Induction of max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis.
Induction of max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis.
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DOI：
10.1210/mend.13.8.0324
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发表时间：
1999-08
影响因子：
--
通讯作者：
Masayoshi Shichiri;Hiroki Kato;Masaru Doi;Fumiaki Marumo;Yukio Hirata
中科院分区：
文献类型：
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作者：
Masayoshi Shichiri;Hiroki Kato;Masaru Doi;Fumiaki Marumo;Yukio Hirata
肾上腺髓质素是一种新的血管舒张肽，最初从嗜铬细胞瘤中分离出来。最近，我们发现肾上腺髓质素作为一种自分泌/旁分泌的大鼠内皮细胞凋亡生存因子。在本研究中，我们表明，肾上腺髓质素诱导的表达最大，异源二聚体的合作伙伴的c-Myc，这可能有助于其拯救内皮细胞凋亡的能力。Max是一种碱性-螺旋-环-螺旋-亮氨酸拉链蛋白，其与其替代伙伴Mad和Mxi-1形成异二聚体，通过竞争常见的DNA靶标而表现为Myc-Max异二聚体的拮抗剂。据报道，Max的表达是组成型的并且比c-Myc更稳定，并且血清诱导立即的c-Myc刺激，随后适度的Max上调。在静止的大鼠内皮细胞，肾上腺髓质素刺激最大的表达，而不影响c-Myc。在ABI Prism 7700序列检测系统上检测的实时定量PCR定量显示，肾上腺髓质素和降钙素基因相关肽（CGRP）以及血清上调Max mRNA水平，并且肾上腺髓质素阻止血清剥夺后Max mRNA的下调。肾上腺髓质素和CGRP均不影响c-Myc表达。将Max表达质粒转染到内皮细胞中可挽救由血清剥夺诱导的细胞凋亡。用抗肾上腺髓质素抗血清中和或用CGRP受体拮抗剂CGRP（8-37）阻断可降低生长中的内皮细胞中的Max mRNA水平，并增强血清饥饿后的细胞凋亡。采用转铁蛋白受体操作转移的反义寡核苷酸对Max mRNA的引入导致肾上腺髓质素诱导的Max转录上调及其细胞存活效应的抑制，而随机、有义或错义寡核苷酸则没有效果。通过使用含有c-Myc-Max结合共有序列的preproendothelin-1启动子证明肾上腺髓质素对E-box驱动的转录的负调控;通过连接Max和Mad-expressing质粒以及添加肾上腺髓质素和CGRP来降低preproendothelin-1的启动子活性。目前的研究结果表明，肾上腺髓质素拮抗血清剥夺诱导的内皮细胞凋亡的最大基因的上调自分泌/旁分泌的方式。
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.