Galpha(13) stimulates Rho-dependent activation of the cyclooxygenase-2 promoter.
Cyclooxygenase-2 (COX-2) gene expression is rapidly increased by cytokines, tumor promoters, and growth factors and is markedly enhanced in various cancer cells. Here, we examine the regulation of COX-2 promoter activity by alpha subunits of heterotrimeric G proteins in NIH 3T3 cells. Using a transient transfection assay with a reporter vector in which the murine COX-2 promoter drives the production of luciferase and expression vectors encoding for alpha subunits of G-proteins, we show that overexpression of wild type and constitutively active Galpha(13) and Galpha(q) induced transcription from the COX-2 promoter. The highest level of induced luciferase activity (5.8-fold) occurred in cells expressing the constitutively active Galpha(13)(Q226L). We also show that expression of a constitutively active mutant of Rho (RhoQ63L) also induced transcription from the COX-2 promoter. Co-expression of Clostridium botulinum C3 toxin specifically blocked induction of the COX-2 promoter by either Galpha(13)Q226L or RhoQ63L but did not prevent the activation of this promoter by Ras, Rac, v-src, or forskolin. We conclude that Galpha(13) signals through a Rho-dependent pathway leading to activation of the COX-2 promoter. This pathway is not inhibited by either cytochalasin D, which disrupts actin filament organization, or genistein, a broad spectrum tyrosine kinase inhibitor, indicating a bifurcation of the signaling pathway used by Galpha(13)/Rho to induce COX-2 expression from that used to induce stress fiber formation and tyrosine phosphorylation of focal adhesion proteins.
Slice
L W
LW
Division of Digestive Diseases, Department of Medicine, CURE, Digestive Diseases Research Center, University of California, Los Angeles, California 90095, USA. lslice@med1.medsch.ucla.edu
Walsh
J H
JH
Rozengurt
E
E
D016475:3T3 Cells; D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins; D003014:Clostridium botulinum / Q000235:genetics; D051546:Cyclooxygenase 2; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection
D020690:GTPase-Activating Proteins; D007527:Isoenzymes; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; C085379:rho GTPase-activating protein; D008156:Luciferases; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins