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Serum withdrawal up-regulates human SIRT1 gene expression in a p53-dependent manner.

Shang L, Zhou H, Xia Y, Wang H, Gao G, Chen B, Liu Q, Shao C, Gong Y - J. Cell. Mol. Med. (2009)

Bottom Line: In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress.There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY.We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Experimental Teratology of the Ministry of Education and Institute of Medical Genetics, Shandong University School of Medicine, Jinan, Shandong, China.

ABSTRACT
SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent histone/protein deacetylase, has been extensively studied recently for its critical role in the regulation of physiology, calorie restriction and aging. Studies on laboratory mice showed that expression of SIRT1 can be induced by starvation in a p53-dependent manner and requires the p53-binding sites present in the Sirt1 promoter. However, it remains to be determined whether these findings based on rodents apply to human beings. In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress. The p53-binding site in the promoter of human SIRT1 is more deviant from the consensus sequence than the corresponding sequence in the mouse Sirt1. There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY. To test whether such sequence change would affect its binding with p53 and the SIRT1 expression under stress, we studied various human cell lines with different p53 status and cells with ectopic expression of functionally distinct p53. We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation. Thus, the mechanism responsible for the regulation of SIRT1 expression by p53 is conserved between mice and human beings.

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Induction of SIRT1 expression by serum starvation in human cell lines is dependent on p53. The cells were grown under normal nutrients (C) or under serum starvation (S) for 16 hrs. Real-time PCR analysis and Western blot were performed as described in the Material and Methods. Significant induction of SIRT1 expression was observed in p53-expressing cells (HEK293, HeLa, U2OS and HepG2), but not p53- cells (Saos-2 and Hep3B). GAPDH RNA and β-actin protein were used as normalizing control for RNA and protein quantification assay, respectively. (A) and (B), mRNA levels quantified with real-time PCR. (C) Protein levels determined by Western blot.
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fig01: Induction of SIRT1 expression by serum starvation in human cell lines is dependent on p53. The cells were grown under normal nutrients (C) or under serum starvation (S) for 16 hrs. Real-time PCR analysis and Western blot were performed as described in the Material and Methods. Significant induction of SIRT1 expression was observed in p53-expressing cells (HEK293, HeLa, U2OS and HepG2), but not p53- cells (Saos-2 and Hep3B). GAPDH RNA and β-actin protein were used as normalizing control for RNA and protein quantification assay, respectively. (A) and (B), mRNA levels quantified with real-time PCR. (C) Protein levels determined by Western blot.

Mentions: A previous study showed that expression of the mouse Sirt1 could be induced by acute nutrient withdrawal, and the induction was mediated via two p53-binding sites present in the Sirt1 promoter in a p53-dependent manner [19]. We therefore reasoned that SIRT1 induction by serum deprivation in human cell lines may similarly be regulated in a p53-dependent manner. To determine whether p53 affected the SIRT1 induction in human cells subjected to serum withdrawal, we examined the SIRT1 levels in six cell lines of different p53 status, four cell lines with wild-type p53 (HEK293, HeLa, HepG2, and U2OS) and two p53- cell lines (Saos-2 and Hep3B), under normal growth condition or under serum starvation. The mRNA and protein levels of SIRT1 were measured by real-time PCR and Western blot, respectively. As shown in Fig. 1A–C, serum withdrawal resulted in a significant induction of SIRT1 at both mRNA and protein levels in the p53-functional, but not p53-, cells, suggesting that SIRT1 induction by serum withdrawal could be p53 dependent. In consistence with a requirement of p53 function for SIRT1 induction, the p53 level was concomitantly increased 16 hrs after serum withdrawal (Fig. 1C).


Serum withdrawal up-regulates human SIRT1 gene expression in a p53-dependent manner.

Shang L, Zhou H, Xia Y, Wang H, Gao G, Chen B, Liu Q, Shao C, Gong Y - J. Cell. Mol. Med. (2009)

Induction of SIRT1 expression by serum starvation in human cell lines is dependent on p53. The cells were grown under normal nutrients (C) or under serum starvation (S) for 16 hrs. Real-time PCR analysis and Western blot were performed as described in the Material and Methods. Significant induction of SIRT1 expression was observed in p53-expressing cells (HEK293, HeLa, U2OS and HepG2), but not p53- cells (Saos-2 and Hep3B). GAPDH RNA and β-actin protein were used as normalizing control for RNA and protein quantification assay, respectively. (A) and (B), mRNA levels quantified with real-time PCR. (C) Protein levels determined by Western blot.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496124&req=5

fig01: Induction of SIRT1 expression by serum starvation in human cell lines is dependent on p53. The cells were grown under normal nutrients (C) or under serum starvation (S) for 16 hrs. Real-time PCR analysis and Western blot were performed as described in the Material and Methods. Significant induction of SIRT1 expression was observed in p53-expressing cells (HEK293, HeLa, U2OS and HepG2), but not p53- cells (Saos-2 and Hep3B). GAPDH RNA and β-actin protein were used as normalizing control for RNA and protein quantification assay, respectively. (A) and (B), mRNA levels quantified with real-time PCR. (C) Protein levels determined by Western blot.
Mentions: A previous study showed that expression of the mouse Sirt1 could be induced by acute nutrient withdrawal, and the induction was mediated via two p53-binding sites present in the Sirt1 promoter in a p53-dependent manner [19]. We therefore reasoned that SIRT1 induction by serum deprivation in human cell lines may similarly be regulated in a p53-dependent manner. To determine whether p53 affected the SIRT1 induction in human cells subjected to serum withdrawal, we examined the SIRT1 levels in six cell lines of different p53 status, four cell lines with wild-type p53 (HEK293, HeLa, HepG2, and U2OS) and two p53- cell lines (Saos-2 and Hep3B), under normal growth condition or under serum starvation. The mRNA and protein levels of SIRT1 were measured by real-time PCR and Western blot, respectively. As shown in Fig. 1A–C, serum withdrawal resulted in a significant induction of SIRT1 at both mRNA and protein levels in the p53-functional, but not p53-, cells, suggesting that SIRT1 induction by serum withdrawal could be p53 dependent. In consistence with a requirement of p53 function for SIRT1 induction, the p53 level was concomitantly increased 16 hrs after serum withdrawal (Fig. 1C).

Bottom Line: In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress.There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY.We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Experimental Teratology of the Ministry of Education and Institute of Medical Genetics, Shandong University School of Medicine, Jinan, Shandong, China.

ABSTRACT
SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent histone/protein deacetylase, has been extensively studied recently for its critical role in the regulation of physiology, calorie restriction and aging. Studies on laboratory mice showed that expression of SIRT1 can be induced by starvation in a p53-dependent manner and requires the p53-binding sites present in the Sirt1 promoter. However, it remains to be determined whether these findings based on rodents apply to human beings. In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress. The p53-binding site in the promoter of human SIRT1 is more deviant from the consensus sequence than the corresponding sequence in the mouse Sirt1. There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY. To test whether such sequence change would affect its binding with p53 and the SIRT1 expression under stress, we studied various human cell lines with different p53 status and cells with ectopic expression of functionally distinct p53. We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53-binding element in SIRT1 is required for the up-regulation. Thus, the mechanism responsible for the regulation of SIRT1 expression by p53 is conserved between mice and human beings.

Show MeSH
Related in: MedlinePlus