HYALURONAN AND THE HYALURONAN RECEPTOR RHAMM PROMOTE FOCAL ADHESION TURNOVER AND TRANSIENT TYROSINE KINASE-ACTIVITY

The molecular mechanisms whereby hyaluronan (HA) stimulates cell motil ity 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 i mmunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and tra nsient with phosphorylation occurring within 1 min 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 wit hin the HA receptor RHAMM. Treatment of cells with tyrosine kinase inh ibitors (genistein, 10 mu g/ml or herbimycin A, 0.5 mu g/ml) or microi njection of anti-phosphotyrosine antibodies inhibited the transient pr otein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimul ated tyrosine phosphorylation and the resulting cell locomotion, cytos keletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapi d assembly and disassembly of focal adhesions as revealed by immunoflu orescent localization of vinculin. The time course with which HA and a ntibody induced focal adhesion turnover exactly paralleled the inducti on of transient protein tyrosine phosphorylation. In addition, phospho tyrosine staining colocalized with vinculin within structures in the l amellapodia of these cells. Notably, the focal adhesion kinase, pp125( FAK), was rapidly phosphorylated and dephosphorylated after HA stimula tion. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAM M. They also provide a possible molecular basis for focal adhesion tur nover, a process that is critical for cell locomotion.