Fibronectin increases RhoA activity through inhibition of PKA in the human gastric cancer cell line SGC-7901.

Fibronectin/integrin-mediated signaling plays a key role in the regulation of adhesion, migration and metastasis of tumors. Numerous studies have addressed the significance of the association between integrin and RhoA, but the exact mechanism is unclear. Results from laboratories, including ours, have demonstrated that PKA inhibits the activity and function of RhoA. This study was designed to investigate the relationships among the fibronectin/integrin-, cAMP/PKA- and RhoA-mediated intracellular signal transduction pathways. Rho activity was detected by pull-down assay. cAMP concentration was measured by radioimmunoassay. The distribution of the PKA catalytic subunit and the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) were detected by fluorescence microscopy and Western blotting, respectively, to examine the activation of PKA. cAMP-mediated gene expression activity was analyzed using a luciferase reporter gene assay. The results revealed that, in SGC-7901 cells, soluble fibronectin increased RhoA activity and blocked the inhibition of RhoA activity by cAMP/PKA. The cAMP level, which was increased by forskolin and pertussis toxin, was decreased by fibronectin. The nuclear location of the PKA catalytic unit, the phosphorylation of VASP and cAMP response element (CRE)-directed reporter gene expression induced by forskolin were blocked by fibronectin. However, fibronectin did not block VASP phosphorylation or CRE-directed reporter gene expression induced by cAMP. These data suggest that fibronectin/integrin induces RhoA activation through the inhibition of cAMP/PKA signal transduction. The possible point of action of fibronectin/integrin is adenylate cyclase.