Rac1 silencing, NSC23766 and EHT1864 reduce growth and actin organization of bladder smooth muscle cells.
Rac1 silencing, NSC23766 and EHT1864 reduce growth and actin organization of bladder smooth muscle cells.
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DOI：
10.1016/j.lfs.2020.118468
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发表时间：
2020-09
期刊：
影响因子：
6.1
通讯作者：
Ruixiao Wang;Qingfeng Yu;Xiaolong Wang;Bingsheng Li;A. Ciotkowska;B. Rutz;Yiming Wang;C. Stief;M. Hennenberg
中科院分区：
文献类型：
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作者：
Ruixiao Wang;Qingfeng Yu;Xiaolong Wang;Bingsheng Li;A. Ciotkowska;B. Rutz;Yiming Wang;C. Stief;M. Hennenberg
最近有人提出，下尿路中 AimsRacGTPase 介导的增殖和平滑肌收缩可能为治疗下尿路症状提供假定的目标。然而，RacGTPase 对逼尿肌平滑肌细胞增殖的功能尚不清楚，Rac 抑制剂的特异性也受到质疑。在这里，我们检查了 Rac1 敲低以及 Rac 抑制剂 NSC23766 和 EHT1864 对人膀胱平滑肌细胞 (hBSMC) 的影响。主要方法通过 shRNA 表达沉默 Rac1 表达。通过 CCK-8 测定、EdU 染色、RT-PCR、集落形成测定、流式细胞术和鬼笔环肽染色评估沉默和 Rac 抑制剂的效果。 主要发现 Rac1 表达沉默会降低活力（与乱序 shRNA 相比高达 83%）和增殖（在增殖测定中几乎完全），增加细胞凋亡 (124%) 和死细胞数量 (51%)，并导致肌动蛋白组织崩溃（与乱序 shRNA 相比，聚合肌动蛋白减少了 56%）。 NSC23766 和 EHT1864 模拟了对增殖、活力和肌动蛋白组织的影响，但这两种化合物对细胞死亡的影响不同（EHT1864 使死亡细胞增加了 32 倍，但 NSC23766 则不然）。 NSC23766 和 EHT1864 对 hBSMC 活力的影响不会因 Rac1 敲低而改变。意义 Rac1 促进增殖、活力和细胞骨架组织，并抑制膀胱平滑肌细胞的凋亡，这可能与膀胱过度活动症或糖尿病相关的膀胱功能障碍有关。 NSC23766 和 EHT1864 模仿这些效应，但可能通过共享和不同的效应独立于 Rac1 发挥作用。
AimsRacGTPase-mediated proliferation and smooth muscle contraction in the lower urinary tract has been recently suggested and may offer putative targets for treamtment of lower urinary tract symptoms. However, RacGTPase function for proliferation of detrusor smooth muscle cells is unknown and the specificity of Rac inhibitors has been questioned. Here, we examined effects of Rac1 knockdown and of the Rac inhibitors NSC23766 and EHT1864 in human bladder smooth muscle cells (hBSMCs).Main methodsRac1 expression was silenced by shRNA expression. Effects of silencing and Rac inhibitors were assessed by CCK-8 assay, EdU staining, RT-PCR, colony formation assay, flow cytometry, and phalloidin staining.Key findingsSilencing of Rac1 expression reduced the viability (up to 83% compared to scramble shRNA) and proliferation (virtually completely in proliferation assay), increased apoptosis (124%) and the number of dead cells (51%), and caused breakdown of actin organization (56% reduction of polymerized actin compared to scramble shRNA). Effects on proliferation, viability, and actin organization were mimicked by NSC23766 and EHT1864, while both compounds showed divergent effects on cell death (32-fold increase of dead cells by EHT1864, but not NSC23766). Effects of NSC23766 and EHT1864 on viability of hBSMCs were not altered by Rac1 knockdown.SignificanceRac1 promotes proliferation, viability, and cytoskeletal organization, and suppresses apoptosis in bladder smooth muscle cells, which may be relevant in overactive bladder or diabetes-related bladder dysfunction. NSC23766 and EHT1864 mimick these effects, but may act Rac1-independently, by shared and divergent effects.