Rho Kinase‐mediated Regulation of Actin Cytoskeleton Dynamics Participates in NFATc3 Nuclear Transport

Abstract

Chronic hypoxia activates the Nuclear Factor of Activated T-cells isoform c3 (NFATc3) via endothelin-1 (ET-1) and NFATc3 is required for chronic hypoxia-induced pulmonary hypertension. ET-1 elevates Ca2+activating calcineurin which dephosphorylates NFATc3 allowing its nuclear import. RhoA/Rho kinase (ROK) has also been implicated in NFAT activation but the mechanism is unknown. We hypothesized that ET-1 via ROK increases actin cytoskeleton polymerization to mediate NFATc3 nuclear transport. We found that ROK blockade (fasudil) and destabilization of actin cytoskeleton (cytochalasin B) inhibited ET-1-induced increases in NFAT transcriptional activity in isolated pulmonary arteries from NFAT-luciferase reporter mice. The same treatments as well as stabilization of actin cytoskeleton (jasplakinolide) also inhibited ET-1-induced NFATc3-GFP nuclear import in pulmonary arterial smooth muscle cells. None of the drugs affected the increase in cytosolic Ca2+ caused by ET-1 as detected by fura 2. ET-1 also increased filamentous actin content and its co-localization with NFATc3. Both responses were inhibited by fasudil and cytochalasin B. Our results suggest NFATc3 nuclear transport depends on Ca2+ and ROK-mediated actin cytoskeleton polymerization. Funding source: R01 HL088151.