Chronic hypoxia (CH) activates the Ca(2+)-dependent transcription factor nuclear factor of activated T cells isoform c3 (NFATc3) in mouse pulmonary arteries. However, the mechanism of this response has not been explored. Since we have demonstrated that NFATc3 is required for CH-induced pulmonary arterial remodeling, establishing how CH activates NFATc3 is physiologically significant. The goal of this study was to test the hypothesis that endothelin-1 (ET-1) contributes to CH-induced NFATc3 activation. We propose that this mechanism requires increased pulmonary arterial smooth muscle cell (PASMC) intracellular Ca(2+) concentration ([Ca(2+)](i)) and stimulation of RhoA/Rho kinase (ROK), leading to calcineurin activation and actin cytoskeleton polymerization, respectively. We found that: 1) CH increases pulmonary arterial pre-pro-ET-1 mRNA expression and lung RhoA activity; 2) inhibition of ET receptors, calcineurin, L-type Ca(2+) channels, and ROK blunts CH-induced NFATc3 activation in isolated intrapulmonary arteries from NFAT-luciferase reporter mice; and 3) both ET-1-induced NFATc3 activation in isolated mouse pulmonary arteries ex vivo and ET-1-induced NFATc3-green fluorescence protein nuclear import in human PASMC depend on ROK and actin polymerization. This study suggests that CH increases ET-1 expression, thereby elevating PASMC [Ca(2+)](i) and RhoA/ROK activity. As previously demonstrated, elevated [Ca(2+)](i) is required to activate calcineurin, which dephosphorylates NFATc3, allowing its nuclear import. Here, we demonstrate that ROK increases actin polymerization, thus providing structural support for NFATc3 nuclear transport.

Endothelin‐1 (ET‐1)‐induced activation of Nuclear Factor of Activated T‐cells isoform c3 (NFATc3) is necessary for the progression of chronic hypoxia (CH)‐induced pulmonary hypertension. Reactive oxygen species (ROS) levels are elevated in CH. ET‐1 increases ROS in pulmonary artery smooth muscle cells (PASMC). ROS participates in NFAT activation in cancer cells. However, it is unknown whether ROS mediates ET‐1‐induced NFATc3 activation in PASMC. Using human PASMC electroporated with a NFATc3‐GFP plasmid, we tested the hypothesis that increased ROS participates in ET‐1‐induced NFATc3 activation. Cells were pretreated with vehicle, a superoxide dismutase mimetic (tempol), a superoxide dismutase inhibitor (diethyldithiocarbamate, DDC) or H2O2. Nuclear fluorescence was measured before and during ET‐1 treatment. To prevent NFATc3 nuclear export, the CRM1 exportin inhibitor leptomycin B was present in all experiments.Our results show that pretreatment with both tempol and H2O2 inhibited ET‐1‐induced NFATc3 nuclear import. On the other hand, DDC enhanced ET‐1‐induced NFATc3 nuclear import.To determine the intracellular source of ROS, cells were pretreated with a xanthine oxidase inhibitor, allopurinol (Allo), or a NADPH oxidase inhibitor, apocynin (Apo). Allo but not Apo inhibited ET‐1‐induced NFATc3 nuclear import.Our results suggest that in human PASMC, ET‐1‐induced NFATc3 nuclear import is dependent on xanthine oxidase‐derived superoxide and/or a reduction in H2O2 levels. Therefore, it is possible that elevated superoxide levels in CH contribute to NFATc3 activation.Funding source: R01 HL088151