SIRT1 Inhibits p53 but not NF-κB Transcriptional Activity during Differentiation of Mouse Embryonic Stem Cells into Embryoid Bodies

Abstract

Background and Objectives

SIRT1, a histone diacetylase, modify transactivation function of various transcription factor including p53 and NF-κB. p53 and NF-κB is involved in in vitro differentiation of mouse embryonic stem cells (mESC) into mouse embryoid body (mEB). These suggest that SIRT1 might affect in vitro differentiation of mESC into mEB by regulation of p53 and NF-κB.

Methods and Results

In this study we analyzed the effect of SIRT1 in in vitro differentiation of mESC into mEB using wild and SIRT1 knockout mESC. To examine SIRT1-specific gene in mESC, this study conducted microarray-based differential gene expression analysis between wild and SIRT1 knockout mESC. Comparing their gene expression patterns, this study determined a list of genes regulated by SIRT1. cDNA microarray data-set analysis revealed that genes associated with transcription and signal transduction are significantly modified in SIRT1 knockout mESC. cDNA microarray data-set analysis between mESC and EB in wild and SIRT1 showed that SIRT1 inhibits p53 signaling pathway but not affect NF-κB signaling pathway.

Conclusions

This study suggests that SIRT1 modify mESC differentiation by regulation of p53 transcriptional activity.

Introduction

SIRT1 is an enzyme that catalyzes deacetylation of acetyl-lysine residues of proteins such as histone, p53 and NF-κB, and plays a role in stress resistance, metabolism, differentiation, and aging (1). SIRT1 extends the life span of Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster (2-4), and may regulate life span in mammals (5). Also, SIRT1 regulates the activity of several important transcription factors, including FOXO1, FOXO3a, and FOXO4 (6-10), HES-1 and HEY-2 (11), MyoD (12), CTIP2 (13), PPARγ (14), NF-κB (15), and PGC1α (16).

It has been known that SIRT1 negatively regulates p53 and NF-κB. SIRT1 physically interacts with p53 and inhibits its function through p53 deacetylation at its C-terminal Lys379 (mouse protein) or Lys382 (human protein) residue (3, 4). SIRT1 overexpression represses p53-dependent cell cycle arrest and apoptosis in response to DNA damage and oxidative stress whereas expression of a point mutant form of SIRT1 that disrupts its deacetyalase activity, increases the sensitivity of cells to a stress response (3, 4). SIRT1 is highly expressed in mESC and inhibits p53-dependent transcription (17). SIRT1 negatively regulates NF-κB in the nucleus by the deacetylation of modified lysine residues (18). SIRT1 regulates NF-κB-dependent transcription and cell survival in response to tumor necrosis factor (TNF)-α (19). Recently, it has been reported that p53 mediates mESC differentiation by suppressing expression of Nanog, a master regulator of mESC (20). Overexpression of NF-κB proteins promoted differentiation, whereas inhibition of NFκB signalling, either by genetic ablation of the Ikbkg gene or overexpression of the IκBα super-repressor, increased expression of pluripotency markers (21), indicating that NF-κB also mediates differentiation of mESC. These suggest that SIRT1 might affect differentiation of mESC by regulation of p53 and NF-κB. Thus, this study analyzed the effect of SIRT1 in in vitro differentiation of mESC into mEB using wild and SIRT1 knockout mESC in the context of modulation of p53 and NF-κB signaling pathway.

Materials and Methods

Cell culture and treatment

mESC line R1 (22) and SIRT1-/- mESC derived from R1 (23) were maintained on mitomycin-treated STO feeder layers in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal calf serum (FCS), 1% GlutaMax (Gibco), 1% nonessential amino acids, antibiotics, 100 μM 2-ME, and 1000 U/ml recombinant LIF.

Western blot

Cells were lysed with M-PER mammalian protein extraction reagent (Pierce, Rockford, USA) supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Roche, Indianapolis, USA) on ice for 30 min. After spin down (12,000 rpm, 10 min), the whole cell lysates were taken from the supernatant, separated by SDS-PAGE, and transferred to PVDF membrane at 60 V for 16 h. The membrane was blocked with 3% fat-free milk in tris-buffered saline/0.05% tween-20 for 1 h, and incubated with primary antibodies (anti-Gadd45a, and anti-SIRT1 antibodies [Santa Cruz Biotechnology, Califonia, USA] anti-p53 and anti-acetyl-p53 [Cell Signaling Technology, Denvers, USA]) for 3 h. After washing, the membrane was further incubated with horseradish peoxidase-conjugated secondary antibodies for 1 h. Proteins were analyzed using an enhanced chemiluminescence detection system (G&E Amersham life science, Uppsala, Sweden).

EB formation

Undifferentiated mESC were trysinzed to obtain a single ESC suspension. A single ESC suspension was placed into 100 mm culture plate with untreated surface (SPL Lifescience, Seoul, Korea). The cells were cultured for 6 day in ESC culture medium without LIF.

Microarray analysis

RNA is prepared from the samples to be compared using the Qiagen RNAeasy Kit, and cDNA is labeled and incorporated with either Cy3 (green) or Cy5 (red) by reverse transcription using Agilent’s Low RNA Input Linear Amplification kit (Agilent Technology, Santa Clara, USA).Microarray hybridizations were performed using Agilent Mouse Oligo Microarray (Agilent Technology, Santa Clara, USA) according to the manufacturer’s instructions. The hybridized microarrays were washed as the manufacturer’s washing protocol (Agilent Technology, Santa Clara, USA). The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technology, Santa Clara, USA). All data normalization and selection of fold-changed genes were performed using Geneplex (Istech, Seoul, Korea). The averages of normalized ratios were calculated by dividing the average of normalized signal channel intensity by the average of normalized control channel intensity.

Results

Identification of differentially expressed genes

To identify differentially expressed genes between wild and SIRT1 knockout mESC, the expression pattern of 30,604 probe sets was analyzed with a microarray system. 195 and 1,291 genes were differentially upregulated and down-regulated, respectively, by SIRT1 knockout (Data not shown). Top 10 specifically upregulated or downregulated genes in SIRT1 knockout mESC is listed in Table 1.

Table 1.

List of 10 specifically upregulated and downregulated genes in SIRT1 knockout mESC

Genbank	Gene symbol	Gene name
Upregulated
NM_010345	Grb10	Growth factor receptor bound protein 10
XM_130038	Cubn	Cubilin (intrinsic factor-cobalamin receptor)
AK044329	A930004D18Rik	RIKEN cDNA A930004D18 gene
AK049087	Cdh4	Cadherin 4
NM_009876	Cdkn1c	Cyclin-dependent kinase inhibitor 1C (P57)
NM_023118	Dab2	Disabled homolog 2 (Drosophila)
NM_009434	Phlda2	Pleckstrin homology-like domain, family A, member 2
BC023079	2900078C09Rik	RIKEN cDNA 2900078C09 gene
NM_011446	Sox7	SRY-box containing gene 7
NM_178703	Slc6a1	Solute carrier family 6 (neurotransmitter transporter, GABA), member 1
Downregulated
AK017935	5830416P10Rik	RIKEN cDNA 5830416P10 gene
NM_029007	AW125753	Expressed sequence AW125753
AK085009	Fut9	Fucosyltransferase 9
AK006636	4930485G23Rik	RIKEN cDNA 4930485G23 gene
NM_027275	Ptcd3	Pentatricopeptide repeat domain 3
NM_173388	Slc43a2	Solute carrier family 43, member 2
NM_007556	Bmp6	Bone morphogenetic protein 6
AK084144		Length enriched library, clone:D230003G10 product
NM_026228	Slc39a8	Solute carrier family 39 (metal ion transporter), member 8

Functional classification of SIRT1 target genes

The probe sets corresponding to all deregulated transcripts were analyzed using David Bioinformatics Tool for molecular and cellular functions. Different categories are ranked according to the numbers of associated genes. SIRT1 knockout deregulates mainly the genes associated with transcription and signal transduction (Data not shown).

SIRT1 inhibits the activation of p53 on the differentiation of mESC

To elucidate whether SIRT1 inhibits p53 or NF-κB function to express their target genes during mESC differentiation, this study also examined changes in p53 and NF-κB target genes during the differentiation of wild and SIRT1 knockout mESC into EB derived from them. The results show that 52% of all genes were upregulated during ESC differentiation in wild mESC whereas 66% of all genes were upregulated in SIRT1 knockout mESC (Fig. 1). Interestingly 29% of p53 target genes were upregulated in wild mESC whereas 79% of p53 target genes were upregulated in SIRT1 knockout mESC (Fig. 1), indicating that SIRT1 inhibits p53 function to express its target genes during ESC differentiation. However, 53% of NF-κB target genes were upregulated in wild mESC whereas 50% of NF-κB target genes were upregulated in SIRT1 knockout mESC (Fig. 1), indicating that SIRT1 has no effect on NF-κB function to express its target genes during ESC differentiation. These results support the conclusion that SIRT1 is involved in the regulation of p53-dependent gene expression, but not, p53-dependent gene expression during ESC differentiation.

Fig.1. The effect of SIRT1 knockout in the expression of p53 or NF-κB target genes during the differentiation of mESC into EB. p53 or NF-κB target genes were mapped to expression ratios for mESC differentiated for 5 day versus undifferentiated ESC. Shown is the distribution of log2-transformed ratios for the p53 or NF-κB target genes that show differential expression more than 1.148 fold in both directions.

SIRT1 inhibits p53 acetylation and its function of inducing Gadd45a expression in response to methyl methanesulfonate in mESC

SIRT1 inhibits p53 transactivation function through deacetylation of p53 at its C-terminal Lys379 residue (4). To confirm more clearly whether SIRT1 inhibits p53 function during ESC differentiation, this study examined the effect of methyl methanesulfonate (MMS), a prototypic stimulant of p53, on p53 acetylation and its target gene expression in wild and SIRT1 knockout mESC. MMS treatment increased p53 acetylation and Gadd45a expression levels in SIRT1-/- mESCs but not wild mESCs (Fig. 2), suggesting that SIRT1 inhibits the ability of p53 to induce Gadd45a in response to MMS. These indicate that SIRT1 inhibits p53 function of inducing its target gene expression in mESC. These are consistent with the above finding that SIRT1 inhibits p53 target gene expression during mESC differentiation.

Fig. 2. The effect of MMS on p53 acetylation and Gadd45a expression. Wild or SIRT1-/- mESCs were treated with 1 mM for 1 h. p53 acetylation and Gadd45a expression levels were determined by western blot analysis using anti-acetyl p53 (Lys379) and anti-Gadd45a antibodies.

SIRT1 has no effect on hypoxia-induced NF-κB activation in mESC

SIRT1 also inhibits NF-κB activation by deacetylation(18). We also analyzed the effect of hypoxia, a prototypic stimulant of NF-κB, on nuclear translocation of NF-κB in wild and SIRT1 knockout mESC. Hypoxia has no effect on nuclear translocation of NF-κB in wild and SIRT1-/- mESCs (Fig. 3), suggesting that SIRT1 has no effect on the ability of NF-κB to induce its target gene. These are consistent with the above finding that SIRT1 has no effect on NF-κB target gene expression during mESC differentiation.

Fig. 3. No effect of SIRT1 on hypoxia-induced nuclear translocation of NF-κB. Wild-and SIRT1 knockout mESC were cultured for 24hr with complete medium under normoxia (20% oxygen) or hypoxia (5%). Cytoplasmic and nuclear fractions were prepared for NF-κB western blotting. The same blot was probed for β-actin (a control for cytoplasmic fractionation) and TBP (a control for nuclear fractionation).

Discussion

This study observed that SIRT1 inhibits p53 signaling pathway during the differentiation of mESC into mEB. SIRT1 is highly expressed in mESC (17, 23, 24) and dis-appears during mESC differentiation into EB (23). This study found that SIRT1 knockout upregulates p53 target genes at day 5 of in vitro differentiaton of mESC into EB. This indicates that SIRT1 may affect early lineage determination in mESC differentiation by regulation of p53 function.

It has been demonstrated that NFκB is inhibited in undifferentiated ESC (25) and is repressed by Nanog (21).It has also been shown that NFκB is activated during ES cell differentiation and that this functions as a positive differentiation stimulus (21). In addition, previous studies have suggested that activation of NFκB may contribute to changes in cell adhesion and cytokine production during ES cell differentiation (26). In contrast to mouse cells,however, NFκB has a positive role in maintaining pluripotency of human ES cells (27), reflecting differences in the self-renewal mechanisms of mouse and human ES cells. However, this study demonstrated that SIRT1 knockout has no effect on NF-κB functions including its target gene expression and nuclear translocation. Thus,this study suggests that SIRT1 modify mESC differentiation independently of NF-κB signaling pathway.

Potential conflict of interest

The authors have no conflicting financial interest.