Cross Talk Between p22phox and ATF4 in the Endothelial Unfolded Protein Response.

BACKGROUND: Cardiovascular diseases have been associated with stress in the endoplasmic reticulum (ER) and accumulation of unfolded proteins leading to the unfolded protein response (UPR). Reactive oxygen species (ROS) such as superoxide and H2O2 derived from NADPH oxidases have been implicated in the pathogenesis of cardiovascular diseases. ROS have also been associated with ER stress. The role NADPH oxidases in the UPR is, however, not completely resolved yet. AIM: In this study, we investigated the role of p22phox, an essential component of most NADPH oxidases, in the UPR of endothelial cells. RESULTS: Induction of ER stress increased p22phox expression at the transcriptional level. p22phox was identified as novel target of the UPR transcription factor ATF4 (activator of transcription factor 4) under ER stress conditions by promoter analyses and ChIP. Depletion of ATF4 and p22phox diminished the levels of superoxide and H2O2 under ER stress conditions. On the contrary, p22phox was instrumental in increasing eIF2α phosphorylation and subsequent ATF4 expression on induction of ER stress by chemicals, oxysterols, or severe hypoxia in vitro and in vivo, leading to increased expression of CHOP and activation of effector caspases. INNOVATION: p22phox is a novel target of ATF4 in response to ER stress, which can promote the PERK-ATF4 branch of the UPR in vitro and in vivo. CONCLUSION: p22phox-dependent NADPH oxidases are important mediators of ER stress driving the UPR.

The ß3-integrin binding protein ß3-endonexin is a novel negative regulator of hypoxia-inducible factor-1.

AIMS: Integrins are multifunctional heterodimeric adhesion receptors that mediate the attachment between a cell and the extracellular matrix or other surrounding cells. In endothelial cells, integrins can modulate cell migration and motility. In particular, ß3-integrin is expressed in angiogenic vessels. Signal transduction by ß3-integrins requires the recruitment of intracellular signaling molecules. ß3-endonexin is a highly spliced molecule that has been identified as a ß3-integrin binding protein. ß3-endonexin isoforms are expressed in endothelial cells and have been suggested to act as shuttle proteins between the membrane and the nucleus. However, their functional role in angiogenesis is unclear. In this study, we investigated whether ß3-endonexin isoforms are involved in endothelial angiogenic processes under hypoxia. RESULTS: The overexpression of ß3-endonexin isoforms decreased endothelial proliferation and tube formation under hypoxia, while the depletion of ß3-endonexin by RNAi promoted angiogenic responses in vitro and in vivo. In hypoxia, ß3-endonexin accumulated in the nucleus, and prevention of this response by depletion of ß3-endonexin increased hypoxic activation and induction of the hypoxia-inducible factor (HIF)-1 and its target genes VEGF and PAI-1. ß3-endonexin diminished nuclear factor kappa B (NFκB) activation and decreased NFκB binding to the HIF-1α promoter under hypoxia, subsequently diminishing NFκB-dependent transcription of HIF-1α under hypoxia. INNOVATION: Our results indicate for the first time that the overexpression of ß3-endonexin can decrease hypoxic induction and activation of HIF-1α and can prevent hypoxic endothelial proliferation and angiogenic responses. CONCLUSION: ß3-endonexin can act as a novel anti-angiogenic factor specifically in the response to hypoxia due to its negative impact on the activation of HIF-1.