Abstract
The up-regulation of transient receptor potential channel 6 (TRPC6) has been found to contribute to the proliferation of pulmonary artery smooth muscle cells (PASMCs), and inhibition of phosphodiesterase-5 (PDE5) has been shown to suppress TRPC6 expression in PASMCs. However, the molecular mechanisms underlying the up-regulation of TRPC6 expression and PDE5 modulation of TRPC6 expression in PASMCs remain largely unclear. The aim of this study is to address these issues. Endothelin-1 (ET-1) dose and time-dependently up-regulated TRPC6 expression in primary cultured rat PASMCs, and this was accompanied with the activation of calcineurin and subsequent translocation of NFATc4 to the nucleus. Further study indicated that inhibition of calcineurin by cyclosporine A or knockdown of NFATc4 using small interfering RNA suppressed ET-1-induced TRPC6 up-regulation. In addition, luciferase reporter assay showed that NFATc4 directly regulated the expression of TRPC6 in PASMCs. Inhibition of PDE5 by sildenafil suppressed ET-1-induced activation of calcineurin/NFATc4 signaling pathway and consequent TRPC6 up-regulation in PASMCs, while these inhibitory effects of sildenafil were abolished by PKG inhibitor Rp-8Br-cGMPs. Taken together, our study indicates that ET-1 stimulates TRPC6 expression by activation of calcineurin/NFATc4 signaling pathway, and inhibition of PDE5 suppresses calcineurin/NFATc4- mediated TRPC6 expression in PASMCs in a cGMP-PKG-dependent manner.
Highlights
Pulmonary hypertension (PH) is a life-threatening disease of various origins characterized by severe remodeling of the pulmonary vascular that leads to increased pulmonary vascular resistance and pulmonary arterial pressure, resulting in right heart failure and premature death[1, 2]
To explore the mechanisms of TRPC6 up-regulation induced by ET-1 in pulmonary artery smooth muscle cells (PASMCs), and to examine the effect of PDE5 inhibition on ET-1-induced TRPC6 expression and its potential mechanisms, primary cultured PASMCs were stimulated with ET-1, TRPC6 expression and activation of the calcineurin/nuclear factors of activated T cells (NFAT) signaling pathway were determined, the effects of sildenafil on these changes and the underlying molecular mechanisms were further investigated
The results showed that Rp-8Br-cyclic guanosine monophosphate (cGMP) abolished the inhibitory effects of sildenafil on calcineurin/NFATc4 activation and TRPC6 up-regulation induced by ET-1 in PASMCs (Fig. 5a–f), the calcineurin activity increased to 2.06-fold over control, the phosphorylation level of NFATc4 declined to 0.43-fold over control, fluorescence-labeled NFATc4 translocated to cell nucleus and the NFATc4 protein level in cell nucleus raised to 1.78-fold over control, TRPC6 mRNA and protein levels increased to 2.99-fold and 1.71-fold over control again, respectively
Summary
Pulmonary hypertension (PH) is a life-threatening disease of various origins characterized by severe remodeling of the pulmonary vascular that leads to increased pulmonary vascular resistance and pulmonary arterial pressure, resulting in right heart failure and premature death[1, 2]. Increased proliferation of PASMCs is the most prominent feature of pulmonary vascular remodeling[4]. TRPC6 expression has been found to be up-regulated in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH)[11]. The mitogenic effect of ET-1 on PASMCs has been confirmed to play a prominent role in pulmonary vascular remodeling in PH17. It has been found that sildenafil can inhibit TRPC6 expression in PASMCs, thereby contributing to the therapeutic effects on chronically hypoxic pulmonary hypertension (CHPH) in rats[23, 24]. Whether inhibition of PDE5 by sildenafil modulates the expression of TRPC6 induced by ET-1 in PASMCs and what are the possible mechanisms for that are still unknown. To explore the mechanisms of TRPC6 up-regulation induced by ET-1 in PASMCs, and to examine the effect of PDE5 inhibition on ET-1-induced TRPC6 expression and its potential mechanisms, primary cultured PASMCs were stimulated with ET-1, TRPC6 expression and activation of the calcineurin/NFAT signaling pathway were determined, the effects of sildenafil on these changes and the underlying molecular mechanisms were further investigated
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