维拉帕米通过阻断 CaMK II 介导的 STAT3 和 Smad3/JunD 通路抑制输尿管狭窄的发展。
Verapamil inhibited the development of ureteral stricture by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.
机构信息
Department of Urology, Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, No. 61, Jiefang Road, Changsha, 410005, Hunan province, People's Republic of China.
出版信息
Int Urol Nephrol. 2022 Nov;54(11):2855-2866. doi: 10.1007/s11255-022-03284-4. Epub 2022 Aug 3.
BACKGROUND
Ureteral stricture (US) is a fibrotic process that leads to urinary tract obstruction and even kidney damage, with the characteristic of reduced extracellular matrix (ECM) degradation and increased collagen synthesis. Verapamil, as a calcium channel blocker, was reported to prevent scar formation. Our work aimed to investigate the biological effects and mechanism of verapamil in US.
METHODS
Fibroblasts were subjected to transforming growth factor-beta 1 (TGF-β1) to stimulate collagen synthesis, and the messenger ribonucleic acid (mRNA) and protein expressions in fibroblasts were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The location of phosphorylation-signal transducer and activator of transcription 3 (p-STAT3) and Jund proto-oncogene subunit (JunD) in fibroblasts were determined by immunofluorescence (IF). The binding relationship between signal transducer and activator of transcription 3 (STAT3) and collagen type I alpha1 (COL1A1)/collagen type III alpha 1 chain (COL3A1) and the binding relationship between JunD and tissue inhibitor of metalloproteinases-1 (TIMP-1) were verified by dual luciferase reporter gene and chromatin Immunoprecipitation (ChIP) assays.
RESULTS
Herein, we found that verapamil could inhibit TGF-β1/Ca2 + ⁄calmodulin-dependent protein kinase II (CaMK II)-mediated STAT3 activation in fibroblasts, and STAT3 inhibition repressed collagen production. In addition, verapamil could inhibit TGF-β1/CaMK II-mediated Mothers against DPP homolog 3 (Smad3)/JunD pathway activation in fibroblasts, and JunD silencing inhibited TIMP1 (a matrix metalloproteinase inhibitor) expression. Our subsequent experiments revealed that STAT3 bound with COL1A1 promoter and COL3A1 promoter and activated their transcription, and JunD bound with TIMP1 promoter and activated its transcription. Moreover, as expected, STAT3 activation could eliminate the inhibitory effect of verapamil treatment on TGF-β1-induced collagen production in fibroblasts, and JunD overexpression reversed the inhibitory effect of verapamil treatment on TGF-β1-induced TIMP1 expression in fibroblasts.
CONCLUSION
Verapamil inhibited collagen production and TIMP-1 expression in US by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.
背景
输尿管狭窄（US）是一种纤维化过程，可导致尿路梗阻甚至肾脏损害，其特征是细胞外基质（ECM）降解减少和胶原合成增加。维拉帕米作为钙通道阻滞剂，被报道可预防瘢痕形成。我们的工作旨在研究维拉帕米在 US 中的生物学作用和机制。
方法
用转化生长因子-β1（TGF-β1）刺激成纤维细胞以刺激胶原合成，并通过定量实时聚合酶链反应（qRT-PCR）和 Western blot 评估成纤维细胞中的信使核糖核酸（mRNA）和蛋白表达。通过免疫荧光（IF）确定磷酸化信号转导和转录激活因子 3（p-STAT3）和 JunD 原癌基因亚基（JunD）在成纤维细胞中的位置。通过双荧光素酶报告基因和染色质免疫沉淀（ChIP）实验验证 STAT3 与胶原类型 I alpha1（COL1A1）/胶原类型 III alpha 1 链（COL3A1）之间的结合关系以及 JunD 与组织金属蛋白酶抑制剂 1（TIMP-1）之间的结合关系。
结果
本文发现维拉帕米可抑制 TGF-β1/Ca2+ /钙调蛋白依赖性蛋白激酶 II（CaMK II）介导的成纤维细胞中 STAT3 的激活，而 STAT3 抑制可抑制胶原产生。此外，维拉帕米可抑制 TGF-β1/CaMK II 介导的成纤维细胞中 Mothers against DPP 同源物 3（Smad3）/JunD 通路的激活，而 JunD 沉默抑制基质金属蛋白酶抑制剂 1（TIMP1）的表达。我们随后的实验表明，STAT3 与 COL1A1 启动子和 COL3A1 启动子结合并激活其转录，而 JunD 与 TIMP1 启动子结合并激活其转录。此外，正如预期的那样，STAT3 的激活可以消除维拉帕米处理对 TGF-β1 诱导的成纤维细胞中胶原产生的抑制作用，而过表达 JunD 则逆转了维拉帕米处理对 TGF-β1 诱导的成纤维细胞中 TIMP1 表达的抑制作用。
结论
维拉帕米通过阻断 CaMK II 介导的 STAT3 和 Smad3/JunD 通路抑制 US 中的胶原产生和 TIMP-1 表达。
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