Calcineurin/NFAT Signaling Modulates Pulmonary Artery Smooth Muscle Cell Proliferation, Migration and Apoptosis in Monocrotaline-Induced Pulmonary Arterial Hypertension Rats

Abstract

Background/Aims: Pulmonary arterial hypertension (PAH) is a severe and debilitating disease characterized by remodeling of the pulmonary vessels, which is driven by excessive proliferation and migration and apoptosis resistance in pulmonary artery smooth muscle cells (PASMCs). The calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling pathway is the most important downstream signaling pathway of store-operated Ca²⁺ entry (SOCE), which is increased in PAH. CaN/NFAT has been reported to contribute to abnormal proliferation in chronic hypoxia (CH)-induced PAH. However, the effect of CaN/NFAT signaling on PASMC proliferation, migration and apoptosis in monocrotaline (MCT)-induced PAH remains unclear. Methods: PAH rats were established by a single intraperitoneal injection of MCT for 21 days. PASMCs were isolated and cultured in normal and MCT-induced PAH Sprague-Dawley rat. PASMCs were treated with CsA targeting CaN and siRNA targeting NFATc2-4 gene respectively by liposome. We investigated the expression of calcineurin/NFAT signaling by immunofluorescence, qRT-PCR and Western blotting methods. Cell proliferation was monitored using MTS reagent or by assessing proliferating cell nuclear antigen (PCNA) expression. Cell apoptosis was evaluated with an Annexin V - FITC/propidium iodide (PI) apoptosis kit by flow cytometry. PASMC migration was assessed with a Transwell chamber. Results: MCT successfully induced PAH and pulmonary vascular remodeling in rats. CaN phosphatase activity and nuclear translocation of NFATc2-4 were increased in PASMCs derived from MCT-treated rats. In addition, CaNBβ/NFATc2-4 expression was amplified at the mRNA and protein levels. PASMC proliferation and migration were markedly inhibited in a dosedependent manner by cyclosporin A (CsA). Furthermore, siRNA targeting NFATc2 and NFATc4 attenuated the excessive proliferation and migration and apoptosis resistance in PASMCs derived from both CON and MCT-treated rats, while NFATc3 knockdown specifically affected MCT-PASMCs. Conclusion: Our results demonstrate that CaN/NFAT signaling is activated and involved in the modulation of PASMC proliferation, migration and apoptosis in MCT-induced PAH.

PDGF mediates pulmonary arterial smooth muscle cell proliferation and migration by regulating NFATc2

“…It is commonly known that intrinsic changes in Ca 2+ homeostasis in PASMCs plays an important role in pulmonary vasoconstriction and vascular reconstruction in PAH ( 32 ) Of note, it was previously discovered that store-operated Ca 2+ entry (SOCE) is the main channel that regulates Ca 2+ influx in PASMCs and regulates PASMC proliferation, apoptosis and migration in PAH ( 33 , 34 ). The CaN/NFAT pathway is the most important downstream signaling pathway of SOCE and is involved in numerous physiological and pathological processes ( 32 ). An increase in cytosolic free Ca 2+ concentration [(Ca 2+ )cyt] in PASMCs is a major trigger for pulmonary vasoconstriction and an important underlying mechanism of pulmonary vascular remodeling via stimulation of PASMC proliferation and inhibition of PASMC apoptosis ( 35 ).…”

BCAT1 binds the RNA-binding protein ZNF423 to activate autophagy via the IRE1-XBP-1-RIDD axis in hypoxic PASMCs

“…Hypoxia occurs in early NSCLC and is related to the increased expression of osteopontin and carbonic anhydrase IX 10 . Importantly, hypoxia is an important pathogenic factor for PASMC proliferation, migration, apoptosis, and pyroptosis 11 . Hypoxia led to the upregulation of mitofusin 1, which maintains mitochondrial homeostasis through mir-125a, and was shown to play an important role in promoting PASMCs 12 .…”

The potential role of TRPV1 in pulmonary hypertension: Angel or demon?

“…The content of CaN and NFAT in PAH PASMCs is highly increased compared with that in the control group, and the inhibitor of CaN/NFAT can attenuate PASMCs proliferation [31]. Therefore, the Ca 2 + /CaN/NFAT pathway can become a central pathway for physical and chemical factors leading to PAH and is an important downstream signaling pathway of SOCE, which is the main candidate for PAH PASMCs contraction, proliferation, and migration in PAH by increasing [Ca 2+ ] i [31][32][33]. TRPV1, as a calcium influx channel, has also been reported to be followed by NFAT nuclear translocation, which may constitute the upstream pathway of diseases [23,34].…”

“…The NFAT family is composed of NFATc1 -NFATc4, which share the property of Ca 2 + /CaN -dependent nuclear translocation, and a fifth member, which is Ca 2+ -independent and distinctly different from the other four members [27]. The content of CaN and NFAT in PAH PASMCs is highly increased compared with that in the control group, and the inhibitor of CaN/NFAT can attenuate PASMCs proliferation [31]. Therefore, the Ca 2 + /CaN/NFAT pathway can become a central pathway for physical and chemical factors leading to PAH and is an important downstream signaling pathway of SOCE, which is the main candidate for PAH PASMCs contraction, proliferation, and migration in PAH by increasing [Ca 2+ ] i [31][32][33].…”

“…NFAT, which were originally identified in T lymphocytes, are also expressed in PASMCs. When dephosphorylated by calcineurin (CaN), a Ca 2 + /CaM-dependent phosphatase, NFAT translocate from the cytoplasm into the nucleus to regulate gene transcription by associating with transcription coactivators and binding cognate DNA motifs at enhancer sites [27,30,31]. The NFAT family is composed of NFATc1 -NFATc4, which share the property of Ca 2 + /CaN -dependent nuclear translocation, and a fifth member, which is Ca 2+ -independent and distinctly different from the other four members [27].…”