Aim

Heat shock protein 60 (Hsp60) is a mediator of stress-induced vascular smooth muscle cell (VSMC) proliferation. This study will determine, first, if the mitochondrial or cytoplasmic localization of Hsp60 is critical to VSMC proliferation and, second, the mechanism of Hsp60 induction of VSMC proliferation with a focus on modification of nucleocytoplasmic trafficking.

Methods and results

Hsp60 was overexpressed in primary rabbit VSMCs with or without a mitochondrial targeting sequence (AdHsp60^mito-). Both interventions induced an increase in VSMC PCNA expression and proliferation. The increase in VSMC PCNA expression and growth was not observed after siRNA-mediated knockdown of Hsp60 expression. Nuclear protein import in VSMC was measured by fluorescent microscopy using a microinjected fluorescent import substrate. Nuclear protein import was stimulated by both AdHsp60 and AdHsp60^mito- treatments. AdHsp60 treatment also induced increases in nucleoporin (Nup) 62, Nup153, importin-α, importin-β and Ran expression as well as cellular ATP levels compared to control. AdHsp60^mito- treatment induced an up-regulation in importin-α, importin-β and Ran expression compared to control. Hsp60 knockdown did not change nuclear protein import nor the expression of any nuclear transport receptors or nucleoporins. Both heat shock treatment and Hsp60 overexpression promoted the interaction of Ran with Hsp60.

Conclusions

VSMC proliferation can be modulated via an Hsp60 dependent, cytosol localized mechanism that in part involves a stimulation of nuclear protein import through an interaction with Ran. This novel cellular signaling role for Hsp60 may be important in growth-based vascular pathologies like atherosclerosis and hypertension.

中文翻译：

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热休克蛋白60参与血管平滑肌细胞增殖

目的

热休克蛋白60（Hsp60）是应激诱导的血管平滑肌细胞（VSMC）增殖的介体。这项研究将首先确定Hsp60的线粒体或细胞质定位对VSMC增殖是否至关重要，其次，确定Hsp60诱导VSMC增殖的机制，重点是修饰核质运输。

方法与结果

Hsp60的过表达在初级兔血管平滑肌细胞具有或不具有线粒体靶向序列（AdHsp60 ^mito-）。两种干预均导致VSMC PCNA表达和增殖增加。siRNA介导的Hsp60表达敲低后，未观察到VSMC PCNA表达和生长的增加。使用显微注射的荧光导入底物，通过荧光显微镜测量VSMC中的核蛋白导入。核蛋白的进口是由两个AdHsp60和AdHsp60刺激^mito-治疗。与对照相比，AdHsp60处理还诱导了核孔蛋白（Nup）62，Nup153，importin-α，importin-β和Ran表达以及细胞ATP水平的增加。AdHsp60 ^mito-与对照组相比，该处理诱导了importin-α，importin-β和Ran表达的上调。Hsp60敲低并没有改变核蛋白的导入，也没有改变任何核转运受体或核孔蛋白的表达。热休克处理和Hsp60过表达均促进Ran与Hsp60的相互作用。

结论

VSMC增殖可以通过Hsp60依赖性的胞浆定位机制进行调节，该机制部分涉及通过与Ran的相互作用刺激核蛋白的导入。Hsp60的这种新的细胞信号传导作用在基于动脉粥样硬化和高血压的基于生长的血管病理中可能很重要。