Research Unit and Nephrology Section, Hospital Universitario Príncipe de Asturias, Alcalá de Henares, Spain.
Arterioscler Thromb Vasc Biol. 2011 Nov;31(11):2577-85. doi: 10.1161/ATVBAHA.111.232025.
Endothelial function depends on the equilibrium in the synthesis of vasoactive endothelial factors. It is well known that endothelin and nitric oxide (NO) exhibit reciprocal regulation. We assessed the ability of NO to regulate endothelin-converting enzyme-1 (ECE-1) expression in vascular endothelial cells.
Bovine aortic endothelial cells were incubated with 2 different NO donors as well as with a cyclic-GMP analog, dibutyryl-cGMP (dB-cGMP). ECE-1 protein content and mRNA expression were evaluated by Western blot and Northern blot, respectively, promoter activity by transfection experiments, ECE-1 activity by ELISA, and cGMP production by radioimmunoassay. Both NO donors decreased ECE-1 protein content, mRNA expression, and ECE-1 activity. ODQ, an inhibitor of soluble guanylyl cyclase, blocked those effects. NO donors raised cGMP levels, and dB-cGMP mimicked their effects on ECE-1 expression, which were blocked by KT5823, a nonspecific PKG inhibitor. The changes on ECE-1 expression were due to a destabilization on 3'-untranslated region (3'-UTR) of this mRNA, because the activity of a luciferase reporter construct containing the 3'-UTR of the ECE-1 gene was reduced by dB-cGMP in a PKG-dependent manner. The biological relevance of this regulation was confirmed in bovine aortic endothelial cells coincubated with macrophages in the presence of lipopolysaccharide, in eNOS-deficient mice, and in Wistar rats treated with NO donors. In every case, an inverse relationship was observed between NO and ECE-1 protein content.
Our results support that NO regulates ECE-1 expression through a cGMP/PKG-dependent regulatory mechanism at the post-transcriptional level via the 3'-UTR of the ECE-1 gene.
内皮功能取决于血管活性内皮因子合成的平衡。众所周知，内皮素和一氧化氮（NO）表现出相互调节。我们评估了 NO 调节血管内皮细胞内皮素转换酶-1（ECE-1）表达的能力。
用 2 种不同的 NO 供体以及环鸟苷酸类似物二丁酰基环鸟苷（dB-cGMP）孵育牛主动脉内皮细胞。通过 Western blot 和 Northern blot 分别评估 ECE-1 蛋白含量和 mRNA 表达，通过转染实验评估启动子活性，通过 ELISA 评估 ECE-1 活性，通过放射免疫法评估 cGMP 产生。两种 NO 供体均降低了 ECE-1 蛋白含量、mRNA 表达和 ECE-1 活性。可溶性鸟苷酸环化酶抑制剂 ODQ 阻断了这些作用。NO 供体增加了 cGMP 水平，dB-cGMP 模拟了它们对 ECE-1 表达的影响，PKG 非特异性抑制剂 KT5823 阻断了这些影响。ECE-1 表达的变化归因于这种 mRNA 的 3'-非翻译区（3'-UTR）的不稳定性，因为含有 ECE-1 基因 3'-UTR 的荧光素酶报告基因构建体的活性在 PKG 依赖性方式下被 dB-cGMP 降低。在存在脂多糖的情况下，在 eNOS 缺陷型小鼠和接受 NO 供体治疗的 Wistar 大鼠中，这种调节在牛主动脉内皮细胞与巨噬细胞共孵育中得到了证实。在每种情况下，NO 和 ECE-1 蛋白含量之间都存在反比关系。
我们的结果支持 NO 通过 cGMP/PKG 依赖性调节机制在转录后水平调节 ECE-1 表达，通过 ECE-1 基因的 3'-UTR。
