Objectives—The NADPH oxidase isoforms Nox2 and Nox4 are coexpressed in many cell types and are implicated in agonist-stimulated redox-sensitive signal transduction. We compared the involvement of Nox2 versus Nox4 in redox-sensitive protein kinase activation after agonist stimulation.

Methods and Results—We transfected HEK293 cells with Nox2 or Nox4 and compared ROS production and activation of mitogen activated protein kinases (MAPKs), Akt, and GSK3 after acute agonist stimulation. Nox4 overexpression substantially increased basal ROS generation whereas ROS generation in response to angiotensin II and tumor necrosis factor (TNF) was enhanced in Nox2-overexpressing cells. Nox4 overexpression induced basal activation of ERK1/2 and JNK whereas Nox2-transfected cells showed a modest increase in p38MAPK activation. After angiotensin II or TNF treatment, JNK activation was augmented in Nox2 but not Nox4-transfected cells, whereas insulin augmented phosphorylation of p38MAPK, Akt, and GSK3 specifically in Nox4-overexpressing cells and JNK specifically in Nox2-overexpressing cells.

Conclusions—These data indicate that Nox2 and Nox4 exhibit distinctive patterns of acute activation by angiotensin II, TNF, and insulin and regulate the activation of distinct protein kinases.