Objective-Insulin promotes differentiation of preadipocytes into adipocytes. Insulin also stimulates reactive oxygen species (ROS) production, and the NADPH oxidases Nox1 and Nox4 are important sources of ROS. We determined in human and mouse preadipocytes whether Nox proteins contribute to ROS formation and differentiation in response to insulin. Methods and Results-The expression of Nox1 and Nox4 was increased during insulin-induced differentiation, and insulin increased ROS production. SiRNA against Nox4 but not Nox1 inhibited insulin-induced differentiation and ROS production but promoted proliferation. Nox4 overexpression yielded the opposite effect. As observed by siRNA and overexpression, Nox4 controlled the expression of MAP kinase phosphatase-1 (MKP-1), which reduces insulin-induced ERK1/2 activation. Consequently, downregulation of Nox4 promoted ERK1/2 signaling: Proliferation was increased and through phosphorylation of the inhibitory site serine612, ERK1/2 inhibited the activation of the insulin-receptor substrate-1 (IRS-1) and thereby prevented differentiation in response to insulin. Inhibition of ERK1/2 or overexpression of MPK-1 promoted insulin-induced differentiation. Accordingly, insulin-induced proliferation was enhanced by siRNA against MKP-1, whereas inhibition of ERK1/2 or overexpression of MKP-1 attenuated proliferation. Key Words: oxidative stress Ⅲ superoxide Ⅲ NADPH oxidase Ⅲ differentiation I nsulin is a major adipogen. It stimulates the uptake of glucose, which is then converted into triglycerides and stored in fat droplets leading to adipocyte maturation; insulin also promotes the differentiation of preadipocytes in the stroma of the adipose tissue into adipocytes. 1 Although paracrine autacoids as well as transcription factors and signaling cascades have been identified to be involved in adipocyte differentiation and insulin signaling, these complex processes are still incompletely understood. 2 Insulin is also known to acutely increase the reactive oxygen species (ROS) production in adipocytes, 3 but the enzymes responsible for ROS formation in these cells have not been extensively characterized. Moreover, it is unknown whether ROS are required for insulin-induced differentiation. Conclusions-Nox4Besides mitochondria, the Nox family of NADPH oxidases is considered the most important source of ROS in the body. 4 It is accepted that Nox1-and Nox2-dependent ROS formation requires activation of the proteins by cytosolic activators, whereas Nox4 is constitutively active and independent of activator proteins. 4,5 It is therefore assumed that Nox1 and -2 mediate short-term effects, whereas Nox4 is responsible for long-lasting events such as controlling cell cycle progression and proliferation. 6 We hypothesize that insulin elicits a prolonged increase in ROS formation, which is mediated by an increase in the expression of constitutively active Nox4. Moreover, we investigated whether or not this induction of Nox4 is required for long-term processes in response to insulin, such as prolifer...

Objectives-Basic fibroblast growth factor (bFGF) stimulates vascular smooth muscle cell (SMC) migration. We determined whether bFGF increases SMC reactive oxygen-species (ROS) and studied the role of ROS for SMC migration. Methods and Results-bFGF rapidly increased rat SMC ROS formation and migration through pathways sensitive to inhibition of NADPH oxidases, PI3-kinase, protein kinase C, and Rac-1. SiRNA directed against the NADPH oxidase Nox4 impaired basal but not bFGF-induced ROS formation and did not affect migration. In contrast, siRNA against Nox1 blocked the agonist-induced ROS generation as well as the bFGF-induced migration. Agonist-induced migration was also attenuated in SMC derived from Nox1 y/Ϫ mice and transduction of Nox1 restored normal migration. Likewise, SMC outgrowth in response to bFGF was attenuated in aortic segments from Nox1 y/Ϫ mice as compared with Nox1 y/ϩ mice. bFGF activated JNK but not Src in a Nox1-dependent manner. Consequently, phosphorylation of the adaptor protein paxillin, which is central for migration and secretion of matrix-metalloproteinases, were dependent on Nox1 as well as JNK but not Src. Conclusions-These

Nox NADPH oxidases differ in their mode of activation, subcellular localization, and physiological function. Nox1 releases superoxide anions (O(2)(-)) and depends on cytosolic activator proteins, whereas Nox4 extracellularly releases hydrogen peroxide (H(2)O(2)), and its activity does not require cotransfection of additional proteins. We constructed chimeric proteins consisting of Nox1 and Nox4 expressed in HEK293 cells. When the cytosolic tail of Nox4 was fused with the transmembrane part of Nox1, Nox1 became constitutively active. The reciprocal construct was inactive, suggesting that cytosolic subunit-dependent activation requires elements in the transmembrane loops. By TIRF-microscopy, Nox1 was observed in the plasma membrane, whereas Nox4 colocalized with proteins of the endoplasmic reticulum. Fusion proteins of Myc and Nox revealed that the N-terminal part of Nox1 but not Nox4 is cleaved. When the potential signal peptide of Nox4 was inserted into Nox1, plasma-membrane localization was lost, and the protein was retained in vesicle-like structures below the plasma membrane. The potential signal peptide of Nox1 failed to translocate Nox4 to the plasma membrane but switched the extracellularly detectable ROS from H(2)O(2) to O(2)(-). Thus, the very N-terminal part of Nox proteins determines subcellular localization and the ROS type released, whereas the cytosolic tail regulates activity.