Nox1/Ref-1-mediated activation of CREB promotes Gremlin1-driven endothelial cell proliferation and migration

Pulmonary arterial hypertension (PAH) is a complex degenerative disorder marked by aberrant vascular remodeling associated with hyperproliferation and migration of endothelial cells (ECs). Previous reports implicated bone morphogenetic protein antagonist Gremlin 1 in this process; however, little is known of the molecular mechanisms involved. The current study was designed to test whether redox signaling initiated by NADPH oxidase 1 (Nox1) could promote transcription factor CREB activation by redox factor 1 (Ref-1), transactivation of Gremlin1 transcription, EC migration, and proliferation. Human pulmonary arterial EC (HPAECs) exposed in vitro to hypoxia to recapitulate PAH signaling displayed induced Nox1 expression, reactive oxygen species (ROS) production, PKA activity, CREB phosphorylation, and CREB:CRE motif binding. These responses were abrogated by selective Nox1 inhibitor NoxA1ds and/or siRNA Nox1. Nox1-activated CREB migrated to the nucleus and bound to Ref-1 leading to CREB:CRE binding and Gremlin1 transcription. CHiP assay and CREB gene-silencing illustrated that CREB is pivotal for hypoxia-induced Gremlin1, which, in turn, stimulates EC proliferation and migration. In vivo, participation of Nox1, CREB, and Gremlin1, as well as CREB:CRE binding was corroborated in a rat PAH model. Activation of a previously unidentified Nox1-PKA-CREB/Ref-1 signaling pathway in pulmonary endothelial cells leads to Gremlin1 transactivation, proliferation and migration. These findings reveal a new signaling pathway by which Nox1 via induction of CREB and Gremlin1 signaling contributes to vascular remodeling and provide preclinical indication of its significance in PAH.