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Nature Immunology
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Gsk3 is a metabolic checkpoint regulator in B cells

Nature Immunologyvolume 18, pages303–312 (2017)Cite this article

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Abstract

B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.

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Figure 1: GC B cells face increased metabolic demands.
Figure 2: Gsk3 promotes B cell quiescence and homeostasis.
Figure 3: Gsk3 is required for T cell–dependent B cell responses.
Figure 4: Gsk3 inhibits CD40-induced B cell proliferation.
Figure 5: Gsk3 limits CD40-induced metabolic activity.
Figure 6: Gsk3 promotes rapamycin sensitivity and c-Myc degradation.
Figure 7: c-Myc as a functional target of Gsk3.
Figure 8: Gsk3 promotes B cell survival under glucose restriction.

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References

  1. Victora, G.D. et al. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter.Cell143, 592–605 (2010).

    Article CAS  Google Scholar 

  2. Calado, D.P. et al. The cell-cycle regulator c-Myc is essential for the formation and maintenance of germinal centers.Nat. Immunol.13, 1092–1100 (2012).

    Article CAS  Google Scholar 

  3. Dominguez-Sola, D. et al. The proto-oncogeneMYC is required for selection in the germinal center and cyclic reentry.Nat. Immunol.13, 1083–1091 (2012).

    Article CAS  Google Scholar 

  4. Doughty, C.A. et al. Antigen-receptor-mediated changes in glucose metabolism in B lymphocytes: role of phosphatidylinositol 3-kinase signaling in the glycolytic control of growth.Blood107, 4458–4465 (2006).

    Article CAS  Google Scholar 

  5. Woodland, R.T. et al. Multiple signaling pathways promote B lymphocyte stimulator–dependent B cell growth and survival.Blood111, 750–760 (2008).

    Article CAS  Google Scholar 

  6. Dufort, F.J. et al. Cutting edge: IL-4-mediated protection of primary B lymphocytes from apoptosis via Stat6-dependent regulation of glycolytic metabolism.J. Immunol.179, 4953–4957 (2007).

    Article CAS  Google Scholar 

  7. Beurel, E., Grieco, S.F. & Jope, R.S. Glycogen synthase kinase 3 (GSK3): regulation, actions and diseases.Pharmacol. Ther.148, 114–131 (2015).

    Article CAS  Google Scholar 

  8. Sutherland, C. What are the bona fide GSK3 substrates?Int. J. Alzheimers Dis.2011, 505607 (2011).

    PubMed PubMed Central  Google Scholar 

  9. McNeill, H. & Woodgett, J.R. When pathways collide: collaboration and connivance among signaling proteins in development.Nat. Rev. Mol. Cell Biol.11, 404–413 (2010).

    Article CAS  Google Scholar 

  10. Cross, D.A., Alessi, D.R., Cohen, P., Andjelkovich, M. & Hemmings, B.A. Inhibition of glycogen synthase kinase 3 by insulin-mediated by protein kinase B.Nature378, 785–789 (1995).

    Article CAS  Google Scholar 

  11. Fang, X. et al. Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A.Proc. Natl. Acad. Sci. USA97, 11960–11965 (2000).

    Article CAS  Google Scholar 

  12. Sutherland, C., Leighton, I.A. & Cohen, P. Inactivation of glycogen synthase kinase 3β by phosphorylation: new kinase connections in insulin and growth-factor signaling.Biochem. J.296, 15–19 (1993).

    Article CAS  Google Scholar 

  13. Zhao, Y. et al. Glycogen synthase kinase 3α and 3β mediate a glucose-sensitive anti-apoptotic signaling pathway to stabilize Mcl-1.Mol. Cell. Biol.27, 4328–4339 (2007).

    Article CAS  Google Scholar 

  14. Jacobs, K.M. et al. GSK-3β: A bifunctional role in cell death pathways.Int. J. Cell Biol.2012, 930710 (2012).

    Article  Google Scholar 

  15. Cato, M.H., Chintalapati, S.K., Yau, I.W., Omori, S.A. & Rickert, R.C. Cyclin D3 is selectively required for proliferative expansion of germinal center B cells.Mol. Cell. Biol.31, 127–137 (2011).

    Article CAS  Google Scholar 

  16. Semenza, G.L., Roth, P.H., Fang, H.M. & Wang, G.L. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1.J. Biol. Chem.269, 23757–23763 (1994).

    CAS PubMed  Google Scholar 

  17. Rickert, R.C. New insights into pre-BCR and BCR signaling with relevance to B cell malignancies.Nat. Rev. Immunol.13, 578–591 (2013).

    Article CAS  Google Scholar 

  18. Amir, A.D. et al. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.Nat. Biotechnol.31, 545–552 (2013).

    Article CAS  Google Scholar 

  19. Doble, B.W., Patel, S., Wood, G.A., Kockeritz, L.K. & Woodgett, J.R. Functional redundancy of GSK-3α and GSK-3β in Wnt–β-catenin signaling shown by using an allelic series of embryonic stem cell lines.Dev. Cell12, 957–971 (2007).

    Article CAS  Google Scholar 

  20. Rickert, R.C., Roes, J. & Rajewsky, K. B lymphocyte–specific, Cre-mediated mutagenesis in mice.Nucleic Acids Res.25, 1317–1318 (1997).

    Article CAS  Google Scholar 

  21. Khalil, A.M., Cambier, J.C. & Shlomchik, M.J. B cell receptor signal transduction in the GC is short-circuited by high phosphatase activity.Science336, 1178–1181 (2012).

    Article CAS  Google Scholar 

  22. Srinivas, S. et al. Cre reporter strains produced by targeted insertion ofEYFP andECFP into theROSA26 locus.BMC Dev. Biol.1, 4 (2001).

    Article CAS  Google Scholar 

  23. Förster, I. & Rajewsky, K. The bulk of the peripheral B cell pool in mice is stable and not rapidly renewed from the bone marrow.Proc. Natl. Acad. Sci. USA87, 4781–4784 (1990).

    Article  Google Scholar 

  24. Nojima, T. et al.In vitro–derived germinal center B cells differentially generate memory B or plasma cellsin vivo.Nat. Commun.2, 465 (2011).

    Article  Google Scholar 

  25. Sengupta, S., Peterson, T.R. & Sabatini, D.M. Regulation of the mTOR complex 1 pathway by nutrients, growth factors and stress.Mol. Cell40, 310–322 (2010).

    Article CAS  Google Scholar 

  26. Gregory, M.A., Qi, Y. & Hann, S.R. Phosphorylation by glycogen synthase kinase 3 controls c-Myc proteolysis and subnuclear localization.J. Biol. Chem.278, 51606–51612 (2003).

    Article CAS  Google Scholar 

  27. Sander, S. et al. Synergy between PI3K signaling and MYC in Burkitt lymphomagenesis.Cancer Cell22, 167–179 (2012).

    Article CAS  Google Scholar 

  28. Hsieh, A.L., Walton, Z.E., Altman, B.J., Stine, Z.E. & Dang, C.V. MYC and metabolism on the path to cancer.Semin. Cell Dev. Biol.43, 11–21 (2015).

    Article CAS  Google Scholar 

  29. Marchi, S. et al. Mitochondria–ROS cross-talk in the control of cell death and aging.J. Signal Transduct.2012, 329635 (2012).

    Article  Google Scholar 

  30. Cho, S.H. et al. Germinal center hypoxia and regulation of antibody qualities by a hypoxia response system.Nature537, 234–238 (2016).

    Article CAS  Google Scholar 

  31. Abbott, R.K. et al. Germinal center hypoxia potentiates immunoglobulin class switch recombination.J. Immunol.197, 4014–4020 (2016).

    Article CAS  Google Scholar 

  32. Caro-Maldonado, A. et al. Metabolic reprogramming is required for antibody production that is suppressed in anergic, but exaggerated in, chronically BAFF-exposed B cells.J. Immunol.192, 3626–3636 (2014).

    Article CAS  Google Scholar 

  33. Dang, C.V., Kim, J.W., Gao, P. & Yustein, J. The interplay between MYC and HIF in cancer.Nat. Rev. Cancer8, 51–56 (2008).

    Article CAS  Google Scholar 

  34. Zhang, H. et al. HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of c-MYC activity.Cancer Cell11, 407–420 (2007).

    Article CAS  Google Scholar 

  35. Inoki, K. et al. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth.Cell126, 955–968 (2006).

    Article CAS  Google Scholar 

  36. Azoulay-Alfaguter, I., Elya, R., Avrahami, L., Katz, A. & Eldar-Finkelman, H. Combined regulation of mTORC1 and lysosomal acidification by GSK3 suppresses autophagy and contributes to cancer cell growth.Oncogene34, 4613–4623 (2015).

    Article CAS  Google Scholar 

  37. Stretton, C. et al. GSK3-mediated raptor phosphorylation supports amino-acid-dependent mTORC1-directed signaling.Biochem. J.470, 207–221 (2015).

    Article  Google Scholar 

Download references

Acknowledgements

We thank the SBP vivarium staff for animal care, M. Shlomchik (University of Pittsburgh) for providing the hCD20-TamCre mice, D. Kitamura (Tokyo University of Science) for providing the CD40LB cell line and C. Lyssiotis (University of Michigan) for discussions. Supported by US National Institutes of Health grant R01AI41649 (R.C.R.), the Lilly Research Award Program (R.C.R.), fellowships from the Deutsche Forschungsgemeinschaft (J.J.) and the Cancer Centers Council (C3) (P.R.-R.) and grants from the Arthritis National Research Foundation (J.J.) and the Canadian Institutes of Health Research (J.W.). The Animal Resources and Cancer Metabolism Cores at SBP are supported by NCI award 5P30CA030199.

Author information

Authors and Affiliations

  1. Tumor Microenvironment and Cancer Immunology Program, Sanford Burnham Prebys Medical Discovery Institute (SBP), La Jolla, California, USA

    Julia Jellusova, Matthew H Cato, John R Apgar, Parham Ramezani-Rad, Charlotte R Leung, Cindi Chen & Robert C Rickert

  2. NCI-designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA

    Julia Jellusova, Matthew H Cato, John R Apgar, Parham Ramezani-Rad, Charlotte R Leung, Cindi Chen, Adam D Richardson & Robert C Rickert

  3. Eli Lilly and Company, La Jolla, California, USA

    Elaine M Conner & Robert J Benschop

  4. Lunenfeld–Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    James R Woodgett

  5. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    James R Woodgett

Authors
  1. Julia Jellusova

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  2. Matthew H Cato

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Contributions

J.J. designed and performed the majority of the experiments, analyzed the data and, together with R.C.R., wrote the manuscript; M.H.C. performed and analyzed the NP–KLH immunization experiment and contributed to the initial phenotypic analysis of Gsk3-deficient mice; J.R.A. performed and analyzed the CyTOF experiments and annexin V stainings; P.R.-R. performed and analyzed thein vivo CD40 stimulations and contributed to the experiments analyzing B cell proliferationin vivo andin vitro; C.R.L. and C.C. provided technical assistance with the experiments; A.D.R. helped perform and interpret the analysis of B cell metabolism; E.M.C. and R.J.B. provided advice, resources and assistance with the CyTOF experiments; J.R.W. provided mice and conceptual input to the manuscript; and R.C.R. conceived of and coordinated the study, interpreted the data and wrote the manuscript.

Corresponding author

Correspondence toRobert C Rickert.

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Competing interests

E.M.C. and R.J.B. are paid employees of Eli Lilly and Company.

Integrated supplementary information

Supplementary Figure 1 Germinal center B cells face increased metabolic demands in a hypoxic environment.

(a-c) Mice were immunized with SRBC, injected with PBS (n=11 mice) or 2DG (n=9 mice) on day 4,5,6 and analyzed on day 7. The percentage of B220+ cells (a), ratio of CD4+/CD8+ cells (b) and the frequency of PD1+ cells (c) in the spleen are shown. (d) Phosphorylation of the signaling molecules BLNK (Y84), Gsk3β (S9), Erk (T202/Y204), S6 (S235/236) and PLCγ2 (Y759) in wild type marginal zone B cells (B220+, CD21hi, IgMhi, CD23lo) at the indicated time points after stimulation with anti-IgM were analyzed by CyTOF Mass Cytometry. (e) c-Myc and β-Catenin expression and GSK3β phosphorylation (S9) in wild-type GC B cells (B220+, CD19+, GL7+, Fas+) were analyzed by CyTOF Mass Cytometry. Data are displayed using the t-Distributed Stochastic Neighbor Embedding (tSNE) algorithm. Horizontal line represents the mean (a, b, c). Data are representative of three (a,b, c) and one (d, e) independent experiments.

Supplementary Figure 2 Gsk3 promotes peripheral B cell quiescence and homeostasis.

(a+b) Total numbers of follicular B cells in the spleen (B220+, CD23+, CD21lo) (n=12 Ctrl mice and 13 dKO mice) (b) and B cells in lymph nodes (B220+) (n=10 Ctrl mice and 7 dKO mice) Mice used:Cd19Cre (Ctrl) andGsk3aL/LGsk3bL/LCd19Cre (dKO) (c) Analysis of frozen spleen sections by histology. Scale bar shows 100μm. Antibodies specific for: B220 or IgM were used to detect B cells, Moma1 to detect metallophilic macrophages, CD5 to detect T cells, and CD35 to detect follicular dendritic cells. Mice used:Cd19Cre (Ctrl) andGsk3aL/LGsk3bL/LCd19Cre (dKO). (d) Flow cytometric analysis of B cell development in the bone marrow. First row shows all live cells in the bone marrow, cells in the second row are pre-gated as: B220+ and CD43-, cells in the third row are pre-gated as: B220+, CD43+. Plots are representative of 3 mice per genotype. Mice shown:Cd19Cre (Ctrl) andGsk3aL/LGsk3bL/LCd19Cre (dKO) (e) Histograms show IgD expression and IgM expression on follicular B cells (B220+, CD23+, CD21-). Plots are representative of 9 mice per genotype. Mice shown:Cd19Cre (Ctrl) andGsk3aL/LGsk3bL/LCd19Cre (dKO) (f) Relative frequency of YFP+ splenic marginal zone B cells fromhCd20-TamCre (Ctrl) andGsk3aL/LGsk3bL/LhCd20-TamCre (dKO) mice 10, 26 and 47 days after tamoxifen injection. The measured frequency of YFP+ cells was normalized to the value obtained in the blood at the peak of induction (d7) Horizontal line represents the mean (a, b, f). Data are representative of thirteen (a), five (b), one (c), three (d), nine (e) and two (f) independent experiments. *P=0.0172, *P=0.0268t test (a,b), *P=0.0181, **P=0.0061 ANOVA (f) and *P=0.0121 Mann Whitney test (f).

Supplementary Figure 3 Gsk3 inhibits CD40-induced B cell proliferation.

(a) Histograms show proliferation of Ctrl and dKO B cells after 3 days of cell culture treated with the indicated amount of anti-CD40. One representative mouse out of four per genotype is shown. (b) Cells were stimulated with anti-IgM for 16h and subsequently with anti-CD40+IL-4+BAFF for 3 days. Histograms show proliferation of Ctrl and dKO B cells on the last day of the experiment. One representative mouse out of four per genotype is shown. (c) B cells from Ctrl and dKO mice were cultured as described in (b), cells size was determined using FSC. Mice used:Gsk3aL/L xGsk3bL/Lx hCd20-TamCre- (Ctrl)Gsk3aL/L xGsk3bL/Lx hCd20-TamCre (dKO). All mice were injected with tamoxifen on 3 consecutive days. Horizontal line represents the mean (c). Data are representative of one (a, b, c) experiment.P=0.0857Mann Whitney test (c).

Supplementary Figure 4 Gsk3 is dispensable for anti-IgM-induced metabolic adaptations.

Basal OCR (left) and ECAR (right) levels in B cells stimulated over night with anti-IgM are shown. Graphs summarize data obtained from 3 independent experiments with 8 mice per genotype in total. For each of the three experiments, the values were normalized to a value obtained from one random wildtype sample. Mice used:hCd20-TamCre orGsk3aL/LGsk3bL/L (Ctrl)Gsk3aL/LGsk3bL/LhCd20-TamCre (dKO) All mice were injected with tamoxifen on 3 consecutive days. Horizontal line represents the mean.

Supplementary Figure 5 Gsk3 promotes c-Myc degradation.

(a) Expression of the indicated proteins in freshly isolated B cells and B cells cultured over night with anti-CD40 and IL4 from Ctrl and dKO mice was analyzed by western blot.Gsk3aL/LGsk3bL/L (Ctrl)Gsk3aL/LGsk3bL/LhCd20-TamCre (dKO). All mice were injected with tamoxifen on 3 consecutive days. (b+c) B cells from Ctrl and dKO mice were cultured with anti-CD40+IL4 (b) or anti-IgM over night (c) and c-Myc expression was analyzed by western blot. Shown are ratios of c-Myc band intensities and the band intensity of the respective loading control. Mice used:hCd20-TamCre orGsk3aL/LGsk3bL/L (Ctrl)Gsk3aL/LGsk3bL/LhCd20-TamCre(dKO). All mice were treated with tamoxifen on 3 consecutive days. Data are representative of one (a), ten (b) and three (c) independent experiments. **P=0.0095 pairedt test (a).

Supplementary Figure 6 Normal B cell development inMyc-tg mice.

Analysis of B cell maturation in the spleen fromR26StopFLMycCd19cre mice (Myc-Tg) andCd19Cre littermates (Ctrl). Plots are representative for 5 mice per genotype analyzed in 5 independent experiments.

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Jellusova, J., Cato, M., Apgar, J.et al. Gsk3 is a metabolic checkpoint regulator in B cells.Nat Immunol18, 303–312 (2017). https://doi.org/10.1038/ni.3664

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