Molecular characterization of the effects of Y-27632
Molecular characterization of the effects of Y-27632
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DOI：
10.1002/cm.20168
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发表时间：
2007-02-01
影响因子：
--
通讯作者：
Mandato, Craig A.
中科院分区：
文献类型：
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作者：
Darenfed, Hassina;Dayanandan, Bama;Mandato, Craig A.
许多关键的细胞功能，如细胞运动和细胞分化是由Rho相关蛋白激酶（ROCK）介导的。已经进行了大量的研究，以检查ROCK信号转导通路参与这些运动和收缩事件的药物抑制剂，如Y-27632的帮助下。然而，Y-27632的分子作用机制尚未完全确定。为了评估这些Rho效应物对Y-27632的作用的相对贡献，我们比较了用Y-27632处理的细胞或用ROCK-I、ROCK-II或siRNA-2特异性敲低的细胞中的细胞骨架表型、伤口愈合和神经突生长。ROCK-I的减少增强了薄的富含肌动蛋白的膜延伸的形成，这是一种与Y-27632的作用非常相似的表型。ROCK-II或ROCK-II-2的敲除分别导致盘状延伸和厚肌动蛋白束的形成。ROCK-I敲低的作用也模拟了Y-27632对伤口闭合率的作用。ROCK-I敲除和Y-27632增强伤口闭合率，而ROCK-II和ROCK-II-2与对照细胞没有明显差异。在神经突生长测定中，ROCK-I、ROCK-II或ROCK-II-2的敲低增强神经突长度，然而没有单独的敲低刺激神经突生长如Y-27632那样强劲。我们的结论是，几种激酶有助于Y-27632对细胞反应的全球影响。
Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2-specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK-II or PRK-2, leads to the formation of disc-like extenstions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses.