Induction of max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis

Mol Endocrinol. 1999 Aug;13(8):1353-63. doi: 10.1210/mend.13.8.0324.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenomedullin
  • Animals
  • Aorta
  • Apoptosis*
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • Basic-Leucine Zipper Transcription Factors
  • Blood
  • Calcitonin Gene-Related Peptide / pharmacology*
  • Cells, Cultured
  • DNA-Binding Proteins / biosynthesis*
  • DNA-Binding Proteins / genetics
  • Endothelium, Vascular / cytology*
  • Endothelium, Vascular / metabolism
  • Gene Expression Regulation / drug effects
  • Helix-Loop-Helix Motifs
  • Male
  • Oligodeoxyribonucleotides, Antisense / pharmacology
  • Peptides / pharmacology*
  • Proto-Oncogene Proteins c-myc / metabolism
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Wistar
  • Transcription Factors*
  • Transfection

Substances

  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • Basic-Leucine Zipper Transcription Factors
  • DNA-Binding Proteins
  • Max protein, rat
  • Myc associated factor X
  • Oligodeoxyribonucleotides, Antisense
  • Peptides
  • Proto-Oncogene Proteins c-myc
  • RNA, Messenger
  • Transcription Factors
  • Adrenomedullin
  • Calcitonin Gene-Related Peptide