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Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells.

Zhang X, Liu L, Chen C, Chi YL, Yang XQ, Xu Y, Li XT, Guo SL, Xiong SH, Shen MR, Sun Y, Zhang CS, Hu KM - Cardiovasc Diabetol (2013)

Bottom Line: The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions.We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions.These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biomedical Engineering, Second Military Medical University, Shanghai, China. zhangxicyl1126@163.com.

ABSTRACT

Background: The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined.

Methods: The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H₂O₂, high glucose/U0126 or normal glucose/H₂O₂/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions.

Results: We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H₂O₂ stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor abolished the proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression under high glucose or normal glucose/H₂O₂ conditions.

Conclusions: These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions. The Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression are associated with ROS/Erk1/2 signaling pathway under high glucose conditions.

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Representative negative or positive staining of phospho-Erk1/2 in VSMCs. High glucose = cells incubated with high glucose for 5 days; normal glucose = cells incubated with normal glucose for 5 days; High glucose/NAC = cells incubated with high glucose and NAC for 5 days; normal glucose/H2O2 = cells incubated with normal glucose and H2O2 for 5 days. Positive staining of phospho-Erk1/2 shows red and transfected VSMCs display green (GFP).
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Figure 7: Representative negative or positive staining of phospho-Erk1/2 in VSMCs. High glucose = cells incubated with high glucose for 5 days; normal glucose = cells incubated with normal glucose for 5 days; High glucose/NAC = cells incubated with high glucose and NAC for 5 days; normal glucose/H2O2 = cells incubated with normal glucose and H2O2 for 5 days. Positive staining of phospho-Erk1/2 shows red and transfected VSMCs display green (GFP).

Mentions: The phosphorylation of ERK1/2 (phospho-Erk1/2) was determined by immunofluorescence staining. The percentage of phospho-Erk1/2 positive cells was low about 2-3% under normal glucose or high glucose/NAC conditions, while the percentage was great about 26-27% under high glucose or normal glucose/H2O2 conditions. Furthermore, VSMCs transfected with pGC-FU-Irf-1 displayed a significantly greater percentage of phospho-Erk1/2 positive cells, while cells transfected with pGCsi-FU-Irf-1 exhibited a lower percentage of phospho-Erk1/2 positive cells compared to untransfected cells under high glucose or normal glucose/H2O2 conditions. In contrast, transfected VSMCs treated with high glucose/NAC or normal glucose showed no significant difference in the percentage of phospho-Erk1/2 positive cells compared to the control cells (Figure 7) (Table 3). These results indicate that overexpression of Irf-1 promoted the activation of Erk1/2 and silencing of Irf-1 caused the opposite effect under high glucose or normal glucose/H2O2 conditions.


Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells.

Zhang X, Liu L, Chen C, Chi YL, Yang XQ, Xu Y, Li XT, Guo SL, Xiong SH, Shen MR, Sun Y, Zhang CS, Hu KM - Cardiovasc Diabetol (2013)

Representative negative or positive staining of phospho-Erk1/2 in VSMCs. High glucose = cells incubated with high glucose for 5 days; normal glucose = cells incubated with normal glucose for 5 days; High glucose/NAC = cells incubated with high glucose and NAC for 5 days; normal glucose/H2O2 = cells incubated with normal glucose and H2O2 for 5 days. Positive staining of phospho-Erk1/2 shows red and transfected VSMCs display green (GFP).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3852693&req=5

Figure 7: Representative negative or positive staining of phospho-Erk1/2 in VSMCs. High glucose = cells incubated with high glucose for 5 days; normal glucose = cells incubated with normal glucose for 5 days; High glucose/NAC = cells incubated with high glucose and NAC for 5 days; normal glucose/H2O2 = cells incubated with normal glucose and H2O2 for 5 days. Positive staining of phospho-Erk1/2 shows red and transfected VSMCs display green (GFP).
Mentions: The phosphorylation of ERK1/2 (phospho-Erk1/2) was determined by immunofluorescence staining. The percentage of phospho-Erk1/2 positive cells was low about 2-3% under normal glucose or high glucose/NAC conditions, while the percentage was great about 26-27% under high glucose or normal glucose/H2O2 conditions. Furthermore, VSMCs transfected with pGC-FU-Irf-1 displayed a significantly greater percentage of phospho-Erk1/2 positive cells, while cells transfected with pGCsi-FU-Irf-1 exhibited a lower percentage of phospho-Erk1/2 positive cells compared to untransfected cells under high glucose or normal glucose/H2O2 conditions. In contrast, transfected VSMCs treated with high glucose/NAC or normal glucose showed no significant difference in the percentage of phospho-Erk1/2 positive cells compared to the control cells (Figure 7) (Table 3). These results indicate that overexpression of Irf-1 promoted the activation of Erk1/2 and silencing of Irf-1 caused the opposite effect under high glucose or normal glucose/H2O2 conditions.

Bottom Line: The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions.We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions.These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biomedical Engineering, Second Military Medical University, Shanghai, China. zhangxicyl1126@163.com.

ABSTRACT

Background: The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined.

Methods: The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H₂O₂, high glucose/U0126 or normal glucose/H₂O₂/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions.

Results: We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H₂O₂ stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor abolished the proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression under high glucose or normal glucose/H₂O₂ conditions.

Conclusions: These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions. The Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression are associated with ROS/Erk1/2 signaling pathway under high glucose conditions.

Show MeSH
Related in: MedlinePlus