title: Galphaq signaling is required for Rho-dependent transcriptional activation of the cyclooxygenase-2 promoter in fibroblasts.
abstract: Previously, we demonstrated that the gastrin releasing peptide (GRP) induces cyclooxygenase-2 (COX-2) expression through a Rho-dependent, protein kinase C (PKC)-independent signaling pathway in fibroblasts (Slice et al., 1999, J Biol Chem 274:27562-27566). However, the specific role of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) that are coupled to the GRP receptor in Rho-dependent COX-2 expression has not been elucidated. In this report, we utilize embryonic fibroblasts from transgenic mice containing double gene knock-outs (DKO) for Galpha(q/11) and Galpha(12/13) to demonstrate that COX-2 promoter activation by GRP requires Galpha(q). Furthermore, we show that GRP-dependent COX-2 gene expression, as assessed by a COX-2 reporter luciferase assay, was induced in cells lacking Galpha(12/13) but was blocked in cells that did not express Galpha(q/11). GRP-dependent COX-2 promoter induction in Galpha(q/11) deficient cells was rescued by expression of wild type Galpha(q) but blocked by inhibition of calcium signaling in calcium-free media or in cells treated with 2-aminoethoxydiphenylborate (2-APB). Co-stimulation of transfected Galpha(q/11) deficient cells with GRP and thapsigargin (TG) induced the COX-2 promoter. Activation of endogenous Rho by expression of Onco-lbc or expression of Rho A Q63L resulted in COX-2 promoter activation in Galpha(q/11) deficient cells. Inhibition of Rho by Clostridium botulinum C3 toxin blocked COX-2 promoter induction. Expression of Galpha(q) Q209L in the well-characterized fibroblast cell line, NIH3T3, induced the COX-2 promoter which was blocked by expression of C3 toxin. These results demonstrate that calcium signaling mediated by Galpha(q) and Rho play critical roles in GRP-dependent COX-2 expression in fibroblasts.
authors.affiliation: Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, Greater Los Angeles VA Medical Center, University of California, Los Angeles, California 90095, USA. lslice@mednet.ucla.edu
authors.forename: Lee W
authors.initials: LW
authors.lastname: Slice
authors.forename: Sang-Kyou
authors.initials: SK
authors.lastname: Han
authors.forename: Melvin I
authors.initials: MI
authors.lastname: Simon
mesh_terms: D051696:Activating Transcription Factors; D000818:Animals; D001798:Blood Proteins / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology; D051546:Cyclooxygenase 2; D005136:Eye Proteins; D005347:Fibroblasts / Q000502:physiology*; D051606:G-Protein-Coupled Receptor Kinase 1; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000235:genetics; D019886:Gastrin-Releasing Peptide / Q000502:physiology; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D007527:Isoenzymes / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000172:deficiency / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012234:Rho Factor / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology*
chemical_list: D051696:Activating Transcription Factors; D001798:Blood Proteins; D005136:Eye Proteins; D007527:Isoenzymes; D020033:Protein Isoforms; D012333:RNA, Messenger; D012234:Rho Factor; D014157:Transcription Factors; D019886:Gastrin-Releasing Peptide; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D011494:Protein Kinases; D011493:Protein Kinase C; D051606:G-Protein-Coupled Receptor Kinase 1; C496762:Grk1 protein, mouse; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins