Chen L S, Cuddy M P, LaVallette L A
Division of Cardiovascular & Metabolic Diseases, Wyeth-Ayerst Research, Princeton, New Jersey, USA.
Kidney Int. 1998 Dec;54(6):2045-55. doi: 10.1046/j.1523-1755.1998.00209.x.
The renin-angiotensin system has been known to regulate blood pressure and body fluid homeostasis. Several lines of evidence have shown that renin gene expression and release are up-regulated by beta-adrenergic stimulation, sodium depletion, and angiotensin converting enzyme inhibition, but down-regulated by cytokines. To further characterize the human renin gene (hREN) promoter structure, its regulation, and to identify an appropriate cell system for study, we examined five cell lines and investigated drug effects on the hREN promoter expression.
Using the hREN-luciferase reporter gene constructs in the DNA transfection assays, approximately 5 kb of the hREN 5' flanking region was assessed for promoter activity in five different cell lines. Regulation of the hREN promoter activity was investigated using Y-1 adrenal cells that were transfected with the hREN-luciferase DNA and were treated with forskolin, calcium ionophore A23187, phorbol ester, angiotensin II (Ang II), or cytokines.
Transient transfection analysis showed that the 5 kb hREN 5' flanking DNA alone was able to confer significant promoter activity in Y-1 adrenal cells. In transfected Y-1 cells, luciferase reporter expression was induced by forskolin, suppressed by the calcium ionophore A23187, and phorbol ester in a dose-dependent manner, but was unaffected by angiotensin II (Ang II). However, when Y-1 reporter cells were transfected with human angiotensin II receptor type 1 (AT1) cDNA, hREN promoter activity was dose-dependently down-regulated by Ang II, which was blockable by losartan, an AT1-selective antagonist. Further studies also showed that hREN promoter activity in Y-1 cells was selectively down-regulated by TNF alpha. Deletion of the hREN promoter sequences between position -3916 and -2822 not only enhanced hREN promoter activity by approximately tenfold, but also caused a failure of down-regulation by TNF alpha. In contrast, neither interleukin (IL)-1 alpha, IL-1 beta, IL-2, nor IL-6 exerted any significant effect.
Together the results suggest that TNF alpha is a negative regulator of the hREN expression in the adrenal cells, and that the TNF alpha responsiveness may be controlled by elements located between the positions -3916 and -2822 of the hREN promoter. Moreover, the Y-1 cell line may provide a valuable model system for studying renin gene regulation.
已知肾素 - 血管紧张素系统可调节血压和体液平衡。多项证据表明，肾素基因的表达和释放可被β - 肾上腺素能刺激、钠耗竭及血管紧张素转换酶抑制上调，但被细胞因子下调。为进一步表征人肾素基因（hREN）启动子结构、其调控机制，并确定合适的研究细胞系统，我们检测了五种细胞系，并研究了药物对hREN启动子表达的影响。
在DNA转染实验中使用hREN - 荧光素酶报告基因构建体，评估约5 kb的hREN 5'侧翼区域在五种不同细胞系中的启动子活性。使用转染了hREN - 荧光素酶DNA并用福斯可林、钙离子载体A23187、佛波酯、血管紧张素II（Ang II）或细胞因子处理的Y - 1肾上腺细胞，研究hREN启动子活性的调控。
瞬时转染分析表明，仅5 kb的hREN 5'侧翼DNA就能在Y - 1肾上腺细胞中赋予显著的启动子活性。在转染的Y - 1细胞中，荧光素酶报告基因表达被福斯可林诱导，被钙离子载体A23187和佛波酯以剂量依赖方式抑制，但不受血管紧张素II（Ang II）影响。然而，当用人类1型血管紧张素II受体（AT1）cDNA转染Y - 1报告细胞时，hREN启动子活性被Ang II以剂量依赖方式下调，这可被AT1选择性拮抗剂氯沙坦阻断。进一步研究还表明，Y - 1细胞中的hREN启动子活性被肿瘤坏死因子α（TNFα）选择性下调。缺失hREN启动子序列中 - 3916至 - 2822位之间的区域不仅使hREN启动子活性增强约十倍，还导致TNFα无法下调其活性。相比之下，白细胞介素（IL）-1α、IL - 1β、IL - 2和IL - 6均未产生任何显著影响。
这些结果共同表明，TNFα是肾上腺细胞中hREN表达的负调节因子，并且TNFα反应性可能受hREN启动子 - 3916至 - 2822位之间元件的控制。此外，Y - 1细胞系可能为研究肾素基因调控提供有价值的模型系统。
