DIFFERENTIAL ACTIVATION OF PROTEIN-KINASE-C ISOZYMES BY PHORBOL ESTERAND COLLAGEN IN HUMAN SKIN MICROVASCULAR ENDOTHELIAL-CELLS

Human dermal microvascular endothelial cells participate in activities including inflammation, wound healing, and angiogenesis (neovasculari zation). Two stages of angiogenesis can be mimicked in vitro by two mo dels of cultured foreskin human dermal microvascular endothelial cells : the differentiation of epithelioid endothelial cells to spindle-shap ed mesenchymal-like cells induced by phorbol ester treatment; and the formation of vascular channels induced by exposing the luminal surface of endothelial cell monolayers to type I collagen gels. The mechanism s underlying these two processes, however, are largely unknown, protei n kinase C isozymes, which are activated by phorbol esters, are import ant mediators in the angiogenic process. In the current work, we ident ified the protein kinase C isozymes present in human dermal microvascu lar endothelial cells and determined which of the isozymes are activat ed in response to phorbol ester or to collagen treatments. Using weste rn blot analysis, we found that microvascular endothelial cells contai n at least six protein kinase C isozymes (alpha, beta, delta, epsilon, zeta, eta). Immunocytochemical studies demonstrated that the isozymes are located in distinct cellular compartments and that following trea tment with phorbol 12-myristate 13-acetate or with a collagen gel over lay, most isozymes (protein kinase C alpha, beta I, beta II, delta, ep silon, eta) translocated to different parts of the cell. Moreover, for two of these isozymes (beta II and eta), the localization differs aft er phorbol 12-myristate 13-acetate treatment as compared with collagen treatment. These results demonstrate that agents that mimic two stage s in the angiogenic process in vitro initiate diverse changes in the s ubcellular localization of specific protein kinase C isozymes and sugg est a role for different isozymes in this process.