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 ch anges in vascular endothelium, including a rapid and significant down- regulation of endothelin 1 (ET-1) mRNA and peptide release in bovine a ortic endothelial cells. We show here that both the cell alignment and ET-1 down-regulation depend on on-going protein synthesis, and that t he latter is the result of a decrease in transcription, as shown by nu clear run-off assay, and not the result of changes in ET-1 mRNA half-l ife. The treatment of endothelial cells with either phorbol 12-myrista te 13-acetate (100 nM) to activate protein kinase C (PKC) or forskolin (10 muM) to stimulate adenylate cyclase sharply decreased ET-1 mRNA. However, the phorbol-induced ET-1 decrease was, unlike the shear-induc ed down-regulation, independent of active protein synthesis. Physiolog ical shear stress (20 dynes/cm2) did not significantly activate PKC, a s assessed by PKC translocation and enzymatic activity assay and faile d to increase intracellular cAMP content. Furthermore treatment with c alphostin C (1 muM) did not prevent the shear-induced down-regulation of ET-1. DNA transfection experiments suggest that the shear stress-re sponsive 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 transcript ion factor AP-1 binding site nor the GATA-2-factor binding site, neces sary for the basal level of transcription of ET-1 gene, is sufficient to confer shear-responsiveness to the reporter gene. These results sug gest that shear stress regulates the transcription of the ET-1 gene vi a an upstream cis element by a distinct mechanism not dependent on the PKC or cAMP pathways.