抑制 SOC/Ca2+/NFAT 通路参与西地那非对肺动脉平滑肌细胞的抗增殖作用。
Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells.
机构信息
Beijing Institute of Respiratory Medicine, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, PR China.
出版信息
Respir Res. 2009 Dec 11;10(1):123. doi: 10.1186/1465-9921-10-123.
BACKGROUND
Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca2+] ([Ca2+]i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca2+]i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca2+ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca2+ influx and increase in [Ca2+]i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca2+]i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca2+/NFAT pathway.
METHODS
Human PASMC were cultured under hypoxia (3% O2) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca2+]i was measured with a dynamic digital Ca2+ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.
RESULTS
Hypoxia induced PASMC proliferation with increases in basal [Ca2+]i and Ca2+ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca2+]i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.
CONCLUSION
The SOC/Ca2+/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.
背景
西地那非是一种有效的磷酸二酯酶 5（PDE5）抑制剂，已被提议用于治疗肺动脉高压（PAH）。其对肺动脉平滑肌细胞（PASMC）的抗增殖作用机制尚不清楚。核因子活化 T 细胞的核转位（NFAT）被认为参与了 PASMC 的增殖和 PAH。细胞浆游离钙浓度（[Ca2+]i）的增加是 NFAT 核转位的前提。已经证明，PAH 患者的 PASMC 中钙库操纵钙通道（SOC）的上调导致 [Ca2+]i 的增加，SOC 是由瞬时受体电位（TRP）通道蛋白编码的。因此，我们研究了以下内容：1）TRPC1 通道表达的上调是否诱导 SOC 介导的 Ca2+内流增强和 [Ca2+]i 的增加，从而参与低氧诱导的 PASMC 增殖；2）低氧诱导的 [Ca2+]i 增加是否导致 NFAT 的核转位，并调节 PASMC 增殖和 TRPC1 表达；3）西地那非的抗增殖作用是否通过抑制该 SOC/Ca2+/NFAT 途径来介导。
方法
在 3% O2 的低氧条件下，用人 PASMC 培养 72 小时，并用或不用西地那非处理。用血细胞计数器和 MTT 测定分别测定细胞数和细胞活力。通过用 fura 2-AM 负载 PASMC，用动态数字钙成像系统测量[Ca2+]i。通过 RT-PCR 和 Western blot 分别检测 TRPC1 mRNA 和蛋白水平。通过免疫荧光显微镜检测 NFAT 的核转位。
结果
低氧诱导 PASMC 增殖，基础 [Ca2+]i 和 SOC（SOCE）介导的 Ca2+内流增加。同时，PASMC 中的 TRPC1 基因和蛋白表达上调。低氧诱导 NFAT 核转位明显增强，这依赖于 SOCE，并对 SOC 抑制剂 SKF96365（SKF）和 cGMP 类似物 8-溴-cGMP 敏感。SKF 和 NFAT 阻滞剂（VIVIT 和环孢菌素 A）抑制低氧诱导的 PASMC 增殖和 TRPC1 上调。西地那非治疗可改善低氧诱导的 PASMC 增殖，并减轻低氧诱导的基础 [Ca2+]i、SOCE、TRPC1 表达上调和 NFAT 核转位增强。
结论
SOC/Ca2+/NFAT 通路至少部分是西地那非抗增殖作用的下游介质，可能具有治疗 PAH 的治疗潜力。
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