Sildenafil inhibits calcineurin/NFATc2-mediated cyclin A expression in pulmonary artery smooth muscle cells
Introduction
Pulmonary hypertension, especially secondary to sustained hypoxia caused by chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis, is a common clinical syndrome that is characterized by increased pulmonary artery resistance, elevated pulmonary artery pressure, and right ventricular hypertrophy, frequently leading to right heart failure and death. The pathological mechanisms underlying this condition consist of persistent pulmonary vasoconstriction, chronic vascular remodeling, and thrombosis (Mam et al., 2009, Oka et al., 2007, Rosenblum, 2010). Among these pathogeneses, vascular remodeling is critical; this remodeling is characterized by vascular wall changes, including intima thickening and media hyperplasia, due to the proliferation/migration of pulmonary artery smooth muscle cells (PASMCs) (Firth et al., 2010, Hanze et al., 2007, Stenmark et al., 2006). Thus, identifying and then inhibiting the molecular mechanisms driving PASMC proliferation is a putative strategy for treating pulmonary hypertension.
Cyclin-dependent kinases (CDKs) play a critical role in mammalian cell cycle progression and cell proliferation (Andres, 2004, Gerard and Goldbeter, 2009, Malumbres and Barbacid, 2009). These CDKs are constitutively expressed in cells but are inactive in the absence of particular partner cyclins. Cyclins are synthesized in the hemodynamics and degraded at specific stages of the cell cycle to precisely control CDK activity and cell cycle progression. Therefore, regulation of transcription and/or translation of cyclins and subsequent CDK activity is a key step in modulating cell proliferation. Protein synthesis in vivo is driven by many transcription factors that are often the downstream targets of a variety of signal transduction pathways that tightly modulate their activity (Chen et al., 2008, Karin and Smeal, 1992). Recent studies have shown that the calcineurin/NFAT signaling pathway is associated with the development of pulmonary hypertension through induction of pulmonary vascular remodeling (de Frutos et al., 2007); however, the detailed molecular mechanisms underlying this process are not completely understood. A study by Wang et al. found that hypoxia induces PASMC proliferation by activating the calcineurin/NFAT signaling pathway, but they did not examine which isoform of NFAT is involved or how activation of this pathway mediates these cells' proliferation (Wang et al., 2009). It has been further reported that the activation of the calcineurin/NFAT signaling cascade upregulates cyclin A expression in aortic smooth muscle cells, leading to cellular proliferation and contributing to the development of restenosis after percutaneous transluminal coronary angioplasty (Karpurapu et al., 2008). Therefore, it would be interesting to know whether the calcineurin/NFAT signaling pathway, and which isoform of NFAT in particular, regulates cyclin A expression in PASMCs and causes pulmonary vascular remodeling and the development of pulmonary hypertension.
Sildenafil is a selective phosphodiesterase 5 (PDE5) inhibitor and is widely used in the clinical treatment of erectile dysfunction (Ghofrani et al., 2005). Sildenafil suppresses the development of pulmonary hypertension in both clinic patients and in animal models by augmenting the cyclic GMP-protein kinase G (cGMP-PKG) signaling cascade. However, it is unknown whether PDE5 inhibition and enhanced cGMP-PKG signaling modulate activation of calcineurin/NFAT and, thus, cyclin A levels in PASMCs to contribute to the overall effects of sildenafil on pulmonary hypertension. To test this putative modulatory role of PDE5 inhibition and cGMP-PKG activation and to examine the mechanisms of PASMC proliferation, primary cultured PASMCs were stimulated with serotonin, an in vivo stimulator of PASMC proliferation, and activation of the calcineurin/NFAT signaling pathway, cyclin A expression and CDK2 activation were examined. Then, we further investigated whether sildenafil inhibits these responses of cells to serotonin and the mechanisms through which such inhibition might occur.
Section snippets
Cell preparation and culture
Primary smooth muscle cells from pulmonary artery were prepared from Sprague–Dawley rats (125–250 g), as described before (Li et al., 2009). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin in a 37 °C, 5% CO2, humidified incubator. Cells were passaged by trypsinization using 0.05% trypsin/EDTA (Invitrogen). All experiments were performed using cells between passages 4 and 8. To test the
Effects of serotonin on cyclin A expression and CDK2 activity in PASMCs
To determine whether serotonin induces cell cycle protein expression and subsequent DNA synthesis in PASMCs, cells were treated with different concentrations of serotonin for 24 h, and cyclin A expression was examined using immunoblotting. As shown in Fig. 1A, serotonin dose-dependently increased cyclin A expression in PASMCs: 1 μM serotonin caused a 3.1-fold increase in the cyclin A protein level (P < 0.01 versus control). We next determined whether CDK2 activity was also elevated in parallel with
Discussion
Calcium signaling plays a critical role in the development of pulmonary hypertension by stimulating smooth muscle cell contraction and proliferation (Shimoda et al., 2006, Wang et al., 2008). The mechanisms underlying calcium induction of smooth muscle cell contraction have been well elucidated (Shimoda et al., 2000). Yet, how calcium signaling triggers smooth muscle cell proliferation – particularly which downstream signaling effectors transduce calcium signals into cell cycle progression, DNA
Conclusion
In the present study, we provide direct evidence that activation of the calcineurin/NFATc2 signaling pathway upregulates cyclin A expression and therefore increases CDK2 activity and DNA synthesis in primary cultured PASMCs. Inhibition of calcineurin by CsA or knockdown of NFATc2 effectively suppressed such responses of cells to serotonin. More importantly, inhibition of PDE5 by sildenafil also abolished serotonin-triggered responses, while the presence of a PKG inhibitor reversed the effects
Conflict of interest
The authors have declared that no conflict of interest exists.
Acknowledgments
This work was supported by the Chinese National Science Foundation (81070045) and the start-up package to Manxiang Li from the Second Affiliated Hospital of Medical College of Xi'an Jiaotong University, P.R. China.
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