Signaling Properties of Hyaluronan Receptors*

In 1979, hyaluronan was demonstrated to bind specifically and with high affinity to intact cells (1), and in 1980, it was shown to enhance cell motility on two-dimensional culture surfaces where the hydrodynamic properties of hyaluronan were not necessary to open spaces for cells to move into (2). These two demonstrations raised the possibility that hyaluronan had the potential to directly modify cell behavior. In 1989, hyaluronan was shown to promote protein tyrosine phosphorylation cascades (3) that were later proven to be required for hyaluronan-mediated motility on planar culture surfaces (4). Since then, small amounts (nanograms) of hyaluronan have been shown to activate a variety of protein tyrosine and serine/threonine kinases. These include the non-receptor protein tyrosine kinase Src (5, 6), HER2/Neu receptor (7), focal adhesion kinase (4, 8–10), protein kinase C (11, 12), and MAP kinases (9, 10). Likely as a consequence of regulating these kinases, hyaluronan promotes expression of specific cytokines and proteins involved in extracellular matrix remodeling (e.g. Ref. 13). The study of murine cardiac cells derived from hyaluronan synthase 2 (HAS2) knockout mice has provided the most convincing evidence for a signaling capability of hyaluronan (14). HAS2 / cardiac cells do not undergo an endothelial-mesenchymal transformation associated with migration from tissue explants whereas wild-type cells do (14). However, the addition of nanogram amounts of exogenous hyaluronan “rescues” knockout cells. Furthermore, a dominant negative mutant of the small GTPase, Ras, blocks the effects of exogenous hyaluronan (14). These results suggest that hyaluronan signals through Ras to regulate motility and are consistent with previous studies showing that exogenous hyaluronanreceptor interactions regulate Ras signaling (4, 8). This ability of hyaluronan to activate intracellular signaling cascades requires interactions with cell-associated hyaluronan-binding proteins or hyaladherins (15) but is additionally modified by the amount and size of hyaluronan present in the environment of the cell. Further, not all cell types activate signaling cascades in response to hyaluronan (11), indicating that cell background is also an important determinant. Here, we review current understanding of the mechanisms by which hyaluronan signals.

In 1979, hyaluronan was demonstrated to bind specifically and with high affinity to intact cells (1), and in 1980, it was shown to enhance cell motility on two-dimensional culture surfaces where the hydrodynamic properties of hyaluronan were not necessary to open spaces for cells to move into (2). These two demonstrations raised the possibility that hyaluronan had the potential to directly modify cell behavior. In 1989, hyaluronan was shown to promote protein tyrosine phosphorylation cascades (3) that were later proven to be required for hyaluronan-mediated motility on planar culture surfaces (4). Since then, small amounts (nanograms) of hyaluronan have been shown to activate a variety of protein tyrosine and serine/threonine kinases. These include the non-receptor protein tyrosine kinase Src (5,6), HER2/Neu receptor (7), focal adhesion kinase (4, 8 -10), protein kinase C (11,12), and MAP 1 kinases (9,10). Likely as a consequence of regulating these kinases, hyaluronan promotes expression of specific cytokines and proteins involved in extracellular matrix remodeling (e.g. Ref. 13).
The study of murine cardiac cells derived from hyaluronan synthase 2 (HAS2) knockout mice has provided the most convincing evidence for a signaling capability of hyaluronan (14). HAS2Ϫ/Ϫ cardiac cells do not undergo an endothelial-mesenchymal transformation associated with migration from tissue explants whereas wild-type cells do (14). However, the addition of nanogram amounts of exogenous hyaluronan "rescues" knockout cells. Furthermore, a dominant negative mutant of the small GTPase, Ras, blocks the effects of exogenous hyaluronan (14). These results suggest that hyaluronan signals through Ras to regulate motility and are consistent with previous studies showing that exogenous hyaluronanreceptor interactions regulate Ras signaling (4,8). This ability of hyaluronan to activate intracellular signaling cascades requires interactions with cell-associated hyaluronan-binding proteins or hyaladherins (15) but is additionally modified by the amount and size of hyaluronan present in the environment of the cell. Further, not all cell types activate signaling cascades in response to hyaluronan (11), indicating that cell background is also an important determinant. Here, we review current understanding of the mechanisms by which hyaluronan signals.

Role of Hyaluronan Receptors in Signaling
The first cell-associated hyaladherin, RHAMM, whose cell surface form is now designated CD168, was isolated from embryonic heart cells (16). 2 Later CD44 was identified as the first integral hyaluronan "receptor." Both RHAMM and CD44 mediate hyaluronan signaling and participate in growth factor-regulated signaling. However, they likely regulate signaling by different mechanisms because they are not homologous proteins, are compartmentalized differently in the cell (17), 2 and differ in the mechanisms by which they bind to hyaluronan (18) (Figs. 1 and 2). Additional cellular hyaladherins have been identified (19 -22), but their role in cell signaling has not yet been reported. Therefore, this review focuses upon the signaling cascades that RHAMM and CD44 regulate.

CD44
CD44 is an integral protein that is subject to extensive alternative splicing (23)(24)(25)(26). All CD44 isoforms contain a link module hyaluronan-binding site in their extracellular domain (see minireview by Day and Prestwich (18) in this series). The binding of CD44 isoforms to hyaluronan affects cell adhesion to extracellular matrix components and is implicated in the stimulation of aggregation, proliferation, migration, and angiogenesis (23-25, 27, 28). The intracellular domain of CD44 isoforms selectively interacts with cytoskeletal proteins and regulates specific signaling (27). Therefore, CD44 isoforms likely provide a direct association between hyaluronan and the cytoskeleton. The mechanisms by which CD44 achieves this association and the signaling cascades that it regulates are summarized in Fig. 1.
CD44 Interaction with Tyrosine Kinases-CD44 is tightly coupled with at least two tyrosine kinases, p185 HER2 (7) and c-Src kinase (6). CD44 and p185 HER2 are physically linked to each other via interchain disulfide bonds; and hyaluronan can stimulate CD44-associated p185 HER2 tyrosine kinase activity that leads to increased tumor cell growth (7). The cytoplasmic domain of CD44 binds to c-Src kinase at a single site with high affinity (6,29). Importantly, hyaluronan interaction with CD44 stimulates c-Src kinase activity, increasing tyrosine phosphorylation of the cytoskeletal protein, cortactin. This attenuates the ability of cortactin to cross-link filamentous actin in vitro ( Fig. 1) (6). Most Src family kinases are modified with specific lipids that direct them to subdomains of the cell membrane called "rafts" that have high cholesterol and glycolipid content. The Src kinases, Lck and Fyn, associate with CD44 in glycosphingolipid-rich plasma membrane domains of human peripheral blood lymphocytes (29). Thus, direct binding of CD44 to c-Src kinase in the membrane "rafts" may facilitate hyaluronan-mediated stimulation of the catalytic activity of c-Src kinase and induce cytoskeleton-regulated tumor cell migration. Therefore, the binding of hyaluronan to CD44 isoforms, which complex with p185 HER2 and c-Src kinase, likely trigger direct "cross-talk" between two tyrosine kinase-linked signaling pathways during tumor progression.
CD44-specific Activation of Rho-like GTPases-Rho GTPases such as RhoA and Rac1 participate in the interaction between CD44 and cytoskeletal proteins. In particular, RhoA is non-covalently linked to a CD44 alternate isoform (e.g. CD44 V3,8 -10 ) in breast tumor cells (30). When complexed with CD44 V3 , RhoA stimulates Rho kinase (ROK) to phosphorylate several cellular proteins including CD44 V3, 8 -10 . This phosphorylation promotes binding of the CD44 variant to ankyrin (Fig. 1). Overexpression of the Rhobinding domain can act as a dominant negative inhibitor of ROK and reverse tumor cell-specific phenotypes (30). Therefore, it has been proposed that CD44v 3,8 -10 and RhoA-mediated signaling are involved in the up-regulation of ROK and that this is necessary for membrane-cytoskeleton interactions and tumor cell migration during the progression of breast cancers (30).
Binding of hyaluronan to some CD44-expressing cells also activates Rac1 signaling, a pathway known to regulate actin assembly that is associated with membrane ruffling, cellular projections, cell motility, and cell transformation (31,32). In particular, the cytoplasmic domain of CD44 binds to guanine nucleotide exchange factors such as Tiam1 and Vav2 that have been shown to catalyze the GDP-GTP exchange leading to hyaluronan-mediated tumor cell migration ( Fig. 1) (32-34). The fact that both Tiam1-Rac1 activa-tion (31,33,34) and RhoA-mediated ROK activity (30) are involved in regulating cytoskeleton function and cell motility in a hyaluronan-and CD44-dependent manner suggests these pathways play a pivotal role in hyaluronan-stimulated CD44 signaling.
CD44 Interaction with the Cytoskeleton-The first 19 residues of the cytoplasmic domain of CD44 interact with the cytoskeleton membrane linker proteins, ezrin/radixin/moesin (ERM) (32), which contain the KKX n (K/R)K motif for phosphatidylinositol 4,5bisphosphate (PIP 2 ) binding (35). Mutation of this motif on ezrin results in the loss of the PIP 2 requirement for optimal binding of ezrin to CD44 but does not influence the complex formation between ezrin and CD44 (35). These findings suggest that the linkage between ERM and CD44 can form in either a PIP 2 -dependent or a PIP 2 -independent manner. An involvement of PIP 2 in regulating CD44-ERM interaction during hyaluronan signaling has not yet been reported.
Ankyrin is also a family of membrane-associated cytoskeletal proteins expressed in a variety of biological systems (24,28). The cytoplasmic domain of CD44 (ϳ70 amino acids) is highly conserved in most of the CD44 isoforms and is directly involved in ankyrin binding (24,28). Deletion mutation analyses indicate that at least two subregions within the CD44 cytoplasmic domain contribute to the ankyrin binding, namely region I (e.g. the high affinity ankyrinbinding region) and region II (e.g. the regulatory region). In particular, region I ( 306 NGGNGTVEDRKPSEL 320 in the mouse CD44 and 304 NSGNGAVEDRKPSGL 318 in the human CD44) is required for hyaluronan-mediated binding and cell adhesion (24). Furthermore, an ankyrin-binding domain of CD44 isoforms has also been shown to be necessary for oncogenic signaling and tumor cell transformation (27). Recently, fragments of the ankyrin repeat domain and/or the subdomain 2 of ankyrin repeat domain have been identified as an ankyrin-binding region for both CD44 (26) and Tiam1 (32). Overexpression of these ankyrin fragments promotes hyaluronan-dependent and CD44-specific tumor cell migration (33). These observations support the notion that CD44-ankyrin interaction is not only very important for presenting CD44 properly for hyaluronan binding but is also required for cytoskeleton activation during hyaluronan signaling (Fig. 1).
The binding of exogenous hyaluronan to cell surface RHAMM plays a key role in activating signaling cascades, probably as a co-receptor for integral membrane proteins. Although the role(s) of intracellular RHAMM protein forms are not yet known, their ability to associate with kinases (5,9), calmodulin (41,42), and the cytoskeleton (41,43) predicts that they play key roles in cytoskeletal assembly. The presence of intracellular hyaladherins typified by RHAMM also raises the interesting possibility that intracellular hyaluronan (48 -50) plays a role in signaling. If so, the separation of cell surface RHAMM from the intracellular RHAMM forms provides the potential for a modular association among hyaluronan, cytoskeleton-signaling complexes, and the cell nucleus (Fig. 2). In this case, hyaladherins such as RHAMM could contribute to the back and forth flow of information between the cell genome and the extracellular environment, a phenomenon that has been termed "dynamic reciprocity" (51). For instance, RHAMM modules could represent a modified version of "inside-outside" signaling characteristic of integrin receptors (52).
RHAMM Interaction with Tyrosine and Serine/Threonine Kinases (see Fig. 2)-Cell surface RHAMM-hyaluronan interactions mediate activation of the protein tyrosine kinases, Src (5) and focal adhesion kinase (4, 8, 10), as well as Erk kinases (10) and protein kinase C (11, 12). Additionally, cell surface RHAMM is required for activation of Erk kinases through PDGF (9), nerve growth factor FIG. 1. A current model for hyaluronan dependent, CD44-specific signaling pathways. CD44-hyaluronan interactions promote tyrosine kinase (TK) activity of HER2 and the non-receptor kinase, Src. Src phosphorylates cortactin, which recruits it to the cell membrane. CD44-hyaluronan interactions also activate RHOA and Rac1, and CD44 binds to Tiam1 and Vav2. Hyaluronan (HA) also promotes the association of CD44 forms with cytoskeletal proteins such as ankyrin and ERM proteins. Activation of these signaling pathways together leads to tumor behavior such as migration and invasion. Our current model suggests that the close interactions between CD44 and its selected binding partners play a pivotal role in coordinating "cross-talk" among various intracellular signaling pathways (e.g. Rho/Ras signaling and receptor-linked (p185 HER2 )/non-receptor-linked (c-Src) tyrosine kinase pathways) leading to the concomitant onset of multiple functions such as tumor cell adhesion, proliferation/growth, migration, and invasion. MLC, myosin light chains.

FIG. 2. A current model for hyaluronan (HA)-dependent, RHAMMmediated signaling pathways.
RHAMM is an itinerant hyaladherin that occurs in multiple subcellular compartments and that can also be exported to the extracellular milieu where it binds to the cell surface. Cell surface RHAMM-hyaluronan interactions regulate signaling through Ras and Src. Cell surface RHAMM modifies the ability of the PDGF receptor to activate Erk kinase, a key map kinase involved in cell motility. Intracellular RHAMM proteins encode multiple kinase docking and recognition sites, and one intracellular form has been shown to physically associate with Erk1 kinase. Intracellular forms also associate with the cytoskeleton, notably interphase and mitotic spindle microtubules. The ability of intracellular RHAMM forms to associate with multiple signaling complexes and to associate with the cytoskeleton suggest that they function as adapter proteins like vinculin and paxillin. FAK, focal adhesion kinase. (53), and after stretch injury (54). Activation of Src through cell surface RHAMM is transient (5) but is nevertheless required for turnover of lamellae focal adhesions and consequently for RHAMM-regulated cell motility (5). Proteins that are phosphorylated on tyrosine as a result of hyaluronan-RHAMM interactions include paxillin, cortactin, focal adhesion kinase, and the MAP kinases, Erk1 and -2 (10).
RHAMM co-associates with ϳ20% of total cellular Erk1 kinase (9) and ϳ10% of total cellular Src (5), as determined by co-immunoprecipitation analyses. Because RHAMM contains recognition sequences for both Src and Erk (MOTIF SCANNER, cansite.bidmc. harvard.edu), it is likely that intracellular RHAMM forms associate directly with these kinases. RHAMM contains multiple SH3and SH2-binding sites as well as putative phosphoacceptor sites for additional serine/threonine kinases. Intracellular forms may therefore participate in linking kinases together as complexes and/or retaining them within the cytoskeleton and the nucleus.
RHAMM Regulation of Ras GTPase-Several studies have indicated that RHAMM regulates Ras (4,5,8,9,37,55), and this regulation likely involves both cell surface and intracellular RHAMM forms. Cell surface RHAMM is required for random motility (4,5), progression through the G 2 M boundary of cell cycle (55), and transformation by oncogenic Ras (8), as determined by antibody blocking and soluble protein competition. Both cell surface and intracellular forms of RHAMM may regulate Ras but at different points along the signaling pathways (e.g. in Fig. 2).
The ability of RHAMM to regulate Ras requires its hyaluronan binding ability. Thus, mutation of the RHAMM hyaluronan-binding sites blocks oncogenic Ras-mediated motility and transformation (8). This is consistent with a requirement for Ras in the hyaluronan-mediated rescue of HAS2Ϫ/Ϫ cell motility (14).
RHAMM Interaction with the Cytoskeleton-Cell surface RHAMM occurs in cell lamellae and podosomes where it co-distributes with cortactin. 2 Exogenous hyaluronan initially promotes spreading of cell lamellae and turnover of focal adhesions in fibroblasts transfected with oncogenic Ras that express cell surface RHAMM and that are plated onto fibronectin-coated surfaces (4). This effect of hyaluronan is proposed to act through cell surface RHAMM because blocking RHAMM antibodies inhibit, whereas agonist RHAMM antibodies mimic this effect (6,7). The rapid formation and then disassembly of focal adhesions precede a hyaluronan-induced increase in cell motility (4). These effects are possibly mediated, either directly or indirectly, through integrin receptors. For example, cell surface RHAMM co-regulates migration of thymocytes on fibronectin substrata in concert with the integrin fibronectin receptors, ␣ 4 ␤ 1 or ␣ 5 ␤ 1 (56).
Intracellular RHAMM forms associate with the actin cytoskeleton and both interphase and mitotic spindle microtubules (41). The ability of RHAMM to form coiled coils as well as its limited homology with proteins such as D-CLIP (57) that link microtubules and actin filaments suggests that intracellular RHAMM proteins may connect actin and microtubule cytoskeleton (58). Because an association of RHAMM with several kinases has already been established, it is likely that intracellular RHAMM proteins act as adapter molecules, much like vinculin and paxillin (59, 60) that connect the cytoskeleton to signaling complexes. Transcriptional profiling of the cell cycle progression reveals enhanced expression of both RHAMM and hyaluronan synthase 2 (HAS2) at the G 2 M boundary (61). These last results and the demonstration that intracellular hyaluronan decorates the mitotic spindle (41) once again raise the possible role of intracellular hyaluronan/RHAMM interactions in signaling.
RHAMM and CD44 can perform separate functions in regulating cell signaling (10,38,62,63). For instance, CD44, but not cell surface RHAMM, can mediate adhesion of endothelial cells and thymocytes to hyaluronan (62) and regulate proliferation (38,63). In contrast, cell surface RHAMM, but not CD44, is required for migration (62,63). As well, cell surface RHAMM but not CD44 appears to be essential for activation of protein tyrosine kinase cascades by endothelial cells responding to hyaluronan (10). Deletion of either CD44 or RHAMM 3 does not result in embryonic lethality and therefore either these two hyaladherins share some functions and/or other cellular hyaladherins are able to compensate for the loss of either CD44 or RHAMM. Phenotypes for CD44Ϫ/Ϫ have been described (Ref. 64 and see below).

Potential Role of Hyaluronan Size in Signaling:
Importance in Response to Injury In physiologic conditions, hyaluronan is a high average molecular mass polymer in excess of 10 6 Da. However, following tissue injury, hyaluronan fragments of lower molecular mass accumulate. A potential functional significance for hyaluronan fragments has been suggested by in vitro studies (e.g. Refs. [65][66][67]. Oligomers of 8 -16 disaccharides prepared by enzymatic digestion of native hyaluronan induce angiogenesis in a chick corneal assay whereas the native hyaluronan molecules do not (68). Small amounts of high molecular weight hyaluronan can activate protein-tyrosine kinase cascades in endothelial cells and Ras-transformed fibroblasts (4,10), although at lower levels than smaller fragments (10). Studies with inflammatory macrophages have shown that fragmented hyaluronan with an average molecular mass of 250,000 Da, but not native hyaluronan from which it was prepared, can induce the expression of inflammatory genes (64,(65)(66)(67)(68)(69). Similar results have been shown with renal tubular epithelial cells (70), T-24 carcinoma cells (71), and eosinophils (72). Smaller hyaluronan oligosaccharides in the 6 -20 kDa size range (but not the 250,000 Da or higher molecular mass hyaluronan) induce inflammatory gene expression in dendritic cells (73). Controls for excluding contaminating substances that can be present in even medical grade hyaluronan preparations are often lacking, as noted in Ref. 75. Nevertheless, biological relevance is suggested by reports showing that fragmented hyaluronan that induces inflammatory gene expression in vitro is in the same size range as hyaluronan that accumulates under inflammatory conditions in vivo (74). A common theme appears to be that low (but not high) molecular weight hyaluronan can initiate gene transcription geared toward cell proliferation and migration. Generation of hyaluronan fragments under conditions of inflammation, tumorigenesis, or tissue injury as a result of hyaluronidases (76) or oxidation (77) may then signal the host that normal homeostasis has been profoundly disturbed.

Role of Hyaladherins in Hyaluronan Internalization
and Host Response to Injury Cellular hyaladherins also bind and internalize hyaluronan (1). Depending on the cell type, the binding affinity and rate of internalization varies. Elegant studies have recently shown that the avidity of binding of hyaluronan oligomers to CD44 increases with an oligomer size of up to 38 sugars (78). Labeled hyaluronan is also observed in the cytoplasm as a diffuse network and in vesicles, in lamellae and the nucleus of smooth muscle cells and fibroblasts (49,50). This unusual pattern of uptake is most obvious following stimulation of serum-starved 3T3 cells or in subconfluent, mutant active Ras-transfected fibroblasts. Hyaluronan uptake into these novel compartments is associated with enhanced cell motility (49). CD44-deficient mice develop normally but exhibit abnormalities in hematopoiesis and lymphocyte recirculation (79,80). Induction of inflammatory gene expression in response to hyaluronan is observed in the absence of CD44 in cultures of bone marrow and dendritic cells (73,81). These data suggest that there are CD44independent mechanisms for induction of gene expression by hyaluronan. In contrast, CD44-deficient mice challenged in models of tissue injury have revealed essential roles for CD44 in regulating pathogenesis of host injury (82,83). Depending on the mechanism of pathogenesis and the predominant cell types that mediate the host injury, differing effects of CD44 have been observed. In a model of endothelial cell injury mediated by interleukin-2, CD44deficient mice were protected from endothelial injury (82). This protection may be because of a decrease in interleukin-2-induced lymphocyte-activated killer cell activity. However, in a model of hepatocellular injury due to administration of concanavalin A, CD44-deficient mice exhibited enhanced hepatitis (83). The increased susceptibility to hepatocyte injury correlated with the observation that T cells from CD44-deficient animals were resistant to activation-induced cell death. The challenge of future studies will be to sort out the CD44-dependent as well as the independent roles in mediating hyaluronan-cell interactions.

Conclusions
Hyaluronan signaling involves cellular hyaladherins such as CD44 and RHAMM that are selectively coupled with their particular downstream signaling pathway(s) leading to the onset of hyaluronan-dependent functions in various cell types and tissues. A signaling response to hyaluronan may be strongly influenced by both the size of hyaluronan and the cell background. Additional studies with defined preparations of hyaluronan of varying size that utilize hyaladherin null mice are clearly required to sort out the cellular conditions that permit hyaluronan-mediated signaling.