Mechanisms of NFATc3 activation by increased superoxide and reduced hydrogen peroxide in pulmonary arterial smooth muscle

We recently demonstrated increased superoxide (O₂^•−) and decreased H₂O₂levels in pulmonary arteries of chronic hypoxia-exposed wild-type and normoxic superoxide dismutase 1 (SOD1) knockout mice. We also showed that this reciprocal change in O₂^•− and H₂O₂ is associated with elevated activity of nuclear factor of activated T cells isoform c3 (NFATc3) in pulmonary arterial smooth muscle cells (PASMC). This suggests that an imbalance in reactive oxygen species levels is required for NFATc3 activation. However, how such imbalance activates NFATc3 is unknown. This study evaluated the importance of O₂^•− and H₂O₂ in the regulation of NFATc3 activity. We tested the hypothesis that an increase in O₂^•− enhances actin cytoskeleton dynamics and a decrease in H₂O₂ enhances intracellular Ca²⁺ concentration, contributing to NFATc3 nuclear import and activation in PASMC. We demonstrate that, in PASMC, endothelin-1 increases O₂^•− while decreasing H₂O₂ production through the decrease in SOD1 activity without affecting SOD protein levels. We further demonstrate that O₂^•− promotes, while H₂O₂ inhibits, NFATc3 activation in PASMC. Additionally, increased O₂^•−-to-H₂O₂ ratio activates NFATc3, even in the absence of a G_q protein-coupled receptor agonist. Furthermore, O₂^•−-dependent actin polymerization and low intracellular H₂O₂ concentration-dependent increases in intracellular Ca²⁺ concentration contribute to NFATc3 activation. Together, these studies define important and novel regulatory mechanisms of NFATc3 activation in PASMC by reactive oxygen species.