Molecular regulation of Nox1 NADPH oxidase in vascular smooth muscle cell activation Jennifer Lee Streeter , University of Iowa Follow

[摘要] Nox1 is of considerable importance because of its involvement in a wide variety of pathologies. Activation of Nox1 induces generation of reactive oxygen species (ROS) and cell migration, events critical for the pathogenesis of cardiovascular disease, amyotropic lateral sclerosis, gastrointestinal disease, immunological disorders, and multiple forms of cancer [1-8]. In order to best determine how to treat Nox1-mediated disease, we must gain a better understanding of the mechanisms that control Nox1 activation. Within the last decade, many studies have found that protein phosphorylation and protein trafficking are critical regulatory mechanisms that control the activation of multiple Nox proteins. Yet, to date, no studies have characterized Nox1 phosphorylation or trafficking. We hypothesized that the activity of Nox1 is controlled by its phosphorylation at specific residues and by its sub-cellular localization; and that modifying Nox1 phosphorylation or localization will alter Nox1-dependent signaling. To test this hypothesis, we utilized both in vivo and in vitro approaches. We found that phosphorylation of Nox1 is significantly increased under pathological conditions in three in vivo models: (1) in atherosclerotic vs. normal aorta from monkey, (2) in neointimal vascular smooth muscle cells (VSMCs) vs. medial VSMCs from rat following aortic balloon injury, and (3) in ligated vs. normal carotid from mouse. Studies using mass spectroscopy, pharmacological inhibition, siRNA, and in vitro phosphorylation identify PKC-βI as a kinase that mediates Nox1 phosphorylation and subsequent ROS production and VSMC migration. Site-directed mutagenesis of predicted Nox1 phospho-residues revealed that cells expressing mutant Nox1 T429A have a significant decrease in TNF-α-stimulated ROS production, VSMC migration and Nox1 NADPH oxidase complex assembly compared to cells expressing wild-type Nox1. Isothermal calorimetry (ITC) revealed that a peptide containing the Activation Domain of NoxA1 (LEPMDFLGKAKVV) binds to phosphorylated Nox1 peptide (KLK-phos-T(429)- QKIYF) but not non-phosphorylated Nox1 peptide. These findings indicate that phosphorylation of Nox1 residue T429 by PKC-βI promotes TNF-α-induced Nox1 NADPH oxidase complex assembly, ROS production, and VSMC migration. Nox1 localization and trafficking studies reveal that Nox1 endocytosis is necessary for TNF-α-induced Nox1 ROS production; and that mutation of a Nox1 VLV motif inhibits Nox1 endocytosis and ROS production. These studies have provided new evidence that phosphorylation and sub-cellular localization are involved in the regulation of Nox1 ROS production and cell migration and offer new insights as to how Nox1 activity can be targeted for the purpose of treating Nox1-mediated diseases.