Insulin-increased prolactin gene expression requires actin treadmilling: potential role for p21 activated kinase.

Insulin-increased prolactin gene transcription in GH4 cells was enhanced by binding on fibronectin. This was mediated by receptor-like protein tyrosine phosphatase alpha, which activated Src, Rho, and phosphatidylinositol 3-kinase. It suggested that insulin signaling to gene transcription was partly dependent on actin rearrangement. This was confirmed through studies using inhibitors of actin treadmilling. Cytochalasin D, jasplakinolide, latrunculin B, and swinholide A altered the actin cytoskeleton of GH4 cells, as assessed by Alexa Fluor phalloidin staining, and inhibited insulin-increased prolactin gene transcription. These reagents did not affect the controls. Nor was it due to a gross defect of insulin signaling because activation/translocation of glycogen synthase kinase 3beta and mammalian target of rapamycin were not affected. Expression of wild-type and mutant actin treadmilling agents, Cdc42, TC10, neuronal Wiskott-Aldrich syndrome protein, and Nck, indicated that they were essential to insulin-increased prolactin gene expression, and suggested that activation of p21 associated kinase (PAK) might also be essential to this process. PAK expression also increased and PAK mutants decreased prolactin promoter activity in insulin-treated cells. The activation of PAK in the presence of inhibitors was also consistent with a role in activation of insulin-increased prolactin gene expression. Finally, small interfering RNA-mediated reduction of PAK decreased the effect of insulin on prolactin gene expression. Thus, it is likely that insulin activation of actin treadmilling through Cdc42/TC10 and neuronal Wiskott-Aldrich syndrome protein activates PAK and prolactin gene transcription.