Hilfiker-Kleiner Denise, Hilfiker Andres, Castellazzi Marc, Wollert Kai C, Trautwein Christian, Schunkert Heribert, Drexler Helmut
Department of Cardiology and Angiology, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany.
Cardiovasc Res. 2006 Jul 1;71(1):108-17. doi: 10.1016/j.cardiores.2006.02.032. Epub 2006 Mar 7.
Mice deficient for the AP-1 transcription factor JunD, the only Jun protein constitutively expressed and clearly detectable in the mammalian heart, develop enhanced cardiac hypertrophy in response to chronic pressure overload. Catecholamines inducing alpha-adrenergic receptor-mediated signaling have been implicated in the neurohumoral response to pressure overload and the development of left ventricular hypertrophy. In the present study we analyzed the mechanistic role of JunD in cardiomyocyte hypertrophy in vitro in response to alpha-adrenergic agonist phenylephrine (PE).
Cardiomyocytes were isolated from 1- to 3-day-old rats and transfected with adenoviruses expressing LacZ or wild-type JunD, or with expression vectors encoding LacZ, wild-type JunD, mutated JunD forming only JunD homodimers (JunDeb1), mutated JunD lacking the JNK site (JunD-Delta 162), or c-Jun. After stimulation with PE (10(-5) mol/L), hypertrophic growth of cardiomyocytes (cross-sectional area and [3H]-leucine incorporation) and mRNA expression of JunD, c-Jun, c-Fos, and atrial natriuretic peptide (ANP) were analyzed. Transcriptional activation was determined by luciferase activity in cardiomyocytes transfected with AP-1 or ANP luciferase reporter plasmids. Gel shift assays with an AP-1 consensus oligonucleotide were performed to analyze AP-1 DNA binding activities.
PE augmented mRNA levels of c-Jun and c-Fos, but decreased JunD transcript levels. Adenoviral over-expression of wild-type JunD blunted PE-induced hypertrophic growth and expression of ANP mRNA. Over-expression of JunD in cardiomyocytes caused enhanced AP-1 protein-DNA binding, without increasing the transcriptional response from AP-1 or ANP luciferase reporter plasmids at baseline or upon PE stimulation. Moreover, over-expression of JunDeb1 attenuated transcription from AP-1 or ANP luciferase reporter plasmids and blunted c-Jun-mediated acceleration of AP-1 transcriptional activity at baseline and in response to PE.
Our observations establish a novel role for JunD as a negative regulator of cardiomyocyte hypertrophy in response to hypertrophic stimuli by inhibiting AP-1 transcriptional activity.
AP-1转录因子JunD基因缺陷的小鼠，是哺乳动物心脏中唯一组成性表达且能清晰检测到的Jun蛋白，其在慢性压力超负荷时会出现心脏肥大增强的情况。诱导α-肾上腺素能受体介导信号传导的儿茶酚胺与压力超负荷的神经体液反应及左心室肥大的发生有关。在本研究中，我们分析了JunD在体外心肌细胞肥大中对α-肾上腺素能激动剂去氧肾上腺素（PE）反应的机制作用。
从1至3日龄大鼠中分离心肌细胞，用表达LacZ或野生型JunD的腺病毒转染，或用编码LacZ、野生型JunD、仅形成JunD同二聚体的突变型JunD（JunDeb1）、缺失JNK位点的突变型JunD（JunD-Delta 162）或c-Jun的表达载体转染。用PE（10⁻⁵ mol/L）刺激后，分析心肌细胞的肥大生长（横截面积和[³H]-亮氨酸掺入）以及JunD、c-Jun、c-Fos和心钠素（ANP）的mRNA表达。通过用AP-1或ANP荧光素酶报告质粒转染的心肌细胞中的荧光素酶活性来测定转录激活。用AP-1共有寡核苷酸进行凝胶迁移试验以分析AP-1 DNA结合活性。
PE增加了c-Jun和c-Fos的mRNA水平，但降低了JunD转录本水平。野生型JunD的腺病毒过表达减弱了PE诱导的肥大生长和ANP mRNA的表达。心肌细胞中JunD的过表达导致AP-1蛋白与DNA的结合增强，而在基线或PE刺激时，AP-1或ANP荧光素酶报告质粒的转录反应并未增加。此外，JunDeb1的过表达减弱了AP-1或ANP荧光素酶报告质粒的转录，并在基线和对PE反应时减弱了c-Jun介导的AP-1转录活性的加速。
我们的观察结果确立了JunD作为心肌细胞肥大负调节因子的新作用，其通过抑制AP-1转录活性来响应肥大刺激。
