Regulation of cyclin D1 expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells

We have shown in bovine tracheal myocytes that extracellular signal-regulated kinase (ERK) and Rac1 function as upstream activators of transcription from the cyclin D-1 promoter. We now examine the role of phosphatidylinositol (PI) 3-kinase in this process. PI 3-kinase activity was increased by platelet-derived growth factor (PDGF) and attenuated by the PI 3-kinase inhibitors wortmannin and LY294002. These inhibitors also decreased cyclin D-1 promoter activity, protein abundance, and DNA synthesis. Overexpression of the active catalytic subunit of PI 3-kinase (p110(PI) (3-K)CAAX) was sufficient to activate the cyclin D-1 promoter. Wortmannin and LY294002 failed to attenuate PDGF-induced ERK activation, and overexpression of p110(PI) (3-K)CAAX was insufficient to activate ERK. p110(PI) (3-K)CAAX-induced cyclin D-1 promoter activity was not blocked by PD98059, an inhibitor of mitogen-activated protein kinase/ERK kinase. We next examined whether PI 3-kinase and the 21-kD guanidine triphosphatase Rad regulate cyclin D-1 promoter activity by similar mechanisms. p110(PI 3-K)CAAX-induced cyclin D-1 promoter activity was decreased by two inhibitors of Rad-mediated signaling, catalase and diphenylene iodonium. Further, PDGF, PI 3-kinase, and Rad each activated the cyclin D-1 promoter at the cyclic adenosine monophosphate response element binding protein (CREB)/activating transcription factor (ATF)-2 binding site, as evidenced by expression of a CREB/ATF-2 reporter plasmid. Finally, PI 3-kinase and Rad-induced CREB/ATF-2 transactivation were each inhibited by catalase. Together, these data suggest that in airway smooth muscle (ASM) cells, PI 3-kinase regulates transcription from the cyclin D-1 promoter and DNA synthesis in an ERK-independent manner. Further, PI 3-kinase and Rac1 regulate ASM cell cycle traversal via a common cis-regulatory element in the cyclin D-1 promoter.