Genomic location and promoter region of rat sirt1 (rSIRT1). A, the rat sirt1 gene (XM_001080493) was mapped on chromosome 20p11 with 11 exons and spanned 61,147 base pairs according to the computer analysis results using the Rat Genome Celera Assembly database. B, the 1.5-kb promoter region of the rat sirt1 gene contains a cluster of five putative FoxO1 core binding motifs (known as insulin-responsive sequence-1 (IRS-1)) (blue), insulin-responsive sequence-2 (IRS-2) (yellow), and a forkhead-like binding element (FKHD-L) (red). aa, amino acids.

Identification of essential FoxO1 DNA-binding elements in the rat sirt1 (rSIRT1) promoter. A, HEK293 cells were transiently transfected with the indicated deletion luciferase constructs or pGL3.0 basic control vector together with FoxO1 expression vector, and the internal control p-RL-TK vector for 24 h. B and C, HEK293 cells were transiently transfected with the indicated single and combined mutations or the 1.5-kb reporter (wild type) together with expression vector encoding FoxO1 or control pcDNA3 and the internal control p-RL-TK vector for 24 h. Luciferase activity was determined. Data from 2–4 independent experiments (each performed in triplicate) are shown as mean ± S.E. (error bars). ns, no significant difference; **, p < 0.01 versus control or wild type or as indicated.

Induction of SIRT1 protein expression by FoxO1. A and B, FoxO1 depletion by siRNA decreases SIRT1 expression. VSMCs (A) or HEK293 cells (B) were transfected with 30 nm FoxO1 or FoxO3a siRNA or scrambled siRNA for 48 h. C and D, FoxO1 overexpression increases SIRT1 protein level. C, VSMCs were infected with adeno-FoxO1 or control adeno-GFP for 24 h. D, HEK293 cells in 100 × 20-mm tissue culture dishes were transiently transfected with 10 μg/dish of FoxO1 expression plasmid or control vector pcDNA3 for 24 h. E, SIRT2 and SIRT3 expression were unaffected by FoxO1 overexpression in VSMCs. Protein expression of the samples from two independent experiments was analyzed by Western blotting. Data shown are from one of 3–4 independent experiments.

Schematic model of an autofeedback regulation of SIRT1 expression via FoxO1. FoxO1 binds to the IRS-1 and FKHD-L sites of the SIRT1 promoter and activates SIRT1 transcription. Newly synthesized SIRT1 interacts with and deacetylates FoxO1 and promotes FoxO1-dependent SIRT1 transcription. Resveratrol (RSV) promotes FoxO1-dependent SIRT1 transcription and thus induces SIRT1 expression.

FoxO1 up-regulates sirt1 gene transcription. A, FoxO1 overexpression increases the rat sirt1 promoter transcriptional activity. HEK293 cells were transfected in 24-well cell culture plates with the 1.5-kb rat SIRT1 luciferase reporter, increasing amounts of expression vectors encoding FoxO1 or FoxO3a (10, 50, and 250 ng/well, respectively), and the internal control p-RL-TK vector for 24 h. Total amounts of DNA for each well were normalized by adding empty vector pcDNA3. Cells were harvested, and luciferase activity was determined as described under “Experimental Procedures.” Data from two independent experiments (each performed in triplicate) are shown as mean ± S.E. (error bars) (*, p < 0.05; **, p < 0.01 versus control vector). B, FoxO1 overexpression increases SIRT1 protein expression. HEK293 cells were transiently transfected in 100 × 20-mm tissue culture dishes with increasing amounts of FoxO1 or FoxO3a expression plasmids (1, 3, and 9 μg/dish, respectively) or control vector pcDNA3 for 24 h. Total equal amounts of DNA for each well were normalized by adding empty vector. Protein expression was analyzed by Western blotting using anti-SIRT1, FLAG, HA, and actin antibodies, respectively. One representative result is shown from three independent experiments. C and D, FoxO1 overexpression increases SIRT1 mRNA levels in VSMCs and HEK293 cells. VSMCs were infected with increasing amounts of adeno-FoxO1 or control adeno-GFP for 24 h. HEK293 cells in 100 × 20-mm tissue culture dishes were transiently transfected with increasing amounts of FoxO1 expression plasmid (1, 3, and 9 μg/dish, respectively) or control vector pcDNA3 for 24 h. Total equal amounts of DNA for each well were normalized by adding empty vector. Total RNA was prepared. One-step RT-PCR products were analyzed by electrophoresis on 1.8% agarose gels containing ethidium bromide (C). D, expression of rat SIRT1 and human SIRT1 mRNA levels was quantified by a two-step quantitative real-time RT-PCR using the same RNA samples from C. E and F, FoxO1 depletion by siRNA reduces SIRT1 mRNA levels in VSMCs and HEK293 cells. VSMCs or HEK293 cells were transfected with increasing concentrations of FoxO1 siRNA (10, 25, and 50 nm, respectively) or scrambled siRNA for 48 h. SIRT1 and GAPDH mRNA levels were analyzed by one-step RT-PCR (E) or quantified by a real-time RT-PCR (F). The results for relative expression were normalized by measuring GAPDH mRNA levels in each sample (n = 3). The expression level of SIRT1 mRNA from scrambled siRNA was assigned the value of 100%. Results are expressed as mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01 versus control vector or scrambled siRNA. rSIRT1, rat SIRT1; hSIRT1, human SIRT1.

Binding of FoxO1 to the sirt1 promoter. A and B, FoxO1 binds directly to the IRS-1bcd and FKHD-L elements in the sirt1 promoter in vitro. EMSA was conducted using in vitro translated FoxO1 proteins as described under “Experimental Procedures.” The partial sequences of the biotin-labeled IRS-1bcd (A) and FKHD-L (B) probes (wild type, underlined) and mutated probes (framed) are shown above the gel shift. The running orientation of the gels and the mobility-shifted bands are indicated by arrows. C and D, FoxO1 associates with the rat sirt1 promoter in VSMCs by ChIP assays. Soluble chromatin from VSMCs was coimmunoprecipitated with anti-FoxO1 or FoxO3a antibodies or an equal amount of rabbit IgG. DNA purified from starting (1% input) and immunoprecipitated samples was subjected to PCR. The amplified region surrounds the IRS-1-binding element (C) (−1035 to −686) or the FKHD-L-binding element (D) (−415 to −89) within the rat sirt1 promoter. One representative result from three independent experiments is shown. E, FoxO1 associates with the human SIRT1 promoter in HEK293 cells by ChIP assays. Soluble chromatin from HEK293 cells was coimmunoprecipitated with anti-FoxO1 or FoxO3a antibodies or an equal amount of rabbit IgG. DNA purified from starting (1% input) and immunoprecipitated samples was subjected to PCR. The amplified region surrounds two putative FoxO1 DNA-binding elements (−587 to −278) within the human SIRT1 promoter. One representative result from three independent experiments is shown. rSIRT1, rat sirt1; hSIRT1, human SIRT1.

A positive feedback regulation of SIRT1 expression via FoxO1. A, SIRT1 promotes FoxO1-mediated SIRT1 promoter activity. VSMCs were transfected with the 1.5-kb SIRT1 reporter and p-RL-TK vector for 24 h and then infected with the indicated Ad.GFP-FoxO1 with or without Ad.SIRT1 (WT) or its dominant negative mutant (H363Y) for 24 h before luciferase activity was determined. B, SIRT1 interacts with and deacetylates FoxO1. VSMCs were infected with the adenovirus encoding the indicated Myc-tagged SIRT1 (WT), SIRT1 (H363Y), GFP-tagged FoxO1, or Ad.GFP (control) for 36 h. Total cell lysates were immunoprecipitated (IP) using anti-GFP polyclonal antibody and blotted (IB) with anti-acetylated lysine (top panel) or anti-Myc monoclonal antibody (second panel). Equal amounts of cell lysates (5%) were loaded as input. Total levels of FoxO1 were analyzed by Western blotting using anti-GFP antibody (bottom panel). C, resveratrol (RSV) increases FoxO1-mediated SIRT1 transcription. VSMCs were transfected with the 1.5-kb SIRT1 reporter, FoxO1 expression plasmids, and p-RL-TK vector for 24 h. Cells were treated with either vehicle (DMSO) or increasing doses of resveratrol for 16 h before luciferase activity was determined. *, p < 0.05; **, p < 0.01 versus vehicle. D and E, resveratrol induces endogenous expression of SIRT1 but not SIRT2 and -3. Growth-arrested VSMCs were treated with 10, 30, and 60 μm resveratrol or vehicle (DMSO) for 24 h. Protein expression was analyzed by Western blotting with the indicated antibodies. F, FoxO1 is required for resveratrol-induced SIRT1 expression. VSMCs transfected with 50 nm FoxO1 siRNA or control scrambled siRNA were treated with 50 μm resveratrol or vehicle (DMSO) for 24 h. Protein expression of the samples from two independent experiments was analyzed by Western blotting.