Characterization of a Rac1 signaling pathway to cyclin D(1) expression in airway smooth muscle cells.
We examined the importance of the Rho family GTPase Rac1 for cyclin D(1) promoter transcriptional activation in bovine tracheal myocytes. Overexpression of active Rac1 induced transcription from the cyclin D(1) 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 D(1) in this system. Rac1 forms part of the NADPH oxidase complex that generates reactive oxygen species such as H(2)O(2). 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 Rac1, attenuated PDGF-induced cyclin D(1) 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 D(1) and smooth muscle growth.
Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.
K
K
Page
J
J
Li
J A
JA
Hodge
P T
PT
Liu
T L
TL
Vanden Hoek
L B
LB
Becker
R G
RG
Pestell
M R
MR
Rosner
M B
MB
Hershenson
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000235:genetics*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D014132:Trachea / Q000166:cytology / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D020744:rac GTP-Binding Proteins
D009861:Onium Compounds; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D019938:Cyclin D1; C007517:diphenyleneiodonium; D008156:Luciferases; D019255:NADPH Oxidases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins