LDL Induces Transcription Factor Activator Protein-1 in Human Endothelial Cells

From the Division of Biomedical Sciences, University of California, Riverside (Y.Z., H.L.L., O.F., L.V., N.W.M., D.S.S., M.B.S.), and the Department of Pathology, New York Medical College, Valhalla, NY (J.H-C.L.).

Abstract—Low density lipoprotein (LDL) has been shown to perturb endothelial cells, with manifestations ranging from alterations in free radicals and arachidonate metabolism to stress fiber formation and monocyte recruitment. Some of these changes are regulated by LDL at the transcriptional level. Using mobility shift assays with consensus sequences for various transcription factors, we have detected an increase in activator protein 1 (AP-1), but not nuclear factor-B (NF-B), binding in human umbilical vein endothelial cells exposed to LDL. Following transfection, AP-1–driven chloramphenicol acetyltransferase and AP-1–driven-luciferase are upregulated by LDL. In contrast, there is no effect on NF-B–driven chloramphenicol acetyltransferase. AP-1 increases in a biphasic fashion, with the first peak occurring 6 hours after and the second 48 hours after exposure to LDL. This AP-1 binding increase involves c-Jun, but not c-Fos, as shown by gel supershift, Northern hybridization, and Western blotting analyses. c-Jun mRNA levels are elevated by 9 hours after and remain so until at least 24 hours after exposure to LDL. c-Jun protein levels increase at 12 hours and continue to rise for 24 hours after exposure to LDL. Moreover, this LDL-increased AP-1 binding is suppressed by several protein kinase (PK) inhibitors: the PKC inhibitor calphostin C, the cAMP-dependent PK inhibitor H89, and the tyrosine PK inhibitors genistein and lavendustin A. This study demonstrates that (1) LDL is an endothelial agonist distinct from other cell stimulators, such as cytokines, endotoxin, and phorbol 12-myristate 13-acetate, because LDL appears to activate human umbilical vein endothelial cells predominantly through the transcription factor AP-1 and not NF-B; and (2) LDL increases AP-1 via mechanisms involving multiple kinase activities and c-Jun transcription.

Key Words: LDL • activator protein 1 • c-Jun • c-Fos • human umbilical vein endothelial cells

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