Aims

RacGTPase-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 methods

Rac1 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 findings

Silencing 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.

Significance

Rac1 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.

中文翻译：

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Rac1沉默，NSC23766和EHT1864会减少膀胱平滑肌细胞的生长和肌动蛋白组织。

目的

最近已经提出了RacGTP酶介导的下尿路增殖和平滑肌收缩，并且可能为治疗下尿路症状提供推定的靶标。然而，RacGTPase在逼尿肌平滑肌细胞增殖中的功能尚不清楚，Rac抑制剂的特异性也受到质疑。在这里，我们检查了Rac1敲除以及Rac抑制剂NSC23766和EHT1864在人膀胱平滑肌细胞（hBSMCs）中的作用。

主要方法

Rac1表达被shRNA表达沉默。通过CCK-8分析，EdU染色，RT-PCR，集落形成分析，流式细胞仪和鬼笔环肽染色评估沉默和Rac抑制剂的作用。

主要发现

Rac1表达的沉默降低了活力（与scramble shRNA相比，高达83％）和增殖（在增殖测定中几乎完全），增加了凋亡（124％）和死细胞数量（51％），并导致肌动蛋白组织破坏（与scramble shRNA相比，聚合的肌动蛋白减少了56％）。NSC23766和EHT1864模仿了对增殖，活力和肌动蛋白组织的影响，而这两种化合物对细胞死亡均表现出不同的作用（EHT1864使死细胞增加了32倍，而NSC23766没有）。Nac23766和EHT1864对hBSMC生存力的影响并未因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模仿这些效果，但可能通过共有和不同的效果独立发挥Rac1作用。