Abstract
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 in vitro. 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 in vivo and in vitro. 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 in vitro, and investigated the activity of SERCA2 and s-SERCA2 in vivo, 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 in vivo and in vitro. 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).
Highlights
Pulmonary arterial hypertension (PAH) is a condition marked by high blood pressure in the lungs and vascular remodeling, which can lead to right heart failure (RHF), and even death
These findings showed that serotonylation of SERCA2 inhibited the activity of SERCA2, and hypoxia induced serotonylation of SERCA2, which was modulated by the key enzyme of TG2 in vivo and in vitro
Our findings demonstrated that the activity of SERCA2 was regulated by a new post-translational modification (PTM) “serotonylation” and SERCA2 serotonylation inhibited activity of SERCA2
Summary
Pulmonary arterial hypertension (PAH) is a condition marked by high blood pressure in the lungs and vascular remodeling, which can lead to right heart failure (RHF), and even death. The abnormal proliferation and migration of pulmonary vein smooth muscle cells (PVSMCs) formed the pathological basis of pulmonary vein remodeling. We indicated that transient receptor potential cation 6 (TRPC6), not TRPC1, was functionally upregulated in rat PVs and PVSMCs in response to chronic hypoxia (CH). SOCE regulates adding Ca2+ back into the ER, which prevents Ca2+ from entering and enables restorative resting of Abbreviations: CAP, cyclopiazonic acid; SOCE, store-operated calcium entry; [Ca2+]i, intracellular Ca2+ concentration; SERCA, sarco-endoplasmic reticulum Ca2+ ATPase; s-SERCA2, SERCA2 serotonination; TG2, transglutaminases; PAH, pulmonary arterial hypertension; RVSP, right ventricular systolic pressure; RVHI, right ventricular hypertrophy index; hPVSMC, human pulmonary venous smooth muscle cell; TRPC, transient receptor potential cation channel; 5-HT, serotonin, 5-hydroxytryptamine; WT, wild type; Tgm2 −/−, TG2 gene knockout
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