Upregulation of Nox4 by TGF{beta}1 oxidizes SERCA and inhibits NO in arterial smooth muscle of the prediabetic Zucker rat

Abstract

Rationale: Vascular smooth muscle cell (SMC) migration is an important pathological process in several vascular occlusive diseases, including atherosclerosis and restenosis, both of which are accelerated by diabetes mellitus.

Objective: To determine the mechanisms of abnormal vascular SMC migration in type 2 diabetes, the obese Zucker rat (ZO), a model of obesity and insulin resistance, was studied.

Methods and results: In culture, ZO aortic SMCs showed a significant increase in Nox4 mRNA and protein levels compared with the control lean Zucker rat (ZL). The sarco-/endoplasmic reticulum Ca(2+) ATPase (SERCA) nitrotyrosine-294,295 and cysteine-674 (C674)-SO(3)H were increased in ZO SMCs, indicating oxidant stress. Unlike ZL SMC, nitric oxide (NO) failed to inhibit serum-induced SMC migration in ZO. Transfection of Nox4 small interference RNA or overexpression of SERCA2b wild type, but not C674S mutant SERCA, restored the response to NO. Knockdown of Nox4 also decreased SERCA oxidation in ZO SMCs. In addition, transforming growth factor-β1 via Smad2 was necessary and sufficient to upregulate Nox4, oxidize SERCA, and block the antimigratory action of NO in ZO SMCs. Corresponding to the results in cultured SMCs, immunohistochemistry confirmed that Nox4 and SERCA C674-SO(3)H were significantly increased in ZO aorta. After common carotid artery injury, knockdown of Nox4 by adenoviral Nox4 short hairpin RNA decreased Nox4 and SERCA C674-SO(3)H staining and significantly decreased injury-induced neointima.

Conclusion: These studies indicate that the upregulation of Nox4 by transforming growth factor-β1 in ZO SMCs is responsible for the impaired response to NO by a mechanism involving the oxidation of SERCA C674. Knockdown of Nox4 inhibits oxidation of SERCA, as well as neointima formation, after ZO common carotid artery injury.

Figure 1

Migration of obese Zucker rat (ZO) smooth muscle cells (SMCs) is not inhibited by nitric oxide (NO), but overexpression of SERCA 2b wild type (WT) restores NO function. A. The NO donor, DETA NONOate, failed to inhibit serum-induced SMC migration in ZO. *P<0.05 compared with serum. N=6, Student t-test. B. Overexpression of WT but not C674S mutant SERCA 2b restored the inhibition of migration by NO in ZO SMCs. *P<0.05 compared with serum alone, N=10, one-way ANOVA. ZL, lean Zucker rat.

Figure 2

Increased oxidant production and SERCA oxidation in ZO SMCs. A. Increased reactive oxygen species production measured by Amplex Red in ZO SMCs compared with ZL SMCs. *P<0.05 compared with ZL SMCs. N=3, Student t-test. B. ZO SMCs have significantly increased SERCA C674SO₃H (N=4) and SERCA nY (N=7) staining compared with ZL SMCs. The upper panel shows a representative western blot. The lower panel shows a summary of band density. *P<0.05 compared with ZL SMCs, Student t-test.

Figure 3

The upregulation of Nox4 NADPH oxidase in ZO SMCs. A. Nox4 mRNA level in ZL and ZO SMCs. *P<0.05 compared with the ZL SMCs, N=4, Student t-test. B. Nox4 protein levels in ZL and ZO SMCs. The upper panel shows a representative western blot. The bar graph in the lower panel shows a summary of band density. *P<0.05 compared with the ZL SMCs, N=3, Student t-test. C. Knockdown of Nox4 by Nox4 siRNA, but not by control siRNA or Nox1 siRNA decreased ROS production in ZO SMCs. *P<0.05 compared with control siRNA, N=3, one-way ANOVA.

Figure 4

Knockdown of Nox4 decreased SERCA oxidation in ZO SMCs and restores the inhibition of cell migration by NO. A. The left panel shows a representative western blot. The right panel shows a summary of band density. *P<0.05 compared with control siRNA. N=3 of Nox4, N=5 of SERCA C674SO₃H/total SERCA, N=6 of 294, 295-nY SERCA/total SERCA, Student t-test. B. Knockdown of Nox4 by Nox4 siRNA but not by control siRNA or Nox1 siRNA restores the inhibition of ZO SMC migration by NO donor, DETA NONOate. *P<0.05 compared with control siRNA, N=4, two-way ANOVA.

Figure 5

The upregulation of Nox4 by TGF-β1 is responsible for the abnormal response to NO in ZO SMCs. A. The expression levels of both TGF-β1(12.5 kDa) and its precursor (45 kDa) are significantly increased in ZO SMCs. The left panel shows a representative western blot. The right panel shows a summary of band density. *P<0.05 compared with the ZL SMCs, N=4, Student t-test. B. Application of TGF-β1 in ZL SMCs increased Nox4 protein level. The left panel shows a representative western blot. The right panel shows a summary of band density. *P<0.05 compared with IgG control, N=3, paired t-test. C. Application of TGF-β1 blocked the inhibition of ZL SMC migration by DETA NONOate. P<0.05 compared with serum alone, N=6, Student t-test. D. Knockdown of Nox4 counteracted the effect of TGF-β1 on NO-induced inhibition of ZL SMC migration. *P<0.05 compared with serum alone, N=6, Student t-test. E. Blockade of TGF-β1 by anti-TGF-β1 antibody restored the inhibition of ZO SMC migration by DETA NONOate. *P<0.05 compared with serum alone, N=6, Student t-test.

Figure 6

Blockade of Smad2 phosphorylation restores the inhibition of cell migration by DETA NONOate and decreases Nox4 and SERCA oxidation. A. The phospho-Smad 2 (Ser245/250/255, 60 kDa) is significantly increased in ZO SMCs compared with ZL SMCs. The upper panel shows a representative western blot. The lower panel shows a summary of band density. *P<0.05 compared with the ZL SMCs, N=4, Student t-test. B. Application of Smad2 phosphorylation inhibitor SB203580 in ZO SMCs decreases both the endogenous and TGF-β1-induced Smad2 phosphorylation. *P<0.05 compared with DMSO, N=3. C. Application of SB203580 in ZO SMCs restores the inhibition of cell migration by DETA NONOate. *P<0.05 compared with serum alone, N=3 in DMSO group and N=6 in SB group, Student t-test. D. Application of SB203580 in ZO SMCs decreases Nox4 and SERCA oxidation. Blot of SMC lysates pooled after the migration assay shown in Figure 6C.

Figure 7

Knockdown of Nox4 by adenovirus Nox4 shRNA inhibits neointima formation after common carotid artery balloon catheter injury. A. Immunohistochemistry of injured and sham common carotid artery. Hematoxylin/Eosin (HE) staining (100×) and others (400×). B. Infection of adenoviral Nox4 shRNA decreases the intima to media ratio. *P<0.05 compared with GFP group, N=5, Student t-test. C. Infection of adenoviral Nox4 shRNA decreases the Nox4 and SERCA C674-SO3H staining. *P<0.05 compared with GFP, N=5, Student t-test.

Figure 8

The proposed mechanisms involved in the abnormal response to NO in ZO SMCs by oxidation of SERCA. Increased TGF-β1 in ZO SMCs activates the phosphorylation of Smad2 which upregulates Nox4-based NADPH oxidase and causes SERCA oxidation. The oxidation of SERCA, especially of the most reactive cysteine 674, inhibits the NO-induced stimulation of SERCA activity and blocks NO-induced inhibition of SMC migration. Blockade of TGF-β1 or Smad2, or knockdown of Nox4, or overexpression of SERCA WT can maintain the ability of NO to inhibit SMC migration.