12): e0143589) Media. doi: 10.3389/978-2-88919-913-6 Frontiers in Immunology 3 July 2016 | Interaction Between Hyaluronic Acid and Its Receptors Table of Contents 06 Editorial: Interaction Between Hyaluronic Acid and Its Receptors (CD44, RHAMM) Regulates the Activity of Inflammation and Cancer David Naor Hyaluronan: the principal player 10 Hyaluronan synthase 1: a mysterious enzyme with unexpected functions Hanna Siiskonen, Sanna Oikari, Sanna Pasonen-Seppänen and Kirsi Rilla 21 The content and size of hyaluronan in biological fluids and tissues Mary K. Cowman, Hong-Gee Lee, Kathryn L. Schwertfeger, James B. McCarthy and Eva A. Turley 29 Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation Olgun Guvench 38 Hyaluronan – a functional and structural sweet spot in the tissue microenvironment James Monslow, Priya Govindaraju and Ellen Puré 57 4-Methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer Nadine Nagy, Hedwich F. Kuipers, Adam R. Frymoyer, Heather D. Ishak, Jennifer B. Bollyky, Thomas N. Wight and Paul L. Bollyky 68 Lipid raft-mediated regulation of hyaluronan–CD44 interactions in inflammation and cancer Toshiyuki Murai 77 Cancer microenvironment and inflammation: role of hyaluronan Dragana Nikitovic, Maria Tzardi, Aikaterini Berdiaki, Aristidis Tsatsakis and George N. Tzanakakis 84 Hyaluronan, inflammation, and breast cancer progression Kathryn L. Schwertfeger, Mary K. Cowman, Patrick G. Telmer, Eva A. Turley and James B. McCarthy Hyaluronan interaction with its receptors – CD44 and RHAMM 96 The role of CD44 in the pathophysiology of chronic lymphocytic leukemia Julia Christine Gutjahr, Richard Greil and Tanja Nicole Hartmann 103 The where, when, how, and why of hyaluronan binding by immune cells Sally S. M. Lee-Sayer, Yifei Dong, Arif A. Arif, Mia Olsson, Kelly L. Brown and Pauline Johnson Frontiers in Immunology 4 July 2016 | Interaction Between Hyaluronic Acid and Its Receptors 115 The hyaluronic acid–HDAC3–miRNA network in allergic inflammation Youngmi Kim, Sangkyung Eom, Deokbum Park, Hyuna Kim and Dooil Jeoung 120 Interactions between CD44 and hyaluronan in leukocyte trafficking Braedon McDonald and Paul Kubes The pathology of hyaluronan interaction with its receptors and therapeutic strategies related to this interaction 126 Selective hyaluronan–CD44 signaling promotes miRNA-21 expression and interacts with vitamin D function during cutaneous squamous cell carcinomas progression following UV irradiation Lilly Y. W. Bourguignon and Daniel Bikle 139 Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer Suniti Misra, Vincent C. Hascall, Roger R. Markwald and Shibnath Ghatak 170 Modulation of CD44 activity by A6-peptide Malcolm Finlayson 178 The role of CD44 in disease pathophysiology and targeted treatment Andre R. Jordan, Ronny R. Racine, Martin J. P. Hennig and Vinata B. Lokeshwar 192 CD44, hyaluronan, the hematopoietic stem cell, and leukemia-initiating cells Margot Zöller 215 CD44 acts as a signaling platform controlling tumor progression and metastasis Véronique Orian-Rousseau Frontiers in Immunology 5 July 2016 | Interaction Between Hyaluronic Acid and Its Receptors Editorial published: 08 February 2016 doi: 10.3389/fimmu.2016.00039 Editorial: Interaction Between Hyaluronic Acid and Its Receptors (CD44, RHAMM) Regulates the Activity of Inflammation and Cancer David Naor* Lautenberg Center of Immunology, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel Keywords: CD44, RHAMM, hyaluronan, inflammation, cancer The Editorial on the Research Topic Interaction Between Hyaluronic Acid and Its Receptors (CD44, RHAMM) Regulates the Activity of Inflammation and Cancer An old Indian legend tells a story about six blind men who touched an elephant. The first man who touched his leg said: “it is a pillar.” The second man who touched the tail said: “it is a rope.” The third man who touched the trunk of the elephant said: “it is a thick branch of a tree.” The fourth man who touched the ear said: “It is a big hand fan.” The fifth man who touched the belly said: “It is a huge wall.” The sixth man who touched the tusk of the elephant said: “It is a solid pipe.” Each one of them loudly insisted that his claim is the right one. Then wise old man arrived to the place of scene, listened to their arguments and said “all of you are right, but all together you identified an elephant.” Similarly, each one of the 18 chapters of this e-book tells a different, fascinating story about the “biological polygamy” of hyaluronan and its receptors. Yet, this story is focused on the author’s Edited and reviewed by: specific field of interest or discipline. On the other hand, when the 18 chapters are collected in one Pietro Ghezzi, e-book, each of them fosters the others and collectively a complete scene is created. Brighton and Sussex Medical School, UK Hyaluronan or hyaluronic acid (HA), which resides in the interstitial collagenous matrices, increases viscosity and hydration and binds to a “link module motif ” of HA-binding proteoglycans *Correspondence: David Naor (e.g., CD44) and link proteins. HA is a non-sulfated, linear glycosaminoglycan (GAG) composed [email protected] of repeating disaccharides of (β, 1–4)-glucuronic acid (GlcUA) and (β, 1-3)-N-acetyl glucosamine (GlcNAc). In most tissues, native HA has a high molecular mass of 1000–10,000 kDa, with extended Specialty section: molecular lengths of 2–20 μm. HA plays crucial roles in structuring tissue architecture, in cell motil- This article was submitted to ity, in cell adhesion, and in proliferation processes (1, 2). Inflammation, Hyaluronic acid is synthesized by three HA synthase (HAS) proteins. These generate predomi- a section of the journal nantly high molecular weight-HA (HMW-HA) of between 200 and 2000 kDa. HA catabolism is Frontiers in Immunology mediated by hyaluronidases, mechanical forces, and oxidative stress (reactive oxygen and nitrogen Received: 10 January 2016 species). The degradation generates different-sized HA polymers (or fragments), abbreviated low Accepted: 25 January 2016 molecular weight-HA (LMW-HA; <200 kDa) and HA oligomers (1). Published: 08 February 2016 In general (exceptions do exist), LMW-HA is pro-inflammatory and pro-cancerous, whereas Citation: HMW-HA is anti-inflammatory and anti-cancerous. In this context, a vicious cycle is generated. Naor D (2016) Editorial: Interaction Inflammatory conditions activate the production of HAS, which synthesizes HA. Subsequently, Between Hyaluronic Acid and Its the HA is degraded by hyaluronidases and reactive oxygen species, the generation of which is also Receptors (CD44, RHAMM) Regulates the Activity of Inflammation induced by the inflammation. The resulting cleaved HA fragments further propagate the inflamma- and Cancer. tion. This perpetuating cycle can be blocked by competition with excess HMW-HA. To this end, Front. Immunol. 7:39. Tian et al. found that naked mole-rat fibroblasts secrete HMW-HA, which is over five times larger doi: 10.3389/fimmu.2016.00039 (6000–12000 kDa), than human or mouse HA (500–2000 kDa). The HMW-HA accumulates in naked Frontiers in Immunology | www.frontiersin.org 6 February 2016 | Volume 7 | Article 39 Naor Hyaluronic Acid and Its Receptor mole-rat tissues. This rodent has a lifespan exceeding 30 years and adult, so that CD44 targeting is not compensated by functional is resistant to cancer. Interestingly, once HMW-HA is removed RHAMM at this phase of life, and the therapeutic effect by CD44 by knocking down HAS-2, or by overexpressing hyaluronidase targeting, can be documented. 2, which cleaves HMW-HA, the naked mole-rat cells become TLR-4, a principle innate receptor of bacterial LPS, is also susceptible to malignant transformation and form tumors (3). an important receptor of HA (1). TLR-4 activates nuclear factor Notably, the pro-inflammatory role of LMW-HA, including HA (NF)-κB protein via two major routes: a myeloid differentiation fragments and oligo-HA, displays not only pathological effects factor (MyD) 88-dependent pathway that acts via NF-κB to but eventually also physiological activities, such as expression of induce pro-inflammatory cytokines and a MyD88-independent β-defensins to combat microbial infections (4) or induction of pathway that acts via type I interferons to increase the expression inflammation to accelerate wound healing (5). of interferon-inducible pro-inflammatory genes. CD44 glycoprotein is expressed on the surface of many mam- Siiskonen et al. describe the mysterious and unexpected malian cells, including leukocytes, endothelial cells, epithelial functions of hyaluronan synthase 1 (HAS-1), which is less cells, fibroblasts, and keratinocytes. Extensive alternative splicing known and less explored than its two HAS-2 and HAS-3 enzyme of nine variable exons and distinct post-translational modifica- “step brothers,” which also engage in HA synthesis. As HAS-1 tions generate many CD44 isoforms. Standard CD44 is the embryonic gene deletion does not influence the normal pheno- smallest and most abundant isoform, whereas the other variants type, this raises the questions: are its functions compensated (by are expressed in a cell-specific manner (e.g., on epithelial cells redundancy) by the two other HAS enzymes, and if so, why has or keratinocytes), as well as in multiple pathologies, including HAS-1 has been preserved in the course of evolution? rheumatoid arthritis, diabetes, multiple sclerosis, and cancer (2, Receptor (e.g., CD44) sensitivity to hyaluronan quantity and 6). The physiological activities of CD44 stem from its multiple size provides a biosensor of the state of the microenvironment functions, including mediating cell–cell and cell–matrix interac- (inflammation, cancer stroma, or wound healing) surrounding tions, cell proliferation, cell adhesion, cell migration, hemat- the cell. Hence, to learn more on the chemical profile of HA in the opoiesis, lymphocyte activation, cell homing, cell extravasation, context of these parameters or on technologies associated with cell survival, and apoptosis, as well as epithelial–mesenchymal its quantification, specification, isolation, and size determination transition (EMT) (7). However, these functions can be converted in both fluids and tissues, it would be highly beneficial to read to pathological activities when exaggerated or they escape out Cowman et al. communication. of control, like in cancer or chronic inflammation. Most of the Readers interested in the structural alternations associated CD44 studies are limited to preclinical models. However, the with the HA-binding domain (HABD) of CD44 after HA binding, use of anti-CD44 antibodies in a few clinical trials resulted in cannot miss Guvench’s chapter. The authors (Guvench et al.) per- life-threatening toxicity (8). Therefore, the risks vs. the benefits formed extensive all-atom explicit-solvent molecular dynamics must be carefully evaluated before CD44-targeting strategies are (MD) simulations of HABD and the conclusions are presented in translated to the clinic. this communication. However, the HABD was analyzed indepen- The receptor for HA-mediated motility (RHAMM or CD168), dently of the rest of the CD44 molecule, while the transmembrane such as CD44, is also alternatively spliced, albeit at a much domain and especially the cytoplasmic tail influence the binding lower intensity. Variant forms of RHAMM are found on both affinity as well (2, 6). Furthermore, it should be recalled that in cell surfaces and inside the cells (9). However, unlike CD44, this study, the conclusions are limited to HABD interaction with RHAMM isoforms do not have the link module domain. Instead, HA oligomers, whereas larger HA molecules were not evaluated. they have a BX7B motif that binds HA, where “B” represents Monslow et al. comprehensively reviewed the role of HA arginine or lysine, and “X” represents any non-acidic amino acid in health and disease, especially in relation to HA size. Their (10). RHAMM supports both malignancy and wound healing size definition for HA is formulated as follows: HMW-HA: processes. >1000 kDa; intermediate (medium) molecular weight-HA As CD44 supports both chronic inflammation and cancer pro- (MMW-HA): 250–1000 kDa; LMW-HA: >10–250 kDa; and gression in many (but not all) experimental models and human oligo-HA (<10 kDa). However, there is no consensus on these diseases, CD44 targeting, e.g., by antibody, was successfully docu- definitions and standardization of these values by an interna- mented in many preclinical studies, such as collagen-induced tional workshop is necessary. In general, there is a consensus arthritis (CIA) (11). Surprisingly, we found that CD44 targeting that HMW-HA controls normal homeostasis and displays by CD44 gene deletion in the embryo aggravates CIA, rather than anti-inflammatory and anti-cancerous effects, with a few excep- ameliorating it. It appears that a CD44 redundancy process in the tions. Many researchers consider LMW-HA and oligo-HA pro- CD44 deleted embryo allows up-regulation of RHAMM, which inflammatory and pro-cancerous GAGs, as well as stimulators of replaces CD44 also during adulthood. The substituting RHAMM pro-inflammatory cytokines. Yet, there are many contradictory supports CIA joint inflammation more effectively than CD44 findings. This confusion is related to the lack of consensus on (11), because it is a better supporter of cell migration. It is not size definition, polydispersity of HA commercial products (differ- surprising that CD44 targeting in the adult is not redundant, like ent HA sizes in the same product), the use of HA from different in the embryo, as CD44 in the embryo displays a survival-sup- animal sources or from different tissues, and, finally, the impurity porting function that generates pressure for ultimate RHAMM of commercial products. These reservations must be taken into replacement. Such a developmental pressure does not exist in the account whenever a new study on HA is undertaken. Frontiers in Immunology | www.frontiersin.org 7 February 2016 | Volume 7 | Article 39 Naor Hyaluronic Acid and Its Receptor Four-methylumbelliferone (4-MU) is an HA-antagonizing If the reader wants to know how HMW-HA and LMW-HA product, described by Nagy et al. The product inhibits HAS are involved in allergic inflammation, he/she should focus on the synthesis by reducing the availability of UDP-GlcUA to the communication by Kim et al. The reader can surmise, following enzyme, thus, interfering with HA synthesis and consequently extrapolation from the inflammation data, that HMW-HA is with HA-related pathologies, such as cancer and autoimmunity. anti-allergic, whereas LMW-HA is pro-allergic. The mechanisms As 4-MU is an already approved drug called “hymecromone” for underlying these effects, including the role of microRNAs, are biliary spasm, the road to 4-MU therapy of inflammatory diseases reported in detail. and malignancy has been largely paved. McDonald and Kubes describe the cell trafficking roles on Hyaluronan and CD44 reside in the lipid rafts, cholesterol- endothelial cells in the liver, which are different than those in and glycosphingolipid-enriched membrane microdomains that other tissues. Recent evidence implicates serum-derived hyalu- regulate the membrane receptors as well as signal delivery from ronan-associated protein (SHAP) as an important co-factor that the cell surface into the cell. Murai et al. examines in particular strengthens the binding of HA to CD44 under shear stress, result- lipid raft regulation of HA binding to the CD44 of T lymphocytes ing in improved cell extravasation. Finally, the authors indicate and malignant cells, binding, which leads to rolling interactions that HA–CD44 interaction supports not only destructive chronic on vascular endothelial cells, an important phase in inflammation inflammation but also the trafficking of stem cells that resolve and cancer development. the inflammation, the balance between the two determining the If the reader centers his/her interest on the inter-relationship tissue’s fate. between the tumor and its inflammatory microenvironment in Bourguignon and Bikle suggest that the interaction of large context to HA, he/she can be referred to the article by Nikitovic HA (>1000 kDa) with cell surface CD44 leads to Rac-signaling et al. The authors focus their discussion on the influence of the and normal keratinocyte differentiation, DNA repair, and survival cancer inflammatory environment on tumor growth, with spe- function. On the other hand, the interaction of small/fragmented cific emphasis on stromal HA. (10–100 kDa) HA (generated by UV irradiation) with cell surface The interplay between the tumor and its stromal microenvi- CD44 stimulates RhoA/ROC activation, NFκB/Stat-3 signaling, ronment is also documented by Schwertfeger et al., using breast and microRNA-21 production, resulting in proliferation and cancer as an example. The generation of a pro-tumorigenic inflammation, as well as in the progression of squamous cell car- inflammatory environment during breast cancer development cinomas (SCC). A balance that favors the “good” Rac-signaling requires LMW-HA-induced recruitment and activation of inflam- over the “bad” RhoA signaling can be generated by Y27623, a matory macrophages. The macrophages release NFkB-regulated ROK inhibitor, vitamin D, or by triggering HAS-2, which acti- pro-inflammatory factors (IL-1β, IL-12, reactive oxygen species), vates the production of large HA. These therapeutic approaches normally involved in tissue repair. Hence, the cancer cells “stole” may be used for therapy of patients with UV irradiation-skin the inflammation supportive machinery from the wound healing diseases (for more details, see the article). process. Misra et al. comprehensively describe technologies that can be Such inter-relationships between the tumor and its micro- used to modulate the signals of HA–HA receptor interactions in environment are described also in hematological tumors. favor of the patient. A sophisticated approach is Misra’s technol- Gutjahr et al. call our attention to the pro-cancerous survival ogy relating to transferrin-coated nanoparticles, which include (or anti-apoptotic) signals delivered by the tumor inflammatory CD44v6 shRNA, to silence the CD44v6 gene in tumor cells environment, focusing on acute lymphocytic leukemia (CLL). expressing transferrin receptor. Readers, who seek information Long-term survival and proliferation of CLL cells requires on this fascinating approach, or to other therapeutic strategies their dynamic interaction with stromal and immune cells in based on disrupting HA–CD44 interactions and subsequent lymphoid organs. Interactions of HA with cell surface CD44 or signaling, are invited to read this chapter. RHAMM contribute to CLL cell localization, and hence to CLL The use of a CD44-targeting peptide, Ac-KPSSPPEE-NH2, pathophysiology. Deep mining of these complex interactions is another therapeutic strategy, documented by Finlayson, to may reveal links more susceptible to therapeutic targeting, such combat CD44-associated pathological activities in experimental as CD44v6, RHAMM, VLA-4, ZAP-70, or HAS (for details, see vascularized eye, tumor xenografts, or in clinical trials. If the this communication). reader wishes to know more on the peptide’s mechanism of Lee-Sayer et al. focus their attention on the inter-relationships action, it is recommended to read this chapter. between HA and CD44 in cells involved in the innate and adop- Jordan et al. focuses our attention on normal and aberrant cel- tive immune system in the context of inflammation. Under innate lular signaling generated after interaction of HA with its receptor inflammatory conditions, dendritic cells express HA on their (mainly CD44), under different physiological and pathological membrane and T cells upregulate CD44. In the adoptive phase, settings. These include bacterial infection, viral infection, inter- interactions between the HA of the antigen-presenting dendritic stitial lung disease, wound healing, chronic inflammation (auto- cells and the activated CD44 of T lymphocytes may allow intimate immunity), and cancer. The outcome of such aberrant signaling is contact between the co-stimulating molecules of the former and uncontrolled cell migration, cell proliferation, cell survival (e.g., accessory molecules of the latter, leading to activation of the of cancer cells), apoptosis [e.g., of β cells in type 1 diabetes; (12)], lymphocyte’s T cell receptor. Going one step further, the HA and angiogenesis, and EMT, leading to different pathologies. the CD44 molecules may also be considered co-stimulating and Both hematopoietic stem cells (HSCs) and leukemia stem accessory molecules. cells (LSCs), also known as leukemia-initiating cells (LICs) seek Frontiers in Immunology | www.frontiersin.org 8 February 2016 | Volume 7 | Article 39 Naor Hyaluronic Acid and Its Receptor a “shelter” called a bone marrow “niche.” The niche maintains the Dr Orian-Rousseau speculates on the function of CD44 in cancer “ stemness” of the host cells, i.e., supports their survival and hom- stem cells (CSCs), which has so far has been studied as a biomarker ing as well as regulates the balance between their quiescence and for these cells, but its role in CSCs remains elusive. Integration growth. Once HSCs are transplanted into a leukemic patients, of the CD44v6 co-receptor (activated by HA ?) and Met-RTK they eventually compete with LICs for lodging in the niche, (activated by hepatocyte growth factor) with Wnt signaling may engaging their cell surface CD44 in interaction with the HA of explain what could be the role of CSC CD44 in colorectal cancer the niche. In this communication, Zöller raises the question: how and perhaps other malignancies, i.e., by promotion of cell migra- can an advantage be imparted to the transplanted HSCs over the tion and metastasis. patient’s LICs in the context of HA–CD44 interaction, in view of In conclusion, the elephant unveiled in this e-book reveals their largely identical biological nature, when they compete for a fascinating story about the HA–CD44 interaction, which not “shelter” in the same niche. The answer to the question may be only exposes the underlying mechanism of this interaction but found in this communication. also allows identification of weak links, which can be targeted by Orian-Rousseau’s communication is focused on the role of various therapeutic approaches in both cancer and inflammatory CD44 isoforms as co-receptors, especially for receptor tyrosine diseases. kinases (RTK). She further calls our attention to the involve- ment of CD44 in Wnt signaling, both as a regulator of the Wnt AUTHOR CONTRIBUTIONS receptor (via interaction with LRP6) or as a Wnt target gene, e.g., for CD44v6 or Met-RTK expression. Involvement of CD44 The author confirms being the sole contributor of this work and in Wnt signaling, leading to EMT, is also discussed. Finally, approved it for publication. REFERENCES 9. Zhang S, Chang MC, Zylka D, Turley S, Harrison R, Turley EA. The hyaluro- nan receptor RHAMM regulates extracellular-regulated kinase. J Biol Chem 1. 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The use, distribution or dose escalation study with anti-CD44v6 bivatuzumab mertansine in patients reproduction in other forums is permitted, provided the original author(s) or licensor with incurable squamous cell carcinoma of the head and neck or esopha- are credited and that the original publication in this journal is cited, in accordance gus. Clin Cancer Res (2006) 12(20 Pt 1):6064–72. doi:10.1158/1078-0432. with accepted academic practice. No use, distribution or reproduction is permitted CCR-06-0910 which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 9 February 2016 | Volume 7 | Article 39 REVIEW ARTICLE published: 05 February 2015 doi: 10.3389/fimmu.2015.00043 Hyaluronan synthase 1: a mysterious enzyme with unexpected functions Hanna Siiskonen 1 , Sanna Oikari 2 , Sanna Pasonen-Seppänen 2 and Kirsi Rilla 2 * 1 Department of Dermatology, Kuopio University Hospital, University of Eastern Finland, Kuopio, Finland 2 Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland Edited by: Hyaluronan synthase 1 (HAS1) is one of three isoenzymes responsible for cellular hyaluro- David Naor, Hebrew University of nan synthesis. Interest in HAS1 has been limited because its role in hyaluronan production Jerusalem, Israel seems to be insignificant compared to the two other isoenzymes, HAS2 and HAS3, which Reviewed by: Alberto Passi, Università degli Studi have higher enzymatic activity. Furthermore, in most cell types studied so far, the expres- dell’Insubria, Italy sion of its gene is low and the enzyme requires high concentrations of sugar precursors Timothy Bowen, Cardiff University, UK for hyaluronan synthesis, even when overexpressed in cell cultures. Both expression and *Correspondence: activity of HAS1 are induced by pro-inflammatory factors like interleukins and cytokines, Kirsi Rilla, Institute of Biomedicine, suggesting its involvement in inflammatory conditions. Has1 is upregulated in states asso- University of Eastern Finland, Yliopistonranta 1 E, Kuopio 70211, ciated with inflammation, like atherosclerosis, osteoarthritis, and infectious lung disease. Finland In addition, both full length and splice variants of HAS1 are expressed in malignancies like e-mail: [email protected] bladder and prostate cancers, multiple myeloma, and malignant mesothelioma. Interest- ingly, immunostainings of tissue sections have demonstrated the role of HAS1 as a poor predictor in breast cancer, and is correlated with high relapse rate and short overall survival. Utilization of fluorescently tagged proteins has revealed the intracellular distribution pattern of HAS1, distinct from other isoenzymes. In all cell types studied so far, a high proportion of HAS1 is accumulated intracellularly, with a faint signal detected on the plasma membrane and its protrusions. Furthermore, the pericellular hyaluronan coat produced by HAS1 is usually thin without induction by inflammatory agents or glycemic stress and depends on CD44–HA interactions. These specific interactions regulate the organization of hyaluronan into a leukocyte recruiting matrix during inflammatory responses. Despite the apparently minor enzymatic activity of HAS1 under normal conditions, it may be an important factor under conditions associated with glycemic stress like metabolic syndrome, inflammation, and cancer. Keywords: hyaluronan, hyaluronan synthase, CD44, inflammation, cytokines, cancer INTRODUCTION Knockout of Has2 results in embryonic lethality with severe car- Hyaluronan is the most abundant matrix polysaccharide, which diac and vascular malformations (2), while the knockout of Has1 maintains tissue homeostasis, gives compressive strength for tis- or Has3 does not have any apparent phenotype under non-stressed sues, acts as an ideal lubricant in body fluids and accelerates growth conditions (3, 4). However, double knockout of Has1 and Has3 and healing. In addition, excess hyaluronan promotes cancer leads to enhanced inflammation and accelerated wound closure progression and mediates inflammation. Therefore, membrane- of mouse skin (5), suggesting that they are necessary for the bound hyaluronan synthases (HAS1–3), special enzymes respon- regulation of acute inflammation following injury. sible for hyaluronan production, have a key role in regulation A number of recent studies have highlighted the role of HAS1 of these conditions. Despite highly homologous amino acid in health and disease. Interestingly, Has1 was the most upreg- sequences, HAS’s differ in subcellular localization, enzymatic ulated gene in aneuploid mouse embryonic fibroblasts (MEFs) activity, and regulation (1). with malignant properties (6) and splice variants of HAS1 are sug- Despite almost 20 years of active research to sequence hyaluro- gested to contribute to genetic instability (7), suggesting that it nan synthase genes, it is not known why vertebrates have three is susceptible to genetic alterations during oncogenic transforma- different isoforms of these enzymes, which are coded by separate tion. Surprisingly, HAS1 immunostainings of breast carcinoma genes on different chromosomes, to synthesize a single sugar poly- cells correlated with hyaluronan staining, estrogen receptor nega- mer. Most research has focused on HAS2 and HAS3, while HAS1 tivity, HER2 positivity, high relapse rate, and short overall survival. has received the least attention and remains the most enigmatic, In stromal cells of tumors from the same patients, the staining level with only a few published reports of its biological effects on cellular of HAS1 was related to obesity and large tumor size (8). Human behavior or association with disease states. mesenchymal stem cells from different donors express HAS1 in Knocking out the activity of hyaluronan synthase genes has variable but significant levels (9), suggesting its contribution to provided a better understanding about normal HAS function. formation of a hyaluronan niche that maintains stemness of the www.frontiersin.org February 2015 | Volume 6 | Article 43 | 10 Siiskonen et al. HAS1 in cancer and inflammation cells. HAS1 is upregulated during human keratinocyte differentia- membrane of fibroblasts (33), requiring a concurrent efflux of tion (10) and its expression correlates with levels of HA synthesis, K+ ions (34). However, ABC transporters do not seem to con- indicating that HAS1 is an important regulator of skin home- tribute to the translocation of hyaluronan in breast cancer cells ostasis. Furthermore, as compared to other isoforms, differences (35). The Has protein has been shown to produce hyaluronan in in HAS1 substrate requirements (11–13), subcellular localization, a combined process of synthesis and membrane translocation, as and the structure of the hyaluronan coat (7, 13, 14) have been demonstrated by Has reconstituted into proteoliposomes in Strep- reported, suggesting an independent role of HAS1 in the regu- tococcus equisimilis (Se) (36). In addition, there is an intraprotein lation of cell and tissue homeostasis. However, a comprehensive pore in Has and the synthase itself is able to translocate hyaluronan review of HAS1 has not been published. Therefore, the purpose in liposomes containing purified Se-Has (37). of this review is to summarize and discuss the current knowledge of this mysterious enzyme. In this review, the abbreviations Has1 REGULATION OF HAS1 EXPRESSION AND ACTIVITY and Has1 are used for non-human gene and protein, and HAS1 The three HAS genes are often regulated in parallel (38, 39) and and HAS1 for human gene and protein, respectively. the synthesis of hyaluronan reflects changes at the mRNA level (40–44). HAS1 expression is transcriptionally regulated by trans- GENETICS AND FUNCTION OF Has1 GENES AND PROTEINS forming growth factor-β (TGF-β) in synoviocytes (45, 46) and by Hyaluronan is synthesized by HAS enzymes found in vertebrates, the pro-inflammatory cytokine interleukin-1β (IL-1β) in fibrob- some bacteria, and a virus (15). The first Has was cloned in Group lasts (44, 47, 48), while these factors may have similar or opposite A Streptococcus pyogenes and it was predicted to be an integral effects on other HASs, depending on cell type. The nuclear factor membrane protein (16). The first human HAS gene was isolated kappa B (NF-κB) (49) and tyrosine kinases (50) have been shown by two research groups almost simultaneously. Shyjan and co- to be important for IL-1β-induced HAS1 activation, while induc- workers used functional expression cloning in Chinese hamster tion of HAS1 by TGF-β seems to act through the p38 MAPK path- ovary (CHO)-cells (17) and Itano and Kimata screened cDNA way (51). There is evidence that some of the effects are mediated libraries of human fetal brain (18). by transcription-factors sp1 (52) and sp3 (53). Table 1 summa- Mammalian cells have three distinct synthase genes, Has1-3 rizes the growth factors and cytokines that regulate Has1/HAS1 (the human genes are abbreviated here as HAS1-3). They are well- expression. In addition to these factors, ultraviolet B radiation conserved with highly homologous amino acid sequences, but induces a fast up-regulation of Has1 expression in rat epidermal located on separate chromosomes. In humans, HAS1 resides in chromosome 19 at q13.3–13.4, HAS2 is located in chromosome Table 1 | Transcriptional regulation of Has1/HAS1 by different growth 8 at q24.12 and HAS3 is in chromosome 16 at q22.1 (19). HAS1 factors and cytokines (↑ increased, ↓ decreased). gene has five exons, whereas HAS2 and HAS3 both have four (20). Several alternative splice variants of HAS1 have been reported in Agent Cell/tissue HAS1 Reference Waldenström’s macroglobulinemia (21), multiple myeloma (22), and bladder cancer (23). In silico, the HAS1 gene has 46 pos- EGF Human fibroblast ↑ (44) sible transcription-factor binding sites 500 bp upstream of the EGF Human oral mucosal cell ↑ (44) transcription start site (20). FGF2 Human dental pulp ↑ (58) Has1 is not essential for embryogenesis. Has2 knockout mice FGF2 Human periodontal ligament ↑ (59) die at embryonic day 9.5 due to cardiovascular defects (2), but mice FGF Human fibroblast ↑ (60) deficient in Has1 (3) or Has3 (4) are viable and fertile. Further- Forskolin Human orbital fibroblast ↑ (48) more, double knockout Has1 and Has3 mice have been developed IGF Human fibroblast ↑ (60) and are phenotypically normal (5). IL-1β Human fibroblast ↑ (44) The three hyaluronan synthase proteins in humans are des- IL-1β Human fibroblast ↑ (61) ignated as HAS1, HAS2, and HAS3. Mammalian hyaluronan IL-1β Murine uterine fibroblast ↑ (47) synthases are integral membrane proteins with 4–6 transmem- IL-1β Human orbital fibroblast ↑ (48) brane domains in addition to 1–2 membrane-associated domains IL-1β Human dermal fibroblast ↑ (53) (15, 24). The synthase enzymes need Mg2+ or Mn2+ to produce PDGF Human fibroblast ↑ (62) hyaluronan, in addition to the uridine diphosphate (UDP) sugar Progesterone Murine uterine fibroblast ↓ (47) precursors, UDP–glucuronic acid (UDP–GlcUA), and UDP–N - Prostaglandin D2 Human orbital fibroblast ↑ (63) acetylglucosamine (UDP–GlcNAc) (15, 25) The synthesis takes Prostaglandin E2 Human synoviocyte ↑ (64) place at the inner surface of the plasma membrane utilizing cyto- TGF-β Human fibroblast ↑ (65) plasmic precursors (26). Human and mouse enzymes add the TGF-β Human keratinocyte ↑ (65) precursor sugars to the reducing end of the growing polymer (27– TGF-β Human synoviocyte ↑ (46) 29), while amphibian Xenopus laevis Has utilizes the non-reducing TGF-β Human synoviocyte ↑ (45) end (30), like the Pasteurella multocida hyaluronan synthase (31). TGF-β Human dermal fibroblast ↑ (53) It has been suggested that the HAS enzymes do not require Estradiol Human vascular smooth muscle cell ↓ (66) any primers for the synthesis of hyaluronan (32). The adenosine 4-MU Human aortic smooth muscle cell ↓ (67) triphosphate-binding cassette (ABC) transporters have been pro- TGF-β1 Human synoviocyte ↓ (68) posed to be important for hyaluronan translocation on the plasma TGF-β Human mesothelial cell ↑ (40) Frontiers in Immunology | Inflammation February 2015 | Volume 6 | Article 43 | 11 Siiskonen et al. HAS1 in cancer and inflammation keratinocytes (54). Additionally, Has1 expression levels are raised by manipulation of its traffic in live cells by factors like 4-MU and in renal (55) and pulmonary (56) ischemia and hyperglycemia brefeldin A (BFA). (57). The synthesis of hyaluronan by HAS1 is also regulated by A typical subcellular localization pattern of GFP-HAS1 is pre- the substrate concentrations of the precursor sugars (discussed in sented in Figure 1. The GFP–HAS1 signal is mainly cytoplasmic, detail later in this review). rather than on the plasma membrane, being distributed either dif- There is evidence that the activities of HAS2 and HAS3 are fusely or in cytoplasmic patches, and partially co-localizing with regulated by posttranslational modifications like phosphorylation the Golgi apparatus (13, 14, 73). Only a small proportion of the (38, 69), ubiquitination (70), or O-GlcNAcylation (71). Whether total cellular pool of HAS1 is located on the plasma membrane, these modifications are involved in the regulation of HAS1 activity even when activated with glucosamine (12), or inflammatory is not completely known. Phosphorylation seems not to regulate cytokines like TNF-α or IL-1β (13). Occasionally, HAS1 signal is HAS1 activation (72), but HAS1 can exist in multimers of full seen on or near the plasma membrane, usually as patches or con- length-HAS1 or its variants, formed by intermolecular disulfide centrated spots (arrows in Figure 1), or on the plasma membrane bonds (73). protrusions (13, 14). The low plasma membrane signal of HAS1 is The reported length of hyaluronan polymers produced by in parallel with the low activity level of HAS1, because latent HAS each of the mammalian Has differs, but the obtained results enzymes are thought to stay in the ER–Golgi compartment. vary depending on the experimental set-up (74–77). For exam- In addition to the full-length form, HAS1 has multiple tran- ple, in membrane preparations from CHO-cells transfected with script variants resulting from alternative splicing. Transfected recombinant Has isoforms, Has2 produced the largest hyaluronan HAS1V–GFP constructs localize with cytoskeletal structures like (over 3.9 × 106 Da), Has3 produced intermediate length hyaluro- microtubules (7, 73). The reticular localization of the standard nan (0.12–1 × 106 Da), and HAS1 produced the smallest polymer form of HAS1 (Figures 1 and 2) suggests that all forms of HAS1 (0.12 × 106 Da). However, all isoforms produced high molecular studied so far are associated with the cytoskeletal network or endo- weight hyaluronan (3.9 × 106 Da) in live cells (76). The size of the plasmic reticulum, which is a distribution that is not typical for growing hyaluronan chain is increased or decreased by mutation HAS2 or HAS3, and indicates different regulation and binding of certain cysteine or serine amino acids in the Has1 protein in X. partners. laevis, suggesting that the size of the hyaluronan chain is affected The size of the pericellular hyaluronan coat correlates with by the ability of the synthase to bind it (74). activity of hyaluronan synthesis. Interestingly, even high over- expression of HAS1 in cell types with little or no endogenous SUBCELLULAR LOCALIZATION AND TRAFFIC OF HAS1 AND hyaluronan production is not enough to produce a clearly visible ITS IMPACT ON FORMATION OF HA-COAT hyaluronan coat (12, 13, 76). Furthermore, like previously pub- Our understanding of the localization and traffic of Has pro- lished (12–14), the coat produced by HAS1 has a clearly different, teins has been deepened after recruitment of fluorescent HAS more“cloudy”structure (Figures 1 and 2), as compared to the tight fusion proteins together with live cell imaging (78–80). All studies and concentrated coat around plasma membrane protrusions pro- reported so far suggest that like other Has/HAS isoforms, Has1 fol- duced by HAS2, and especially HAS3 (Figure 2). However, the size lows the normal intracellular route from rER to Golgi (78), and its of the coat produced by HAS1 can be induced upon induction traffic is regulated similarly to other HAS isoforms (13), as shown by inflammatory agents or glucosamine (12, 13). The effect of FIGURE 1 | Intracellular localization of GFP–HAS1 and structure of hyaluronan coat (red). Localization of EGFP–HAS1 is shown in pericellular hyaluronan coat induced by GFP–HAS1 overexpression. (A), fHABC in (B), and merged images in (C). Arrows in (A) point patches Confocal optical sections of live MCF-7 breast cancer cells transfected of signal near the plasma membrane. Scale bar 10 µm. Original data with EGFP–HAS1 (green) and stained with fHABC to visualize the published in Ref. (13). www.frontiersin.org February 2015 | Volume 6 | Article 43 | 12 Siiskonen et al. HAS1 in cancer and inflammation FIGURE 2 | Comparison of the structure and intensity of the horizontal optical sections are shown in (D–F) to show the dorsal pericellular hyaluronan coat in MCF-7 cells overexpressing the three protrusions (arrows). The integrated intensity (mean intensity × area) of HAS isoenzymes. Structure of the hyaluronan coat of live MCF-7 cells hyaluronan coat probed with fHABC in the three HAS transfectants was transfected with fusion proteins Dendra2–HAS1 (A,D), Dendra2–HAS2 measured in thresholded area of optical sections through the center of (B,E), and Dendra2–HAS3 (C,F) and labeled with fHABC (red). Single nucleus (G). Mean of three independent experiments is represented (total confocal sections obtained from the middle level of nucleus (blue) are number of measured cells in each group = 92). Magnification bar in (F), shown in (A–C). Vertical views created from compressed image stacks of 10 µm. Original data published in Ref. (12, 81). glucosamine is presented in Figure 3. Additionally, the hyaluro- and oral mucosa (87) have been published recently. In tumor tis- nan coat synthesized by HAS1 is largely dependent on hyaluronan sues, HAS1 is typically expressed in tumor cells (8, 83–85), as well interactions with CD44 (13). as in stromal fibroblasts (Figure 5). The localization of HAS1 is Detailed studies on tissue distribution and subcellular local- mainly intracellular, corresponding to the staining observed in cell ization of endogenous HAS’s have been challenging due to the cultures. Typical staining patterns vary from diffuse to granular lack of reliable antibodies and apparently low expression level with deposits next to the nucleus, which suggests HAS1 accumu- of HAS’s in many cell types. Subcellular localization of endoge- lation in the Golgi area (arrowheads in Figure 5), similar to that nous HAS1 detected with affinity purified polyclonal antibodies seen in cell cultures. shows a similar pattern to exogenously expressed HAS1 fusion proteins (14). HAS1 immunostainings have shown notable levels HAS1 REQUIRES HIGH CELLULAR CONTENT OF UDP-SUGARS of HAS1 in mesothelial cells, fibroblasts (14), and human chon- FOR ACTIVATION drosarcoma cells (9). Furthermore, MEFs have prominent Has1 An important factor affecting activity of all HAS enzymes is the staining (6). Examples of HAS1 immunostainings in cultured cells cytoplasmic availability of substrates, namely, UDP–GlcUA and are summarized in Figure 4. These results are in line with the UDP–GlcNAc. Many studies have shown that treatments influenc- notable mRNA levels of Has1/HAS1 observed in these cell types ing either UDP–GlcUA or UDP–GlcNAc levels regulate hyaluro- (6, 12, 13). nan production [reviewed by Vigetti et al. (88)]. This role of Staining patterns of HAS1 in tissue sections is in line with cell substrates is particularly interesting in regulation of HAS1 as its culture studies. Immunostainings of Has1 in developing tissues activity of hyaluronan production in many cell models is low or (14) and HAS1 in tumor tissues (8, 82–85), endometrium (86), absent unless stimulated. Frontiers in Immunology | Inflammation February 2015 | Volume 6 | Article 43 | 13 Siiskonen et al. HAS1 in cancer and inflammation FIGURE 3 | Glucosamine induces the growth of hyaluronan coat after 6 h incubation with 1 mM glucosamine (D–F). Green, Dendra2–HAS1; produced by HAS1. Confocal optical sections of pericellular hyaluronan coats red, hyaluronan coat; blue, nuclei. Magnification bars 20 µm. Original data on COS-1 cells expressing Dendra2–HAS1 without glucosamine (A–C) and published in Ref. (12, 13). FIGURE 5 | Localization of HAS1 in breast cancer tissue. A paraffin section of breast carcinoma immunostained with HAS1 polyclonal antibody (brown). Nuclei are labeled blue. A mainly cytoplasmic localization of HAS1 is detected in carcinoma cells (asterisks) and in stromal fibroblasts (arrows). Special accumulation of staining is seen intracellularly (arrowheads). FIGURE 4 | Subcellular localization of endogenous HAS1 detected by Magnification bar 50 µm. Original data published in Ref. (8). immunostainings. MCF-7 cells transiently transfected with empty vector (A) and HAS1 expressing plasmid (B), followed by immunostaining with polyclonal HAS1 antibodies (brown color). Arrows in (B) show the HAS1 overexpressing cells. A 3D confocal projection of human chondrosarcoma In order to study the effect of UDP–GlcUA on hyaluro- cell (HCS) (C) and transformed mouse embryonic fibroblast (MEF) (D) nan production, 4-methylumbelliferone (4-MU) and overex- stained with HAS1 immunofluorescence (green). Arrows in (C,D) point pression of enzymes involved in either UDP-Glucose (UDP- plasma membrane protrusions. Blue, nuclei. Magnification bars in (B,D) = 20 µm. Original data published in Ref. (6, 9, 14). glucose pyrophosphorylase) or UDP–GlcUA (UDP–glucose 6-dehydrogenase) production have mainly been used (39, 89, 90). www.frontiersin.org February 2015 | Volume 6 | Article 43 | 14 Siiskonen et al. HAS1 in cancer and inflammation These reports rely mostly on mRNA data to explain the altered Therefore, Has1/HAS1 up-regulation has been noted in many dis- hyaluronan production. The effect of UDP–GlcUA fluctuations eases associated with inflammation such as murine atherosclerosis on HAS1–3 expression levels vary considerably from one cell line (100), human osteoarthritis (101), murine infectious lung disease to another and it is often impossible to reveal the exact role of (102), and human rheumatoid arthritis (45). HAS1 expression is HAS1 during these changes. A recent investigation demonstrated also increased, among several other genes, in osteophytic chondro- that availability of UDP–GlcUA can have a direct effect on HAS1 cytes (103). Interestingly, the expression of both HAS1 and HAS2 activity, as treatment of MCF-7 cells overexpressing HAS1 with an was reduced in the synovium of patients with osteoarthritis or inducing agent and 4-MU significantly decrease hyaluronan coat rheumatoid arthritis compared to healthy controls (104). More- compared to cells treated with the inducing agent only (13). It has over, elevated HAS1 expression is observed in oral lichen planus, been reported that Has1 has a lower affinity for UDP–GlcUA than which is a chronic inflammatory disease of the oral mucosa (87). other Has’s, and the Km of Has1 is about double that of Has2–3. It is worth noting that in oral lichen planus the increased HAS1 Interestingly, availability of the other substrate, UDP–GlcNAc, did expression is detected in the basal layers of the epithelium, which not considerably influence the Km of Has1 toward UDP–GlcUA, is the most affected, inflamed area in lichen planus. whereas levels of UDP–GlcUA did have a significant effect of the It is not known whether the product of HAS1 enzyme of certain Km toward UDP–GlcNAc (11). polymer length, HAS1 enzyme itself or hyaluronan with HAS1 The affinity of Has1 for UDP–GlcNAc is lower than the affinity and hyaluronan binding proteins like CD44 mediate the pro- of Has2–3 as with UDP–GlcUA. The Km toward UDP–GlcNAc of inflammatory responses. One explanation for HAS1 involvement Has1 is about two to three times higher that of the other Has’s. in inflammation might be that HAS1 is associated with production Interestingly, all Has enzymes exhibit lower affinity toward UDP– of a special type of pericellular hyaluronan coat, which is pro- GlcNAc than for UDP–GlcUA (11). Treatments with compounds inflammatory. Recently, Siiskonen and co-workers showed that like mannose and glucosamine that regulate UDP–GlcNAc content inflammatory agents and glycemic stress induce HAS1 to pro- also affect cellular hyaluronan secretion levels (12, 91). Simi- duce an expanded pericellular hyaluronan coat (13). Compared to lar to the level of UDP–GlcUA, the availability of UDP–GlcNAc Has3-induced hyaluronan coat, which is rather tight and formed influences both mRNA levels and activity of all HAS’s. The differ- around microvillus protrusions (105), HAS1 produces a looser, ences in substrate affinities are well demonstrated in intact cells but extensive pericellular hyaluronan coat, which is dependent on using HAS1 overexpressing cell lines. Both COS-1 and MCF-7 CD44. In several cell types, these types of hyaluronan coats have cell lines have negligible endogenous hyaluronan production, and been shown to associate with monocyte binding (106, 107). It has even overexpression of HAS1 enzymes does not cause prominent even shown that hyaluronan produced by Has1 binds mononu- changes in it. Upon treatment with glucose or glucosamine, com- clear cells more effectively than hyaluronan produced by the two pounds that increase the amounts of hyaluronan substrates, the other Has enzymes (77). This could provide an explanation for the HAS1 enzyme is able to produce significant amounts of hyaluro- central role of HAS1 in inflammation. nan (12, 13). Furthermore, this effect of substrate availability on In rheumatoid arthritis, the rate of hyaluronan synthesis is HAS1 activity is dose dependent (12). altered. Hyaluronan accumulates in joints affected by rheumatoid The above mentioned findings on the regulation of HAS1 arthritis, which causes periarticular swelling and morning stiffness activity point out that although HAS1 has a minor role in total cel- (108). In synoviocytes isolated from RA patients, HAS2 and HAS3 lular hyaluronan production, it may have significant effects when are constitutively activated, but HAS1 is the gene that responds induced by increased substrate availability. Since the affinity of readily to pro-inflammatory cytokines like IL-1β (49) and TGF- HAS1 for its substrates is lower compared to the two other HAS’s, β (45). However, IL-1β is not able to stimulate Has1 expression the fluctuations in UDP–GlcNAc and UDP–GlcUA levels can have in healthy synoviocytes like in type-B synoviocytes isolated from a more significant effect on HAS1 than on HAS2–3. rheumatoid arthritis patients (49, 109). This IL-1β-induced HAS1 up-regulation is dependent on the activation of the transcription- HAS1 AS A MEDIATOR IN INFLAMMATION factor NF-κB (49), like many other pro-inflammatory molecules. Many recent results suggest HAS1 may play a pivotal role during In type-B synoviocytes, IL-1β stimulation induces the transloca- cell stress, such as inflammation. Earlier in this frontiers review tion of NF-κB into the nucleus, which results in up-regulation series, Petrey and de la Motte comprehensively discussed the role of HAS1 mRNA expression (49). Similarly, in fibroblast-like syn- of hyaluronan in inflammation (92). Whether hyaluronan acts oviocytes, viral infection causes NF-κB activation and increased as a pro- or anti-inflammatory molecule is highly dependent HA release due to HAS1 up-regulation. This HAS1 up-regulation on its molecular size. Generally, low-molecular weight hyaluro- is reversed with mitogen-activated protein kinase p38 and JNK nan fragments mediate pro-inflammatory responses (93) such as inhibitors indicating that viral RNA activates HAS1 through these recruitment of macrophages and other leukocytes to the injured signaling pathways (110). Moreover, HAS1 activation is blocked or inflamed tissue (94, 95) and stimulate transcription of genes with commonly used anti-inflammatory drugs, hydrocortisone, related to inflammation including several cytokines and matrix and dexamethasone, in TGF-β stimulated synoviocytes (51). In metalloproteinases (96). Growth factors and pro-inflammatory these cells, glucocorticoids block p38 activation, which results in cytokines (Table 1) released during inflammation, like TGF-β, IL- suppressed HAS1 expression (51). Interestingly, sodium salicylate 1β, and TNF-α, which stimulate inflammatory cells also induce inhibits IL-1β induced HAS1 activation and HA release in type-B expression of HAS1 (44, 45, 64) and Has1 (97). Expression of synoviocytes (64). This might explain some of the beneficial effects HAS1 is also upregulated in response to prostaglandins (98, 99). of sodium salicylate in the treatment of rheumatoid arthritis. Frontiers in Immunology | Inflammation February 2015 | Volume 6 | Article 43 | 15 Siiskonen et al. HAS1 in cancer and inflammation In addition to its role in rheumatoid inflammation, altered so far is the artificial overexpression of fluorescently tagged HAS1 HAS1 levels contribute to other inflammation-related states. In in cells with low levels of HAS enzymes (12–14). murine models of asthma, Has1 mRNA is increased at an early Interestingly, HAS1 overexpression in many epithelial cell types stage, but later decreased (111, 112). In thyroid dysfunction asso- has shown a low activity in normal culture conditions, without ciated with activation of the thyrotropin receptor, hyaluronan is addition of glucosamine or inflammatory cytokines. This suggests accumulated through up-regulation of HAS1 and HAS2 (113). that these cell types may lack factors that are crucial for HAS1 Taken together, HAS1 seems to be fundamentally involved in the activity. Several studies suggest that HAS1 has a low capacity to inflammatory processes. However, many questions are still waiting retain hyaluronan chains on the plasma membrane, thus other for an answer. molecules may be required to retain hyaluronan chains on the plasma membrane and assemble the hyaluronan coat. A potential HAS1 AS A PREDICTOR OF CANCER PROGRESSION molecule for these interactions is CD44, which seems to play a Hyaluronan content is known to be increased in many cancers, special role in the formation of the HAS1-induced coat (13). which may be altered due to hyaluronan synthase expression. The complexity of hyaluronan metabolism, existence of three Few studies have shown a direct association of HAS’s with can- isoenzymes, and the crucial role of HAS2 make it complicated cer progression in vivo, but interestingly, HAS1 associates with to study the biological effects of HAS1 in animal models. Further- tumor progression and prognostic factors in many cases. Increased more, since most human tissues and cells express all HAS isoforms, expression of HAS1 is associated with poor patient survival in it is impossible to get comprehensive answers and make conclu- ovarian cancer (114, 115), colon cancer (116), Waldenström’s sions on the role of a single isoenzyme. Furthermore, many cells macroglobulinemia (21), and multiple myeloma (22). In multiple and tissues express low or negligible levels of HAS1 mRNA. How- myeloma and Waldenström’s macroglobulinemia, the occurrence ever, variable sensitivity of the methods used and other limitations of HAS1 splice variants, rather than the full length HAS1, is may explain the low or absent HAS1 levels detected in some cases. related to cancer prognosis. HAS1 expression is also increased Several trials have been done to solve the function and reg- in bladder cancer, correlating with increased hyaluronan levels ulation of this puzzling enzyme. Evidently, HAS1 is an impor- (23), and predicting metastasis (117). In bladder cancer, HAS1 has tant regulator during inflammation and in states with altered been shown to modulate HA and CD44 levels, affecting tumor sugar metabolism. However, contradictory results raise several growth and progression (118). Accumulation of hyaluronan is new questions, which need to be resolved before we can elucidate associated with poor patient survival in breast cancer (119, 120). the exact role of HAS1. Recently, HAS1 and HA stainings were found to correlate with each other in breast carcinoma cells of these tumors, and HAS1 ACKNOWLEDGMENTS was associated with estrogen receptor negativity, HER2 positivity, The authors gratefully acknowledge financial support from the high relapse rate, and short overall survival. In addition, expres- Academy of Finland (grant 276426), Spearhead Funds from the sion levels of stromal HAS1 and HAS2 were related to obesity, University of Eastern Finland (Cancer Center of Eastern Finland), large tumor size, lymph node positivity, and estrogen receptor Saimaa Cancer Foundation, Cancer Foundation of Northern Savo, negativity (8). and the Special Government Funding of the Kuopio University In serous ovarian tumors, HAS1 has been shown to be very low Hospital. or totally absent, whereas the levels of HAS2 and HAS3 mRNA REFERENCES or staining levels are not elevated compared to normal ovaries or 1. Tammi RH, Passi AG, Rilla K, Karousou E, Vigetti D, Makkonen K, et al. Tran- benign tumors (83). Interestingly, the levels of HAS1 and HAS2 scriptional and post-translational regulation of hyaluronan synthesis. FEBS J immunostainings are decreased in melanomas, correlating with (2011) 278(9):1419–28. doi:10.1111/j.1742-4658.2011.08070.x 2. 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Am J Pathol (2000) 156(2):529–36. 2014.04.003 doi:10.1016/S0002-9440(10)64757-8 103. Gelse K, Ekici AB, Cipa F, Swoboda B, Carl HD, Olk A, et al. Molecu- 120. Auvinen P, Tammi R, Kosma VM, Sironen R, Soini Y, Mannermaa A, et al. lar differentiation between osteophytic and articular cartilage – clues for Increased hyaluronan content and stromal cell CD44 associate with HER2 a transient and permanent chondrocyte phenotype. Osteoarthritis Cartilage positivity and poor prognosis in human breast cancer. Int J Cancer (2013) (2012) 20(2):162–71. doi:10.1016/j.joca.2011.12.004 132(3):531–9. doi:10.1002/ijc.27707 Frontiers in Immunology | Inflammation February 2015 | Volume 6 | Article 43 | 19 Siiskonen et al. HAS1 in cancer and inflammation 121. Karjalainen JM, Tammi RH, Tammi MI, Eskelinen MJ, Ågren UM, Parkki- Citation: Siiskonen H, Oikari S, Pasonen-Seppänen S and Rilla K (2015) Hyaluronan nen JJ, et al. Reduced level of CD44 and hyaluronan associated with unfavor- synthase 1: a mysterious enzyme with unexpected functions. Front. Immunol. 6:43. doi: able prognosis in clinical stage I cutaneous melanoma. Am J Pathol (2000) 10.3389/fimmu.2015.00043 157(3):957–65. doi:10.1016/S0002-9440(10)64608-1 This article was submitted to Inflammation, a section of the journal Frontiers in Immunology. Conflict of Interest Statement: The authors declare that the research was conducted Copyright © 2015 Siiskonen, Oikari, Pasonen-Seppänen and Rilla. This is an open- in the absence of any commercial or financial relationships that could be construed access article distributed under the terms of the Creative Commons Attribution License as a potential conflict of interest. (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this Received: 08 December 2014; accepted: 22 January 2015; published online: 05 February journal is cited, in accordance with accepted academic practice. No use, distribution or 2015. reproduction is permitted which does not comply with these terms. www.frontiersin.org February 2015 | Volume 6 | Article 43 | 20 REVIEW published: 02 June 2015 doi: 10.3389/fimmu.2015.00261 The content and size of hyaluronan in biological fluids and tissues Mary K. Cowman 1 *, Hong-Gee Lee 1 , Kathryn L. Schwertfeger 2 , James B. McCarthy 2 and Eva A. Turley 3,4,5 1 Department of Chemical and Biomolecular Engineering, Biomatrix Research Center, New York University Polytechnic School of Engineering, New York, NY, USA, 2 Department of Laboratory Medicine and Pathology, Masonic Comprehensive Cancer Center, University of Minnesota, Minneapolis, MN, USA, 3 Department of Oncology, London Health Sciences Center, Schulich School of Medicine, Western University, London, ON, Canada, 4 Department of Biochemistry, London Health Sciences Center, Schulich School of Medicine, Western University, London, ON, Canada, 5 Department of Surgery, London Health Sciences Center, Schulich School of Medicine, Western University, London, ON, Canada Hyaluronan is a simple repeating disaccharide polymer, synthesized at the cell surface by integral membrane synthases. The repeating sequence is perfectly homogeneous, and is Edited by: the same in all vertebrate tissues and fluids. The polymer molecular mass is more variable. David Naor, Most commonly, hyaluronan is synthesized as a high-molecular mass polymer, with an Hebrew University of Jerusalem, Israel average molecular mass of approximately 1000–8000 kDa. There are a number of studies Reviewed by: Feng Gao, showing increased hyaluronan content, but reduced average molecular mass with a Shanghai Sixth People’s Hospital, broader range of sizes present, in tissues or fluids when inflammatory or tissue-remodeling China processes occur. In parallel studies, exogenous hyaluronan fragments of low-molecular Shibnath Ghatak, Medical University of South Carolina, mass (generally, <200 kDa) have been shown to affect cell behavior through binding USA to receptor proteins such as CD44 and RHAMM (gene name HMMR), and to signal Robert Stern, Touro College of Osteopathic either directly or indirectly through toll-like receptors. These data suggest that receptor Medicine, USA sensitivity to hyaluronan size provides a biosensor of the state of the microenvironment Antonio Rosato, surrounding the cell. Sensitive methods for isolation and characterization of hyaluronan University of Padova, Italy and its fragments have been developed and continue to improve. This review provides an *Correspondence: Mary K. Cowman, overview of the methods and our current state of knowledge of hyaluronan content and Biomatrix Research Center, New York size distribution in biological fluids and tissues. University, 433 First Avenue, New York, NY 10010 USA Keywords: hyaluronan, quantification, assay, molecular mass, molecular weight [email protected] Specialty section: Introduction This article was submitted to Inflammation, a section of the journal Hyaluronan (hyaluronic acid, HA) is found in vertebrate tissues, as a key component of the Frontiers in Immunology extracellular matrix. It has a simple covalent structure consisting of alternating β--glucuronate Received: 16 February 2015 and N-acetyl-β--glucosamine sugars. The linear anionic polymer has a semi-flexible structure, Accepted: 11 May 2015 causing it to adopt an expanded wormlike random coil. The domain of a coiled chain can be Published: 02 June 2015 conceptually described as a sphere [mostly filled with (unbound) solvent], with a dynamically Citation: changing chain configuration. The apparent volume occupied by a single isolated molecule depends Cowman MK, Lee H-G, strongly on the chain length, and thus the molecular mass, M. The volume increases approximately Schwertfeger KL, McCarthy JB and as M raised to the 1.8 power, as dictated by polymer chain statistics (1). Where HA chains Turley EA (2015) The content and size of hyaluronan in biological fluids and are crowded together, their domains are forced to interpenetrate, and this leads to severe non- tissues. ideality in behavior. The non-ideality determines such properties as the large colloid osmotic Front. Immunol. 6:261. pressure, viscoelasticity, and effect on partition of other macromolecules (excluded volume) in the doi: 10.3389/fimmu.2015.00261 biomatrix. Since the molecular mass of HA in normal biological fluids and tissues is normally Frontiers in Immunology | www.frontiersin.org 21 June 2015 | Volume 6 | Article 261 Cowman et al. Hyaluronan content and size very high (ca. 1000–8000 kDa), the non-ideality effects domi- Isolation of HA nate the physicochemical properties of HA in the extracellular matrix (2–4). There are a number of methods appropriate for assaying the In addition to the physicochemical effects due to mutual macro- content and size of HA in biological fluids and tissues. Depending molecular crowding, HA has important binding interactions. At on the method, it may be necessary to purify HA to remove/digest the cell surface, HA provides a protective coat that is tethered bound proteins and sulfated glycosaminoglycans prior to assay. to receptors embedded in the cell membrane (5–7). Beyond the The isolation of HA follows protocols (25, 30, 32–35) that are cell surface, HA organizes proteoglycans (e.g., versican, aggre- quite similar to those historically employed in the purification of can) and other binding proteins via specific non-covalent inter- DNA. The requirement for specific steps depends on the nature actions, creating a further highly hydrated and charged domain of the sample: fluid tissue vs. conditioned medium from cell (8–10). In inflammation and other specific tissue-remodeling culture vs. solid tissue. For solid tissues, the HA is extracted into processes, covalent transfer of the heavy chain domains of IαI soluble form, and liberated from proteins. Protein removal can to HA can be catalyzed by TSG-6 protein (11–14). The HA- be accomplished by digestion with a protease, or by denaturing protein assemblies, whether covalently or non-covalently medi- the protein by gentle mixing with chloroform. Lipids are removed ated, are integral to maintenance of an expanded pericellular with acetone or other organic solvent mixtures. Removal of low- matrix. molecular mass contaminants may require dialysis, or precipita- Normally, HA has a high turnover rate (15, 16). Thus, the extra- tion of the HA with ethanol or isopropyl alcohol. DNA and RNA cellular environment is constantly renewed. The need for renewal can be enzymatically digested. There are many variations on these may reside in the protective role HA fulfills. Facile degradation steps. A sample protocol for extraction of HA from solid tissue of HA by reactive oxygen and nitrogen species (ROS/RNS) (17, might include the following steps: digestion with a protease such as 18) during active inflammation can weaken the protective HA proteinase K, boiling to denature enzyme, centrifugation, extrac- coat that usually protects the cell. The HA acts as a scavenger of tion with chloroform, centrifugation, dialysis, precipitation with damaging free radicals and other chemical agents. If the rate of HA ethanol, centrifugation, re-dissolution, digestion with Benzonase synthesis keeps pace with the rate of degradation and turnover, the (or DNase plus RNase), boiling to denature enzyme, and repeat of homeostatic environment is maintained (19). steps starting with chloroform extraction. Abbreviated protocols When the rate of HA degradation is not adequately com- can be used for fluid samples, or where the HA needs to be pensated by its synthesis, fragments of the polymer might be liberated but not purified because specific assay will be employed. present at significant levels and consequently cells are poorly The above purification will not remove other glycosamino- protected. Changes in the physicochemical control of the pericel- glycans. Sulfated glycosaminoglycans can be removed by anion lular environment take place. HA fragments compete in binding exchange chromatography. Unsulfated or undersulfated chon- interactions with proteins, altering the integrity of the bioma- droitin, which is rare in normal tissues but may be significant in trix. Fragments can displace high M HA in interactions with remodeling tissues, is not removed by this process. At this point cell surface receptors, resulting in changes in receptor clus- in the procedure, specific isolation of HA can be accomplished by tering and altered signaling (20). Fragments can also signal affinity methods, such as use of a biotinylated HA-specific binding through alternate receptors (21–23). In these ways, HA may protein and streptavidin-coated magnetic beads, or other similar be regarded as a biosensor of damaging processes in the cel- medium such as gel beads (36, 37). lular microenvironment. Altering the balance of high and low It is worth noting that most isolation methods in current use M HA is a stimulus that sets in motion multiple cellular have not been validated with respect to quantitative yield of HA, or response mechanisms. These can be purely defensive, such as HA preferential extraction/isolation of specific HA sizes. In particular, fragment-induced expression of β-defensins to combat micro- losses of very low M HA may be significant in some procedures. bial infection in the gut (24, 25). But sustained responses can It is also possible to degrade HA during isolation. Endogenous also lead to chronic inflammation via aberrant signaling through enzymes may cause some of this degradation. However, most receptors and consequently increased expression of inflammation degradation is the result of ROS generation catalyzed by contami- mediators (26). nating iron (II) or copper (I), and molecular species that regen- Tissue-remodeling processes, including wound healing and erate the active metal ion oxidation states. Thus, use of papain tumor progression, are associated with changes in HA content and activated with cysteine can lead to HA degradation (38). The pres- size (27–31). HA synthesis is usually increased during remodeling, ence of EDTA can also enhance the ability of contaminating iron but increased expression of hyaluronidases may also occur, and to catalyze formation of hydroxyl radicals. Iron contamination is together with macrophage-generated ROS/RNS, degrade HA. The better inactivated by chelation with deferoxamine (34). Also, of balance of high and low M HA may differ from the homeostatic note, EDTA and phosphates can be co-precipitated with HA using case, thus altering both the physicochemical and signaling effects ethanol. of HA. To understand HA biology, we are faced with multiple Testing for degradation of HA during isolation can be easily questions: (1) What is the content of HA present, (2) What is the accomplished by “spiking” the initial fluid or tissue with a pure molecular mass distribution of the HA, and (3) Can we control HA sample of known M and low polydispersity in M, and then pathological processes by altering the content, size, and binding testing its size in the final isolate. Spiking samples with known interactions of HA? amounts of HA can also be used to detect losses during isolation, Frontiers in Immunology | www.frontiersin.org 22 June 2015 | Volume 6 | Article 261 Cowman et al. Hyaluronan content and size including losses due to non-specific interactions with surfaces or usually a protein or proteoglycan. The soluble HA competes with other macromolecules that HA may not normally contact. immobilized surface HA for the specific binding agent, so that the resulting surface-bound amount of the binding agent is a measure of the amount of soluble HA in the sample being analyzed (41, 44, Methods to Analyze Content of HA 45). There are multiple possible detection schemes to quantify the bound agent. For example, if aggrecan proteoglycan is the specific The most simple and historical assay for HA is measurement of binding agent, it can be quantified with an antibody to the keratan uronic acid content. The assay involves hydrolysis in concentrated sulfate chains of aggrecan, and a suitably labeled second antibody. sulfuric acid, so that protein content is not a problem. Other When the specific binding agent is a labeled (e.g., biotinylated) glycosaminoglycans that contain uronic acid will contribute to the HABP, it can be quantified by binding of the label to a specific result, and should be separated or removed from HA if possible. agent such as streptavidin, which is, in turn, conjugated to an The uronic acid assay has been widely employed, especially in enzyme or other detectable species. Radiolabeled HABP may also analysis of fluids (synovial fluid, vitreous) with high HA and low be used but is less desirable on the basis of safety and disposal. sulfated glycosaminoglycan content. Because the recognition step in a competitive binding assay occurs Hyaluronic acid content can be also determined by analysis in solution, HA chains as short as approximately decasaccharides of the oligosaccharide products of enzymatic digestion. Quan- can be accurately detected, depending on the labeled binding tification is accomplished by methods such as HPLC, capillary protein used. The results of sandwich and competitive assays have electrophoresis (CE), mass spectrometry, or fluorophore-assisted been shown to be in good agreement for high M HA (46). carbohydrate electrophoresis. These methods have primarily been employed to determine relative amounts of different glycosamino- glycans in a sample, rather than absolute quantities. HA Content in Biological Fluids and Tissues The most sensitive, specific, and accurate methods for determi- nation of HA content are based on enzyme-linked sorbent assays The content of HA in many normal biological fluids has been (ELSA, ELISA-like assays) (39–47). The specific detection of HA determined. Here, we cite a few relevant results. HA is a major is an important step of these methods, because purification of low component of articular joint synovial fluid, where it provides the M HA is difficult, and contaminants interfere with non-specific viscoelasticity and lubrication necessary for protection of cartilage detection modes. The specificity is based on the use of molecular surfaces. Its concentration in the human knee joint is approxi- species such as proteins or proteoglycans that recognize and bind mately 2–3 mg/ml, being slightly higher in younger adults than in HA but no other biological molecules. For example, the aggrecan older adults (48–50). HA is also a major component of the vitreous proteoglycan binds HA specifically (8, 9). The intact proteoglycan body of the eye, but at a lower concentration of approximately may be used, or a terminal fragment called globular domain 1 – 200 μg/ml, in the phakic human eye vitreous (51). The concentra- interglobular domain – globular domain 2 (G1–IGD–G2), often tion in the aqueous humor is lower still, being only about 1 μg/ml referred to as HA-binding protein (HABP) or HA-binding region (52). Human lymph fluid contains HA at a concentration of about (HABR). The link protein, also called CRTL1 or HAPLN1, is 0.1–18 μg/ml (36). In the blood serum of healthy human adults, similar to the G1 domain of aggrecan, and is another suitable the concentration of HA is lower still, being usually between 10 protein for specific detection of HA. Isolated HABP, usually a and 100 ng/ml, mostly 20–40 ng/ml, and averaging about 30 ng/ml mixture of the aggrecan HABR and the link protein, may also (36, 40, 44). Normal human urine also contains a low level of HA, be used. Similarly, versican proteoglycan G1 domain is useful. around 100–300 ng/ml (44), and human milk similarly contains Hyaluronectin, a HA-specific binding protein isolated from brain, HA at about 200–800 ng/ml (25). may be used. Recently, a recombinant fusion protein of human The HA content of solid tissues varies widely. Bovine nasal TSG-6 and the Fc domain of human IgG, and a second variant of cartilage contains approximately 1200 μg HA/g wet tissue weight the fusion protein in which the heparin-binding region of TSG- (44). The HA content of human articular cartilage is similar, being 6 was mutated to become inactive, were found to be suitable about 500–2500 μg/g (53). Human skin contains approximately for development of a specific HA assay (47). Other HA-specific 400–500 μg HA/g tissue, mostly in the dermis (54). Fetal skin binding molecules could be used. and young skin have higher HA contents than older skin. Other There are two types of ELSA. The first type, sandwich assays, organs have much less HA. Laurent and Tengblad (44) reported are sensitive and reproducible, but fail to adequately quantify low HA contents of approximately 1–100 μg HA/g wet tissue weight M HA (39–42). This is because the plate surface, coated with an for most organs. Rabbit kidney had 103 μg/g, brain had 65 μg/g, HABP, strongly binds HA, but does not allow further probing of muscle had 27 μg/g, liver had 1.5 μg/g, and cornea had 1.3 μg/g. short HA chains by the detector protein (43). Longer HA chains Armstrong and Bell (34) also reported rabbit tissue HA contents of have accessibility as a result of looped sections above the surface. 500 μg/g for skin, 200 μg/g for large intestine and heart, 130 μg/g (The same problem occurs in HA blotted to positively charged for small intestine, and 80–90 μg/g for lung and muscle tissues. nylon membranes after electrophoresis, from which short HA Measurement of HA content is of continuing high interest, chains cannot be detected.) The second type of ELSA is com- because there are multiple studies correlating changes in HA con- petitive assays. In these, HA is usually immobilized on a surface tent with tissue remodeling and pathological processes. While the such as the wells of a plastic 96-well plate. Alternative surfaces normal HA concentration in human serum is usually <40 ng/ml, it are suitably modified magnetic beads. Soluble HA samples, either is elevated (>46.5 ng/ml) in hepatic cirrhosis (55), in rheumatoid standards or unknowns, are mixed with the specific binding agent, arthritis (56, 57) (highly variable; reports up to nearly 200 μg/ml, Frontiers in Immunology | www.frontiersin.org 23 June 2015 | Volume 6 | Article 261 Cowman et al. Hyaluronan content and size but more generally between 0.07 and 6.4 μg/ml), in ankylos- uses of solutions of high molecular mass HA as a viscosurgical ing spondylitis (57) (7–13 μg/ml), and in osteoarthritis (57, 58) tool in ophthalmic surgery, and as an analgesic treatment for (0.04–2.3 μg/ml). The elevated HA concentration in serum of osteoarthritis, are based on this understanding. More recently, patients with hepatic cirrhosis is utilized as one component of the discovery that exogenous HA fragments can alter cellular a diagnostic assay. A small but significant elevation (frequently, behavior by signaling through multiple receptor proteins, and that about twofold) of HA in serum is found in multiple types of the existence of such fragments in vivo is likely, based on increased untreated cancer (59–61). Radical surgery to remove the tumor hyaluronidase levels and reactive oxygen and nitrogen species in causes the HA concentration in serum to return to the normal tissue remodeling and pathological processes, has led to increased range. Most interestingly, it was found that the low-molecular interest in measuring the size distribution of HA in biological mass component of serum HA can be used to differentiate fluids and tissues. metastatic from non-metastatic breast cancer (62), which may Many current methods for determination of the M distribution form the basis of a new diagnostic test. of HA from tissues and biological fluids have been optimized In solid tissues, many but not all cancers progress in a tumor for highly purified HA. A commonly employed method used microenvironment of increased HA content (28). Further, some commercially is size exclusion chromatography with multiangle non-aggressive cancer types such as non-malignant fibroadenoma laser light scattering (SEC-MALLS) (76, 77). However, detection produce elevated HA (63, 64). The presence of HA may therefore of very low M HA by light scattering is inherently insensitive, and not be sufficient by itself to promote tumorigenesis. However, the SEC-MALLS method requires a highly purified HA sample. high levels of HA accumulate in lung, colorectal, prostate, bladder, CE (78) is similarly limited to pure HA samples. MALDI-TOF and breast carcinomas and in these cancers are linked to tumor mass spectrometry (79, 80) has high sensitivity, but requires a pure aggression (28). For example, the HA content of human lung tissue sample and HA with M larger than about 10 kDa becomes difficult increases 4- to 200-fold in lung carcinoma (65), 100-fold in grade to analyze. A new method that has extremely high sensitivity and 3 ovarian cancer (66) and 7-fold in prostate cancer (67). Increased works best for low M HA is gas-phase electrophoretic mobility tumor HA accumulation is also linked to tumor aggression. The molecular analysis (GEMMA), but it still requires pure HA (81). HA content of malignant ovarian epithelial tumor correlates with The most widely used methods, to date, for size distribution tumor grade and with metastasis. Elevated HA accumulation analysis of imperfectly pure HA isolated from biological samples within the stroma or tumor parenchyma of breast cancer is associ- are size exclusion chromatography with enzyme-linked sorbent ated with unfavorable prognosis of the patient. Recent studies have assay (SEC-ELSA) (36, 56, 82), and agarose or polyacrylamide further linked high stromal HA staining to HER2 positive tumors gel electrophoresis (83–86) with staining or with blotting and and poor outcome parameters including time to relapse, large specific detection. Both methods are capable of detecting a wide tumor size, lymph node positivity, hormone receptor negativity, range of HA sizes. Gel electrophoresis with staining can analyze high body mass, and shortened overall survival (68). Elevated samples on the microgram scale, and can tolerate some impurities HA in the tumor microenvironment is linked to inflammation in the sample, but non-specific staining by those impurities can (69). Thus, high amounts of both tumor-associated macrophages interfere with size distribution analysis of the HA. Blotting of gels and HA are concurrent in breast carcinoma. High macrophage to positively charged nylon and detection of HA using a labeled numbers correlate with high tumor HA, HAS expression and poor specific binding protein works only for HA with M >100 kDa, as a outcome, suggesting that HA facilitates a macrophage tumor sup- result of strong surface binding (43). To address the issues of lim- porting function in breast cancer. The link between inflammation ited sample amount, purification difficulty, and the importance of and cancer has led to recent interest in HA as a contributor to analyzing both high and low M HA simultaneously, we recently a pro-tumorigenic inflammatory environment, as detailed in a developed a method using size-dependent fractionation of HA by companion article in this issue (70). anion exchange on a spin column, and quantification of HA in As for cancer, wound healing and fibrosis are associated with the fractions using a competitive ELSA assay (IEX-ELSA) (37). All inflammation and increased HA content (71). An approximately of these methods require calibration with purified HA samples of twofold increase was observed in HA content of rat skin during known size. healing of excisional wounds (30). Similarly, scleroderma patients with early stage disease have an approximately twofold increase in serum HA (72). Many other pathological states characterized HA Size in Biological Fluids and Tissues by inflammation similarly have elevated HA, as estimated by The average M and distribution of M for HA present in biological immunohistochemical analyses (73, 74). sources have been studied primarily for fluid tissues such as synovial fluid, vitreous, serum, lymph, and milk. Until recently, Methods to Analyze HA Molecular Mass the emphasis has been on documenting reduction of the average Distribution M, which strongly affects the biomechanical properties of HA solutions (48, 87–89). This has been done using physicochemical It has long been appreciated that degradation of HA negatively methods such as viscometry, light scattering, and sedimenta- affects its biomechanical properties. For example, degradation of tion–diffusion. Interest in the distribution of sizes present, and HA in articular joint synovial fluid can reduce the viscosity and the possibility that specific sizes have unique biological effects, has elasticity of the synovial fluid, and has also been shown to reduce led to an increasing number of studies by chromatographic and its lubricating ability (49, 75). The widespread and successful electrophoretic separation methods. Frontiers in Immunology | www.frontiersin.org 24 June 2015 | Volume 6 | Article 261 Cowman et al. Hyaluronan content and size stimulating the expression of human β-defensin 2 in the infant intestinal epithelium (24). Human amniotic fluid contains HA with an average M of about 330 kDa at 16 weeks gestation, but the M distribution changes to a mixture of high and very low M HA by 40 weeks gestation (92). HA in lymph fluid is variable in size, and can occur as a mixture of high and lower M compo- nents (36), or as a broad distribution of moderate M, ca. 800 kDa average (93). HA in normal blood serum is mainly relatively low M (ca. 100–300 kDa) (36, 56). It is also low in M in saliva and urine (94, 95). Tumors have been proposed to shed very low M HA into associ- ated body fluids. The quantity of such very low (but undetermined size) M HA in patient serum, obtained by centrifugal filtration, has been reported to be associated with metastatic breast cancer (62). It has also been reported in saliva of patients with head and neck tumors (95), and in the urine of patients with bladder cancers (94). The precise size of all such HA has not yet been determined, but should be accessible using recent improvements in methods. Rarely, high M HA is found in serum, as, for example, associated with Wilm’s tumor (96). For solid tissues, the pattern is a bit simpler. Normal healthy tissues are almost always associated with high M HA. HA with average M > 2000 kDa is found in young human cartilage (53). Larger HA averaging closer to 4000–6000 kDa is found in human skin (54), in rabbit skin (34), and in rat skin (30, 97). High M HA is FIGURE 1 | Example molecular mass distributions of human synovial found in rooster combs (32). High M HA is also found in skeletal fluid (SF) HA determined by agarose gel electrophoresis. From top to muscle, lung, heart, ileum, and colon of the rabbit (34). Little if bottom: (A) normal human SF obtained from young healthy volunteers. any low M HA is found in these healthy tissues. (B–D) Representative osteoarthritis (OA) patient SF. The dashed vertical line corresponds to the migration position of 4000 kDa HA. The fraction of HA Remodeling tissues and tumors show evidence of some lower having slower electrophoretic migration, and thus higher M than 4000 kDa, is M HA. Reduction in HA M occurs in older human cartilage a measure of the high M HA content. In normal human SF, the portion of HA (53). Low M HA also occurs in healing rat skin wounds (30), in with M > 4000 kDa averaged 61%. OA patients varied in the extent of HA human skin following irradiation with UVB (74), and in mouse degradation. The OA-HIGH profile, similar to that seen in normal SF and cervix undergoing postpartum remodeling (98). It is found in rat representative of OA samples with more than 60% of HA having M > 4000 kDa, was found for 26% of patients. The OA-MEDIUM profile, kidney after ischemia–reperfusion injury (99). Human prostate representative of OA samples with approximately 41–60% of HA having tumor HA has also been reported to contain some low M HA of M > 4000 kDa, was found for 41% of patients. The OA-LOW profile, indeterminate size (67). Many of the above-described studies of representative of OA samples with <40% of HA having M > 4000 kDa, was reduced M HA should be regarded as indicative but not conclu- found for 33% of patients. From Lee (90). sive proof of the presence of specific low M HA species. Recent improvements in techniques for analysis of very low quantities of polydisperse HA will allow this uncertainty to be addressed. In normal human synovial fluid, most of the HA is very high in Future studies should also include spiking samples with multi- molecular mass. Gel filtration chromatography with HA-specific ple monodisperse HA species to show that the isolation meth- detection (50) and agarose gel electrophoresis with staining (84, ods cause no degradation, or preferential isolation of high or 90) show the average M to be approximately 6000–7000 kDa, low M HA. with little if any HA <1000 kDa. In rheumatoid arthritis and It is interesting to consider that all efforts to determine the in osteoarthritis, HA can be partially degraded, resulting in a content and size of HA in biological tissues and fluids have made broad distribution of sizes, extending perhaps down to a few the tacit assumption that the HA has a constant chemical struc- hundred kilodaltons (90, 91) (Figure 1). Normal rabbit vitreous ture, except for variation in size. Since degradation by ROS/RNS HA has mostly high M (2000–3000 kDa), but bovine vitreous HA can cause chemical changes including ring opening reactions, it has mostly moderate M (500–800 kDa) (82). Owl monkey vitreous is possible that HA assays and size analyses may be influenced by has very high M HA (84). such changes, if present at significant levels. Further examination For fluids containing HA at very low concentrations, determi- of this possibility is warranted. nation of the M distribution is correspondingly difficult. Despite this, evidence for the occurrence of HA below 100 kDa (<250 disaccharides) in M is accumulating. Human milk contains Conclusion mainly HA with an average M of about 440 kDa, and also has been definitively shown to have approximately 5% of HA with It is now well established that HA synthesis is significantly M < 100 kDa (37). The low M HA is proposed to participate in increased in remodeling tissues and tumors. The concomitant Frontiers in Immunology | www.frontiersin.org 25 June 2015 | Volume 6 | Article 261 Cowman et al. Hyaluronan content and size presence of hyaluronidases and ROS/RNS makes it likely that frag- Author Contributions ments of HA can be created by degradation of high M polymers. The balance of synthetic and degradative activities, coupled with MC, HL, KS, JM, and ET contributed to the drafting and revising turnover through outflow or internalization, will determine the of this manuscript. All authors approved this manuscript. steady state M distribution of the tissue HA. HA shed into lymph or blood from a tumor may represent only the lowest M fraction Acknowledgments of that present. Whether HA fragments of particular sizes exist in sufficient amounts within a tissue or tumor environment to trigger This work was supported in part by The Endre A. Balazs Founda- specific cellular responses is not yet clear. The fact that exogenous tion (MC), the Fund for Neurodegeneration and Inflammation at HA fragments can elicit such effects is suggestive but not yet a New York University (MC), NIH R01 CA132827 (KS), Chairman’s proof of their role in vivo. There is good reason to expect clarity Fund Professor in Cancer Research (JM), and the Prostate Cancer on these issues in the near future. Society of Canada (ET). References Kinetics and Thermodynamics for Chemistry and Biochemistry. Hauppauge, NY: Nova Science (2009). p. 181–99. 1. Cowman MK, Matsuoka S. 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J Equine Vet Copyright © 2015 Cowman, Lee, Schwertfeger, McCarthy and Turley. This is an open- Sci (1985) 5:217–28. doi:10.1016/S0737-0806(85)80102-7 access article distributed under the terms of the Creative Commons Attribution License 90. Lee HG. An Agarose Gel Electrophoretic Method for Hyaluronan Molecu- (CC BY). The use, distribution or reproduction in other forums is permitted, provided lar Weight Analysis and its Application to Osteoarthritic Synovial Hyaluro- the original author(s) or licensor are credited and that the original publication in this nan. [Ph.D. Dissertation], Polytechnic University (1994) Available through journal is cited, in accordance with accepted academic practice. No use, distribution ProQuest LLC, Ann Arbor, MI. or reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 28 June 2015 | Volume 6 | Article 261 Review published: 16 June 2015 doi: 10.3389/fimmu.2015.00305 Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation Olgun Guvench* Department of Pharmaceutical Sciences, University of New England College of Pharmacy, Portland, ME, USA The extracellular N-terminal hyaluronan binding domain (HABD) of CD44 is a small globular domain that confers hyaluronan (HA) binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-sol- uble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD Edited by: David Naor, distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, Hebrew University of Jerusalem, and such glycosylation can diminish HA binding when the associated N-glycans are Israel capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of Reviewed by: Barry C. Finzel, N-glycans makes it difficult to apply experimental structural biology approaches to probe University of Minnesota, USA the molecular mechanisms of how the order-to-disorder transition and N-glycosylation Ichio Shimada, can modulate HA binding by HABD. We review recent results from molecular dynamics The University of Tokyo, Japan simulations that provide atomic-resolution mechanistic understanding of such modulation *Correspondence: Olgun Guvench, to help bridge gaps between existing experimental binding and structural biology data. Department of Pharmaceutical Findings from these simulations include: Tyr42 may function as a molecular switch that Sciences, University of New England College of Pharmacy, 716 Stevens converts the HA-binding site from a low affinity to a high affinity state; in the partially Avenue, Portland, ME 04103, USA disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient [email protected] mobility to form direct contacts with bound HA to further stabilize binding; and terminal Specialty section: sialic acids on covalently attached N-glycans can form charge-paired hydrogen bonding This article was submitted to interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA Inflammation, a section of the journal binding to glycosylated CD44 HABD. Frontiers in Immunology Received: 15 February 2015 Keywords: CD44, hyaluronan, binding, free energy, molecular dynamics, glycosylation, inhibition, unfolding Accepted: 28 May 2015 Published: 16 June 2015 Citation: Guvench O (2015) Revealing the Introduction mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan The structure of the cell surface protein CD44, from its N-terminus to its C-terminus, consists binding using molecular simulation. of a globular hyaluronan binding domain (HABD), a stalk domain, a single-pass transmembrane Front. Immunol. 6:305. domain, and a cytoplasmic domain (1, 2). Amino acids located N-terminal to the transmembrane doi: 10.3389/fimmu.2015.00305 domain are on the extracellular side of the cell membrane, and amino acids located C-terminal to the Frontiers in Immunology | www.frontiersin.org 29 June 2015 | Volume 6 | Article 305 Guvench CD44 molecular simulation transmembrane domain are on the intracellular side (Figure 1). sample is identical throughout (19). The carbohydrate component Post-translational modifications to CD44 include glycosylation is also flexible (20, 21), especially in comparison to globular proteins of the extracellular portion (3–5), palmitoylation of amino acids with their well-defined tertiary (three-dimensional) structures, immediately C-terminal to the transmembrane domain (6–9), and which therefore were crystallized early in the development of and phosphorylation of the cytoplasmic domain (10–12). The the field of structural biology (22) and still compose the majority already-complex structural biology of CD44 is further complicated of publicly available experimental atomic-resolution structures. by variable splicing of the RNA transcript of the CD44 gene, which In contrast to globular proteins, which exist in an aqueous envi- yields a variety of different patterns of amino acid insertion into ronment, transmembrane proteins are located in biological lipid the stalk domain and which modulates CD44 function (1, 13, 14), bilayers. Therefore, this environment must be suitably reproduced and by shedding that produces soluble CD44 (15). in samples in order to do experimental structural biology, which Atomic-resolution structures can lead to substantial insight can be very challenging (23, 24). Additionally, heterologous expres- into the function of a biomolecule. Such high-resolution structural sion of transmembrane proteins and subsequent purification can information is typically obtained from X-ray crystallography or be more difficult than for water-soluble globular proteins because NMR spectroscopy experiments, with examples being as small as of the limited surface area of the cell membrane for expression, a single zinc finger domain (16) and as large as ribosomes (17) the resulting toxicity to the organism being used for expression, and virus capsids (18). However, not all biomolecules are equally and the subsequent need to reconstitute the protein in a lipid amenable to having their structures solved by these methods. environment after extraction and purification (25, 26). Glycoproteins and proteoglycans are particularly challenging Intrinsically disordered proteins provide another contrast to because of the difficulty in obtaining pure samples and the inherent globular proteins in that the former lack well-defined unique flexibility of these two types of biomolecules. Sample homogeneity stable three-dimensional structures (27–29). In X-ray experiments, is a challenge for glycoproteins and proteoglycans because the car- this results in crystallographic disorder, diffuse scattering, and bohydrate component results from non-template-based enzymatic therefore undefined atomic coordinates (30). While NMR has synthesis, leading to carbohydrate microheterogeneity at a given been used extensively to study intrinsically disordered proteins, glycosylation site even though the protein component for a given solution NMR experiments yield data that represent ensemble averages, which can limit understanding of the various discrete conformations that such proteins may assume (31). CD44, with its multiple domains, poses a number of challenges for characterization by X-ray crystallography or NMR spectros- copy. The N-terminal HABD, which is similar to globular proteins, is in its biologically relevant form a glycoprotein that has numerous glycosylation sites (1). Furthermore, HABD in the presence of hyaluronan (HA) has characteristics of an intrinsically disordered protein (32, 33). The stalk domain that connects the extracellular HABD to the cell membrane has both N- and O-glycosylation sites (34). And, with alternative splicing, the stalk domain can have proteoglycan characteristics, namely a protein core with glycosa- minoglycan (GAG) attachments in the form of chondroitin sulfate and heparan sulfate (35). Recalling that CD44 is a transmembrane protein, in addition to the challenge associated with being located in the cell membrane, the transmembrane domain can be post- translationally modified by the attachment of lipids, which further complicates its structural biology since this modification can alter its interactions with the membrane bilayer (9, 36, 37). Finally, the cytoplasmic domain is likely disordered when not non-covalently bound to intracellular adapter proteins (12). Atomic-resolution experimental structural biology on CD44 has been largely limited to the ~150 amino acid HABD in its non-glycosylated form because of the many specific challenges above. X-ray structures for HABD exist for both human and mouse isoforms. Human HABD has been reproducibly crystallized in its apo form (i.e., not bound to HA, inhibitors, or peptides) (38, 39), as FIGURE 1 | CD44 structure. The four different structural/functional regions are denoted by different colors, and labels include the number of amino acids well as with unidentified peptide (39) found to be non-covalently in each region for the standard splice variant “CD44s.” Results of variation in bound to the face of HABD opposite that of the location of the RNA splicing include additional amino acids in the stalk region and loss of HA-binding site. However, there are no publicly available X-ray amino acids composing the cytoplasmic region. Amino acid numbering for structures of human HABD complexed with either HA or with the human isoform begins with residue 21 because of cleavage of a inhibitors of HA binding. In contrast, mouse HABD has been 20-residue N-terminal signal peptide. co-crystallized with both oligomeric HA (oHA) (40, 41) and Frontiers in Immunology | www.frontiersin.org 30 June 2015 | Volume 6 | Article 305 Guvench CD44 molecular simulation small-molecule inhibitors (40), as well as in its apo form (41). These Conformational Switching at the CD44 co-crystals reveal the binding mode of HA with HABD, which is HABD Binding Site presumably the same for human HABD given the ~85% sequence identity between the two forms, and the 100% sequence identity of Two lines of experimental evidence lead to the hypothesis that the HA-binding site (42). NMR structures exist for human HABD, direct contact between CD44 Arg 41 and HA is a major source both in its apo form (38) and bound to oHA (32). However, unlike of binding affinity (“Arg41” reflects amino acid numbering in X-ray structures with HA, NMR structural characterization of the the human form of CD44; the equivalent amino acid is Arg45 bound form lacks atomic coordinates for HA and therefore does is the mouse form. For simplicity, the human numbering will be not provide comprehensive information into the non-covalent used throughout this text.). From X-ray crystallography, Arg41 atomic contacts between HABD and bound HA. All of these and the loop that contains it change conformation depending previous structural biology examples are of CD44 HABD; the upon binding of oHA. In the apo form, the loop is in an open only non-HABD example of experimental CD44 structure is a conformation that locates this sidechain too far away to contact complex consisting of a nine amino acid long portion of the 72 oHA if it were present, and HABD is said to be in the “A” state (38, amino acid cytoplasmic domain complexed with the radixin FERM 41). In the complexed form, the loop can be either in the open domain (12). conformation, or in a closed conformation that facilitates direct Molecular dynamics (MD) is a physics-based approach to contact between the positively charged guanidinium group of the the modeling and simulation of biomolecules (43). In all-atom Arg41 sidechain and bound oHA, in which case HABD is in the explicit-solvent MD simulations, all the atoms of the system, “B” state (40, 41) (Figure 2, bottom row). From mutation data, the including those for the solvent, are included as interaction sites Arg41Ala single point mutation essentially destroys the ability of for computing the forces in the system. The values of the forces as HABD to associate with oHA (41, 55, 56). This thermodynamic a function of atomic positions are determined by a combination information demonstrates the critical nature of Arg41 in binding. of a mathematical expression and parameters, commonly called a However, the X-ray crystallographic data do not say anything “force field,” that encodes properties such as the energetic cost to about the thermodynamics of the A and B states, other than they stretch a bond or the energetic benefit of a van der Waals or charge- are both sufficiently stable to be trapped as crystals when oHA is pairing interaction (44). These forces are numerically integrated bound. And the mutation data do not provide information about to propagate the system, and this is done in an iterative manner the conformation of Arg41 when oHA is bound. We therefore to generate a trajectory, analogous to a movie, that shows how the applied MD simulations in an attempt to tie these two previous positions of the atoms in the system evolve with the passing of time findings together. (45). Typical present-day simulations involve tens of thousands All-atom explicit-solvent MD simulations can be used to to hundreds of thousands of atoms with trajectory lengths of tens determine true thermodynamic free energies for a variety of to hundreds of nanoseconds, which require tens to hundreds of biomolecular processes (46, 57–60). This is because of two reasons: millions of consecutive integration steps. MD simulations can (1) a model system under study includes water molecules and be used to determine not only the time-evolution (dynamics) of allows for full conformational flexibility of the included biomol- the system but also the relative probabilities, and therefore free ecules, which means both solvent effects and entropic effects energies, associated with different states (thermodynamics) (46). are explicitly included (that is, not as an approximation, but as As such, MD is an especially useful tool for studying flexible part of the system under investigation); and (2) there exist exact biomolecules at an atomic level of resolution, which makes it an mathematical expressions to determine thermodynamic quantities ideal complement to experimental structural biology techniques directly from simulation data. One approach to determining the like X-ray crystallography and NMR spectroscopy (47). free-energy difference between two conformational states x and y Over the past several years, our research group has applied all- of a biomolecule from MD simulation is to compute the reversible atom explicit-solvent MD simulations to extend the understanding work required to transition the system from state x to state y by of the function of the CD44 HABD. These efforts have aimed to integrating the measured average force along the transition path address the following scientific questions: (1) what is the mecha- (61). Since free energy is a thermodynamic state function, it does nism and associated thermodynamics of a conformational change not depend on the actual path used to convert the system from in an arginine-containing loop at the HABD binding site that is state x to state y. However, in practice obtaining good numerical associated with HA binding? (41); (2) why does HABD transition convergence as well as plausible biological insight both depend from a well-ordered (folded) three-dimensional structure to one upon determining a physically reasonable transition path. that is partially disordered when it binds to HA? (32, 33, 48); and For CD44 HABD, the two states are A, having an open loop (3) why do covalently attached sialyated N-glycans inhibit HA and the Arg41 sidechain separated from bound oHA, and B, with binding while unsialyated ones do not? (49–51) This article reviews a closed loop and the Arg41 sidechain in direct contact with oHA. the contribution MD simulations have made toward developing While a simple distance between Arg41 and oHA can be used to answers to these questions. We note that others have also recently discriminate between different conformations of the sidechain, it is applied MD simulation to the study of CD44 HABD, with topics not immediately obvious that a similarly simple metric can be used including conformational flexibility and the microscopic struc- to discriminate between the open vs. closed loop conformations. In ture and dynamics of water surrounding HABD (52, 53), and an effort to identify such a metric, we first compared the conforma- the mechanism and thermodynamics of the ordered to partially tions of the backbone dihedral angles φ, ψ for Arg41 and three disordered transition (54). amino acids on either side: -Lys-Asn-Gly-Arg41-Tyr-Ser-Ile-. This Frontiers in Immunology | www.frontiersin.org 31 June 2015 | Volume 6 | Article 305 Guvench CD44 molecular simulation contact between Arg41 and oHA. In neither case was any force applied to directly affect the Arg41 to oHA distance; rather, changes in this distance spontaneously resulted from changes in the loop backbone conformation. Furthermore, a similar effect could not be achieved using the backbones of other amino acids in the loop (62). Thus, the reaction path for interconversion between the A and B states was defined in terms of two progress variables: the value of the Tyr42 backbone dihedral angle φ, and the distance r between the Arg41 sidechain guanidinium central Cζ atom and the ether oxygen atom in the glycosidic linkage connecting GlcNAc3 to GlcUA4 in the bound oHA. Extensive simulations were subsequently done to compute the free energy of the system as a function of the progress variables φ and r (42). All simulations were of the human HABD (hHABD) complexed with [-4GlcUAβ1-3GlcNAcβ1-]4 (“HA8”), which was computationally constructed by combining information from mouse and human structures. The four hHABD-HA8 systems considered were: wildtype, ordered; wildtype, partially disordered; Arg41Ala, ordered; and Arg41Ala, partially disordered. Figure 2, middle row shows the data for both wildtype systems, and the major free-energy minima, which are the most stable states for each system, correspond to A and B. Wildtype simulation data demonstrate that the B state of the hHABD-HA8 complex is more stable than the A state by ~8–9 kcal/mol. This is true for both the ordered and partially disordered forms of hHABD (Figure 2, top row). Additional simulation data demonstrate that the analogous transition for the Arg41Ala mutant is substantially less favorable at ~6 kcal/ mol. From these data, it is possible to calculate the loss in bind- ing affinity associated with the point mutation, with values of 2.2 kcal/mol for the ordered form and 2.3 kcal/mol for the partially disordered form (42). These simulation data are in close agreement with existing experimental data measuring the loss in binding affinity to be 2.5 kcal/mol (41), which helps validate both the force field and the convergence of the simulations. Taken together, these FIGURE 2 | Thermodynamics of the transition between the A and B results support the idea that formation of direct contact between states of CD44 HABD. Bottom row: In the A state, the Arg41 sidechain HA and the Arg41 sidechain is a substantial source of favorable (tubes) is not in direct contact with oHA (balls-ands-sticks), whereas in the B binding free energy (41). state, the Arg41-containing loop is in a different conformation that facilitates In contrast to some of these findings, Plazinski and Knys- direct contact. r is the distance between the Arg41 sidechain guanidinium central Cζ atom and the ether oxygen atom in the glycosidic linkage Dzieciuch, in their simulation studies, found that the φ-related free- connecting GlcNAc3 to GlcUA4 in the bound oHA. Middle row: energy barrier was not correlated with the Arg41-HA distance (54, conformational free energies for the wildtype, ordered (left) and wildtype, 63). The authors also observed a low free-energy barrier associated partially disordered (right) forms of HABD. Free-energy values are in kcal/mol, with separation of Arg41 from HA, with the A and B states reducing contours are every 1 kcal/mol, and data are from Ref. (42). Top row: ordered to an average dynamic structure (54, 63). They suggested that a (left) and partially disordered (right) forms of HABD (ribbons) with bound HA (balls-and-sticks). possible explanation might be differences in the force fields used in their studies (54), since the previous analogous work found the A and B states to be distinct (62). Differences in force fields, which are the underlying physical models used to represent the bonded revealed a difference in the Tyr42 φ dihedral where in the A state and non-bonded interactions in such simulations, can indeed cause φ = −60° and in the B state φ = +60° (62). This was followed by such differing results. When such differences are inferred based on MD simulation where force was applied to this dihedral angle to differing simulation results and conclusions, one possibility is to gradually convert it from one value of φ to the other. In one case, review the methodology involved in the force field development starting from the A state and increasing φ converted the system (44, 64). Another is to compare the outcomes of the particular to the B state, not just with respect to the Tyr42 backbone but simulations with the existing experimental data for inconsisten- also with respect to the separation distance between Arg41 and cies. In this particular case, relevant experimental data include oHA. In the other case, starting from the B state and decreasing the crystal structures of the A and B states of HABD complexed φ converted the system to the A state, including breaking of the with oHA, and the mutation data showing loss of binding affinity Frontiers in Immunology | www.frontiersin.org 32 June 2015 | Volume 6 | Article 305 Guvench CD44 molecular simulation in the Arg41Ala mutant. Dynamic averaging would manifest as conformational switching at the binding site appears no more or crystallographic disorder with poorly resolved electron density for less favorable if the HABD C-terminal region is folded or unfolded. Arg41, which is in contrast to the existing crystallographic data. Analysis of the MD trajectories that were generated as part of Additionally, rapid equilibrium between short and long separation the free-energy experiments yielded a result that, in hindsight, distances between Arg41 and oHA suggests a weak interaction is obvious: flexibility from partial unfolding permits favorable between them, which is in contrast to the mutation data. electrostatic interactions between HA and the C-terminal HABD amino acids that cannot occur when the domain is fully ordered. Binding and Unfolding in CD44 HABD In the ordered form of HABD, the amino acids in question are locked into a folded conformation that keeps them far from bound Early experimental studies of the Arg41Ala mutation that predated oHA, while in the partially disordered form, this is no longer the the CD44 structural studies also probed the contribution of other case (Figure 2, top row). Because this span of amino acids is no basic amino acids by both point and truncation mutations (55). longer in consistent contact with the rest of the HABD domain, it This was a logical course of action in the absence of HABD three- assumes the properties of a random coil peptide, which through dimensional structure since HA contains a negatively charged random fluctuations can collide with bound oHA. If this collision carboxylate group every other monosaccharide and Arg and Lys happens in a way that brings one of the basic sidechains into close sidechains are positively charged, suggesting the possibility of proximity with oHA, a favorable contact can be formed. Unlike charge-charge interaction as a mechanism of binding. From that the Arg41 interaction, which has a well-defined mechanism based perspective, it is not surprising that these mutations all decrease the on specific interactions of the sidechain with a particular limited strength of binding of HA oligomers. But, taken in the context of section of bound oHA, the flexibility of the disordered amino acids the subsequent structural information, the explanation is less obvi- and the repeating nature of the HA polymer permit the possibility ous, since these additional amino acids, in contrast to Arg41, are of a wide range of basic sidechain interactions with bound HA located spatially far from the now-known binding site (41). Further (42), consistent with the long-standing finding that a 13-amino complicating the situation is the observation that the portion of acid CD44 peptide spanning Arg150–Arg162 itself will bind HA HABD that contains these amino acids 153-169 goes from having (65). This wide range may conceivably include interaction with well-defined three-dimensional structure to becoming unfolded in HA bound to an adjacent molecule of CD44, though the present conjunction with HA binding (32, 33, 48). That is, the change in the simulations had a single copy of the complex and therefore could conformation, which correlates with the binding of HA, in this span not directly address the possibility of such trans association. of amino acids located in the C-terminal most region of HABD is The above computational experiments do suggest a plausible what defines the ordered-to-partially disordered HABD transition. molecular mechanism by which the ordered to partially disordered The above suggests two questions: why do basic amino acids transition confers increased binding affinity to oHA. However, the distant from the binding site affect affinity? And why is the affinity simulations involved either the ordered or the partially disordered greater when the sequence containing these amino acids unfolds form of CD44 hHABD, and therefore do not provide any insight and becomes disordered? To answer these two questions, we return into the mechanism of the ordered to partially disordered transi- to the previous set of free-energy data from MD simulations. tion itself. Independent work has been done toward this end, and For wildtype hHABD, the free-energy data associated with the with the additional aims of estimating the free-energy profile of the interconversion between the A and B states are qualitatively the transition and clarifying the role of select amino acids in the transi- same regardless of whether hHABD is in the ordered or partially tion (54). Connected with the proposed transition mechanism disordered form (Figure 2, middle row). The same is also the was a free-energy change of +25 kcal/mol, implying the partially case for the Arg41Ala mutant, where the free-energy change in disordered form in the absence of HA is very unstable relative to going from A to B is independent of whether those distant amino the ordered form. While the sign of the free-energy change, associ- acids are folded or not (42) (data not shown here). Not only are ated with loss of a single beta strand at the edge of a beta sheet, there strong qualitative similarities in free-energy data between agrees with experiment (32), the magnitude is substantially larger the ordered and partially disordered forms, but the quantitative than the folding free-energy values for entire small single-domain values are also very similar. For the A→B transition of the bind- proteins, which are typically <10 kcal/mol, including for proteins ing site in the wildtype, ordered form, the associated free-energy consisting exclusively of beta strands (66). A further difficulty is change is −8.7 kcal/mol, and for this transition in the wildtype, that the simulation free-energy data for the analogous transition in partially disordered form the value is a very similar −7.8 kcal/ the Tyr161Ala mutant are identical to those for the wild type. This mol (42). Likewise for the Arg41Ala mutant, in the ordered form is in contrast to experimental data, where the Tyr161Ala mutant the value is −6.5 kcal/mol and in the partially disordered form constitutively exhibits the partially disordered conformation (33, it is −5.5 kcal/mol (42). The small difference of ~1 kcal/mol is 48). One possible explanation for these apparent inconsistencies within the precision that can be expected from these particular is that the study represents the initial steps of the transition (54), computational experiments. such that extending simulations further along the selected reac- In an allosteric mechanism, a conformational change distant tion coordinate may result in subsequent decreases in free energy. from the site affects the energetics at the binding site. In the case Another possibility is lack of convergence of simulations given of HABD, the independence of the energetics of the binding site the large scale of the transition (54). This was the case for the free A→B transition from the ordered vs. partially disordered form of energy for the A→B transition, which involves a much smaller HABD contradicts the hypothesis that allostery is at work. That is, conformational change than the ordered to partially disordered Frontiers in Immunology | www.frontiersin.org 33 June 2015 | Volume 6 | Article 305 Guvench CD44 molecular simulation transition. The first study suggested that the A and the B states were essentially equally stable (62), while subsequent work that extended the timescale of the simulations by 40-fold showed the B state to be substantially more stable than the A (42). Importantly, in the case of the ordered to partially disordered transition, the partially disordered form is not a single, well-defined conforma- tion. Rather, the disordered C-terminal HABD amino acids are free to take on a multitude of conformations. Therefore the partially disordered form is actually an ensemble of diverse conformations, and this further complicates computational experiments toward understanding the transition mechanism. Inhibition by Glycosylation N-glycosylation of CD44 HABD is known to have variable effects FIGURE 3 | Molecular dynamics snapshots demonstrating ordered on CD44 function depending on the nature of the N-glycans. HABD (ribbons) residue Arg41 (van der Waals spheres) forming a One effect is to block HABD binding to HA (49–51). Another charge-paired hydrogen bonding interaction with complex-type N-glycan (balls-and-sticks; sialic acid atoms in purple) attached to is to make CD44 itself a ligand that binds to lectins (67, 68). Asn25. Data are timepoints from a single 100-nanosecond (ns) trajectory The biochemistry behind both of these contrasting functions is from Ref. (70). related and is modulated by N-acetylneuraminic acid (Neu5Ac) monosaccharide, which is commonly called “sialic acid.” In one case, HABD N-glycans are capped with sialic acids, and this both the positively charged sidechains of HABD amino acids and the blocks HA binding and makes CD44 a selectin ligand. In the other negatively charged carboxylate groups of the terminal sialic acids case, sialidase activity removes these terminal sialic acids, leaving (Figure 3). In contrast, asialo glycans form only brief contacts, the bulk of the N-glycan structures intact, and this change restores which is understandable since they lack the negative charge of the both HA-binding and removes selectin ligand functionality. While sialo form. Long-lasting contacts in the sialoglycan simulations sialidase treatment removes only the terminal monosaccharide involve Arg41 and Arg154, and these contacts form spontaneously from the attached N-glycan, the functional result is the same as during the simulation and can last for 40–50% of the simulation removal of the entire N-glycan. For example, Asn point mutation length (70). Both of these amino acids can directly associate with precludes N-glycan attachment and heterologous expression yields HA8 when it is bound, based on findings from the computational non-glycosylated protein. In both of these cases, the functional experiments on partially disordered HABD, as summarized in outcome is the same as sialidase treatment, which implies that the previous section. However, their binding with sialic acid is an inhibition of HA-binding cannot be explained as a consequence interaction that would directly compete with their binding with of steric blockage of the HABD binding site, since the de-sialylated HA8. Therefore, the view that emerges is that, in the sialo form, the N-glycan has nearly the same bulk as the sialo-glycan. This covalently attached N-glycans will form charge-paired hydrogen mechanism of regulation is not unique to CD44, as the related bonding interactions with Arg sidechains known to be important hyaladherin LYVE-1 demonstrates similar behavior (69). for HA binding. As further evidence, free-energy simulations, The sialidase data immediately suggest two sets of simulations similar to the ones for Arg41–HA8 association described in the of glycosylated HABD: one set with sialylated N-glycan and a previous section, demonstrate that the Arg–sialic acid association second set with asialo N-glycan. Asn25 and Asn120 were selected is indeed thermodynamically favorable by ~1 kcal/mol (70). We do for computational N-glycosylation based on previous mutagenesis note that these simulations were limited to only the CD44 HABD, studies showing that cells expressing Asn25Ser and Asn120Ser which is present in all splice variants of CD44, and that the simula- mutants constitutively bind HA (50). Complex-type N-glycans that tions did not include HA. Clearly, additional work needs to be done were selected (70) based on the existing finding that blocking the to understand the molecular mechanisms by which glycosylation metabolic pathway for processing complex N-glycans restores HA alters binding, since it has been shown that N-glycosylation of binding (71, 72). In conjunction with the two different glycosyla- CD44 can also facilitate HA binding (73). tion sites and the sialo and asialo forms of the N-glycan, both the ordered and partially disordered forms of CD44 HABD were studied in the simulations. A representative starting conformation Conclusion of ordered HABD with a sialo glycan attached to Asn25 is shown in the left frame of Figure 3; the analogous asialo form would be A subset of the computational experiments above suggests the missing only the atoms colored purple. following four conclusions. The first is that the Tyr42 backbone The key finding from the comprehensive set of simulations dihedral angle φ can act as a molecular switch to convert the covering the ordered and disordered HABD paired with sialo HABD HA-binding site from the open A state to the closed B and asialo N-glycans is that only in the sialo form do stable, state, which includes the formation of direct contact between HA long-lasting non-covalent contacts form between the protein and the Arg41 sidechain (62). The second is that the B state is and glycan components. Furthermore, these contacts involve more thermodynamically stable, and this stability is due to direct Frontiers in Immunology | www.frontiersin.org 34 June 2015 | Volume 6 | Article 305 Guvench CD44 molecular simulation Arg41-HA contact (42). The third is that basic amino acids located The concept of doing computational experiments to distant from the HA-binding site in the ordered form of HABD gain address biological questions is appealing, but the technique sufficient mobility in the partially disordered form to be able to form used here, namely all-atom explicit-solvent MD simulations, direct contacts with oHA and further stabilize binding (42). And the requires significant resources. The most obvious resource is fourth is that terminal sialic acids on covalently attached N-glycans computing capacity, since the computing demands are quite can form charge-paired hydrogen bonding interactions with basic large. It is not uncommon for a set of simulations to utilize the amino acids that could otherwise bind to HA, thereby blocking equivalent of hundreds to thousands of personal computers HA binding to glycosylated CD44 HABD (70). In addition to con- running at full speed around the clock for weeks at a time. In tributing to the mechanistic understanding of CD44-HA binding, practice, this type of computing power tends to be restricted these conclusions may be of utility in the future development of to nationally funded supercomputing centers (77). A second small-molecule modulators of CD44 function (40), especially given is the development of software capable of making optimal the potential for CD44 as a therapeutic target (74–76). use of modern supercomputers (78–81). And a third is the However, it should be kept in mind that the role of Tyr42 as development of accurate models (i.e., force fields) for the a molecular switch, and the discrete nature of the A and B states types of molecules that make up the biological systems under of the Arg41-containing loop is contradicted by other computa- study (44, 64, 82). For example, the development of just the tional work (54, 63) Furthermore, there do exist experimental carbohydrate component of the force field used in our studies data that are in apparent conflict with the above conclusions. As of CD44 HABD involved a collaborative effort spanning over mentioned previously, N-glycosylation of CD44 can facilitate HA half a decade (83–91). Fortunately, the technique continues binding (73). And, mutation of positively charged amino acids to mature, resulting in an increasingly reliable analytical in the disordered region has been found to enhance HA-binding scientific methodology capable of providing accurate and affinity in the context of both purified HABD and cell surface CD44 direct insight into questions that could be addressed only (41). Given these differences, further investigation is warranted indirectly and with great technical difficulty using other to achieve a comprehensive consistent view. 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Frontiers in Immunology | www.frontiersin.org 37 June 2015 | Volume 6 | Article 305 REVIEW published: 15 May 2015 doi: 10.3389/fimmu.2015.00231 Hyaluronan – a functional and structural sweet spot in the tissue microenvironment James Monslow, Priya Govindaraju and Ellen Puré * Department of Biomedical Sciences, University of Pennsylvania, Philadelphia, PA, USA Transition from homeostatic to reactive matrix remodeling is a fundamental adaptive tissue response to injury, inflammatory disease, fibrosis, and cancer. Alterations in architecture, physical properties, and matrix composition result in changes in biomechanical and biochemical cellular signaling. The dynamics of pericellular and extracellular matrices, including matrix protein, proteoglycan, and glycosaminoglycan modification are continu- ally emerging as essential regulatory mechanisms underlying cellular and tissue function. Nevertheless, the impact of matrix organization on inflammation and immunity in particular and the consequent effects on tissue healing and disease outcome are arguably under- studied aspects of adaptive stress responses. Herein, we review how the predominant Edited by: glycosaminoglycan hyaluronan (HA) contributes to the structure and function of the tissue David Naor, microenvironment. Specifically, we examine the evidence of HA degradation and the Hebrew University of Jerusalem, Israel generation of biologically active smaller HA fragments in pathological settings in vivo. We Reviewed by: Alberto Passi, discuss how HA fragments versus nascent HA via alternate receptor-mediated signaling Università degli Studi dell’Insubria, influence inflammatory cell recruitment and differentiation, resident cell activation, as well Italy as tumor growth, survival, and metastasis. Finally, we discuss how HA fragmentation Linda M. Pilarski, University of Alberta, Canada impacts restoration of normal tissue function and pathological outcomes in disease. *Correspondence: Keywords: hyaluronan, remodeling, matrix, homeostasis, pathogenesis Ellen Puré, Department of Biomedical Sciences, University of Pennsylvania, 380 S. University Avenue, Philadelphia, PA Introduction 19104, USA [email protected] In the 80 years that have passed since hyaluronan (HA - also known as hyaluronic acid or hyaluronate) was first isolated and purified from the vitreous humor of the eye (1), the perception Specialty section: of this structurally seemingly simple molecule has changed dramatically. From simple beginnings, This article was submitted to and being thought of merely as a “space-filler,” our understanding of its role grew slowly at first, Inflammation, a section of the journal steadily gathered steam and has now entered its exponential phase. HA is now recognized as a Frontiers in Immunology molecular powerhouse with critical roles in homeostasis, pathological disease onset, progression, Received: 15 February 2015 and recovery or decline. This is none more so evident than in the number of review articles of Accepted: 29 April 2015 which the biological role of HA has been the focus over the last few years alone [2012-2014 nearly 40 Published: 15 May 2015 reviews, including an entire edition dedicated to its role in cancer (2)]. It is well established that native Citation: HA matrix found in homeostasis plays important biomechanical and biophysical roles as a hydrated Monslow J, Govindaraju P and Puré cushioning agent and/or molecular filter in connective tissue, joints, and skin (3, 4). Furthermore, E (2015) Hyaluronan – a functional and structural sweet spot in the tissue increased HA accumulation is a hallmark of almost all diseases in which inflammation and/or microenvironment. fibrosis occur, especially tumor growth and metastasis (2, 4–10). Importantly, HA polymer length Front. Immunol. 6:231. (and thus its molecular weight, MW) plays a significant part in the nature of its interactions with doi: 10.3389/fimmu.2015.00231 the extracellular matrix (ECM), cell surface receptors (including its major receptor, CD44) on both Frontiers in Immunology | www.frontiersin.org 38 May 2015 | Volume 6 | Article 231 Monslow et al. Hyaluronan; simple structure – complex function resident and recruited cells, and influences how cells in tissue the extent of HA fragmentation is greatly enhanced, causing respond to extracellular cues under these conditions (4, 10–15). significant changes in the distribution and size of biologically The existing literature clearly pinpoints that the MW character- active HA products, including the accumulation of HA oligomers istics of HA are important determinants of its biological activity, [<10 kDa or <20 monomers – oligo-HA (25, 26)]. Collectively, through in vitro and in vivo studies describing how exogenously these bioactive HA fragments serve to interact with cells and added HA of different MW affects cellular signaling and function influence behavior in different ways to HMW-HA (27–31). (4, 8, 11, 12, 16–19). There, however, has been limited work to elucidate the distribution of varying sizes of endogenous HA HA MW Distribution in Health Versus Disease – It in the tissue in question, the alterations to HA MW that occur is the Small (HA) Things that Matter during disease progression and how these HA fragments change A correlation of increased HA levels in the pathological setting is the biomechanical and biophysical properties of the tissue in vivo. now par for the course. However, understanding the MW distri- Nor have many of the reports where exogenous HA was added, bution of HA in vivo, how it varies between different tissues, and elucidated how this effected the size distribution of endogenous how the ratio of HMW-, MMW-, LMW-, and oligo-HA changes HA, and over time, its effects on tissue architecture and cellular during disease progression is also paramount when developing signaling that translate to either recovery of homeostasis or pro- treatment regimens that target HA. Surprisingly, measurements gression of disease. This is especially important in the context of HA MW distribution in vivo have only occasionally been of cancer progression, as the effects of altering HA MW may investigated; these are summarized in Table S1 in Supplementary have varying and opposing effects depending on the origin of the Material. cancer, the tissue in which it resides, and the stage of the disease Upon review of the literature, it became clear that there was no (20). The recent findings in the naked mole rat that suggest a link consensus for what was termed HMW- versus MMW-, LMW- and between the animals’ resistance to cancer and the extraordinarily oligo-HA. To better understand and compare the roles of HA of high MW (HWA) HA in its tissues have brought this subject different sizes under various biological settings going forward, we, into the limelight (21). For the above reasons, we have confined for the purpose of this review, categorize the various MW forms this review to focus on (i) a summary of the existing knowledge of HA as follows; HMW-HA (>1000 kDa), MMW-HA (250- about HA MW distribution in vivo under homeostasis and disease, 1000 kDa), LMW-HA (10-250 kDa), and oligo-HA (<10 kDa). (ii) mechanisms responsible for alterations in HA MW and the These groups are by no means distinctly distributed; in many occurrence of these mechanisms in pathological settings, and settings, HA MW is polydisperse, encompassing more than one (iii) the opposing effects of HMW-HA versus HA fragments on size category. In contrast, specific properties of HA are in certain ECM function, receptor-mediated cellular signaling and disease instances associated with a defined and narrow spectrum of its outcome. MW (12). A total of 65 studies reported analysis of HA MW in an array HA Molecular Weight Distribution in of tissues including skin, brain, eye, prostate, blood, circulating Homeostasis and Disease leukocytes, synovial tissue and fluid, cartilage, amniotic fluid, lymphatics, kidney, aorta, gums, lung and lung fluid, heart, larynx, HA Molecular Weight – Why Do We Care? liver, cervix, skeletal muscle, and urine across a variety of species Hyaluronan is a polysaccharide of repeating units of -glucuronic (see Table S1 in Supplementary Material for references). Nineteen acid and N-acetyl-glucosamine. This highly charged, hydrophilic of the studies analyzed HA MW under homeostatic conditions molecule is among the largest polysaccharides in nature, and exclusively. Surprisingly, we only found eight studies that analyzed in mammals one of the simplest with regards to structure. It HA in the context of cancer. The remaining studies reported HA is the major, non-proteinaceous component of the ECM, struc- size in a number of pathological settings, including cardiovascular turally distinct from other glycosaminoglycans (GAGs) in that it disease (atherosclerosis and vascular injury), arthritis (rheuma- is unmodified (i.e., non-sulfated) and linear [non-branching (22)]. toid and osteoarthritis), liver disease (septic shock and chronic In its most common, homeostatic, and native form, HA polymer liver fibrosis), vanishing white matter disease, skeletal ischemia, chain length exists as a HMW molecule, with sizes commonly lung disease (asphyxia, cigarette smoke exposure, asthma, fibro- above 1000 kDa. In this form, HMW-HA possesses biophysical sis, ischemia, and hypertension), skin wounding/healing, kidney properties that serve as a lubricant to hydrate tissue and create disease, development, pregnancy, inflammation, and aging. HA a matrix that sequesters growth factors and cytokines (23). It is exists in a HMW form under homeostatic conditions in almost uniquely synthesized at the plasma membrane with the completed all of the tissues where it was analyzed, with subtle yet possi- polymer extruded to the extracellular space by the hyaluronan bly significant differences depending on the tissue and species synthase enzymes (HASs). Increased HAS synthesis and HA accu- (1000-7000 kDa). Notably, increased HA fragmentation was evi- mulation are hallmarks of many pathological conditions (24). dent under pathological conditions, occurring in both inflam- HMW-HA is degraded in vivo by hyaluronidases (Hyals), a family matory and fibrotic diseases. HA MW analysis in lung and skin of enzymes that hydrolyze HA chains into intermediate (medium pathologies had been more extensively analyzed compared to MW, MMW) or short (low MW, LMW) fragments (18). Changes other tissues. A small amount of HA was detected in lungs under in HA synthesis and degradation in part mediate the biochemical homeostatic conditions, found predominantly in the HMW form. and rheological alterations to reactive matrices that occur dur- Following insult or injury, a dramatic increase in total HA as ing disease progression. Under certain pathological conditions, well as fragmentation yielding LMW-HA species was observed. Frontiers in Immunology | www.frontiersin.org 39 May 2015 | Volume 6 | Article 231