Published online before print October 24, 2002, doi: 10.1161/01.RES.0000042703.39845.B4
© 2002 American Heart Association, Inc.
Molecular Medicine |
Phorbol Ester Induction of Angiotensin-Converting Enzyme Transcription Is Mediated by Egr-1 and AP-1 in Human Endothelial Cells via ERK1/2 Pathway
From the Institut National de la Santé et de la Recherche Médicale, Unit 525, Faculté de Médecine Pitié-Sapétrière, Paris, France.
Correspondence to Florent Soubrier, INSERM U525, Faculté de Médecine Pitié-Salpétrière-6e Étage, 91, Boulevard de l’Hôpital, 75013 Paris, France. E-mail florent.soubrier{at}chups.jussieu.fr
Angiotensin-converting enzyme (ACE) is an enzyme that plays a major role in vasoactive peptide metabolism, and it has been implicated in various cardiovascular diseases. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, has been shown to increase ACE mRNA at the transcriptional level in human umbilical vein endothelial cells. We have investigated the transcriptional mechanism involved in protein kinase C induction of the ACE gene. Deletion and transfection analyses have revealed that two regions are required for PMA-inducible gene expression. The first is a G+C-rich region located in the proximal ACE promoter bearing overlapping consensus recognition sequences for stimulatory protein-1 (Sp1) and early growth response gene 1 (Egr-1). Electrophoretic mobility shift assay and supershift experiments have shown that Egr-1 is present in the specific nucleoprotein complex induced by PMA in human umbilical vein endothelial cells. The second region is located in the distal ACE promoter. DNase I footprinting analysis restricted this region to a 21-bp element containing a cAMP-responsive element/12-O-tetradecanoylphorbol 13-acetate–responsive element sequence. Electrophoretic mobility shift assays and supershift analyses have revealed that activating protein 1 (AP-1) is the transcription factor binding the cAMP-responsive element/12-O-tetradecanoylphorbol 13-acetate–responsive element located in the ACE promoter after PMA stimulation. Mutations of either Egr-1 or AP-1 binding sites partially abrogate ACE expression induced by PMA, whereas mutation of both sites totally abrogates PMA-induced ACE expression. Treatment of cells with PD98059, a mitogen-activated protein kinase kinase-1–specific inhibitor, inhibited PMA-induced ACE expression. Our results demonstrate that the two transcription factors, Egr-1 and AP-1, are involved in the PMA-induced ACE transcriptional activation in human endothelial cells via the activation of the extracellular signal–regulated kinase 1/2 signaling pathway.
Key Words: mitogen-activated protein kinase • angiotensin-converting enzyme • transcriptional regulation • site-directed mutagenesis
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