Ghosh Science and Technology Center, Department of Biology, Worcester State University, Worcester, Massachusetts, USA.
Curr Mol Med. 2013 Jan;13(1):205-19. doi: 10.2174/1566524011307010205.
The serine/threonine protein kinase paralogs ROCK1 & 2 have been implicated as essential modulators of angiogenesis; however their paralog-specific roles in endothelial function are unknown. shRNA knockdown of ROCK1 or 2 in endothelial cells resulted in a significant disruption of in vitro capillary network formation, cell polarization, and cell migration compared to cells harboring non-targeting control shRNA plasmids. Knockdowns led to alterations in cytoskeletal dynamics due to ROCK1 & 2-mediated reductions in actin isoform expression, and ROCK2-specific reduction in myosin phosphatase and cofilin phosphorylation. Knockdowns enhanced cell survival and led to ROCK1 & 2-mediated reduction in caspase 6 and 9 cleavage, and a ROCK2-specific reduction in caspase 3 cleavage. Microarray analysis of ROCK knockdown lines revealed overlapping and unique control of global transcription by the paralogs, and a reduction in the transcriptional regulation of just under 50% of VEGF responsive genes. Finally, paralog knockdown in xenograft angiosarcoma tumors resulted in a significant reduction in tumor formation. Our data reveals that ROCK1 & 2 exhibit overlapping and unique roles in normal and dysfunctional endothelial cells, that alterations in cytoskeletal dynamics are capable of overriding mitogen activated transcription, and that therapeutic targeting of ROCK signaling may have profound impacts for targeting angiogenesis.
丝氨酸/苏氨酸蛋白激酶同源物 ROCK1 和 ROCK2 被认为是血管生成的重要调节因子；然而，它们在血管内皮细胞功能中的特异性作用尚不清楚。与携带非靶向对照 shRNA 质粒的细胞相比，内皮细胞中 ROCK1 或 ROCK2 的 shRNA 敲低导致体外毛细血管网络形成、细胞极化和细胞迁移的显著破坏。由于 ROCK1 和 ROCK2 介导的肌动蛋白同工型表达减少，以及 ROCK2 特异性的肌球蛋白磷酸酶和原肌球蛋白磷酸化减少，导致细胞骨架动力学发生改变。敲低增强了细胞存活，并导致 ROCK1 和 ROCK2 介导的半胱天冬酶 6 和 9 切割减少，以及 ROCK2 特异性的半胱天冬酶 3 切割减少。ROCK 敲低系的微阵列分析显示，同源物对全局转录具有重叠和独特的控制作用，并且 VEGF 反应基因的转录调控减少了近 50%。最后，异种移植血管肉瘤肿瘤中同源物的敲低导致肿瘤形成显著减少。我们的数据表明，ROCK1 和 ROCK2 在正常和功能失调的血管内皮细胞中表现出重叠和独特的作用，细胞骨架动力学的改变能够覆盖有丝分裂原激活的转录，靶向 ROCK 信号的治疗可能会对靶向血管生成产生深远的影响。
