Voelkl Jakob, Castor Tatsiana, Musculus Katharina, Viereck Robert, Mia Sobuj, Feger Martina, Alesutan Ioana, Lang Florian
Cell Physiol Biochem. 2015;37(2):603-14. doi: 10.1159/000430380.
BACKGROUND/AIMS: The serum- and glucocorticoid-inducible kinase SGK1 participates in the orchestration of cardiac hypertrophy and remodeling. Signaling linking SGK1 activity to cardiac remodeling is, however, incompletely understood. SGK1 phosphorylation targets include cyclin-dependent kinase inhibitor 1B (p27), a protein which suppresses cardiac hypertrophy. The present study explored how effects of SGK1 on nuclear p27 localization might modulate the hypertrophic response in cardiomyocytes.
Experiments were performed in HL-1 cardiomyocytes and in SGK1-deficient (sgk1-/-) and corresponding wild-type (sgk1+/+) mice following pressure overload by transverse aortic constriction (TAC). Transcript levels were quantified by RT-PCR, protein abundance by Western blotting and protein localization by confocal microscopy.
In HL-1 cardiomyocytes, overexpression of constitutively active SGK1 (SGK1S422D) but not of inactive SGK1 (SGK1K127N) increased significantly the cell size and transcript levels encoding Acta1, a molecular marker of hypertrophy. Those effects were paralleled by almost complete relocation of p27 in the cytoplasm. Treatment of HL-1 cardiomyocytes with isoproterenol was followed by up-regulation of SGK1 expression. Moreover, isoproterenol treatment stimulated the hypertrophic response and was followed by disappearance of p27 from the nuclei, effects prevented by the SGK1 inhibitor EMD638683. The effect of SGK1S422D overexpression on Acta1 mRNA levels was disrupted by overexpression of p27 and of the p27T197A mutant lacking the SGK1 phosphorylation site, but not of the phosphomimetic p27T197D mutant. In sgk1+/+ mice, TAC increased significantly SGK1 and Acta1 mRNA levels and decreased the nuclear to cytoplasmic protein ratio of p27 in cardiac tissue, effects blunted in the sgk1-/- mice.
SGK1-induced hypertrophy of cardiomyocytes involves p27 phosphorylation at T197, which fosters cytoplasmic p27 localization.
背景/目的：血清和糖皮质激素诱导激酶SGK1参与心脏肥大和重塑的调控。然而，将SGK1活性与心脏重塑联系起来的信号传导尚不完全清楚。SGK1的磷酸化靶点包括细胞周期蛋白依赖性激酶抑制剂1B（p27），一种抑制心脏肥大的蛋白质。本研究探讨了SGK1对核p27定位的影响如何调节心肌细胞的肥大反应。
在HL-1心肌细胞以及经横向主动脉缩窄（TAC）造成压力过载后的SGK1缺陷型（sgk1-/-）和相应野生型（sgk1+/+）小鼠中进行实验。通过逆转录聚合酶链反应（RT-PCR）定量转录水平，通过蛋白质印迹法定量蛋白质丰度，通过共聚焦显微镜法定量蛋白质定位。
在HL-1心肌细胞中，组成型活性SGK1（SGK1S422D）而非无活性SGK1（SGK1K127N）的过表达显著增加了细胞大小以及编码Acta1（肥大的分子标志物）的转录水平。这些效应伴随着p27几乎完全重新定位于细胞质中。用异丙肾上腺素处理HL-1心肌细胞后，SGK1表达上调。此外，异丙肾上腺素处理刺激了肥大反应，并随后导致p27从细胞核中消失，而SGK1抑制剂EMD638683可阻止这些效应。p27和缺乏SGK1磷酸化位点的p27T197A突变体的过表达破坏了SGK1S422D过表达对Acta1 mRNA水平的影响，但模拟磷酸化的p27T197D突变体则没有。在sgk1+/+小鼠中，TAC显著增加了心脏组织中SGK1和Acta1 mRNA水平，并降低了p27的核与细胞质蛋白比率，而在sgk1-/-小鼠中这些效应减弱。
SGK1诱导的心肌细胞肥大涉及T197位点的p27磷酸化，这促进了p27在细胞质中的定位。
