Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells.
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.


CONCLUSIONS
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.
Wang
Cong
C
Beijing Institute of Respiratory Medicine, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, PR China.
Li
Ji-Feng
JF
Zhao
Lan
L
Liu
Jie
J
Wan
Jun
J
Wang
Yue Xiu
YX
Wang
Jun
J
Wang
Chen
C
D002118:Calcium / Q000037:antagonists & inhibitors*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006801:Humans; D006976:Hypertension, Pulmonary / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D000860:Hypoxia / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D010879:Piperazines / Q000494:pharmacology* / Q000627:therapeutic use; D011651:Pulmonary Artery / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011687:Purines / Q000494:pharmacology / Q000627:therapeutic use; D015398:Signal Transduction / Q000187:drug effects*; D000068677:Sildenafil Citrate; D013450:Sulfones / Q000494:pharmacology* / Q000627:therapeutic use; D050052:TRPC Cation Channels / Q000378:metabolism
D015220:Calcium Channels; D050778:NFATC Transcription Factors; C494008:NFATC3 protein, human; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D000068677:Sildenafil Citrate; D002118:Calcium