Hypoxia increases AP-1 binding activity by enhancing capacitative Ca2+ entry in human pulmonary artery endothelial cells.
Activating protein (AP)-1 transcription factors modulate expression of genes involved in cell proliferation and migration. Chronic hypoxia increases pulmonary artery smooth muscle cell proliferation by upregulating AP-1-responsive genes encoding for endothelium-derived vasoactive and mitogenic factors implicated in pulmonary hypertension development. The expression of AP-1 transcription factors is sensitive to changes in cytosolic free [Ca2+] ([Ca2+]cyt). Capacitative Ca2+ entry (CCE) via store-operated Ca2+ channels (SOC) is an important mechanism for raising [Ca2+]cyt in pulmonary artery endothelial cells (PAEC). Using combined molecular biological, fluorescence microscopy, and biophysical approaches, we examined the effect of chronic hypoxia (3% O2, 72 h) on AP-1 DNA binding activity, CCE, and transient receptor potential (TRP) gene expression in human (h) PAEC. EMSA showed that AP-1 binding to hPAEC nuclear protein extracts was significantly enhanced by hypoxia, the increase being dependent on store-operated Ca2+ influx and sensitive to La3+, an SOC inhibitor. Hypoxia also increased basal [Ca2+]cyt, the amount of CCE produced by store depletion with cyclopiazonic acid, and the amplitude of SOC-mediated currents (ISOC). The increases of CCE amplitude and ISOC current density by hypoxia were paralleled by enhanced TRPC4 mRNA and protein expression. Hypoxia-enhanced CCE and TRPC4 expression were also attenuated by La3+. These data suggest that hypoxia increases AP-1 binding activity by enhancing Ca2+ influx via La3+-sensitive TRP-encoded SOC channels in hPAEC. The Ca2+-mediated increase in AP-1 binding may play an important role in upregulating AP-1-responsive gene expression, in stimulating pulmonary vascular cell proliferation and, ultimately, in pulmonary vascular remodeling in patients with hypoxia-mediated pulmonary hypertension.
Fantozzi
Ivana
I
Division of Pulmonary and Critical Care Medicine, Department of Medicine, School of Medicine, University of California, San Diego, CA 92103-8382, USA.
Zhang
Shen
S
Platoshyn
Oleksandr
O
Remillard
Carmelle V
CV
Cowling
Randy T
RT
Yuan
Jason X-J
JX
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002908:Chronic Disease; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020868:Gene Silencing; D006801:Humans; D006976:Hypertension, Pulmonary / Q000378:metabolism; D000860:Hypoxia / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D015220:Calcium Channels; D004791:Enzyme Inhibitors; D007211:Indoles; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; D018808:Transcription Factor AP-1; D002118:Calcium; C000543:cyclopiazonic acid