Induction of max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis.
Adrenomedullin is a novel vasodilatory peptide originally isolated from pheochromocytoma. Recently, we found that adrenomedullin acts as an autocrine/paracrine apoptosis survival factor for rat endothelial cells. In the present study, we show that adrenomedullin induces the expression of Max, a heterodimeric partner of c-Myc, which may contribute to its ability to rescue endothelial cells from apoptosis. Max is a basic-helix-loop-helix-leucine zipper protein that forms heterodimers with its alternative partners, Mad and Mxi-1, to behave as an antagonist for Myc-Max heterodimer through competition for common DNA targets. The expression of Max is reported to be constitutive and more stable than c-Myc, and serum induces immediate c-Myc stimulation followed by modest Max up-regulation. In quiescent rat endothelial cells, adrenomedullin stimulated the expression of Max without affecting c-Myc. Quantitation with real-time quantitative PCR detected on the ABI Prism 7700 Sequence Detection System revealed that adrenomedullin and calcitonin gene-related peptide (CGRP), as well as serum, up-regulated Max mRNA levels and that down-regulation of Max mRNA after serum deprivation was prevented by adrenomedullin. Neither adrenomedullin nor CGRP affected c-Myc expression. Transfection of a Max-expressing plasmid into endothelial cells rescued the apoptosis induced by serum deprivation. Neutralization with anti-adrenomedullin antiserum or blockade with a CGRP receptor antagonist, CGRP(8-37), reduced Max mRNA levels in growing endothelial cells and enhanced apoptosis after serum starvation. Introduction of an antisense oligodeoxynucleotide against Max mRNA using transferrin receptor-operated transfer led to inhibition of both adrenomedullin-induced up-regulation of Max transcripts and its cell survival effect, whereas random, sense, or missense oligonucleotides were without effect. The negative regulation of E-box-driven transcription by adrenomedullin was demonstrated by using preproendothelin-1 promoter containing c-Myc-Max binding consensus sequence; the promoter activity of preproendothelin-1 was reduced by cotransfecting Max- and Mad-expressing plasmids as well as addition of adrenomedullin and CGRP. The present results demonstrate that adrenomedullin antagonizes serum deprivation-induced endothelial apoptosis by up-regulation of the max gene in an autocrine/ paracrine manner.
Shichiri
M
M
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan. mshichiri.med2@med.tmd.ac.jp
Kato
H
H
Doi
M
M
Marumo
F
F
Hirata
Y
Y
D053607:Adrenomedullin; D000818:Animals; D001011:Aorta; D017209:Apoptosis; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D001769:Blood; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D018257:Helix-Loop-Helix Motifs; D008297:Male; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014157:Transcription Factors; D014162:Transfection
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C499329:Max protein, rat; C067850:Myc associated factor X; D020319:Oligodeoxyribonucleotides, Antisense; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D014157:Transcription Factors; D053607:Adrenomedullin; D015740:Calcitonin Gene-Related Peptide