Liu Bo, Wang Dong, Luo Erfei, Hou Jiantong, Qiao Yong, Yan Gaoliang, Wang Qingjie, Tang Chengchun
Department of Cardiology, Zhongda Hospital, Southeast University, Nanjing, China.
Department of Cardiology, Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, China.
Front Pharmacol. 2020 Feb 11;10:1611. doi: 10.3389/fphar.2019.01611. eCollection 2019.
Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pumps take up Ca2+ from the cytoplasm to maintain the balance of intracellular Ca2+. A decline in expression or activity of SERCA results in persistent store-operated calcium entry (SOCE). In cardiomyocytes as well as vascular smooth muscle cells (SMCs), SERCA2 acts as an important regulator of calcium cycling. The purpose of this study is to identify and better understand the role of transglutaminases2 (TG2) as a key factor involved in SERCA2 serotonination (s-SERCA2) and to elucidate the underlying mechanism of action. Human pulmonary venous smooth muscle cell in normal pulmonary lobe were isolated and cultured . Establishment of hypoxic pulmonary hypertension model in wild type and TG2 knockout mice. SERCA2 serotonylation was analyzed by co-(immunoprecipitation) IP when the TG2 gene silenced or overexpressed under normoxia and hypoxia and . Intracellular calcium ion was measured by using Fluo-4AM probe under normoxia and hypoxia. Real-time (RT)-PCR and Western blot analyzed expression of TG2, TRPC1, and TRPC6 under normoxia and hypoxia. Bioactivity of cells were analyzed by using Cell Counting Kit (CCK)-8, flow cytometry, wound healing, RT-PCR, and Western blot under PST-2744 and cyclopiazonic acid. We confirmed that 1) hypoxia enhanced the expression and activity of TG2, and 2) hypoxia increased the basal intracellular Ca2+ concentration ([Ca2+]i) and SOCE through activating TRPC6 on human pulmonary vein smooth muscle cells (hPVSMC). Then, we investigated the effects of overexpression and downregulation of the TG2 gene on the activity of SERCA2, s-SERCA2, basal [Ca2+]i, and SOCE under normoxia and hypoxia , and investigated the activity of SERCA2 and s-SERCA2 , respectively. We confirmed that SERCA2 serotonylation inhibited the activity of SERCA2 and increased the Ca2+ influx, and that hypoxia induced TG2-mediated SERCA2 serotonylation both and . Furthermore, we investigated the effect of TG2 activity on the biological behavior of hPVSMC by using an inhibitor and agonist of SERCA2, respectively. Finally, we confirmed that chronic hypoxia cannot increase vessel wall thickness, the right ventricular systolic pressure (RVSP), and right ventricular hypertrophy index (RVHI) of vascular smooth muscle-specific Tgm2-/- mice. These results indicated that hypoxia promoted TG2-mediated SERCA2 serotonylation, thereby leading to inhibition of SERCA2 activity, which further increased the calcium influx through the TRPC6 channel. Furthermore, tissue-specific conditional TG2 knockout mice prevents the development of pulmonary hypertension caused by hypoxia. In summary, we uncovered a new target (TG2) for treatment of chronic hypoxic pulmonary hypertension (CHPH).
肌浆网钙ATP酶（SERCA）泵从细胞质中摄取Ca2+以维持细胞内钙平衡。SERCA表达或活性下降会导致持续性钙库操纵性钙内流（SOCE）。在心肌细胞和血管平滑肌细胞（SMC）中，SERCA2是钙循环的重要调节因子。本研究的目的是鉴定并更好地理解转谷氨酰胺酶2（TG2）作为参与SERCA2血清素化（s-SERCA2）的关键因子的作用，并阐明其潜在作用机制。分离并培养正常肺叶中的人肺静脉平滑肌细胞。在野生型和TG2基因敲除小鼠中建立缺氧性肺动脉高压模型。当TG2基因在常氧和缺氧条件下沉默或过表达时，通过免疫共沉淀（co-IP）分析SERCA2血清素化情况。在常氧和缺氧条件下使用Fluo-4AM探针测量细胞内钙离子。通过实时（RT）-PCR和蛋白质免疫印迹法分析常氧和缺氧条件下TG2、瞬时受体电位通道蛋白1（TRPC1）和瞬时受体电位通道蛋白6（TRPC6）的表达。在PST-2744和环匹阿尼酸作用下，使用细胞计数试剂盒（CCK）-8、流式细胞术、伤口愈合实验、RT-PCR和蛋白质免疫印迹法分析细胞的生物学活性。我们证实：1）缺氧增强了TG2的表达和活性；2）缺氧通过激活人肺静脉平滑肌细胞（hPVSMC）上的TRPC6增加了基础细胞内Ca2+浓度（[Ca2+]i）和SOCE。然后，我们研究了TG2基因过表达和下调对常氧和缺氧条件下SERCA2活性、s-SERCA2、基础[Ca2+]i和SOCE的影响，并分别研究了SERCA2和s-SERCA2的活性。我们证实SERCA2血清素化抑制了SERCA2的活性并增加了Ca2+内流，并且缺氧在常氧和缺氧条件下均诱导TG2介导的SERCA2血清素化。此外，我们分别使用SERCA2的抑制剂和激动剂研究了TG2活性对hPVSMC生物学行为的影响。最后，我们证实慢性缺氧不会增加血管平滑肌特异性Tgm2-/-小鼠的血管壁厚度、右心室收缩压（RVSP）和右心室肥厚指数（RVHI）。这些结果表明缺氧促进了TG2介导的SERCA2血清素化，从而导致SERCA2活性受到抑制，进而通过TRPC6通道进一步增加了钙内流。此外，组织特异性条件性TG2基因敲除小鼠可预防缺氧引起的肺动脉高压的发展。总之，我们发现了治疗慢性缺氧性肺动脉高压（CHPH）的新靶点（TG2）。
