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J Biol Chem, Vol. 274, Issue 31, 22065-22071, July 30, 1999

Characterization of a Rac1 Signaling Pathway to Cyclin D1 Expression in Airway Smooth Muscle Cells

Kristen PageDagger , Jing LiDagger , Joshua A. HodgeDagger , Pai T. LiuDagger , Terry L. Vanden Hoek, Lance B. Becker, Richard G. Pestell, Marsha R. Rosnerparallel , and Marc B. HershensonDagger

From the Dagger  Department of Pediatrics, § Department of Medicine, parallel  Department of Pharmacological and Physiological Sciences and the Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637 and the  Albert Einstein Cancer Center, Department of Medicine and Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461

We examined the importance of the Rho family GTPase Rac1 for cyclin D1 promoter transcriptional activation in bovine tracheal myocytes. Overexpression of active Rac1 induced transcription from the cyclin D1 promoter, whereas platelet-derived growth factor (PDGF)-induced transcription was inhibited by a dominant-negative allele of Rac1, suggesting that Rac1 functions as an upstream activator of cyclin D1 in this system. Rac1 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 D1 promoter activation, while having no effect on the induction of cyclin D1 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 D1 protein expression and DNA synthesis. Overexpression of an N-terminal fragment of p67phox, a component of NADPH oxidase which interacts with Rac1, attenuated PDGF-induced cyclin D1 promoter activity, whereas overexpression of the wild-type p67 did not. Finally, Rac1 was neither required nor sufficient for ERK activation. Taken together, these data suggest a model by which two distinct signaling pathways, the ERK and Rac1 pathways, positively regulate cyclin D1 and smooth muscle growth.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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