Activation of the EGFR/p38/JNK pathway by mitochondrial-derived hydrogen peroxide contributes to oxygen-induced contraction of ductus arteriosus.

Abstract

Oxygen-induced contraction of the ductus arteriosus (DA) involves a mitochondrial oxygen sensor, which signals pO2 in the DA smooth muscle cell (DASMC) by increasing production of diffusible hydrogen peroxide (H2O2). H2O2 stimulates vasoconstriction by regulating ion channels and Rho kinase, leading to calcium influx and calcium sensitization. Because epidermal growth factor receptor (EGFR) signaling is also redox regulated and participates in oxygen sensing and vasoconstriction in other systems, we explored the role of the EGFR and its signaling cascade (p38 and c-Jun N-amino-terminal kinase (JNK)) in DA contraction. Experiments were performed in DA rings isolated from full-term New Zealand white rabbits and human DASMC. In human DASMCs, increasing pO2 from hypoxia to normoxia (40 to 100mmHg) significantly increased cytosolic calcium, p < 0.01. This normoxic rise in intracellular calcium was mimicked by EGF and inhibited by EGFR siRNA. In DA rings, EGF caused contraction while the specific EGFR inhibitor (AG1478) and the tyrosine kinase inhibitors (genistein or tyrphostin A23) selectively attenuated oxygen-induced contraction (p < 0.01). Conversely, orthovanadate, a tyrosine phosphatase inhibitor known to activate EGFR signaling, caused dose-dependent contraction of hypoxic DA and superimposed increases in oxygen caused minimal additional contraction. Anisomycin, an activator of EGFR's downstream kinases, p38 and JNK, caused DA contraction; conversely, oxygen-induced DA contraction was blocked by inhibitors of p38 mitogen-activated protein kinases (MAPK) (SB203580) or JNK (JNK inhibitor II). O2-induced phosphorylation of EGFR occurred within 5min of increasing pO2 and was inhibited by mitochondrial-targeted overexpression of catalase. AG1478 prevented the oxygen-induced p38 and JNK phosphorylation. In conclusion, O2-induced EGFR transactivation initiates p38/JNK-mediated increases in cytosolic calcium and contributes to DA contraction. The EGFR/p38/JNK pathway is regulated by mitochondrial redox signaling and is a promising therapeutic target for modulation of the patent ductus arteriosus. Oxygen activates epidermal growth factor receptor (EGFR) in ductus arteriosus (DA) smooth muscle cells. EGFR inhibition selectively attenuates O2-induced DA constriction. pO2-induced EGFR activation is mediated by mitochondrial-derived hydrogen peroxide. p38 MAPK and JNK mediated EGFR's effects on oxygen-induced DA contraction. Tyrosine kinases and phosphatases participate in oxygen sensing in the DA. The EGFR pathway offers new therapeutic targets to modulate patency of the ductus arteriosus.