Transcriptional mechanism of protein kinase C-induced isoform-specific expression of the gene for endothelin-converting enzyme-1 in human endothelial cells.
Isoform-specific expression of endothelin-converting enzyme (ECE)-1, the major big endothelin-processing enzyme, is controlled by alternative promoters. Signaling pathways and transcriptional mechanisms of ECE-1 mRNA expression are largely unknown. To investigate ECE-1 isoform expression after protein kinase C (PKC) activation, we used phorbol 12-myristate 13-acetate (PMA) to stimulate primary cultured human umbilical vein endothelial cells and the related EA.hy926 cell line. ECE-1a mRNA was up-regulated (approximately 3-fold), whereas mRNA of alternative isoforms (b, c, and d) was unchanged, which was confirmed on the protein level. PMA effects on mRNA expression were suppressed by the PKC inhibitors H-7 and Calphostin C. Because increased ECE-1a expression was preceded by induction of the transcription factor Ets-1, we performed gel shift assays and demonstrated specific DNA/protein interactions involving the ETS binding motif GGAA. Luciferase reporter assays showed that PMA induced ECE-1a promoter activity about 2.5-fold in EA.hy926 cells. Similarly, coexpression of Ets-1 protein resulted in a dose-dependent increase in ECE-1a promoter activity (more than 8-fold). Using gel shift assays and mutation analysis, we identified two tandemly arranged Ets-1 binding sites (EBS) at -638 and -658, respectively, that are involved in transcriptional activation of the ECE-1a promoter by PMA or Ets-1. Moreover, we also found evidence for binding of a transcriptional repressor to EBS -638. The inhibitor of mitogen-activated protein kinase kinase, PD98059, inhibited PMA effects on ECE-1a mRNA expression and promoter activity, respectively. Our results provide the first detailed analysis of signaling pathways and transcriptional mechanisms involved in isoform-specific ECE-1 gene expression.
Orzechowski
H D
HD
Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Freie Universität, Berlin, Germany.
Günther
A
A
Menzel
S
S
Zimmermann
A
A
Funke-Kaiser
H
H
Real
R
R
Subkowski
T
T
Zollmann
F S
FS
Paul
M
M
D000906:Antibodies; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008666:Metalloendopeptidases; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology*; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000502:physiology; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D000906:Antibodies; D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; D007527:Isoenzymes; D009687:Nuclear Proteins; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D014157:Transcription Factors; D004247:DNA; D011493:Protein Kinase C; D020929:Mitogen-Activated Protein Kinase Kinases; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D013755:Tetradecanoylphorbol Acetate