THE CIS-ACTING PHORBOL ESTER 12-O-TETRADECANOYLPHORBOL 13-ACETATE-RESPONSIVE ELEMENT IS INVOLVED IN SHEAR STRESS-INDUCED MONOCYTE CHEMOTACTIC PROTEIN-1 GENE-EXPRESSION

Vascular endothelial cells, serving as a barrier between vessel and bl ood, are exposed to shear stress in the body. Although endothelial res ponses to shear stress are important in physiological adaption to the hemodynamic environments, they can also contribute to pathological con ditions-e.g., in atherosclerosis and reperfusion injury. We have previ ously shown that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells and that the regulation is at the transcriptional level. These observations led us to functionally analyze the 550-bp promoter region of the MCP-l-encoding gene to define the cis element responding to sh ear stress. The shear stress/luciferase assay on the deletion construc ts revealed that a 38 bp segment (-53 to -90 hp relative to the transc ription initiation site) containing two divergent phorbol ester ''12-O -tetradecanoylphorbol 13-acetate'' (TPA)-responsive elements (TRE) is critical for shear inducibility. Site-specific mutations on these two sites further demonstrated that the proximal one (TGACTCC) but not the distal one (TCACTCA) was shear-responsive. Shear inducibility was los t after the mutation or deletion of the proximal site. This molecular mechanism of shear inducibility of the MCP-1 gene was functional in bo th the epithelial-like HeLa cells and bovine aortic endothelial cells (BAEC). In a construct with four copies of the TRE consensus sequences TGACTACA followed by the rat prolactin minimal promoter and luciferas e gene, shear stress induced the reporter activities by 35-fold and 7- fold in HeLa cells and BAEC, respectively. The application of shear st ress on BAEC also induced a rapid and transient phosphorylation of mit ogen-activated protein kinases. Pretreatment of BAEC with TPA attenuat ed the shear-induced mitogen-activated protein kinase phosphorylation, suggesting that shear stress and TPA share a similar signal transduct ion pathway in activating cells. The present study provides a molecula r basis for the transient induction of MCP-1 gene by shear stress.