Hyaluronan Minireview Series*

The birth announcement for hyaluronan occurred in a Journal of Biological Chemistry article by Karl Meyer (Fig. 1) and his laboratory assistant John Palmer in 1934 (1). They isolated a “polysaccharide acid of high molecular weight” from bovine vitreous that contained a “uronic acid, an amino sugar, and possibly a pentose” but no sulfates (and as it turned out, no pentoses either). They proposed “for convenience, the name ‘hyaluronic acid,’ from hyaloid (vitreous) uronic acid.” It is fitting, therefore, that this minireview series on this fascinating, often enigmatic macromolecule is being inaugurated by reproducing this classic paper from the Journal. This seminal work also established the research direction for the rest of Karl Meyer’s long and highly productive career, namely isolating and characterizing glycosaminoglycans. It is interesting to pause and reflect on the combination of circumstances that brought this study to fruition. Karl Meyer first came to the United States in 1930 after accepting an offer from Herbert Evans to work on anterior pituitary hormones as an assistant professor at Berkeley. After two productive years, he returned to Europe to attend a scientific conference only to learn that his position at Berkeley was being terminated. Thus, he was faced with the difficult decision of whether to remain in Germany or to return to the United States. He decided on the latter, perhaps sensing the storm clouds on the horizon of World War II in the turmoil of his native country. After arriving in New York, friends put him in contact with Hans Clarke at Columbia University who provided him with an interim fellowship until a position as assistant professor was arranged for him in the Department of Ophthalmology in 1933. Under some pressure to work on relevant tissue, Karl Meyer initiated studies on lysozyme in tears and sought another, more relevant source for a “mucoid” substrate for the enzyme. He considered the highly viscous vitreous humor as a likely candidate, and the discovery of hyaluronan quickly followed. It would take almost 25 years before his studies would link the two sugars together correctly in the disaccharide repeat unit that forms the glycosaminoglycan we now call hyaluronan (see Ref. 2 for a finale): glucuronic acid1,3-N-acetylglucosamine-1,4-. Along the way, classic studies on the mechanisms of action of hyaluronidases, both hydrolases and lyases, would prove essential in defining the structures of hyaluronan. A true gem of a paper published in Nature in 1954 (3) defined the structure of the disaccharide (Fig. 2) isolated from bacterial hyaluronidase digests of hyaluronan. The 4,5-unsaturated bond on the uronic acid provided the key to unraveling the lyase mechanism. Those of us who have labored hard to purify hyaluronan oligosaccharides can only admire the profile in Fig. 3, taken from another Journal of Biological Chemistry classic paper (4), that shows separation of hyaluronan oligosaccharides in a testicular hyaluronidase digest by ion exchange chromatography in the days when fraction collectors were homemade, if available at all. Today, research on hyaluronan is a growth industry, and this minireview series reflects the diverse biological functions that are continuously emerging from its simple, repetitive structure. The first minireview in this series, “Hyaluronan and Homeostasis: a Balancing Act,” by Markku I. Tammi, Anthony J. Day, and Eva A. Turley discusses the structure and metabolism of hyaluronan. Significant milestones include the identification of hyaluronan-binding molecules with signaling properties, including CD44 and RHAMM, and the molecular cloning of the family of prokaryotic and eukaryotic hyaluronan synthases. Gene deletion by homologous recombination in embryonic stem cells demonstrates the essential roles of hyaluronan in vertebrate development and in the expansion of extracellular spaces. Studies with cells that are deficient in hyaluronan synthase (and thus deficient in hyaluronan) provide dramatic confirmation of the requirement for hyaluronan in developmentally regulated transformation of epithelial cells to invasive mesenchymal cells in morphogenesis of the heart. This process requires Ras activation and argues compellingly for a hyaluronan-mediated mechanism involving activation of intracellular signaling pathways. This minireview also appraises the important role of hyaladherins, proteins that bind to hyaluronan, in the formation of the hyaluronan-rich pericellular matrix and in signaling responses to hyaluronan. The provocative discovery of intracellular hyaluronan and associated hyaluronan-binding proteins opens a new chapter in hyaluronan biology. In order for hyaluronan to function as more than simply a viscous, space-filling glycosaminoglycan, it must interact with the specific binding domains in hyaladherins. The second minireview, “Hyaluronan-binding Proteins: Tying up the Giant,” by Anthony J. Day and Glenn D. Prestwich reviews the current status of the structures of proteins and proteoglycans that bind hyaluronan. The tertiary structure of the link module, the best characterized hyaluronan-binding domain, has been solved for one member of the family of proteins that contain this domain, namely tumor necrosis factor-stimulated gene-6 (TSG-6). This module is found in extracellular matrix molecules such as the proteoglycans aggrecan and versican and on cell surface receptors such as CD44. However, a number of other proteins without the link module also bind to hyaluronan with high specificity. It remains to be seen if these proteins have tertiary structural topographies similar to the link module family that underlie their ability to interact with hyaluronan. Exposure of certain cells to hyaluronan results in activation of intracellular signaling pathways. The third minireview, “Signaling Properties of Hyaluronan Receptors,” by Eva A. Turley, Paul W. Noble, and Lilly Y. W. Bourguignon delves inside the cell to begin to unravel these pathways and their consequences. CD44 is the best understood hyaluronan receptor and can engage Rho and Ras signaling pathways, interact with c-Src tyrosine kinase, and recruit ankyrin and ezrin/radixin/moesin cytoskeleton proteins. Thus, multiple cellular behaviors ranging from a highly structured cytoskeleton typical of an organized epithelium to ruffling and migration can potentially be induced by hyaluronan. For example, low, physiological concentrations (nanomolar) of hyaluronan can increase cell motility with both Ras-mitogen-activated protein kinase and phosphoinositide 3-kinase pathways implicated. Similarly, we have found that exposure of endocardial cells from animals deficient in hyaluronan synthase 2 to very low concentrations of hyaluronan stimulates their migration and subsequent transformation to mesenchymal phenotype. This suggests that hyaluronan can act as a co-stimulatory molecule with growth factors resulting in pluripotent cellular outcomes depending upon the repertoire of co-receptors present. Cells and animal models derived from hyaluronan-synthase null mice will be valuable tools in unraveling the signaling mechanisms underlying these outcomes. Another hyaluronan receptor, RHAMM, has a peripatetic existence inside and on cells, complicating elucidation of its protean signaling properties and other potential roles, and this minireview presents a current view of its localization and functions. * These minireviews will be reprinted in the 2002 Minireview Compendium, which will be available in December, 2002. 1 This “classic” paper is reprinted at the end of this Minireview Prologue. Minireview Prologue THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 7, Issue of February 15, pp. 4575–4579, 2002 Printed in U.S.A.

The birth announcement for hyaluronan occurred in a Journal of Biological Chemistry article by Karl Meyer (Fig. 1) and his laboratory assistant John Palmer in 1934 (1). 1 They isolated a "polysaccharide acid of high molecular weight" from bovine vitreous that contained a "uronic acid, an amino sugar, and possibly a pentose" but no sulfates (and as it turned out, no pentoses either). They proposed "for convenience, the name 'hyaluronic acid,' from hyaloid (vitreous) ϩ uronic acid." It is fitting, therefore, that this minireview series on this fascinating, often enigmatic macromolecule is being inaugurated by reproducing this classic paper from the Journal.
This seminal work also established the research direction for the rest of Karl Meyer's long and highly productive career, namely isolating and characterizing glycosaminoglycans. It is interesting to pause and reflect on the combination of circumstances that brought this study to fruition. Karl Meyer first came to the United States in 1930 after accepting an offer from Herbert Evans to work on anterior pituitary hormones as an assistant professor at Berkeley. After two productive years, he returned to Europe to attend a scientific conference only to learn that his position at Berkeley was being terminated. Thus, he was faced with the difficult decision of whether to remain in Germany or to return to the United States. He decided on the latter, perhaps sensing the storm clouds on the horizon of World War II in the turmoil of his native country. After arriving in New York, friends put him in contact with Hans Clarke at Columbia University who provided him with an interim fellowship until a position as assistant professor was arranged for him in the Department of Ophthalmology in 1933. Under some pressure to work on relevant tissue, Karl Meyer initiated studies on lysozyme in tears and sought another, more relevant source for a "mucoid" substrate for the enzyme. He considered the highly viscous vitreous humor as a likely candidate, and the discovery of hyaluronan quickly followed.
It would take almost 25 years before his studies would link the two sugars together correctly in the disaccharide repeat unit that forms the glycosaminoglycan we now call hyaluronan (see Ref. 2 for a finale): glucuronic acid-␤1,3-N-acetylglucosamine-␤-1,4-.
Along the way, classic studies on the mechanisms of action of hyaluronidases, both hydrolases and lyases, would prove essential in defining the structures of hyaluronan. A true gem of a paper published in Nature in 1954 (3) defined the structure of the disaccharide ( Fig. 2) isolated from bacterial hyaluronidase digests of hyaluronan. The 4,5-unsaturated bond on the uronic acid provided the key to unraveling the lyase mechanism. Those of us who have labored hard to purify hyaluronan oligosaccharides can only admire the profile in Fig. 3, taken from another Journal of Biological Chemistry classic paper (4), that shows separation of hyaluronan oligosaccharides in a testicular hyaluronidase digest by ion exchange chromatography in the days when fraction collectors were homemade, if available at all.
Today, research on hyaluronan is a growth industry, and this minireview series reflects the diverse biological functions that are continuously emerging from its simple, repetitive structure. The first minireview in this series, "Hyaluronan and Homeostasis: a Balancing Act," by Markku I. Tammi, Anthony J. Day, and Eva A. Turley discusses the structure and metabolism of hyaluronan. Significant milestones include the identification of hyaluronan-binding molecules with signaling properties, including CD44 and RHAMM, and the molecular cloning of the family of prokaryotic and eukaryotic hyaluronan synthases. Gene deletion by homologous recombination in embryonic stem cells demonstrates the essential roles of hyaluronan in vertebrate development and in the expansion of extracellular spaces. Studies with cells that are deficient in hyaluronan synthase (and thus deficient in hyaluronan) provide dramatic confirmation of the requirement for hyaluronan in developmentally regulated transformation of epithelial cells to invasive mesenchymal cells in morphogenesis of the heart. This process requires Ras activation and argues compellingly for a hyaluronan-mediated mechanism involving activation of intracellular signaling pathways. This minireview also appraises the important role of hyaladherins, proteins that bind to hyaluronan, in the formation of the hyaluronan-rich pericellular matrix and in signaling responses to hyaluronan. The provocative discovery of intracellular hyaluronan and associated hyaluronan-binding proteins opens a new chapter in hyaluronan biology.
In order for hyaluronan to function as more than simply a viscous, space-filling glycosaminoglycan, it must interact with the specific binding domains in hyaladherins. The second minireview, "Hyaluronan-binding Proteins: Tying up the Giant," by Anthony J. Day and Glenn D. Prestwich reviews the current status of the structures of proteins and proteoglycans that bind hyaluronan. The tertiary structure of the link module, the best characterized hyaluronan-binding domain, has been solved for one member of the family of proteins that contain this domain, namely tumor necrosis factor-stimulated gene-6 (TSG-6). This module is found in extracellular matrix molecules such as the proteoglycans aggrecan and versican and on cell surface receptors such as CD44. However, a number of other proteins without the link module also bind to hyaluronan with high specificity. It remains to be seen if these proteins have tertiary structural topographies similar to the link module family that underlie their ability to interact with hyaluronan.
Exposure of certain cells to hyaluronan results in activation of intracellular signaling pathways. The third minireview, "Signaling Properties of Hyaluronan Receptors," by Eva A. Turley, Paul W. Noble, and Lilly Y. W. Bourguignon delves inside the cell to begin to unravel these pathways and their consequences. CD44 is the best understood hyaluronan receptor and can engage Rho and Ras signaling pathways, interact with c-Src tyrosine kinase, and recruit ankyrin and ezrin/radixin/moesin cytoskeleton proteins. Thus, multiple cellular behaviors ranging from a highly structured cytoskeleton typical of an organized epithelium to ruffling and migration can potentially be induced by hyaluronan. For example, low, physiological concentrations (nanomolar) of hyaluronan can increase cell motility with both Ras-mitogen-activated protein kinase and phosphoinositide 3-kinase pathways implicated. Similarly, we have found that exposure of endocardial cells from animals deficient in hyaluronan synthase 2 to very low concentrations of hyaluronan stimulates their migration and subsequent transformation to mesenchymal phenotype. This suggests that hyaluronan can act as a co-stimulatory molecule with growth factors resulting in pluripotent cellular outcomes depending upon the repertoire of co-receptors present. Cells and animal models derived from hyaluronan-synthase null mice will be valuable tools in unraveling the signaling mechanisms underlying these outcomes. Another hyaluronan receptor, RHAMM, has a peripatetic existence inside and on cells, complicating elucidation of its protean signaling properties and other potential roles, and this minireview presents a current view of its localization and functions.
Alterations in hyaluronan production and organization are widely implicated in pathologies. The fourth minireview, "Hyaluronan-Cell Interactions in Cancer and Vascular Disease," by Bryan P. Toole, Thomas N. Wight, and Markku I. Tammi explores the roles of hyaluronan in these processes. Increased production of hyaluronan is associated with tumors, both in the surrounding stroma and with the malignant cells; with vascular lesions, such as in restenosis following angioplasty where the hydrated matrix resembles that found in large vessels during development; and with the response of smooth muscle cells in inflammatory diseases such as Crohn's disease as indicated in the confocal micrograph on the cover of this issue. Hyaluronan accumulation is often a predictor of patient survival, particularly in tissues normally low in hyaluronan such as breast and ovary. The relationship between hyaluronan and cell migration and proliferation is discussed in the context of metastasis and of atherosclerosis of vascular smooth muscle cells. The emerging instructive role of hyaluronan-based extracellular structures in trafficking of inflammatory cells is also noted.
Collectively, these minireviews by established experts in hyaluronan biology set the stage for an exciting era of discovery. Many seminal questions remain, including elucidation of the structural basis of hyaluronan binding by proteins and proteoglycans lacking link modules; the molecular basis for signaling initiated by cellular interactions with hyaluronan; the basis for the disparate biological responses to high and low molecular weight hyaluronan; and the identity of additional receptors for hyaluronan that mediate cell migration and invasion. This knowledge will serve as a basis for further rational manipulation of hyaluronan-mediated events central to morphogenesis, extracellular matrix biology, and human health.