Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity.

Abstract

Abstract. The molecular mechanisms whereby hyaluronan (HA) stimulates cell motility was investigated in a C-H-ras transformed 10T 1/2 fibroblast cell line (C3). A significant (p < 0.001) stimulation of C3 cell motility with HA (10 ng/ml) was accompanied by an increase in protein tyrosine phosphorylation as detected by anti-phosphotyrosine antibodies using immunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and transient with phosphorylation occurring within 1 rain of HA addition and dissipating below control levels 10-15 min later. These responses were also elicited by an antibody generated against a peptide sequence within the HA receptor RHAMM. Treatment of cells with tyrosine kinase inhibitors (genistein, 10 #g/ml or herbimycin A, 0.5 /zg/ml) or microinjection of anti-phosphotyrosine antibodies inhibited the transient protein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimulated tyrosine phosphorylation and the resulting cell locomotion, cytoskeletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapid assembly and disassembly of focal adhesions as revealed by immunofluorescent localization of vinculin. The time course with which HA and antibody induced focal adhesion turnover exactly paralleled the induction of transient protein tyrosine phosphorylation. In addition, phosphotyrosine staining colocalized with vinculin within structures in the lamellapodia of these cells. Notably, the focal adhesion kinase, pp125 F~, was rapidly phosphorylated and dephosphorylated after HA stimulation. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAMM. They also provide a possible molecular basis for focal adhesion turnover, a process that is critical for cell locomotion.

Hyaluronan-dependent pericellular matrix☆

“…As mentioned above, removal of the pericellular coat of human smooth muscle cells by competition with oligosaccharides resulted in a flatter, more adherent phenotype, again consistent with an anti-adhesive property of the coat. Evidence of increased focal adhesion turnover following addition of hyaluronan to migrating cells highlights the dynamic role of the pericellular matrix [87]. Rapid uptake of the hyaluronan and translocation to the nucleus, with signaling mediated through RHAMM, was associated with increased motility.…”

Inhibition of tumor growth in vivo by hyaluronan oligomers

“…This result suggests that both oligomers and polymer act by similar mechanisms, possibly inhibiting a cellular event essential for tumor cell proliferation. Evidence for signal transduction in response to interactions of hyaluronan with cell surface CD44 (Lesley et al, 1993;Bourguignon et al, 1993;Galluzo et al, 1995;Taher et al, 1996) or RHAMM (Entwistle et al, 1996;Hall et al, 1994Hall et al, , 1995 has been reported. However, in some cases, the effects of hyaluronan oligomers have been shown to differ markedly from those of hyaluronan polymer (West and Kumar, 1989;Noble et al, 1993).…”

During human thymic development, β1 integrins regulate adhesion, motility, and the outcome of RHAMM/hyaluronan engagement

“…Previous work demonstrated that motility of human thymocytes was mediated by RHAMM [2,34,39], an HA receptor involved in signal transduction and turnover of focal adhesions [57]. Ex vivo, thymocytes bind HA almost exclusively via RHAMM despite their abundant expression of CD44 [S. L. Gares, M. Crainie, and L. M. Pilarski, unpublished results].…”