Nikitenko Leonid L, Smith Dave M, Bicknell Roy, Rees Margaret C P
NDOG, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK.
FASEB J. 2003 Aug;17(11):1499-501. doi: 10.1096/fj.02-0993fje. Epub 2003 Jun 17.
Adrenomedullin is a 52 amino acid peptide that shows a remarkable range of effects on the vasculature that include inter alia, vasodilatation, regulation of permeability, inhibition of endothelial cell apoptosis, and promotion of angiogenesis. Recently the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CRLR), and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signaling system. However, mechanisms of regulation of CRLR expression are still largely unknown. This is in part due to lack of information on the gene promoter. In this study we have determined the transcriptional start of human CRLR cDNA by 5'-RACE and cloned the proximal 5'-flanking region of the gene by PCR. The 2318 bp genomic fragment contains the basal promoter of human CRLR, including potential TATA-boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp-1, Pit-1, glucocorticoid receptor, and hypoxia-inducible factor-1 alpha (HIF-1alpha) were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5'-flanking region isolated by PCR contains the gene promoter of human CRLR. Of significance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site-directed mutagenesis of the consensus hypoxia-response element (HRE) in the 5'-flanking region abolished such a response. We also demonstrated by semi-quantitative RT-PCR that transcription of the gene is activated by hypoxia in microvascular endothelial cells. In contrast, expression of RAMPs 1, 2, and 3 was unaffected by low oxygen tension. We conclude that simultaneous transcriptional up-regulation of CRLR and its ligand adrenomedullin in endothelial cells could lead to a potent survival loop and therefore might play a significant role in vascular responses to hypoxia and ischemia.
肾上腺髓质素是一种由52个氨基酸组成的肽，它对脉管系统具有一系列显著作用，其中包括血管舒张、通透性调节、抑制内皮细胞凋亡以及促进血管生成等。最近，G蛋白偶联受体（GPCR）降钙素受体样受体（CRLR）和受体活性修饰蛋白（RAMP）已被确认为肾上腺髓质素信号系统的重要组成部分。然而，CRLR表达的调控机制在很大程度上仍不明确。部分原因是缺乏关于该基因启动子的信息。在本研究中，我们通过5'-RACE确定了人CRLR cDNA的转录起始位点，并通过PCR克隆了该基因的近端5'-侧翼区域。这个231bp的基因组片段包含人CRLR的基础启动子，其中包括潜在的TATA盒和几个GC盒。还鉴定出了与已知转录因子如Sp-1、Pit-1、糖皮质激素受体和缺氧诱导因子-1α（HIF-1α）结合的调控元件。当克隆到报告基因载体中时，该基因组片段显示出显著的启动子活性，表明通过PCR分离的5'-侧翼区域包含人CRLR的基因启动子。重要的是，当在原代微血管内皮细胞中进行转染时，克隆的启动子片段会被缺氧激活。对5'-侧翼区域中共有缺氧反应元件（HRE）进行定点诱变消除了这种反应。我们还通过半定量RT-PCR证明，在微血管内皮细胞中该基因的转录会被缺氧激活。相比之下，RAMP 1、2和3的表达不受低氧张力的影响。我们得出结论，内皮细胞中CRLR及其配体肾上腺髓质素的同时转录上调可能导致一个有效的存活环，因此可能在血管对缺氧和缺血的反应中发挥重要作用。
