Lady Davis Institute, McGill University, 3755, Chemin de la cote-Ste-Catherine, Montreal, QC H3T 1E2, Canada.
School of Medicine, McGill University, 3605 Rue de la Montagne, Montréal, QC H3G 2M1, Canada.
Cell Signal. 2021 May;81:109936. doi: 10.1016/j.cellsig.2021.109936. Epub 2021 Jan 30.
The nerve growth factor precursor (proNGF) activates p75 receptor and promotes cell death in different tissues, yet this pathophysiological effect is not fully described in the bladder. The aim of this study was to identify the biological effect of proNGF/p75 activation on urothelial and smooth muscle (SM) cells of rodents' bladder. Cell viability was assessed by MTT assay which showed a significant reduction in urothelial viability after 24 h of incubation with proNGF in culture medium [5 or 10 nM], an effect not seen in SM cells. Western blot analysis on cellular protein extracts showed increased expression of the transmembrane TNF-α and activation of RhoA in urothelial cells exposed to proNGF with no evidence of a nuclear translocation of NF-κB assessed by western blotting on nuclear extracts and immunofluorescence. The activation of p75-death domain related pathways in urothelial cells such as TNF-α or RhoA had a downstream effect on NO release and the junctional protein occludin, as estimated respectively by colorimetric and western blotting. On the other hand, proNGF did not induce TNF-α or RhoA expression in SM cells, but induced a significant NF-κB nuclear translocation. ProNGF had a different impact on SM as evidenced by a significant dose- and time-dependent increase in SM proliferation and migration examined by MTT test and cell migration assay. Together, our results indicate that activation of proNGF/p75 axis induces degenerative changes to the urothelial layer impacting its barrier and signaling integrity, while promoting adaptive proliferative changes in detrusor SM cells that can interfere with the contractile phenotype essential for proper bladder function.
神经生长因子前体 (proNGF) 激活 p75 受体并促进不同组织中的细胞死亡，但这一病理生理效应在膀胱中尚未完全描述。本研究旨在确定 proNGF/p75 激活对啮齿动物膀胱上皮细胞和平滑肌 (SM) 的生物学效应。通过 MTT 测定评估细胞活力，结果显示在培养物中孵育 24 小时后，上皮细胞活力在 proNGF 存在下显著降低 [5 或 10 nM]，而 SM 细胞则未见此现象。细胞蛋白提取物的 Western blot 分析显示，暴露于 proNGF 的上皮细胞中转膜 TNF-α表达增加，RhoA 被激活，但核提取物 Western blot 和免疫荧光分析均未显示 NF-κB 核易位。上皮细胞中 p75 死亡结构域相关通路的激活，如 TNF-α或 RhoA，对 NO 释放和连接蛋白 occludin 有下游影响，分别通过比色法和 Western blot 进行评估。另一方面，proNGF 未诱导 SM 细胞中 TNF-α或 RhoA 的表达，但诱导了 NF-κB 的核易位。proNGF 对 SM 的影响不同，这表现在 MTT 试验和细胞迁移试验显示 SM 增殖和迁移的剂量和时间依赖性显著增加。总之，我们的结果表明，proNGF/p75 轴的激活会导致上皮层发生退行性变化，影响其屏障和信号完整性，同时促进逼尿肌 SM 细胞的适应性增殖变化，这可能会干扰对膀胱功能至关重要的收缩表型。
