Phorbol Ester Induction of Angiotensin-Converting Enzyme Transcription Is Mediated by Egr-1 and AP-1 in Human Endothelial Cells via ERK1/2 Pathway

Abstract

Abstract-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 GϩC-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 Ⅲ angiotensin-converting enzyme Ⅲ transcriptional regulation Ⅲ site-directed mutagenesis A ngiotensin I-converting enzyme 1 (ACE; DCP1, EC 3.4.15.1) is a dipeptidyl carboxypeptidase that plays an important role in the renin-angiotensin system and kallikreinkinin system. It is a zinc metallopeptidase that generates the vasoconstrictor peptide and growth factor angiotensin II from angiotensin I, inactivates the vasodilator peptide bradykinin, and is able to cleave several other peptides. Two isoenzymes of ACE are found in mammalian tissues, transcribed from a single gene by two alternate promoters. 1 Somatic ACE (170 kDa) is synthesized by vascular endothelial cells and several epithelial cells, whereas testis ACE (110 kDa) is expressed exclusively by male germinal cells. 2,3 Modulation of ACE expression has been observed in response to various stimuli. ACE expression is induced by steroid hormones in macrophages and bovine pulmonary artery endothelial cells 4,5 or by vas...

20-HETE Activates the Transcription of Angiotensin-Converting Enzyme via Nuclear Factor- B Translocation and Promoter Binding

“…Studies in animal models showing that increased vascular 20-HETE production is associated with upregulation of vascular ACE (Sodhi et al, 2010) further support the notion that 20-HETE is an endogenous modulator of the ACE. Gene expression of endothelial ACE has been shown to be increased by phorbol 12-myristate 13-acetate (Villard et al, 1998;Eyries et al, 2002) and isoproterenol (Xavier-Neto et al, 1999). Interestingly, there are no known endogenous inducers of ACE expression, although activation of protein kinase C and the transcription factors EGR-1, AP-1, and ETS-1 has been demonstrated to mediate ACE upregulation (Villard et al, 1998;Mungunsukh et al, 2008).…”

Characterization of the human CREB3L2 gene promoter

“…PMA induced promoter activity of CREB3L2 irrespective of the presence or absence of the CRE site. Indeed, PMA function might involve Egr1, AP1 and Sp1 transcription factor sites found in the promoter of CREB3L2 (19). Interestingly, forskolin and PMA have opposite effects on the CREB3L2 promoter.…”

Signaling via the Angiotensin-Converting Enzyme Enhances the Expression of Cyclooxygenase-2 in Endothelial Cells

“…Because the expression of COX-2, like that of ACE, is reported to be regulated by mechanisms involving c-Jun 20 and the transcription factor AP-1, 12,20 we determined whether the activation of ACE signaling can affect the expression and activity of COX-2. We found that ramiprilat increases the expression of COX-2 in different experimental models; in primary cultures of human umbilical endothelial cells that endogenously express ACE; in a porcine aortic endothelial cell line overexpressing human somatic ACE; and in the lungs of mice treated with ramipril for 5 days.…”

“…Increased ACE levels have been demonstrated in lung tissue and plasma from ACE inhibitor-treated rats 7,8 and in the serum from patients who distinctly benefit from ACE inhibitor therapy. 9,10 The finding that ACE inhibitors enhance the phosphorylation of ACE on Ser 1270 within the short cytoplasmic tail of the enzyme and thus induce the phosphorylation and nuclear translocation of c-Jun, 11 together with the report that the increase in ACE expression detected in phorbol esterstimulated endothelial cells can be attributed to the activity of an activator protein-1 (AP-1) complex containing a c-Jun homodimer, 12 suggests that a novel ACE-dependent signaling cascade can affect gene expression.…”