背景:前列腺平滑肌收缩和基质生长可能导致下尿路症状提示良性前列腺增生,但不完全理解。单体GTP酶CDC42对平滑肌收缩和增殖的作用似乎是可能的,但前列腺未知。这里,我们沉默了前列腺基质细胞(WPMY-1)中CDC42的表达,并检查了收缩性,与生长相关的功能和对假定的CDC42抑制剂的反应,ML141.
方法:用乱序或CDC42特异性siRNA转染WPMY-1细胞,并表征GTPase活性,收缩,扩散,菌落形成,凋亡,细胞死亡和活力。在有和没有沉默的细胞中检查ML141的作用。
结果:CDC42沉默通过减少的mRNA和蛋白表达得到证实,和降低CDC42活性。沉默受损收缩(23-47%),肌动蛋白组织(25%),增殖(17-63%),集落形成和生存力(64-89%),并增加死亡细胞的百分比(2.6倍)。ML141模拟了在乱序siRNA转染的对照中沉默的表型,在未转染的WPMY-1细胞中,包括抑制收缩,扩散,集落形成和生存力,肌动蛋白组织的破坏和细胞死亡的增加。在CDC42沉默的细胞中,ML141仍然影响成团组织,增殖和细胞死亡,效果大小类似于没有沉默的对照。ML141抑制CDC42沉默细胞中的RhoA活性,但不是在没有沉默的细胞中。
结论:CDC42促进前列腺基质细胞的收缩,并通过CDC42介导的增殖和抑制非凋亡性细胞死亡来驱动基质生长。ML141模仿CDC42沉默的所有效果,但其特异性可能是有限的,取决于细胞的GTP酶表型。
BACKGROUND: Prostate smooth muscle contraction and stromal growth may contribute to lower urinary tract symptoms suggestive of benign prostatic hyperplasia, but are incompletely understood. A role of the monomeric GTPase CDC42 for smooth muscle contraction and proliferation appears possible, but is unknown for the prostate. Here, we silenced CDC42 expression in prostate stromal cells (WPMY-1), and examined contractility, growth-related functions and responses to the presumed CDC42 inhibitor, ML141.
METHODS: WPMY-1 cells were transfected with scrambled or CDC42-specific siRNA, and characterized for GTPase activities, contraction, proliferation, colony formation, apoptosis, cell death and viability. Effects of ML141 were examined in cells with and without silencing.
RESULTS: CDC42 silencing was confirmed by reduced mRNA and protein expression, and reduced CDC42 activity. Silencing impaired contraction (23-47 %), actin organization (25 %), proliferation (17-63 %), colony formation and viability (64-89 %), and increased the percentage of dead cells (2.6-fold). ML141 mimicked the phenotype of silencing in scrambled siRNA-transfected controls, and in non-transfected WPMY-1 cells, including inhibition of contraction, proliferation, colony formation and viability, breakdown of actin organization and increased cell death. In CDC42-silenced cells, ML141 still affected phalloiding organization, proliferation and cell death, with effect sizes resembling controls without silencing. ML141 inhibited RhoA activity in CDC42-silenced cells, but not in cells without silencing.
CONCLUSIONS: CDC42 promotes contraction of prostate stromal cells, and drives stromal growth by CDC42-mediated proliferation and suppression of apoptosis-independent cell death. ML141 mimicks all effects of CDC42 silencing, but its specificity may be limited and depends on GTPase phenotypes of cells.