Regulation of endothelin 1 gene by fluid shear stress is transcriptionally mediated and independent of protein kinase C and cAMP.
Fluid shear stress induces a number of morphological and functional changes in vascular endothelium, including a rapid and significant down-regulation of endothelin 1 (ET-1) mRNA and peptide release in bovine aortic endothelial cells. We show here that both the cell alignment and ET-1 down-regulation depend on on-going protein synthesis, and that the latter is the result of a decrease in transcription, as shown by nuclear run-off assay, and not the result of changes in ET-1 mRNA half-life. The treatment of endothelial cells with either phorbol 12-myristate 13-acetate (100 nM) to activate protein kinase C (PKC) or forskolin (10 microM) to stimulate adenylate cyclase sharply decreased ET-1 mRNA. However, the phorbol-induced ET-1 decrease was, unlike the shear-induced down-regulation, independent of active protein synthesis. Physiological shear stress (20 dynes/cm2) did not significantly activate PKC, as assessed by PKC translocation and enzymatic activity assay and failed to increase intracellular cAMP content. Furthermore treatment with calphostin C (1 microM) did not prevent the shear-induced down-regulation of ET-1. DNA transfection experiments suggest that the shear stress-responsive element of the ET-1 gene is contained in the sequence between -2.5 kb and -2.9 kb of the 5'-upstream region. Neither the transcription factor AP-1 binding site nor the GATA-2-factor binding site, necessary for the basal level of transcription of ET-1 gene, is sufficient to confer shear-responsiveness to the reporter gene. These results suggest that shear stress regulates the transcription of the ET-1 gene via an upstream cis element by a distinct mechanism not dependent on the PKC or cAMP pathways.
Harvard Medical School-Massachusetts Institute of Technology, Division of Health Sciences and Technology, Boston 02215.
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AM
Malek
A L
AL
Greene
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Izumo
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D015536:Down-Regulation; D016232:Endothelins / Q000235:genetics*; D005786:Gene Expression Regulation; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D013314:Stress, Mechanical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D016232:Endothelins; D012333:RNA, Messenger; D005576:Colforsin; D000242:Cyclic AMP; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate