Abstract
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.
Highlights
Prostaglandins play a pivotal role in a broad range of physiological and pathological processes including inflammation, pain transmission, maintenance of gastrointestinal integrity, and progression of colorectal cancer (1–3)
COX-2 expression by G␣13/Rho can be dissociated from stress fiber formation, focal adhesion assembly, and tyrosine phosphorylation of focal adhesion proteins, indicating a bifurcation of the signaling pathway used by G␣13/Rho to induce COX-2 expression from that used to induce actin cytoskeleton reorganization
G␣13 Induces COX-2 Promoter Activity—To determine whether G␣13 can regulate COX-2 expression, NIH 3T3 cells were co-transfected with a reporter plasmid in which the production of luciferase is driven by the COX-2 promoter (Ϫ371 to ϩ70) and expression vectors encoding wild type G␣13 or constitutively activated G␣13(G␣13Q226L)
Summary
Prostaglandins play a pivotal role in a broad range of physiological and pathological processes including inflammation, pain transmission, maintenance of gastrointestinal integrity, and progression of colorectal cancer (1–3). Plateletderived growth factor, serum, and v-src promote COX-2 expression through Ras-mediated increases in c-Jun NH2-terminal kinase and extracellular signal-related kinase pathways in NIH 3T3 cells (22–24). Our results show that mutationally activated or aluminum fluoride-stimulated G␣13 induces activation of the COX-2 promoter through a Rho-dependent pathway.
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