NADPH oxidase 4 mediates insulin-stimulated HIF-1α and VEGF expression, and angiogenesis in vitro

Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-β and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis in vitro induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment

NADPH Oxidase 4 Mediates Insulin-Stimulated HIF-1α and VEGF Expression, and Angiogenesis <em>In Vitro</em>

<div><p>Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-β and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis <em>in vitro</em> induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment.</p> </div

Nox4 mediates insulin-induced VEGF transcriptional activation through HIF-1.

<p>(A) HMVECs were co-transfected with a VEGF reporter containing a 2.65-kb human VEGF promoter fragment or pGL2-basic luciferase reporter and β-gal plasmid. In 24 hours the cells were growth-arrested, followed by stimulation with insulin (100 nM) for 24 hours in the presence or absence of DPI (5 μM). (B) VEGF reporter (2.65-kb) or (C) VEGF reporter containing the HIF-1 binding site (46-bp) and β-gal plasmids were cotransfected with the control siRNA or Nox4 siRNA. In 24 hours, the cells were growth-arrested and then treated with insulin (100 nM) for 24 hours. (D) VEGF reporter (2.65-kb) and β-gal plasmids were cotransfected with Nox4 or pcDNA3.1 plasmid. In 24 hours, the cells were growth-arrested and then treated with insulin (100 nM) for 24 hours. Relative luciferase activity was determined by the ratio of luciferase/β-galactosidase activity, and expressed as the ratio of VEGF/pGL2-basic. **<i>P</i><0.01 vs. the control siRNA group; ##<i>P</i><0.01 vs. the insulin+control siRNA group.</p

Nox4 mediates insulin-induced VEGF mRNA and protein expression.

<p>HMVECs were transfected with Nox4 siRNA or the control siRNA for 24 h. The cells were growth arrested and then treated with 100 nM insulin. (A) In 12 hours, VEGF 165 mRNA level was analyzed by real time-PCR. (B) In 24 hours, the VEGF protein levels in the supernatants were determined by ELISA assay. *<i>P</i><0.05, **<i>P</i><0.01 vs. the control siRNA group; #<i>P</i><0.05, ##<i>P</i><0.01 vs. the insulin+control siRNA group.</p

Nox4 is involved in insulin-stimulated insulin receptor and IRS tyrosine phosphorylation, ERK1/2, and AKT activation.

<p>HMVECs were treated with Nox4 siRNA or scrambled control siRNA for 24 hours, and then incubated with serum-free medium for 16 hours. (A) The tyrosine phosphorylated and total insulin receptor, (B) The tyrosine phosphorylated, the serine phosphorylated, and total IRS-1 protein levels were measured after stimulation with 100 nM insulin for 5 minutes. (C) Phosphorylated and total ERK1/2, (D) phosphorylated and total AKT, and (E) SHIP-1, MKP-1, Nox4, and β-actin protein expressions were determined after stimulation with 100 nM insulin for 30 minutes. Densitometry values (bottom) were normalized as the indicated. *<i>P</i><0.05, **<i>P</i><0.01 vs. the control siRNA group; #<i>P</i><0.05, ##<i>P</i><0.01 vs. the insulin+control siRNA group.</p

Nox4 mediates insulin-induced HMVEC migration, proliferation, and angiogenesis <i>in vitro</i>.

<p>HMVECs were transfected with the control or Nox4 siRNA for 24 hours. (A) The cells were growth arrested followed by stimulation with insulin (100 nM) for 10 hours. Cell migration was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048393#s2" target="_blank">Methods</a>. The data are expressed as the fold change relative to the control. (B) Growth-arrested cells were treated with insulin (100 nM) for 24 hours. Cell proliferation was measured using CCK-8 assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048393#s2" target="_blank">Methods</a>. (C) and (D) HMVECs transfected with the control or Nox4 siRNA were seeded onto Matrigel and incubated with or without insulin (100 nM), treated or not with H₂O₂ (0.1 µM) for 12 hours. The relative tube lengths were normalized by the negative control. Bar = 1000 µm. **<i>P</i><0.01 vs. the control siRNA group; #<i>P</i><0.05, ##<i>P</i><0.01 vs. the insulin+control siRNA group.</p

Nox4 mediates insulin-induced HIF-1α mRNA and protein expression.

<p>HMVECs were transfected with Nox4 siRNA or the control siRNA for 24 hours. The cells were growth arrested and then treated with 100 nM insulin for 12 hours. (A) HIF-1α mRNA levels were analyzed by real time-PCR. (B) HIF-1α, PHD2, Nox4, and β-actin protein levels were determined by immunoblotting (*<i>P</i><0.05, **<i>P</i><0.01 vs. the control siRNA group; #<i>P</i><0.05, ##<i>P</i><0.01 vs. the insulin+control siRNA group). (C) Cycloheximide (CHX) was added to a final concentration of 50 µg/ml, and the cells were harvested after being incubated for the indicated time in the presence of CHX and insulin. HIF-1α, Nox4, and β-actin protein levels were determined by immunoblotting (*<i>P</i><0.05, **<i>P</i><0.01 vs. zero-time control in insulin+con siRNA group; #<i>P</i><0.05, ##<i>P</i><0.01 vs. zero-time control in insulin+Nox4 siRNA group).</p

Genetic Variants in Caveolin-1 and RhoA/ROCK1 Are Associated with Clear Cell Renal Cell Carcinoma Risk in a Chinese Population.

The RhoA/ROCK pathway and Caveolin-1 (Cav-1) participate in the process of tumorigenesis in numerous types of cancer. Up-regulation of RhoA/ROCK and Cav-1 expression is considered to be associated with the development and progression of clear cell renal cell carcinoma (ccRCC). We investigated the association between genetic variations of RhoA/ROCK and Cav-1 and the risk of ccRCC in the Chinese population.Between May 2004 and March 2014, a total of 1,248 clear cell renal cell carcinoma cases and 1,440 cancer-free controls were enrolled in this hospital-based case-control study. Nine SNPs in RhoA/ROCK and Cav-1 were genotyped using the TaqMan assay.We found two SNPs (Cav-1 rs1049334 and ROCK1 rs35996865) were significantly associated with the increasing risk of ccRCC (P = 0.002 and P < 0.001 respectively). The analysis of combined risk alleles revealed that patients with 2-4 risk alleles showed a more remarkable growth of ccRCC risk than the patients with 0-1 risk alleles(OR = 1.66, 95%CI = 1.31-2.11, P < 0.001). Younger subjects (P = 0.001, OR = 1.83, 95%CI = 1.30-2.57), higher weight subjects (P = 0.001, OR = 1.76, 95%CI = 1.25-2.47), female subjects (P = 0.007, OR = 1.75, 95% CI = 1.17-2.62), nonsmokers (P < 0.001, OR = 1.67, 95%CI = 1.26-2.23), drinkers (P = 0.025, OR = 1.75, 95% CI = 1.07-2.85), subjects with hypertension (P = 0.025, OR = 1.75, 95% CI = 1.07-2.85) and diabetes (P = 0.026, OR = 4.31, 95% CI = 1.19-15.62) showed a stronger association between the combined risk alleles and the risk of ccRCC by using the stratification analysis. Furthermore, we observed higher Cav-1 mRNA levels in the presence of the rs1049334 A allele in normal renal tissues.Our results indicate that the two SNPs (Cav-1 rs1049334 and ROCK1 rs35996865) and genotypes with a combination of 2-4 risk alleles were associated with the risk of ccRCC. The functional SNP rs1049334 may affect the risk of ccRCC by altering the expression of Cav-1 and the relevance between the risk effects and the functional impact of this polymorphism needs further validation