Eva A. Turley

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.

HA receptors: Regulators of signalling to the cytoskeleton

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix (ECM) and occurs transiently in both the cell nucleus and cytoplasm. It has been shown to promote cell motility, adhesion, and proliferation and thus it has an important role in such processes as morphogenesis, wound repair, inflammation, and metastasis. These processes require massive cell movement and tissue reorganization and are always accompanied by elevated levels of HA. Many of the effects of HA are mediated through cell surface receptors, three of which have been molecularly characterized, namely CD44, RHAMM, and ICAM‐1. Binding of the HA ligand to its receptors triggers signal transduction events which, in concert with other ECM and cytoskeletal components, can direct cell trafficking during physiological and pathological events. The HA mediated signals are transmitted, at least in part, by the activation of protein phosphorylation cascades, cytokine release, and the stimulation of cell cycle proteins. A variety of extracellular signals regulate the expression of both HA and the receptors necessitating that HA‐receptor signalling is a tightly controlled process. Regulated production of soluble forms of the receptors, alternately spliced cell surface isoforms, and glycosylation variants of these receptors can dramatically modulate HA binding, ligand specificity, and stimulation of the signalling pathway. When these processes are deregulated cell behaviour becomes uncontrolled leading to developmental abnormalities, abnormal physiological responses, and tumorigenesis. The elucidation of the molecular mechanisms regulating HA‐mediated events will not only contribute greatly to our understanding of a variety of disease processes but will also offer many new avenues of therapeutic intervention.

Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl‐terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA‐‐Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site‐directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino‐terminus of RHAMM (amino acids 1‐238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses. Site‐directed mutations of these motifs in CD44 sequences abolished HA binding. Collectively, these results predict that the motif of B(X7)B as a minimal binding requirement for HA in RHAMM, CD44 and link protein, and occurs in all HA binding proteins described to date.

Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation

Overexpression of the RHAMM gene by transfection into fibroblasts is transforming and causes spontaneous metastases in the lung. H-ras-transformed fibrosarcomas transfected with a dominant suppressor mutant of RHAMM exhibit a so-called revertant phenotype and are completely nontumorigenic and nonmetastatic. Conversely, fibroblasts stably expressing low levels of RHAMM as a result of antisense transfection are resistant to ras transformation. Collectively, these results indicate that RHAMM acts downstream of ras. The loss of functional RHAMM ablates signaling within focal adhesions, in particular changes in focal adhesion kinase phosphorylation, and as a result these focal adhesions are unable to turn over in response to hyaluronan. These results provide evidence of the oncogenic potential of a novel extracellular matrix receptor and establish a functional link between transformation by ras and signaling within focal adhesions that are required for transformation by this oncogene.

The Hyaluronan Receptor RHAMM Regulates Extracellular-regulated Kinase

We have identified two RHAMM (receptor for hyaluronan-mediated motility) isoforms that encode an alternatively spliced exon 4 (Hall, C. L., Yang, B., Yang, X., Zhang, S., Turley, M., Samuel, S., Lange, L. A., Wang, C., Curpen, G. D., Savani, R. C., Greenberg, A. H., and Turley, E. A. (1995) Cell 82, 19–26 and Wang, C., Entwistle, J., Hou, G., Li, Q., and Turley, E. A. (1996) Gene 174, 299–306). One of these, RHAMM variant 4 (RHAMMv4), is transforming when overexpressed and regulates Ras signaling (Hall et al.). Here we note using flow cytometry and confocal analysis that RHAMM isoforms encoding exon 4 occur both on the cell surface and in the cytoplasm. Epitope-tagging experiments indicate that RHAMMv4 occurs only in the cytoplasm. Several observations suggest that both cell surface RHAMM isoforms and RHAMMv4 are involved in regulating extracellular-regulated kinase (ERK) activity. Affinity-purified anti-RHAMM exon 4 antibodies block the ability of platelet-derived growth factor to activate ERK, and these reagents modify the protein tyrosine phosphorylation profile of proteins resulting from treatment with platelet-derived growth factor. A dominant negative form of RHAMMv4 inhibits mutant active Ras activation of ERK and coimmunoprecipitates with both mitogen-activated protein kinase kinase and ERK, suggesting that the intracellular RHAMMv4 acts downstream of Ras, possibly at the level of mitogen-activated protein kinase kinase-ERK interactions. Consistent with this, overexpression of RHAMMv4 constitutively activates ERK. These results identify a novel mechanism for the regulation of the Ras-ERK signaling pathway and suggest that RHAMM plays multiple roles in this regulation.

Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM.

The migration of smooth muscle cells is a critical event in the pathogenesis of vascular diseases. We have investigated the role of hyaluronan (HA) and the hyaluronan receptor RHAMM in the migration of adult bovine aortic smooth muscle cells (BASMC). Cultured BASMC migrated from the leading edge of a single scratch wound with increased velocity between 1 and 24 h. Polyclonal anti-RHAMM antisera that block HA binding with this receptor abolished smooth muscle cell migration following injury. HA stimulated the random locomotion of BASMC and its association with the cell monolayer increased following wounding injury. Immunoblot analysis of wounded monolayers demonstrated a novel RHAMM protein isoform that appeared within one hour after injury. At the time of increased cell motility after wounding, FACS analysis demonstrated an increase in the membrane localization in approximately 25% of the cell population. Confocal microscopy of injured monolayers confirmed that membrane expression of this receptor was limited to cells at the wound edge. Collectively, these data demonstrate that RHAMM is necessary for the migration of smooth muscle cells and that expression and distribution of this receptor is tightly regulated following wounding of BASMC monolayers.

Autocrine induction of tumor protease production and invasion by a metallothionein-regulated TGF-beta 1 (Ser223, 225).

An expression vector was constructed in which TGF‐beta 1 was placed under the control of the metallothionein promoter. Cys223 and Cys225 in the TGF‐beta 1 propeptide were converted to serines, mutations which result in dissociation of the pro‐peptide and secretion of bioactive TGF‐beta 1 [Brunner, A.M., Marquardt, H., Malacko, A.R., Lioubin, M.N. and Purchio, A.F. (1989) J. Biol. Chem., 264, 13660–13664]. A fibrosarcoma was transfected with this plasmid and a clone (17.18) was selected in which TGF‐beta 1 mRNA was able to be induced six‐fold following zinc sulphate treatment. These cells increased the secretion of bioactive TGF‐beta 1 14‐fold and exhibited a coincidental increase in jun‐B mRNA expression, suggesting that secreted TGF‐beta 1 was acting to induce this early response gene by autocrine activation. Following zinc sulphate induction, the tumor cells became progressively more motile and able to invade collagen gels. In contrast to parental tumor not bearing the TGF‐beta 1 expression vector, zinc sulphate stimulation of clone 17.18 enhanced collagenase IV and procathepsin L mRNA levels and enhanced the secretion of many collagenolytic proteases into the medium. Since the action of TGF‐beta generally decreases proteolysis by suppression of protease transcription, we compared the response of normal parental fibroblasts to ras‐transformed fibrosarcomas and confirmed that TGF‐beta could greatly enhance collagenase IV and procathepsin L mRNA levels while having little effect on non‐transformed fibroblasts. These experiments indicate that induction of TGF‐beta secretion can enhance motility and protease production through autocrine activation, thus increasing the invasion potential of fibrosarcomas.

Purification of a hyaluronate-binding protein fraction that modifies cell social behavior

Abstract A hyaluronate-binding protein fraction (HABP) has been purified from the supernatant media of chick fibroblas cultures by ultrafiltration and Dowex-hyaluronate affinity chromatography. This protein fraction binds more avidly to Dowex-hyaluronate than to other Dowex-glycosaminoglycans. It can be inserted into the cell layer of urea-pretreated fibroblast monolayers where it specifically increases the amount of exogenous hyaluronate, but not sulfated glycosaminoglycans, attached to the cell glycocalyx. This interaction promotes a culture morphology and nuclear overlap ratio similar to virally-transformed cells. The principal hyaluronate-binding protein in the affinity purified fraction has a m.w. app. of 60,000–63,000 daltons.

Localization of hyaluronate and hyaluronate-binding protein on motile and non-motile fibroblasts☆

The distribution of a hyaluronate-binding (HABP) and rhodamine B-isothiocyanate (RITC)-labeled hyaluronate (HA) were studied on both actively motile and stationary chick heart fibroblasts to assess the relationship of these molecules to each other, to other extracellular matrix molecules, to membrane protrusions and to adhesion sites. RITC-HA and HABP, detected by indirect immunofluorescence, were concentrated in the perinuclear region, the leading lamella and retraction processes of actively motile cells, although RITC-HA also occurred diffusely over the rest of the cell body. Double immunofluorescence confirmed that HA and HABP co-localized in the former three regions, suggesting that, at these locations, the HABP may act as a cell surface-binding site for HA. With increasing culture confluency and consequent slowing of fibroblast motility, the localization of both polymers changed to a uniform and diffuse distribution over the cell body and processes. On actively motile cells, RITC-HA and HABP did not co-distribute with fibronectin, heparan sulfate proteoglycan or laminin. Areas coated with RITC-HA and HABP often contained specialized adhesion sites as determined by interference reflection microscopy (IRM) but neither polymer appeared to particularly localize to adhesion sites. However, the occurrence of RITC-HA and HABP in the leading lamellae of motile cells consistently coincided with ruffling activity. These results are discussed with respect to a possible instructive role of HA in cell motility.

RHAMM, a receptor for hyaluronan-mediated motility, on normal human lymphocytes, thymocytes and malignant B cells : a mediator in B cell malignancy?

RHAMM (Receptor for HA Mediated Motility) is a novel HA receptor that has been linked to regulating cell locomotion and density dependent contact inhibition of fibroblasts, smooth muscle cells, macrophages, lymphocytes, astrocytes and sperm. The ubiquitous expression of RHAMM suggests the existence of multiple isoforms, and indeed, RHAMM is found in various cellular compartments, namely nuclear, cytosolic, membrane-bound and extracellular. In this review, we emphasize the evolving role of RHAMM in B cell malignancies, and examine the function of RHAMM in T cell development in the thymic microenvironment. Both the motile behaviour of progenitor thymocytes (CD3-CD4-CD8-) and malignant B cells from multiple myeloma (MM), plasma cell leukemia, and hairy cell leukemia was blocked by monoclonal antibodies to RHAMM, suggesting that motility may correlate with increased expression of RHAMM at the cell surface. Interestingly, the soluble form of RHAMM is able to inhibit fibroblast locomotion, and it is likely that a balance between expression of both forms determines, in part the motility of cells. RHAMM appears to play a fundamental role in the immune system and the ability of RHAMM to function as a motility receptor is likely to be due to complex variables including the extent to which soluble RHAMM is secreted. RHAMM expression characterizes circulating monoclonal B cells as abnormal. potentially invasive and/or metastatic components of myeloma and may underlie the malignant behavior of these cells.