ADP Ribosylation Factor 6 Promotes Contraction and Proliferation, Suppresses Apoptosis and Is Specifically Inhibited by NAV2729 in Prostate Stromal Cells
ADP Ribosylation Factor 6 Promotes Contraction and Proliferation, Suppresses Apoptosis and Is Specifically Inhibited by NAV2729 in Prostate Stromal Cells
复制标题
DOI：
10.1124/molpharm.121.000304
复制
发表时间：
2021-08
影响因子：
3.6
通讯作者：
Ruixiao Wang;S. Schneider;O. Keppler;Bingsheng Li;B. Rutz;A. Ciotkowska;C. Stief;M. Hennenberg
中科院分区：
文献类型：
--
作者：
Ruixiao Wang;S. Schneider;O. Keppler;Bingsheng Li;B. Rutz;A. Ciotkowska;C. Stief;M. Hennenberg
推测的 ADP 核糖基化因子 (ARF) 6 抑制剂 NAV2729 抑制人前列腺平滑肌收缩和基质细胞增殖，这是良性前列腺增生 (BPH) 排尿症状的驱动因素。然而，其特异性和 ARF6 对平滑肌收缩的作用尚未得到证实。在这里，我们在人前列腺基质细胞 (WPMY-1) 中产生了单克隆 ARF6 敲除，并表征了敲除克隆和对照克隆中的收缩性、生长相关功能和对 NAV2729 的敏感性的表型。通过蛋白质印迹验证ARF6敲除。敲除克隆显示收缩和肌动蛋白组织受损，增殖和活力降低，细胞凋亡和细胞死亡增加。在表达 ARF6 的对照克隆中，NAV2729 (5 µM) 强烈抑制收缩（所有三个对照克隆的抑制率为 67%）、肌动蛋白组织（72%）、增殖（97%）和活力（高达 82%），并增加细胞凋亡（5 倍）和细胞死亡（6 倍）。在 ARF6 敲除中，NAV2729 (5 µM) 的作用大大降低，包括对收缩（所有三个敲除克隆的抑制均为 0%）、肌动蛋白（18%）和增殖（13%）缺乏或轻微影响，并且缺乏细胞凋亡和细胞死亡的增加。 NAV2729 降低了活力，所有三个 ARF6 对照克隆的 IC50 均为 3.3 µM，但在 ARF6 敲除中为 4.5–8.2 µM。总之，ARF6促进前列腺平滑肌收缩和基质细胞增殖。两者均被 NAV2729 抑制，NAV2729 对 ARF6 表现出高达 5 µM 的高度特异性，是治疗 BPH 的一种有吸引力的化合物。考虑到基于平滑肌的疾病的相关性，ARF6 在其他平滑肌类型中的共同作用值得进一步研究。意义声明 通过敲除前列腺基质细胞中的 ARF6，本研究证明了 ARF6 参与促进前列腺平滑肌收缩和基质生长，并定义了 NAV2729 对其 ARF6 特异性抑制的浓度范围。除了良性前列腺增生和下尿路症状外，类似的 ARF6 收缩和生长功能在其他富含平滑肌的器官中似乎也可能存在，考虑到基于平滑肌的疾病的高度临床相关性，这一点值得进一步关注。
The presumed ADP ribosylation factor (ARF) 6 inhibitor NAV2729 inhibits human prostate smooth muscle contraction and proliferation of stromal cells, which are driving factors of voiding symptoms in benign prostatic hyperplasia (BPH). However, its specificity and a confirmed role of ARF6 for smooth muscle contraction are still pending. Here, we generated monoclonal ARF6 knockouts in human prostate stromal cells (WPMY-1), and characterized phenotypes of contractility, growth-related functions, and susceptibility to NAV2729 in knockout and control clones. ARF6 knockout was verified by Western blot. Knockout clones showed impaired contraction and actin organization, reduced proliferation and viability, and increased apoptosis and cell death. In ARF6-expressing control clones, NAV2729 (5 µM) strongly inhibited contraction (67% inhibition across all three control clones), actin organization (72%), proliferation (97%), and viability (up to 82%), and increased apoptosis (5-fold) and cell death (6-fold). In ARF6 knockouts, effects of NAV2729 (5 µM) were widely reduced, including lacking or minor effects on contractions (0% inhibition across all three knockout clones), actin (18%) and proliferation (13%), and lacking increases of apoptosis and cell death. Viability was reduced by NAV2729 with an IC50 of 3.3 µM across all three ARF6 control clones, but of 4.5–8.2 µM in ARF6 knockouts. In conclusion, ARF6 promotes prostate smooth muscle contraction and proliferation of stromal cells. Both are inhibited by NAV2729, which showed high specificity for ARF6 up to 5 µM and represents an attractive compound in the context of BPH. Considering the relevance of smooth muscle-based diseases, shared roles of ARF6 in other smooth muscle types merit further investigation. SIGNIFICANCE STATEMENT By knockout of ARF6 in prostate stromal cells, this study demonstrates the involvement of ARF6 in promotion of prostate smooth muscle contraction and stromal growth, and defines concentration ranges for their ARF6-specific inhibition by NAV2729. Besides the context of benign prostatic hyperplasia and lower urinary tract symptoms, analog ARF6 functions in contraction and growth appear possible in other smooth muscle-rich organs, which merits further attention considering the high clinical relevance of smooth muscle-based diseases.