Homocysteine-responsive ATF3 gene expression in human vascular endothelialcells: activation of c-Jun NH2-terminal kinase and promoter response element

Activating transcription factor (ATF)3 is a member of ATF/cyclic adenosine monophosphate (cAMP)-responsive element binding protein (ATF/CREB) family o f transcription factors and functions as a stress-inducible transcriptional repressor. To understand the stress-induced gene regulation by homocystein e, we investigated activation of the ATF3 gene in human endothelial cells. Homocysteine caused a rapid induction of ATFB3 at the transcriptional level . This induction was preceded by a rapid and sustained activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK), and dominan t negative mitogen-activated protein kinase kinase 4 and 7 abolished these effects. The effect of homocysteine appeared to be specific, because cystei ne or homocystine had no appreciable effect, but it was mimicked by dithiot hreitol and P-mercaptoethanol as well as tunicamycin, The homocysteine effe ct was not inhibited by an active oxygen scavenger. Deletion analysis of th e 5' flanking sequence of the ATF3 gene promoter revealed that one of the m ajor elements responsible for the induction by homocysteine is an ATF/cAMP responsive element (CRE) located at -92 to -85 relative to the transcriptio nal start site. Gel shift, immunoprecipitation, and cotransfection assays d emonstrated that a complex (or complexes) containing ATF2, c-Jun, and ATF3 increased binding to the ATF/CRE site in the homocysteine-treated cells and activated the ATF3 gene expression, while ATF3 appeared to repress its own promoter. These data together suggested a novel pathway by which homocyste ine causes the activation of JNK/SAPK and subsequent ATF3 expression throug h its reductive stress. Activation of JNK/SAPK and ATF3 expression in respo nse to homocysteine may have a functional role in homocysteinemia-associate d endothelial dysfunction. (Blood, 2000;96: 2140-2148) (C) 2000 by The Amer ican Society of Hematology.