Characterization of a rac1 signaling pathway to cyclin D1 expression in airway smooth muscle cells

We examined the importance of the Rho family GTPase Rad for cyclin D-1 promoter transcriptional activation in bovine tracheal myocytes. Overexpression of active Rad induced transcription from the cyclin D-1 promoter, whereas platelet-derived growth factor (PDGF)-induced transcription was inhibited by a dominant-negative allele of Rad, suggesting that Rad functions as an upstream activator of cyclin D-1 in this system. Rad forms part of the NADPH oxidase complex that generates reactive oxygen species such as H2O2. PDGF stimulated a substantial increase in intracellular reactive oxygen species, as measured by the fluorescence of dichlorofluorescein-loaded cells, and this was blocked by the glutathione peroxidase mimetic ebselen. Pretreatment with ebselen, catalase, and the flavoprotein inhibitor diphenylene iodonium each attenuated PDGF- and Rac1-mediated cyclin D-1 promoter activation, while having no effect on the induction of cyclin D-1 by mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase-1 (MEK1), the upstream activator of ERKs. Antioxidant treatment also inhibited PDGF-induced cyclin D-1 protein expression and DNA synthesis. Overexpression of an N-terminal fragment of p67(phox), a component of NADPH oxidase which interacts with Rad, attenuated PDGF-indueed cyclin D-1 promoter activity, whereas overexpression of the wild-type p67 did not. Finally, Rad was neither required nor sufficient for ERK activation. Taken together, these data suggest a model by which two distinct signaling pathways, the ERK and Rad pathways, positively regulate cyclin D-1 and smooth muscle growth.