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SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2 - PubMed

. 2016 Feb;26(2):190-205.

doi: 10.1038/cr.2016.4. Epub 2016 Jan 15.

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SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2

Huize Pan et al. Cell Res. 2016 Feb.

Abstract

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD(+)-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.

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Figures

Figure 1
Figure 1

SIRT6-deficient hMSCs exhibit accelerated cell attrition. (A) Schematic representation of deletion of SIRT6 by removing exon 1 of SIRT6 gene via TALEN-based gene targeting technique. The donor vector contains a neomycin-resistant cassette (neo) allowing for positive selection, and the neo cassette was then removed from the SIRT6 gene locus. (B) Left panel: western blotting analysis of SIRT6 protein in hESCs. Protein extracts from wild-type (WT, SIRT6+/+) and SIRT6-deficient (SIRT6−/−) hESCs were analyzed by western blotting using an anti-SIRT6 antibody. β-actin was used as the loading control. Right panel: RT-PCR analysis of SIRT6 mRNA in hESCs. A pair of PCR primers spanning the junction region of SIRT6 mRNA exon 1 and exon 2 was used. 18S rRNA was used as the loading control. (C) Bright-field and SIRT6 immunofluorescence micrographs of WT and SIRT6-deficient hESCs. DNA was stained by Hoechst 33342. Bright-field scale bar, 200 μm; immunofluorescence scale bar, 20 μm; zoom-field immunofluorescence scale bar, 10 μm. (D) Bright-field micrographs and FACS analysis of the surface markers CD105, CD73, and CD90 in WT and SIRT6-deficient hMSCs. Scale bar, 100 μm. (E) Western blotting analysis of SIRT6 protein in hMSCs. Protein extracts from WT and SIRT6-deficient hMSCs were analyzed by western blotting using anti-SIRT6 antibody. β-actin was used as the loading control. (F) Immunofluorescence analysis showing the absence of SIRT6 protein in the nuclei of SIRT6-deficient hMSCs. Scale bar, 10 μm. (G) SA-β-GAL staining from passage 6-8 showing an accelerated senescence in SIRT6-deficient hMSCs. Percentages of SA-β-GAL-positive cells were calculated. Data were presented as mean ± SEM, n = 5, NS, not significant, **P < 0.01. (H) Western blotting analysis of P16 and P21 protein in hMSCs. Protein extracts from WT and SIRT6-deficient hMSCs at late passage (LP, passage 9) were analyzed by western blotting. β-actin was used as the loading control. (I) Analysis of luciferase activity in the TA muscles of immunodeficient mice by in vivo imaging system (IVIS) demonstrating premature attrition of SIRT6-deficient hMSCs after implantation. WT (1 × 106, left) and SIRT6-deficient (1 × 106, right) hMSCs (passage 6) pretransduced with luciferase were implanted into the muscles of mice. Luciferase activities were imaged and quantified 1 week after implantation. Data were presented as mean ± SEM, n = 4, ***P < 0.001.

Figure 2
Figure 2

Ablation of SIRT6 in hMSCs results in elevated ROS levels and increased vulnerability to oxidative injury. (A) WT and SIRT6-deficient hMSCs were treated with vehicle (DMSO) or 50 μM PX-12 for 24 h, and the apoptotic cells were determined by Annexin V-PI staining via FACS. (B) Statistical analysis of A. Apoptotic cell percentage in vehicle-treated WT hMSCs was normalized to 1. Data were presented as mean ± SEM, n = 3, NS, not significant, *P < 0.05. (C, D) Cellular reactive oxygen species (ROS) and 8-oxodG levels were determined by staining with H2DCFDA probe (C) and an anti-8-oxodG antibody (D), respectively, and measured by FACS. (E) SIRT6-deficient hMSCs were pretreated with H2O (control) or 1 mM NAC for 1 week, and then were treated with vehicle (DMSO) or 50 μM PX-12 for 24 h. Cellular apoptosis was measured by Annexin V-PI staining. Data were presented as mean ± SEM, n = 3, **P < 0.01. (F) Overexpression of WT SIRT6 (SIRT6 (WT)), not SIRT6 H133Y mutant (SIRT6 (HY)), in SIRT6-deficient hMSCs partially restored cellular ROS to normal levels. A luciferase (Control)-expressed vector was used as control. (G, H) SIRT6-deficient hMSCs were transduced with SIRT6 (WT), SIRT6 (HY), or Control vector, and then cells were treated with vehicle (DMSO) or 50 μM PX-12 for 24 h. Cell viability (G) and cellular apoptosis (H) were measured by MTS assay and Annexin V-PI staining, respectively. Data in G and H were presented as mean ± SEM, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3
Figure 3

SIRT6 is required for NRF2-depedent HO-1 expression in hMSCs. (A) Volcano plot showing significantly altered genes (q-value < 0.05, FC[SIRT6-deficient/WT] < 0.5 or FC [SIRT6-deficient/WT] > 2) between WT and SIRT6-deficient hMSCs. Representative NRF2 target genes were highlighted (indicated by arrows). FC, fold change. (B) Gene ontology (GO) analysis (biological process) of significantly downregulated genes in hMSCs upon SIRT6 depletion. (C) Venn diagram showing that early passage (EP, passage 6) and late passage (LP, passage 9) hMSCs shared 183 significantly downregulated genes in SIRT6-deficient hMSCs compared with WT hMSCs. NRF2 target genes shared in EP and LP were indicated. (D) RT-qPCR analysis of NRF2 target genes in WT and SIRT6-deficient hMSCs. Values were normalized against 18S rRNA. Data were presented as mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. (E) Average profile of the H3K4me3 histone modification around the gene body regions of NRF2 target genes in SIRT6-deficient and WT hMSCs. TSS, transcription start site; TTS, transcription termination site. (F, G) RT-qPCR (F) and western blotting (G) analyses of HO-1 expression in WT and SIRT6-deficient hMSCs treated with 25 μM PX-12 for the indicated times. Relative mRNA and protein expressions were presented as fold induction. For RT-qPCR (F), values were normalized against 18S rRNA. Data were presented as mean ± SEM, n = 3, *P < 0.05, **P < 0.01. (H) Overexpression of SIRT6 (WT), not SIRT6 (HY), in SIRT6-deficient hMSCs partially restored HO-1 transcript. Values were normalized against 18S rRNA. Data were presented as mean ± SEM, n = 3, **P < 0.01, ***P < 0.001.

Figure 4
Figure 4

SIRT6 interacts with NRF2 and positively regulates NRF2-ARE pathway. (A) Transcriptional activity of NRF2 in WT and SIRT6-deficient hMSCs was measured by ARE-driven luciferase reporter assay. WT and SIRT6-deficient hMSCs were transfected with pcDNA3.1 (vector) or pcDNA3.1-NRF2 (NRF2), together with ARE-luciferase and Renilla plasmids. Data were presented as mean ± SEM, n = 3, *P < 0.05. (B) Plasmid expressing GFP, SIRT6 (WT), or SIRT6 (HY) was transfected into hMSCs, together with NRF2 or vector, and then NRF2 activity was measured using ARE-driven luciferase reporter. Data were presented as mean ± SEM, n = 3, NS, not significant, *P < 0.05. (C) Effect of SIRT6 overexpression on activation of NRF2 target genes in primary hMSCs. hMSCs were transduced with luciferase (control), SIRT6 (WT), SIRT6 (HY), or NRF2, and then the HO-1 and AKR1C1 transcripts were determined by RT-qPCR. Data were presented as mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. (D) WT and SIRT6-deficient hMSCs were transfected with a (UAS)5-TATA-luciferase plasmid together with GAL4 or GAL4-NRF2, and then the NRF2 transactivity was measured. Data were presented as mean ± SEM, n = 3, NS, not significant, **P < 0.01. (E) Luciferase analysis of NRF2 transactivity in WT and SIRT6−/− hMSCs in the presence of overexpressed GFP, SIRT6 (WT), or SIRT6 (HY). Data were presented as mean ± SEM, n =3, *P < 0.05, **P < 0.01. (F) ChIP-qPCR analysis of (UAS)5-associated SIRT6 in hMSCs co-expressing (UAS)5-TATA-luciferase, GAL4-NRF2, and Flag-SIRT6 using an anti-Flag antibody. Data were presented as mean ± SEM, n = 3, **P < 0.01. (G) Co-immunoprecipitation (Co-IP) showing that SIRT6 and NRF2 formed a protein complex. Exogenous (upper and middle panels) and endogenous (lower panel) Co-IPs were performed with the indicated antibodies. (H) GST-NRF2 or GST protein expressed from E. coli was incubated with Flag-SIRT6 expressed from HEK293T cells. The GST pull-down assay indicated an in vitro interaction between NRF2 and SIRT6.

Figure 5
Figure 5

SIRT6 deacetylates H3K56 and is required for recruiting RNAP II to the HO-1 gene promoter. (A) Co-IP assay using protein extracts from HEK293T cells expressing Flag-SIRT6 indicated that SIRT6 formed a protein complex with RNAP II and TAF II-p135. (B) ChIP-qPCR performed in SIRT6−/− hMSCs transduced with Flag-SIRT6 or Flag-luciferase (control) indicated association of SIRT6 with HO-1 promoter and enhancers. Data were presented as mean ± SEM, n = 3, *P < 0.05, **P < 0.01. (C) ChIP-qPCR assay showing SIRT6-dependent recruitment of RNAP II at HO-1 promoter. Data were presented as mean ± SEM, n = 3, *P <0.05. (D) Western blotting analyses of H3K56Ac, H3K9Ac, and H3K4me3 in WT and SIRT6-deficient hMSCs. Histone 3 (H3) was used as the loading control. (E) Immunofluorescence (left) and statistical (right) analyses of H3K56Ac levels in WT and SIRT6-deficient hMSCs. Scale bar, 100 μm. (F) ChIP-qPCR analysis of the enrichment of H3K56Ac and H3K9Ac at HO-1 promoter in WT and SIRT6-deficient hMSCs. Data were presented as mean ± SEM, n = 3, NS, not significant, **P < 0.01. (G) ChIP-qPCR analysis of H3K56Ac at HO-1 promoter in WT or SIRT6-deficient hMSCs transduced with lentiviral vector encoding SIRT6 (WT), SIRT6 (HY), or luciferase (control). Data were presented as mean ± SEM, n = 3, **P < 0.01.

Figure 6
Figure 6

Compromised NRF2-HO-1 axis accounts for redox dysregulation in SIRT6-deficient hMSCs. (A) FACS analyses of ROS level in hMSCs transduced with lentiviral vector encoding luciferase (control) or HO-1. (B) Lactate dehydrogenase (LDH) detection in the indicated hMSCs transduced with lentiviral vector encoding luciferase (control) or HO-1 in the presence of 50 μM PX-12 treatment. Data were presented as mean ± SEM, n = 3, *P < 0.05. (C) FACS analyses of PX-12-induced cytotoxicity in hMSCs transduced with lentiviral vector encoding luciferase (control) or HO-1 (left panel). Cells were treated with vehicle (DMSO) or 50 μM PX-12 for 24 h. Statistical analysis of apoptotic cells (right panel) was presented as mean ± SEM. n = 6, **P < 0.01. (D) Measurement of luciferase activity in immunodeficient mice with IVIS. SIRT6-deficient hMSCs overexpressing GFP plus luciferase (control group, left) and SIRT6-deficient hMSCs overexpressing HO-1 plus luciferase (right) were implanted into the TA muscles of mice. Six days after implantation, mice were intraperitoneally injected with 20 mg/kg PX-12 for 24 h, and then luciferase activity was measured. Data were presented as mean ± SEM, n = 4, *P < 0.05. (E) A putative model for SIRT6-mediated redox regulation in hMSCs. In WT hMSCs, SIRT6 is a key regulator of the cellular redox homeostasis by co-activating NRF2 antioxidant pathway. SIRT6 associates with NRF2 and deacetylates H3K56 at the promoter of NRF2 target genes (i.e., HO-1), which is required for the recruitment of RNAP II complex and subsequent transactivation of NRF2. In SIRT6-deficient hMSCs, SIRT6 deficiency causes increased level of H3K56Ac and impaired recruitment of RNAP II complex to HO-1 promoter, resulting in decrease in HO-1 expression and compromised cellular redox homeostasis.

Comment in

  • SIRT6, oxidative stress, and aging.

    Liao CY, Kennedy BK. Liao CY, et al. Cell Res. 2016 Feb;26(2):143-4. doi: 10.1038/cr.2016.8. Epub 2016 Jan 19. Cell Res. 2016. PMID: 26780861 Free PMC article.

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