背景:先前的证据表明,G蛋白信号调节因子6(RGS6)在响应细胞毒性应激时易位到核仁,尽管这种现象的功能意义仍然未知。
方法:利用小鼠体内基因操作,原代鼠心肌细胞,我们剖析了RGS6-核仁素复合物在化疗依赖性心脏毒性中的参与情况。
结果:这里我们证明了RGS6与一个关键的核仁蛋白结合,核仁素,并控制其在心肌细胞中的表达和活性。在人肌细胞AC-16细胞系中,诱导多能干细胞来源的心肌细胞,原代鼠心肌细胞,和完整的鼠心肌通过过表达或敲低调节RGS6水平导致对核仁素mRNA的完全相反的影响,蛋白质,和磷酸化。RGS6耗竭在体外提供了明显的保护,防止核仁应激介导的细胞死亡,and,相反,RGS6过表达抑制核糖体RNA的产生,核仁的关键输出,并引发肌细胞死亡.重要的是,核仁素或核仁素效应子miRNA-21的过表达抵消了RGS6的促凋亡作用。在人类和小鼠的心脏组织中,暴露于基因毒性应激源阿霉素与RGS6/Nucleolin比值增加相关.通过心内注射引入RGS6指导的shRNA来预防RGS6诱导,在小鼠中证明了心脏保护作用,并伴有恢复的Nucleolin/miRNA-21表达,减少的核仁应力,促凋亡的表达减少,肥大,和心脏中的氧化应激标志物。
结论:一起,这些数据暗示RGS6通过其调节核仁素的能力作为心肌细胞核仁应激依赖性细胞死亡的驱动因素。这项工作首次证明了RGS蛋白在核仁中的功能作用,并确定了RGS6/Nucleolin相互作用作为预防心脏毒性的可能的新治疗靶标。
Prior evidence demonstrated that Regulator of G protein Signaling 6 (RGS6) translocates to the nucleolus in response to cytotoxic stress though the functional significance of this phenomenon remains unknown.
Utilizing in vivo gene manipulations in mice, primary murine cardiac cells, human cell lines and human patient samples we dissect the participation of a RGS6-nucleolin complex in chemotherapy-dependent cardiotoxicity.
Here we demonstrate that RGS6 binds to a key nucleolar protein, Nucleolin, and controls its expression and activity in cardiomyocytes. In the human myocyte AC-16 cell line, induced pluripotent stem cell derived cardiomyocytes, primary murine cardiomyocytes, and the intact murine myocardium tuning RGS6 levels via overexpression or knockdown resulted in diametrically opposed impacts on Nucleolin mRNA, protein, and phosphorylation.RGS6 depletion provided marked protection against nucleolar stress-mediated cell death in vitro, and, conversely, RGS6 overexpression suppressed ribosomal RNA production, a key output of the nucleolus, and triggered death of myocytes. Importantly, overexpression of either Nucleolin or Nucleolin effector miRNA-21 counteracted the pro-apoptotic effects of RGS6. In both human and murine heart tissue, exposure to the genotoxic stressor doxorubicin was associated with an increase in the ratio of RGS6/Nucleolin. Preventing RGS6 induction via introduction of RGS6-directed shRNA via intracardiac injection proved cardioprotective in mice and was accompanied by restored Nucleolin/miRNA-21 expression, decreased nucleolar stress, and decreased expression of pro-apoptotic, hypertrophy, and oxidative stress markers in heart.
Together, these data implicate RGS6 as a driver of nucleolar stress-dependent cell death in cardiomyocytes via its ability to modulate Nucleolin. This work represents the first demonstration of a functional role for an RGS protein in the nucleolus and identifies the RGS6/Nucleolin interaction as a possible new therapeutic target in the prevention of cardiotoxicity.