Protein Disulfide Isomerase increases Nox1 activation through EGFR signaling in hypertension

Abstract

We have previously shown that expression of protein disulfide isomerase (PDI) and Nox1 in resistance arteries correlates with the development of hypertension. Epidermal growth factor (EGF)-like ligands are increased in vascular disease and their levels correlate with higher levels of vascular Nox1. EGFR can be activated through metalloproteinase (MMP) mediated cleavage and release of EGF-ligands. Here, we sought to investigate the molecular mechanisms through which PDI regulates Nox1 expression in hypertension. Pro-and mature heparin bound (HB)-EGF ligands were increased in the aorta of 8-week-old spontaneously hypertensive rats (SHR) as compared to control (Wistar) rats. In contrast, the mature form of the ligand was lower in aortae, and increased in the plasma, of 12-week-old SHR. We next assessed if shedding of EGF ligands was responsible for the increase in Nox1 expression. Overexpression of PDI increased the expression of Nox1 in rat VSMCs and this effect was inhibited by the EGFR inhibitor AG1478, the MMP inhibitor TAP and the flavoenzyme inhibitor diphenylene iodonium (DPI). In contrast, overexpression of a PDI mutated in its 4 redox active cysteines (PDI mut) failed to increase Nox1 expression. We confirmed these observations following expression of a Nox1-promoter luciferase plasmid in mouse VSMCs. HB-EGF stimulated cells transfected with WT PDI showed an increase in Nox1 promoter activity as compared to cells transfected with PDI mut. Finally, cells deficient in Nox1 did not respond to HB-EGF when transfected with PDI WT, suggesting that Nox1 activation and PDI redox cysteines are essential for inducing the activation of EGFR. Altogether, our findings indicate that PDI increases the redox-mediated expression of Nox1 via EGFR signaling in hypertension.