Abstract
Manganese-superoxide dismutase (MnSOD) is one of the major cellular antioxidant defense systems. To study the effect of age on the regulation of MnSOD in the vasculature, we compared MnSOD expression and its transcriptional regulation in explanted vascular smooth muscle cells (VSMC) isolated from old (24 months old) versus young (6 months old) rats and grown in a normal (5 mM) or high (12.5 and 25 mM) glucose or tumor necrosis factor alpha (5 ng/ml) environment to induce oxidative stress. Both MnSOD protein and activity were reduced in VSMC from old compared with young animals. FOXO3a, a member of the family of Forkhead transcription factors, interacted with the promoter of the rat MnSOD gene at a specific binding site. Inhibition of FOXO3a transcription with small interfering RNA led to a reduction in MnSOD gene expression. VSMC from old rats had increased phosphorylated FOXO3a at Ser(253), which paralleled the reduction of MnSOD protein. Treatment of VSMC with 5 nm insulin-like growth factor-1 induced phosphorylation of Akt and FOXO3a over time, repressing FOXO3a DNA binding and consequently MnSOD gene expression. Furthermore, Akt activity was selectively increased in VSMC from the old, supporting the hypothesis that increased age-related Akt activity might be responsible for the phosphorylation and inactivation of FOXO3a, which in turn down-regulates MnSOD transcription.
Highlights
The transcriptional regulation of the Manganese-superoxide dismutase (MnSOD) gene is complex
We hypothesized that MnSOD expression in vascular smooth muscle cells (VSMC) from young and old rats might differ as a consequence of differences in age-related regulation of FOXO3a phosphorylation by Akt during oxidative stress that was simulated by exposure to high glucose (23, 24) or the addition of TNF-␣ (25, 26)
We demonstrate that there is a FOXO3A-binding site, located 1272 bp upstream of the coding region of the rat SOD2 promoter, by the DNA binding assay and the Chromatin Immunoprecipitation (ChIP) approach
Summary
The transcriptional regulation of the MnSOD gene is complex. A series of transcription factors, such as specificity protein-1, activator protein-2, and NF-B, appear to regulate MnSOD gene expression (10 –12). A model depicting the FOXO shuttling system was initially described to explain the mechanism of transcriptional regulation by this family member of transcription factors (19) This shuttling system changes the intracellular localization of FOXO through phosphorylation of three Akt sites in the Forkhead domain, primarily at Ser253. Understanding the role of Akt and FOXO3a in the regulation of MnSOD in VSMC with advancing age or under conditions known to promote atherosclerosis would be important as future targets for attenuating oxidative stress and its consequences. We hypothesized that MnSOD expression in VSMC from young and old rats might differ as a consequence of differences in age-related regulation of FOXO3a phosphorylation by Akt during oxidative stress that was simulated by exposure to high glucose (23, 24) or the addition of TNF-␣ (25, 26)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
