Lavall Daniel, Schuster Pia, Jacobs Nadine, Kazakov Andrey, Böhm Michael, Laufs Ulrich
From the Universität des Saarlandes, Klinik für Innere Medizin III-Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarlandes, D-66421 Homburg/Saar, Germany
From the Universität des Saarlandes, Klinik für Innere Medizin III-Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarlandes, D-66421 Homburg/Saar, Germany.
J Biol Chem. 2017 May 5;292(18):7542-7553. doi: 10.1074/jbc.M116.764449. Epub 2017 Mar 20.
The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11β-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11β-HSD2 expression ( = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11β-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11β-HSD2 expression ( = 0.788 and = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11β-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11β-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11β-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11β-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling.
本研究的目的是阐明Rac1 GTP酶在盐皮质激素受体（MR）介导的促纤维化重塑中的作用。心脏中组成型活性Rac1过表达的转基因小鼠（RacET）会出现与年龄相关的表型，包括心房扩张、纤维化和心房颤动。RacET小鼠和野生型（WT）小鼠中MR的表达相似。在RacET小鼠的心房和左心室中，2型11β羟类固醇脱氢酶（11β-HSD2）的表达在mRNA和蛋白质水平上均呈年龄依赖性上调。抑制Rac1香叶基香叶基化的他汀类药物治疗可降低11β-HSD2的上调。人类左心房心肌样本显示Rac1活性与11β-HSD2表达之间呈正相关（r = 0.7169）。免疫沉淀显示，相对于Rac1表达，RacET小鼠中与Rac1结合的11β-HSD2增强，而他汀类药物治疗可使其减少。RacET小鼠的基础和佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）诱导的NADPH氧化酶活性均增加，且与11β-HSD2表达呈正相关（分别为r = 0.788和r = 0.843）。在培养的H9c2心肌细胞中，用L-丁硫氨酸亚砜胺激活Rac1会增加；用NSC23766抑制Rac1会降低11β-HSD2的mRNA和蛋白质表达。NSC23766可阻止醛固酮诱导的结缔组织生长因子（CTGF）上调。用11β-HSD2小干扰RNA转染心肌细胞可消除醛固酮诱导的CTGF上调。醛固酮刺激的MR核转位被11β-HSD2抑制剂甘草次酸阻断。在心脏成纤维细胞中，NSC23766和螺内酯可抑制醛固酮诱导的MR核转位。NSC23766可阻止醛固酮诱导的心脏成纤维细胞增殖和迁移以及CTGF和纤连蛋白的上调。总之，Rac1 GTP酶调节11β-HSD2表达、MR激活以及MR介导的促纤维化信号传导。
