Presentation Authors: Ruixiao Wang*, Xiaolong Wang, Anna Ciotkowska, Beata Rutz, Alexander Tamalunas, Christian Gratzke, Christian G. Stief, Martin Hennenberg, Munich, Germany
Introduction: Lower urinary tract symptoms (LUTS) include irritative symptoms caused by overactive bladder (OAB) and voiding symptoms caused by benign prostatic hyperplasia. Recently, a role of the small monomeric GTPase Rac1 has been suggested for prostate smooth muscle contraction and prostate growth, which are key factors contributing to voiding symptoms. A similar role of Rac1 appears possible for growth and contraction of smooth muscle cells in the bladder, which may contribute to LUTS attributed to OAB. Recently, pharmacologic studies using small molecule inhibitors suggested a function of Rac1 in detrusor contraction, but target validation by knockout or the role of Rac1 in detrusor growth have not been addressed so far. Here, we examined effects of Rac inhibition or Rac1 knockout on growth and actin organization in bladder smooth muscle cells.
Methods: Experiments were carried out in cultured human bladder smooth muscle cells (hBSMCs). Rac inhibition was induced by small molecule inhibitors (NSC23766, EHT1864), or knockdown of Rac expression by lentiviral-based shRNA. Different readouts were applied to assess viability (CCK8 assay), proliferation activity (EdU assay, colony formation, Ki-67 expression, FACS), actin organization (phalloidin staining), or to confirm knockout.
Results: In wildtype hBSMCs, NSC23766 and EHT1864 reduced viability concentration-dependently. Low concentrations (25 ÂµM EHT 1864, 50 ÂµM NSC23766) showed only moderate effects on viability, but caused strong reductions of proliferation activity and increased apoptosis, as reflected by reduced proliferation index in EdU assay, reduced colony formation, and annexin-v FACS assay. Knockdown of Rac1 expression was induced by lentiviral shRNA, which was confirmed by RT-PCR and Western blot. Similar to small molecule inhibitors with assumed specificty for Rac, Rac1 knockdown resulted in reduced proliferation activity and increased apoptosis, as shown by EdU assay, analysis of Ki-67 content, and annexin-v FACS analysis. This was paralleled by reduced viability in CCK8 assay. In control and wildtype cells, actin was organized to filaments forming long protrusion. Filament organization was widely reduced by NSC23766, EHT, and Rac1 knockdown.
Conclusions: Rac1 promotes proliferation and actin organization in human bladder smooth muscle cells. Effects of the small molecule inhibitors NSC23766 and EHT1864 are mimicked by specific knockdown of Rac1 expression. A role of Rac1 for detrusor growth and detrusor contraction in OAB appears possible.
Source of Funding: Deutsche Forschungsgemeinschaft (DFG), Chinese Scholarship Council (CSC)