JAK/STAT Signaling Pathway Regulates Nox1 and Nox4-Based NADPH Oxidase in Human Aortic Smooth Muscle Cells

Abstract

Objective-Oxidative stress mediated by Nox1-and Nox4-based NADPH oxidase (Nox) plays a key role in vascular diseases. The molecular mechanisms involved in the regulation of Nox are not entirely elucidated. Because JAK/STAT regulates many genes linked to inflammation, cell proliferation, and differentiation, we questioned whether this pathway is involved in the regulation of Nox1 and Nox4 in human aortic smooth muscle cells (SMCs). Methods and Results-Cultured SMCs were exposed to interferon ␥ (IFN␥) for 24 hours. Using lucigenin-enhanced chemiluminescence and dihydroethidium assays, real-time polymerase chain reaction, and Western blot analysis, we found that JAK/STAT inhibitors significantly diminished the IFN␥-dependent upregulation of Nox activity, Nox1 and Nox4 expression. In silico analysis revealed the presence of highly conserved GAS elements within human Nox1, Nox4, p22phox, p47phox, and p67phox promoters. Transient overexpression of STAT1/STAT3 augmented the promoter activities of each subunit. JAK/STAT blockade reduced the Nox subunits transcription. Chromatin immunoprecipitation demonstrated the physical interaction of STAT1/STAT3 proteins with the predicted GAS elements from Nox1 and Nox4 promoters. Key Words: NADPH oxidase Ⅲ JAK/STAT Ⅲ oxidative stress Ⅲ atherosclerosis E merging clinical and experimental evidence demonstrate the role of oxidative stress in the development of cardiovascular disorders. Reactive oxygen species (ROS) are implicated in different cell processes associated to vascular plaque formation such as growth, proliferation, differentiation, and apoptosis of smooth muscle cells (SMCs). 1,2 Deciphering the molecular mechanisms underlying the regulation of ROS production may lead to new therapeutic approaches. Conclusions-JAK/STATNADPH oxidases (Nox) are regulated by a plethora of stimuli and represent a major source of ROS in the vasculature. Depending on the cell type, the vascular Nox comprises 3 distinct catalytic subunits (Nox1, Nox2, Nox4) and 6 cytosolic regulatory components (p47phox, p67phox, NoxO1, NoxA1, Rac1/2). The p22phox component is essential for Nox activity forming a bound complex with 4 SMCs express predominantly Nox1 and Nox4 isoforms, which are differentially distributed in the cellular compartments and direct several redox-dependent processes. 5 It was postulated that Nox1 associates with and promotes SMCs proliferation, whereas Nox4 is required for the maintenance of differentiated phenotype. 6 Changes in gene expression of the Nox isoforms are critical for its function. In previous studies we showed that the expression of p22phox subunit and the ensuing superoxide production is regulated by NF-kB and AP-1 in human aortic SMCs. 7,8 However, the transcriptional regulatory mechanisms of oxidase components are not entirely elucidated.Activation of Janus tyrosine kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is an essential pathogenic mechanism leading to SMC hypertrophy and hyperplasia. It was shown that STAT1 and STAT...

Myocardin-related Transcription Factor Regulates Nox4 Protein Expression

“…Several transcription factors and the corresponding cis-elements have been implicated in transcriptional regulation of Nox4, implying the multifactorial nature of this process. These include Smad3 (39,45), which through AP1/Smad-binding elements (39) couples Nox4 to TGF␤ signaling and thereby fibrogenesis; Hif1␣ (58), Nrf2 (59) and Oct-1 (60), which link Nox4 expression to ROS generation per se under conditions of hypoxia, hyperoxia, and shear stress; NFB (61) and STAT proteins (62), mediators of inflammatory gene expression; and E2F (63), a regulator of proliferation. This study enriches this picture by two important findings indicating that MRTF and TAZ/YAP, two main cytoskeleton-regulated transcrip-FIGURE 7.…”

STAT-Dependent Upregulation of 12/15-Lipoxygenase Contributes to Neuronal Injury after Stroke

“…The increased 12/15-LOX mRNA levels point to a dominant effect of transcriptional events, however. (2) Both STAT1 and STAT6 will likely regulate additional genes in the ischemic brain, including the NADPH oxidases NOX1 and NOX4, 38 and the cyclooxygenase COX-2. 39 Furthermore, AKT phosphorylation was increased, and caspase-3 cleavage decreased in STAT1 knockouts subjected to focal ischemia, 14 and the impact of 12/15-LOX on these events should be explored.…”

Reactive Oxygen Species, Vascular Noxs, and Hypertension: Focus on Translational and Clinical Research

“…Nox4 appears to generate H 2 O 2 , although the primary product is probably O 2 -, which is rapidly dismutated to H 2 O 2 (202,232). Nox4 contributes to basal ROS production through its constitutive activity and to increased ROS generation when induced by Ang II, glucose, tumor necrosis factor a, and growth factors (90,127). Recent studies have identified a 28-kDa Splice Variant of Nox4 located in the nucleus of vascular cells, which may be important in pathophysiologic effects through modulation of nuclear signaling and DNA damage (9).…”