Wang Yan, Li Naijian, Wang Yingfeng, Zheng Guobing, An Jing, Liu Chang, Wang Yajie, Liu Qicai
The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Department of Cardiology, Laboratory of Heart Center, Zhujiang Hospital of Southern Medical University, Guangzhou, China.
Front Cell Dev Biol. 2021 Dec 7;9:656625. doi: 10.3389/fcell.2021.656625. eCollection 2021.
Peroxisome proliferator-activated receptor gamma (PPARγ) has an anti-proliferation effect on pulmonary arterial smooth muscle cells (PASMCs) via the transient receptor potential channel (TRPC) and protects against pulmonary artery hypertension (PAH), whereas nuclear factor-kappa B (NF-κB) has pro-proliferation and pro-inflammation effects, which contributes to PAH. However, the association between them in PAH pathology remains unclear. Therefore, this study aimed to investigate this association and the mechanisms underlying TRPC1/6 signaling-mediated PAH. Human pulmonary arterial smooth muscle cells (hPASMCs) were transfected with p65 overexpressing (pcDNA-p65) and interfering plasmids (shp65) and incubated in normal and hypoxic conditions (4% O and 72 h). The effects of hypoxia and p65 expression on cell proliferation, invasion, apoptosis, [Ca]i, PPARγ, and TRPC1/6 expression were determined using Cell Counting Kit-8 (CCK-8), Transwell, Annexin V/PI, Fura-2/AM, and western blotting, respectively. In addition, the binding of p65 or PPARγ proteins to the TRPC6 promoter was validated using a dual-luciferase report assay, chromatin-immunoprecipitation-polymerase chain reaction (ChIP-PCR), and electrophoretic mobility shift assay (EMSA). Hypoxia inhibited hPASMC apoptosis and promoted cell proliferation and invasion. Furthermore, it increased [Ca]i and the expression of TRPC1/6, p65, and Bcl-2 proteins. Moreover, pcDNA-p65 had similar effects on hypoxia treatment by increasing TRPC1/6 expression, [Ca]i, hPASMC proliferation, and invasion. The dual-luciferase report and ChIP-PCR assays revealed three p65 binding sites and two PPARγ binding sites on the promoter region of TRPC6. In addition, hypoxia treatment and shPPARγ promoted the binding of p65 to the TRPC6 promoter, whereas shp65 promoted the binding of PPARγ to the TRPC6 promoter. Competitive binding of NF-κB p65 and PPARγ to TRPC6 produced an anti-PAH effect.
过氧化物酶体增殖物激活受体γ（PPARγ）通过瞬时受体电位通道（TRPC）对肺动脉平滑肌细胞（PASMCs）具有抗增殖作用，并可预防肺动脉高压（PAH），而核因子-κB（NF-κB）具有促增殖和促炎作用，会导致PAH。然而，它们在PAH病理过程中的关联仍不清楚。因此，本研究旨在探究这种关联以及TRPC1/6信号介导的PAH的潜在机制。将人肺动脉平滑肌细胞（hPASMCs）用p65过表达质粒（pcDNA-p65）和干扰质粒（shp65）转染，并在正常和低氧条件下（4%氧气，72小时）孵育。分别使用细胞计数试剂盒-8（CCK-8）、Transwell、Annexin V/PI、Fura-2/AM和蛋白质印迹法测定低氧和p65表达对细胞增殖、侵袭、凋亡、细胞内钙离子浓度（[Ca]i）、PPARγ以及TRPC1/6表达的影响。此外，使用双荧光素酶报告基因检测、染色质免疫沉淀-聚合酶链反应（ChIP-PCR）和电泳迁移率变动分析（EMSA）验证p65或PPARγ蛋白与TRPC6启动子的结合。低氧抑制hPASMC凋亡，促进细胞增殖和侵袭。此外，它增加了[Ca]i以及TRPC1/6、p65和Bcl-2蛋白的表达。而且，pcDNA-p65通过增加TRPC1/6表达、[Ca]i、hPASMC增殖和侵袭，对低氧处理有类似的作用。双荧光素酶报告基因检测和ChIP-PCR分析显示TRPC6启动子区域有三个p65结合位点和两个PPARγ结合位点。此外，低氧处理和shPPARγ促进p65与TRPC6启动子的结合，而shp65促进PPARγ与TRPC6启动子的结合。NF-κB p65和PPARγ与TRPC6的竞争性结合产生了抗PAH作用。
