Department of Pharmacology, And Cardiac & Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 510080, China; Department of Neurosurgery, Shenzhen Second People's Hospital/the First Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen, 518035, China.
Department of Pharmacology, And Cardiac & Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 510080, China; Department of Molecular Medicine, School of Medicine, Sun Yat-Sen University, Guangzhou, 510006, China.
Eur J Pharmacol. 2021 May 5;898:173997. doi: 10.1016/j.ejphar.2021.173997. Epub 2021 Mar 4.
Hypotonic stimulus enlarges cell volume and increased cell proliferation with the exact mechanisms unknown. Glucocorticoid-induced kinase-1 (SGK1) is a serine/threonine kinase that can be regulated by osmotic pressure. We have revealed that SGK1 was activated by hypotonic solution-induced lowering of intracellular Cl concentration. Therefore, we further examined whether SGK1 mediated hypotonic solution-induced proliferation and the internal mechanisms in basilar smooth muscle cells (BASMCs). In the present study, BrdU incorporation assay, flow cytometry, western blotting were performed to evaluate cell viability, cell cycle transition, and the expression of cell cycle regulators and other related proteins. We found that silence of SGK1 largely blunted hypotonic challenge-induced increase in cell viability and cell cycle transition from G/G phase to S phase, whereas overexpression of SGK1 showed the opposite effects. The effect of SGK1 on proliferation was related to the upregulation of cyclin D1 and cyclin E1, and the downregulation of p27 and p21, which is mediated by the interaction between SGK1 and cAMP responsive element-binding protein (CREB). Moreover, we overexpressed ClC-3 Cl channel to further verify the role of SGK1 in low Cl environment-induced proliferation. The results revealed that overexpression of ClC-3 further enhanced hypotonic solution-induced cell viability, cell cycle transition, and CREB activation, which were alleviated or potentiated by silencing or overexpression of SGK1. In summary, this study provides compelling evidences that SGK1, as a Cl-sensitive kinase, is a critical link between low osmotic pressure and proliferation in BASMCs, and shed a new light on the treatment of proliferation-associated cardiovascular diseases.
低渗刺激会扩大细胞体积并增加细胞增殖，但具体机制尚不清楚。糖皮质激素诱导的激酶-1（SGK1）是一种丝氨酸/苏氨酸激酶，可受渗透压调节。我们已经揭示，低渗溶液诱导的细胞内氯离子浓度降低会激活 SGK1。因此，我们进一步研究了 SGK1 是否介导低渗溶液诱导的增殖以及基底动脉平滑肌细胞（BASMC）中的内部机制。在本研究中，通过 BrdU 掺入测定、流式细胞术和 Western blot 分析来评估细胞活力、细胞周期转换以及细胞周期调节剂和其他相关蛋白的表达。我们发现，沉默 SGK1 可显著减弱低渗刺激引起的细胞活力增加和细胞周期从 G1/G0 期向 S 期的转换，而 SGK1 的过表达则表现出相反的效果。SGK1 对增殖的影响与细胞周期蛋白 D1 和 E1 的上调以及 p27 和 p21 的下调有关，这是由 SGK1 与 cAMP 反应元件结合蛋白（CREB）之间的相互作用介导的。此外，我们过表达了 ClC-3 Cl 通道，以进一步验证 SGK1 在低 Cl 环境诱导增殖中的作用。结果表明，过表达 ClC-3 进一步增强了低渗溶液诱导的细胞活力、细胞周期转换和 CREB 激活，而沉默或过表达 SGK1 则减轻或增强了这些作用。总之，本研究提供了有力的证据表明，作为一种 Cl 敏感激酶，SGK1 是 BASMC 中低渗透压与增殖之间的关键环节，为治疗与增殖相关的心血管疾病提供了新的思路。
