Endothelin-2 synthesis is stimulated by the type-1 tumour necrosis factor receptor and cAMP: comparison with endothelin-converting enzyme-1 expression.
ABSTRACT The synthesis of the vasoconstrictor peptide endothelin-2 (ET-2) is dependent on hydrolysis of the biologically inactive intermediate big ET-2 by an endothelin-converting enzyme (ECE). Here, mechanisms inducing ET-2 synthesis have been investigated using the human renal adenocarcinoma cell line (ACHN). Synthesis of ET-2 by ACHN cells was inhibited by phosphoramidon (IC(50( congruent with11 microM). To determine whether ET-2 synthesis occurs in parallel with the metallopeptidase ECE-1, a putative processing peptidase for big ET-2, changes in the levels of their mRNAs were compared by semi-quantitative RT-PCR under conditions causing the upregulation of ET-2 synthesis. Tumour necrosis factor-alpha (TNFalpha), forskolin and a cell-permeable cAMP analogue (dibutyryl cAMP) caused concentration-dependent increases in ET-2 synthesis. Combination of forskolin or dibutyryl cAMP with TNFalpha produced a significantly greater increase in ET-2 production than these agents alone, indicating that adenylate cyclase and TNFalpha induce ET-2 synthesis by separate signalling pathways. Studies using receptor selective TNFalpha mutants, (125(I-TNFalpha binding and TNF receptor mRNA showed that type-1 TNF receptors mediate the ET-2 response to TNFalpha. PreproET-2 mRNA levels were increased by TNFalpha at 1 h and 2 h, but returned to control levels at 4 h. Treatment with forskolin significantly increased preproET-2 mRNA levels after 1 h and 4 h. ACHN cells expressed ECE-1b and ECE-1c, but not the ECE-1a isoform of this peptidase. RT-PCR for the combined isoforms ECE-1b/c/d showed TNFalpha to increase mRNA levels at 2 h and 4 h. Forskolin had no effect on ECE-1b/c/d mRNA levels. Thus, expression of ET-2 and ECE-1b/c/d mRNAs in ACHN cells do not display the co-ordinated regulation observed with typical peptide prohormone processing enzymes and their substrates.
Lambert
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The William Harvey Research Institute, St Bartholomew's and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, Charterhouse Square Campus, London EC1M 6BQ, UK.
Barker
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Lees
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Corder
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D000230:Adenocarcinoma; D015703:Antigens, CD / Q000502:physiology*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D000242:Cyclic AMP / Q000502:physiology*; D019333:Endothelin-2 / Q000096:biosynthesis / Q000235:genetics*; D000072396:Endothelin-Converting Enzymes; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology*; D006020:Glycopeptides / Q000494:pharmacology*; D006801:Humans; D007680:Kidney Neoplasms; D008666:Metalloendopeptidases / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology*; D047888:Receptors, Tumor Necrosis Factor, Type I; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D019333:Endothelin-2; D006020:Glycopeptides; D011480:Protease Inhibitors; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D000242:Cyclic AMP; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon