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Metabolic control of primed human pluripotent stem cell fate and function by the miR-200c–SIRT2 axis

Nature Cell Biology volume 19, pages 445–456 (2017)Cite this article

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Abstract

A hallmark of cancer cells is the metabolic switch from oxidative phosphorylation (OXPHOS) to glycolysis, a phenomenon referred to as the ‘Warburg effect’, which is also observed in primed human pluripotent stem cells (hPSCs). Here, we report that downregulation of SIRT2 and upregulation of SIRT1 is a molecular signature of primed hPSCs and that SIRT2 critically regulates metabolic reprogramming during induced pluripotency by targeting glycolytic enzymes including aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase. Remarkably, knockdown of SIRT2 in human fibroblasts resulted in significantly decreased OXPHOS and increased glycolysis. In addition, we found that miR-200c-5p specifically targets SIRT2, downregulating its expression. Furthermore, SIRT2 overexpression in hPSCs significantly affected energy metabolism, altering stem cell functions such as pluripotent differentiation properties. Taken together, our results identify the miR-200c–SIRT2 axis as a key regulator of metabolic reprogramming (Warburg-like effect), via regulation of glycolytic enzymes, during human induced pluripotency and pluripotent stem cell function.

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Figure 1: SIRT2 downregulation and SIRT1 upregulation is a molecular signature of human pluripotency.
Figure 2: SIRT2 regulates acetylation and enzymatic activity of glycolytic enzymes.
Figure 3: Acetylation status of K322 regulates AldoA activity.
Figure 4: SIRT2 influences metabolism and cell survival of hPSCs.
Figure 5: SIRT2 influences metabolism during early in vitro differentiation of hESCs.
Figure 6: SIRT2KD facilitates metabolic reprogramming in fibroblasts during the induced pluripotency.
Figure 7: SIRT2 influences somatic nuclear reprogramming through metabolic changes.
Figure 8: miR-200c directly targets SIRT2.

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Acknowledgements

This work was supported by NIH grants (NS084869, NS070577, and GM101420) and the National Research Foundation of Korea (2011-0030043). The authors are grateful to members of the Kim laboratory for critical discussions.

Author information

Author notes

  1. Min-Joon Han & Janet Zoldan

    Present address: Present addresses: Department of Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA (M.-J.H.); Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA (J.Z.).,

  2. Young Cha, Min-Joon Han and Hyuk-Jin Cha: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Psychiatry and McLean Hospital, Molecular Neurobiology Laboratory, Harvard Medical School, 115 Mill Street, Belmont, Massachusetts 02478, USA

    Young Cha, Min-Joon Han, Jin Hyuk Jung, Yongwoo Jang, Chun-Hyung Kim & Kwang-Soo Kim

  2. Department of Life Sciences, College of Natural Sciences, Sogang University, Seoul 04107, Korea

    Hyuk-Jin Cha & Ho-Chang Jeong

  3. Department of Chemical Engineering, David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts 02139, USA

    Janet Zoldan & Robert Langer

  4. Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215, USA

    Alison Burkart & C. Ronald Kahn

  5. Center for Genomic Integrity Institute for Basic Science, Ulsan National Institute of Science and Technology, Ulsan 44919, Korea

    Byung-Gyu Kim

  6. Department of Biology, MIT, Cambridge, Massachusetts 02139, USA

    Leonard Guarente

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Contributions

Y.C. and M.-J.H.: concept and design, collection and/or assembly of data, data analysis and interpretation, and writing. L.G., H.-J.C. and K.-S.K.: concept and design, data analysis and interpretation, and writing. J.Z., A.B., J.H.J., Y.J. and H.-C.J.: collection and/or assembly of data. C.-H.K., B.-G.K., R.L. and C.R.K.: data analysis and interpretation.

Corresponding author

Correspondence to Kwang-Soo Kim.

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Competing interests

K.-S.K. is co-founder of NurrON Pharmaceuticals and the remaining authors have no financial conflict to disclose.

Integrated supplementary information

Supplementary Figure 1 Warburg-like effect in hESCs and hiPSCs compared to hDFs.

(a) Human ESCs (H9) and hiPSCs cultured under feeder-free condition were stained with specific antibodies against pluripotency markers (e.g., Oct4, Nanog, SSEA4, and TRA1-60) along with Hoechst nuclear staining. Scale bar, 100 μm. (b) Representative pictures of hESCs and hiPSCs. Scale bar, 100 μm. (c) In vitro spontaneous differentiation of hESCs and hiPSCs by culturing in serum-free ITSFn medium for 7 days. Immunostaining images (first and second row panels) show lineage specific markers for ectoderm (Otx2), mesoderm (Brachyury; B-T), and endoderm (Sox17). Scale bar, 100 μm. (d) Intracellular ATP levels were significantly lower in hiPSCs and hESCs than in the original fibroblasts. (mean ± s.e.m., n=3 biologically independent experiments, ∗∗∗p < 0.005, two-tailed unpaired Student’s t-test). (e) Mitochondrial bioenergetics of parental hDFs and hiPSCs as well as hESCs assessed by Seahorse XF analyzer. (mean ± s.d., n=4 biologically independent experiments). (f) Expression levels of glucose transporters (GLUTs) including GLUT1 to GLUT7 in hDFs and hiPSCs as well as hESCs. (mean ± s.e.m., n=3 biologically independent experiments, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005; ∗∗∗∗p < 0.001, one-way ANOVA with Bonferroni post-test). (g) Immunoprecipitation of hDF and hESCs proteins using antibodies against acetyl-Lys, followed by LC-MS/MS analyses to identify acetylated proteins. Statistics source data are in Supplementary Table 9. Unprocessed original scans of blots (d,g) are shown in Supplementary Fig. 9.

Supplementary Figure 2 CID spectra for the acetylated proteins shown in Supplementary Fig. 1g and Supplementary Table 1.

Peptides for tubulin, Fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase1, enolase, pyruvate kinase isozymes M1/M2 and ATP synthase were detected via combination of IP and LC-MS/MS analyses. IP was performed with anti-acetyl-Lys antibody.

Supplementary Figure 3 Meta-analysis of Sirtuin family expression in hESCs.

(a) Representative data showing SIRT2 expression changes between different cells. SIRT2 downregulation was observed in hPSCs compared to differentiated cells and original fibroblasts. (b–f) Mean value scatter plot of expression levels of SIRT3 (b), SIRT4 (c), SIRT5, (d) SIRT6 (e), and SIRT7 (f) in hESC lines (n=25) and normal somatic cell lines (n=15) using results from a Database search (http://www.nextbio.com). All cell lines information is shown in Supplementary Table 5. (mean ± s.e.m., two-tailed unpaired Student’s t-test).

Supplementary Figure 4 Characterization of inducible SIRT2-GFP H9 hESCs.

(a) Plasmid map of doxycycline (Dox) inducible SIRT2-EGFP overexpression vector. (b) Expression levels of pluripotency markers in wild-type hESCs, hDFs, and SIRT2-GFP hESCs with or without Dox. (mean ± s.d., n=3 biologically independent experiments, ∗∗∗p < 0.005, one-way ANOVA with Bonferroni post-test). (c) Effects of TGF-β on spontaneous differentiation of SIRT2-GFP hESCs. Cells were maintained in hESC culture conditions with or without TGF-β. TGF-β was added for 4 days and cells were immunostained for expression of the endodermal marker Sox17; Scale bar, 100 μm. (d) Expression levels of glycolytic enzymes in SIRT2-GFP hESCs with or without Dox analyzed by qRT-PCR. (mean ± s.d., n=3 biologically independent experiments, ∗∗∗p < 0.005, two-tailed unpaired Student’s t-test). Statistics source data are in Supplementary Table 9.

Supplementary Figure 5 Effects of altered SIRT2 expression on acetylation of AldoA.

(a–d) Detection of AldoA K111 (a, b) and K322 (c,d) acetylation by mass spectrometry analysis. Symbol @ indicates the acetylation site. (e) Myc-tagged AldoA, AldoAK111Q, and AldoAK322Q were each expressed in 293T cells. AldoA proteins were purified by IP with Myc antibody, and specific activity for AldoA was determined. (mean ± s.d., n=3 biologically independent experiments, ∗∗∗p < 0.005, one-way ANOVA with Bonferroni post-test). (f) Myc-tagged AldoA, AldoAK111R, and AldoAK322R were each expressed in 293T cells co-expressing SIRT2 shRNA (SIRT2KD). AldoA proteins were purified by IP with Myc antibody and specific activity for AldoA was determined. (mean ± s.d., n=3 biologically independent experiments, ∗∗∗p < 0.005, one-way ANOVA with Bonferroni post-test). Statistics source data are in Supplementary Table 9.

Supplementary Figure 6 Metabolic and functional characterization of hPSC lines following SIRT2 overexpression.

(a,c,e) Glycolytic bioenergetics of wild type (Mock) and inducible SIRT2-GFP cell lines from H7 hESCs (a) and two iPSC lines (c,e) with or without Dox were assessed by XF analyzer. (mean ± s.d., n=3 biologically independent experiments). (b,d,f) Basal glycolytic rate, glycolytic capacity and glycolytic reserve of mock and SIRT2OE from H7 hESCs (b) and two iPSC lines (d,f) with or without Dox are shown in (a, c, e), respectively. (mean ± s.d., n=3 biologically independent experiments, ∗p < 0.05; ∗∗p < 0.01, one-way ANOVA with Bonferroni post-test). (g) OCRs for two hESC lines (H9 and H7) and the hiPSC-1 line with or without Dox are shown. 1: Mock w/o Dox, 2: Mock with Dox, 3: SIRT2OE w/o Dox, 4: SIRT2OE with Dox. (mean ± s.d., n=3 biologically independent experiments, ∗p < 0.05; ∗∗∗p < 0.005, one-way ANOVA with Bonferroni post-test). (h) Cell proliferation of mock and SIRT2OE from H7 hESCs and two independent iPSC lines (hiPSC-1 and hiPSC-2) with or without Dox were analyzed by determining cell numbers every 2 days under ESC culture conditions. (mean ± s.d., n=3 biologically independent experiments, ∗∗p < 0.01; ∗∗∗p < 0.005, two-way ANOVA with Bonferroni post-test). Statistics source data are in Supplementary Table 9.

Supplementary Figure 7 SIRT2 influences metabolic signatures of early differentiation potential of hiPSCs.

(a–c) Inducible SIRT2OE hiPSC-1 cells with or without Dox were induced to differentiate spontaneously by culturing in serum-free ITSFn medium for up to 4 days, and gene expression levels of pluripotency markers (Oct4, Nanog, and Rex1) (a), early-differentiation markers (Pax6, Brachyury (B-T), and Sox17) (b) and SIRT2 (c) were determined by qRT-PCR. (mean ± s.d., n=3 biologically independent experiments, ∗p < 0.05, one-way ANOVA with Bonferroni post-test). (d) Glycolytic bioenergetics of mock and SIRT2OE hiPSC-1 cells with or without Dox were assessed using the Seahorse XF analyzer. (mean ± s.d., n=3 biologically independent experiments, ∗p < 0.05, one-way ANOVA with Bonferroni post-test). (e) Extracellular lactate production of mock and SIRT2OE hiPSC-1 cells with or without Dox. (mean ± s.d., n=3 biologically independent experiments, ∗p < 0.05; ∗∗p < 0.01, one-way ANOVA with Bonferroni post-test). (f) Heatmaps depicting gene expression levels of markers representing ectoderm (Pax6, Map2, GFAP and AADC), endoderm (Foxa2, Sox17, AFP, CK8 and CK18), and mesoderm (Msx1 and B-T) in wild type (Mock) and inducible SIRT2OE hiPSC lines including hiPSC-1 and hiPSC-2 with or without Dox for up to 12 days under differentiation conditions. (n=2 biologically independent experiments). 1: Mock w/o Dox, 2: Mock with Dox, 3: SIRT2OE w/o Dox, 4: SIRT2OE with Dox. Statistics source data are in Supplementary Table 9.

Supplementary Figure 8 Effects of altered SIRT1 expression on metabolic reprogramming and iPSC generation.

(a) Plasmid map of doxycycline (Dox) inducible SIRT1-EGFP overexpression vector. (b) OCR for hDFs infected with wild type (Mock) or inducible SIRT1-GFP (SIRT1OE) with or without Dox at day 3 post transfection. (mean ± s.d., n=3 biologically independent experiments). (c,d) OCR/ECAR ratio (c), and relative OCR changes after FCCP injection (d) from Mock and SIRT1OE with or without Dox are shown in (b). (mean ± s.d., n=3 biologically independent experiments). (e,f) Effects of SIRT1KD or OE on iPSC generation. Upper: Efficiency of SIRT1KD or OE was confirmed by western blotting with anti-SIRT1 antibody. Lower: Representative pictures of AP-positive colonies at day 14 post-transduction. (mean ± s.e.m., n=3 biologically independent experiments, ∗∗∗p < 0.005, two-tailed unpaired Student’s t-test). (g,h) OCR in hDF infected with Y4 and/or SIRT1OE at 3 (g) or 6 (h) days after transfection. (mean ± s.d., n=3 biologically independent experiments). Statistics source data are in Supplementary Table 9. Unprocessed original scans of blots (e,f) are shown in Supplementary Fig. 9.

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Cha, Y., Han, MJ., Cha, HJ. et al. Metabolic control of primed human pluripotent stem cell fate and function by the miR-200c–SIRT2 axis. Nat Cell Biol 19, 445–456 (2017). https://doi.org/10.1038/ncb3517

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