Regulation of the small GTPase RhoA by syndecan-4 and integrins in focal adhesion formation

Introduction  The transmembrane heparan sulfate proteoglycan, syndecan-4, and integrins are important receptors for focal adhesion (FA) formation on fibronectin (FN) substrates. The small GTPase RhoA is also known to regulate FA and stress fiber formation. It has been suggested that syndecan-4 and integrins co-operatively regulate the assembly of FA in a Rho-dependent manner, but the mechanism is unclear. Here, we examined the function of RhoA and the Rho effector kinases ROCKs in syndecan-4 signalling on the process of FA formation and the possible mechanism by which syndecan-4 may regulate RhoA activity.

Methods  Primary rat embryonic fibroblasts (REFs) were seeded on FN or ‘RGD’-containing integrin-binding domain of FN and lysed at various time points. The amount of active form of RhoA in each lysate was analysed by pull-down experiments.

Results and discussion  The relative activities of RhoA showed one peak in the process of FA formation on FN, whereas no peak was obtained on the integrin-binding domain. The one peak of RhoA activity on integrin-binding domain was restored by addition of heparin-binding domain into medium. These results suggested that a signal through syndecan-4 link to the Rho pathway. Both ROCK-I and -II isozymes were present in REF cell lysates and each could be specifically immunoprecipitated. The ROCK kinase activities in immunoprecipitates were analysed using GST-myosin light chain as a substrate. The amount of ROCK-I and -II activities changed through the adhesion process on FN and appeared to be independently regulated. Therefore, one or both ROCKs may be downstream of a syndecan-4-mediated signalling response through RhoA. The core protein of syndecan-4 can directly bind to and activate PKC-α. We found that PKC-α could phosphorylate Rho-Guanine Nucleotide Dissociation Inhibitor (GDI) in vitro. It has been suggested that PKC-α-mediated phosphorylation of Rho GDI stimulates GDI dissociation, thereby resulting in Rho activation. It is possible that syndecan-4 regulates Rho/ROCK pathway through PKC-α activation on the process of FA formation.