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STEM CELL BIOLOGY

Transcription factor Nkx2-3 maintains the self-renewal of hematopoietic stem cells by regulating mitophagy

Leukemia volume 37, pages 1361–1374 (2023)Cite this article

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Abstract

Hematopoietic stem cells (HSCs) reside at the top of the hematopoietic hierarchy, exhibiting a unique capacity to self-renew and differentiate into all blood cells throughout the lifetime. However, how to prevent HSC exhaustion during long-term hematopoietic output is not fully understood. Here, we show that the homeobox transcription factor Nkx2-3 is required for HSC self-renewal by preserving metabolic fitness. We found that Nkx2-3 is preferentially expressed in HSCs with excessive regenerative potential. Mice with conditional deletion of Nkx2-3 displayed a reduced HSC pool and long-term repopulating capacity as well as increased sensitivity to irradiation and 5-flurouracil treatment due to impaired HSC quiescence. In contrast, overexpression of Nkx2-3 improved HSC function both in vitro and in vivo. Furthermore, mechanistic studies revealed that Nkx2-3 can directly control the transcription of the critical mitophagy regulator ULK1, which is essential for sustaining metabolic homeostasis in HSCs by clearing activated mitochondria. More importantly, a similar regulatory role of NKX2-3 was observed in human cord blood-derived HSCs. In conclusion, our data demonstrate an important role of the Nkx2-3/ULK1/mitophagy axis in regulating the self-renewal of HSCs, therefore providing a promising strategy to improve the function of HSCs in the clinic.

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Fig. 1: Nkx2-3 is preferentially expressed in HSCs with great regenerative potential.
Fig. 2: Conditional knockout of Nkx2-3 results in a reduction in the HSC pool in mice.
Fig. 3: Deletion of Nkx2-3 intrinsically compromises the long-term repopulation ability of HSCs.
Fig. 4: Nkx2-3 deficiency significantly impairs the quiescence of HSCs.
Fig. 5: Ablation of Nkx2-3 leads to the accumulation of activated mitochondria in HSCs due to attenuated mitophagy.
Fig. 6: Nkx2-3 regulates mitophagy in HSCs by transcriptionally controlling the expression of Ulk1.
Fig. 7: The NKX2-3/ULK1/mitophagy axis maintains the self-renewal of human HSCs.

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Data availability

The raw data of scRNA-seq were deposited in the NCBI Gene Expression Omnibus (GEO) database (no. GSE221907). The raw data of bulk RNA-seq were deposited in the NCBI Sequence Read Archive (SRA) database (no. PRJNA891790).

References

  1. Sanjuan-Pla A, Macaulay IC, Jensen CT, Woll PS, Luis TC, Mead A, et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature. 2013;502:232–6.

    Article  CAS  PubMed  Google Scholar 

  2. Cheng H, Zheng Z, Cheng T. New paradigms on hematopoietic stem cell differentiation. Protein Cell. 2020;11:34–44.

    Article  PubMed  Google Scholar 

  3. Sinha S, Dwivedi TR, Yengkhom R, Bheemsetty VA, Abe T, Kiyonari H, et al. Asrij/OCIAD1 suppresses CSN5-mediated p53 degradation and maintains mouse hematopoietic stem cell quiescence. Blood. 2019;133:2385–400.

    Article  CAS  PubMed  Google Scholar 

  4. Pinho S, Frenette PS. Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol. 2019;20:303–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chen Z, Guo Q, Song G, Hou Y. Molecular regulation of hematopoietic stem cell quiescence. Cell Mol Life Sci. 2022;79:218.

    Article  CAS  PubMed  Google Scholar 

  6. Nakamura-Ishizu A, Ito K, Suda T. Hematopoietic stem cell metabolism during development and aging. Dev Cell. 2020;54:239–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Suda T, Takubo K, Semenza GL. Metabolic regulation of hematopoietic stem cells in the hypoxic niche. Cell Stem Cell. 2011;9:298–310.

    Article  CAS  PubMed  Google Scholar 

  8. Hu M, Wang J. Mitochondrial metabolism and the maintenance of hematopoietic stem cell quiescence. Curr Opin Hematol. 2019;26:228–34.

    Article  PubMed  Google Scholar 

  9. Hu M, Zeng H, Chen S, Xu Y, Wang S, Tang Y, et al. SRC-3 is involved in maintaining hematopoietic stem cell quiescence by regulation of mitochondrial metabolism in mice. Blood. 2018;132:911–23.

    Article  CAS  PubMed  Google Scholar 

  10. Zheng J, Lu Z, Kocabas F, Bottcher RT, Costell M, Kang X, et al. Profilin 1 is essential for retention and metabolism of mouse hematopoietic stem cells in bone marrow. Blood. 2014;123:992–1001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jin G, Xu C, Zhang X, Long J, Rezaeian AH, Liu C, et al. Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells. Nat Immunol. 2018;19:29–40.

    Article  CAS  PubMed  Google Scholar 

  12. Luchsinger LL, de Almeida MJ, Corrigan DJ, Mumau M, Snoeck HW. Mitofusin 2 maintains haematopoietic stem cells with extensive lymphoid potential. Nature. 2016;529:528–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Qian P, He XC, Paulson A, Li Z, Tao F, Perry JM, et al. The Dlk1-Gtl2 locus preserves LT-HSC function by inhibiting the PI3K-mTOR pathway to restrict mitochondrial metabolism. Cell Stem Cell. 2016;18:214–28.

    Article  CAS  PubMed  Google Scholar 

  14. de Almeida MJ, Luchsinger LL, Corrigan DJ, Williams LJ, Snoeck HW. Dye-independent methods reveal elevated mitochondrial mass in hematopoietic stem cells. Cell Stem Cell. 2017;21:725–9.e4.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Holland PW. Evolution of homeobox genes. Wiley Interdiscip Rev Dev Biol. 2013;2:31–45.

    Article  CAS  PubMed  Google Scholar 

  16. Pabst O, Zweigerdt R, Arnold HH. Targeted disruption of the homeobox transcription factor Nkx2-3 in mice results in postnatal lethality and abnormal development of small intestine and spleen. Development. 1999;126:2215–25.

    Article  CAS  PubMed  Google Scholar 

  17. Tarlinton D, Light A, Metcalf D, Harvey RP, Robb L. Architectural defects in the spleens of Nkx2-3-deficient mice are intrinsic and associated with defects in both B cell maturation and T cell-dependent immune responses. J Immunol. 2003;170:4002–10.

    Article  CAS  PubMed  Google Scholar 

  18. Robles EF, Mena-Varas M, Barrio L, Merino-Cortes SV, Balogh P, Du MQ, et al. Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics. Nat Commun. 2016;7:11889.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dovey OM, Cooper JL, Mupo A, Grove CS, Lynn C, Conte N, et al. Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia. Blood. 2017;130:1911–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Huang L, Yu X, Jiang Z, Zeng P. Novel autophagy-related gene signature investigation for patients with oral squamous cell carcinoma. Front Genet. 2021;12:673319.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Guo R, Hu F, Weng Q, Lv C, Wu H, Liu L, et al. Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors. Cell Res. 2020;30:21–33.

    Article  CAS  PubMed  Google Scholar 

  22. Hu M, Lu Y, Zeng H, Zhang Z, Chen S, Qi Y, et al. MicroRNA-21 maintains hematopoietic stem cell homeostasis through sustaining the NF-kappaB signaling pathway in mice. Haematologica. 2021;106:412–23.

    CAS  PubMed  Google Scholar 

  23. Hu M, Lu Y, Wang S, Zhang Z, Qi Y, Chen N, et al. CD63 acts as a functional marker in maintaining hematopoietic stem cell quiescence through supporting TGFbeta signaling in mice. Cell Death Differ. 2022;29:178–91.

    Article  CAS  PubMed  Google Scholar 

  24. Zhang Z, Lu Y, Qi Y, Xu Y, Wang S, Chen F, et al. CDK19 regulates the proliferation of hematopoietic stem cells and acute myeloid leukemia cells by suppressing p53-mediated transcription of p21. Leukemia. 2022;36:956–69.

    Article  CAS  PubMed  Google Scholar 

  25. Rodriguez-Fraticelli AE, Wolock SL, Weinreb CS, Panero R, Patel SH, Jankovic M, et al. Clonal analysis of lineage fate in native haematopoiesis. Nature. 2018;553:212–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Doherty J, Baehrecke EH. Life, death and autophagy. Nat Cell Biol. 2018;20:1110–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ho TT, Warr MR, Adelman ER, Lansinger OM, Flach J, Verovskaya EV, et al. Autophagy maintains the metabolism and function of young and old stem cells. Nature. 2017;543:205–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mortensen M, Soilleux EJ, Djordjevic G, Tripp R, Lutteropp M, Sadighi-Akha E, et al. The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance. J Exp Med. 2011;208:455–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Man N, Tan Y, Sun XJ, Liu F, Cheng G, Greenblatt SM, et al. Caspase-3 controls AML1-ETO-driven leukemogenesis via autophagy modulation in a ULK1-dependent manner. Blood. 2017;129:2782–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gong J, Gu H, Zhao L, Wang L, Liu P, Wang F, et al. Phosphorylation of ULK1 by AMPK is essential for mouse embryonic stem cell self-renewal and pluripotency. Cell Death Dis. 2018;9:38.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Iorio R, Celenza G, Petricca S. Mitophagy: molecular mechanisms, new concepts on parkin activation and the emerging role of AMPK/ULK1 axis. Cells. 2021;11:30.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Bellio C, DiGloria C, Spriggs DR, Foster R, Growdon WB, Rueda BR. The metabolic inhibitor CPI-613 negates treatment enrichment of ovarian cancer stem cells. Cancers. 2019;11:1678.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Guo P, Liu Y, Geng F, Daman AW, Liu X, Zhong L, et al. Histone variant H3.3 maintains adult haematopoietic stem cell homeostasis by enforcing chromatin adaptability. Nat Cell Biol. 2022;24:99–111.

    Article  CAS  PubMed  Google Scholar 

  34. Bagger FO, Kinalis S, Rapin N. BloodSpot: a database of healthy and malignant haematopoiesis updated with purified and single cell mRNA sequencing profiles. Nucleic Acids Res. 2019;47:D881–D5.

    Article  CAS  PubMed  Google Scholar 

  35. Nagel S, Pommerenke C, Scherr M, Meyer C, Kaufmann M, Battmer K, et al. NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia. PLoS One. 2017;12:e0171164.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Zhou F, Li X, Wang W, Zhu P, Zhou J, He W, et al. Tracing haematopoietic stem cell formation at single-cell resolution. Nature. 2016;533:487–92.

    Article  CAS  PubMed  Google Scholar 

  37. Pabst O, Forster R, Lipp M, Engel H, Arnold HH. NKX2.3 is required for MAdCAM-1 expression and homing of lymphocytes in spleen and mucosa-associated lymphoid tissue. EMBO J. 2000;19:2015–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Dinh TT, Xiang M, Rajaraman A, Wang Y, Salazar N, Zhu Y, et al. An NKX-COUP-TFII morphogenetic code directs mucosal endothelial addressin expression. Nat Commun. 2022;13:7448.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Singh SK, Singh S, Gadomski S, Sun L, Pfannenstein A, Magidson V, et al. Id1 ablation protects hematopoietic stem cells from stress-induced exhaustion and aging. Cell Stem Cell. 2018;23:252–65.e8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kataoka K, Sato T, Yoshimi A, Goyama S, Tsuruta T, Kobayashi H, et al. Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity. J Exp Med. 2011;208:2403–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kocabas F, Zheng J, Thet S, Copeland NG, Jenkins NA, DeBerardinis RJ, et al. Meis1 regulates the metabolic phenotype and oxidant defense of hematopoietic stem cells. Blood. 2012;120:4963–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ficara F, Murphy MJ, Lin M, Cleary ML. Pbx1 regulates self-renewal of long-term hematopoietic stem cells by maintaining their quiescence. Cell Stem Cell. 2008;2:484–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Freire PR, Conneely OM. NR4A1 and NR4A3 restrict HSC proliferation via reciprocal regulation of C/EBPalpha and inflammatory signaling. Blood. 2018;131:1081–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Min IM, Pietramaggiori G, Kim FS, Passegue E, Stevenson KE, Wagers AJ. The transcription factor EGR1 controls both the proliferation and localization of hematopoietic stem cells. Cell Stem Cell. 2008;2:380–91.

    Article  CAS  PubMed  Google Scholar 

  45. Lehnertz B, Chagraoui J, MacRae T, Tomellini E, Corneau S, Mayotte N, et al. HLF expression defines the human hematopoietic stem cell state. Blood. 2021;138:2642–54.

    Article  CAS  PubMed  Google Scholar 

  46. Li C, Wu B, Li Y, Liu Y, Wang J, Xie J, et al. Loss of sphingosine kinase 2 promotes the expansion of hematopoietic stem cells by improving their metabolic fitness. Blood. 2022;140:1686–701.

    Article  CAS  PubMed  Google Scholar 

  47. Garcia-Prat L, Kaufmann KB, Schneiter F, Voisin V, Murison A, Chen J, et al. TFEB-mediated endolysosomal activity controls human hematopoietic stem cell fate. Cell Stem Cell. 2021;28:1838–50.e10.

    Article  CAS  PubMed  Google Scholar 

  48. Warr MR, Binnewies M, Flach J, Reynaud D, Garg T, Malhotra R, et al. FOXO3A directs a protective autophagy program in haematopoietic stem cells. Nature. 2013;494:323–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Palikaras K, Lionaki E, Tavernarakis N. Mechanisms of mitophagy in cellular homeostasis, physiology and pathology. Nat Cell Biol. 2018;20:1013–22.

    Article  CAS  PubMed  Google Scholar 

  50. Ito K, Turcotte R, Cui J, Zimmerman SE, Pinho S, Mizoguchi T, et al. Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance. Science. 2016;354:1156–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Murakami K, Kurotaki D, Kawase W, Soma S, Fukuchi Y, Kunimoto H, et al. OGT regulates hematopoietic stem cell maintenance via PINK1-dependent mitophagy. Cell Rep. 2021;34:108579.

    Article  CAS  PubMed  Google Scholar 

  52. Lee EJ, Tournier C. The requirement of uncoordinated 51-like kinase 1 (ULK1) and ULK2 in the regulation of autophagy. Autophagy. 2011;7:689–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Nah J, Shirakabe A, Mukai R, Zhai P, Sung EA, Ivessa A, et al. Ulk1-dependent alternative mitophagy plays a protective role during pressure overload in the heart. Cardiovasc Res. 2022;118:2638–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Dhingra R, Rabinovich-Nikitin I, Kirshenbaum LA. Ulk1/Rab9-mediated alternative mitophagy confers cardioprotection during energy stress. J Clin Invest. 2019;129:509–12.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Kundu M, Lindsten T, Yang CY, Wu J, Zhao F, Zhang J, et al. Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation. Blood. 2008;112:1493–502.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Lechauve C, Keith J, Khandros E, Fowler S, Mayberry K, Freiwan A, et al. The autophagy-activating kinase ULK1 mediates clearance of free alpha-globin in beta-thalassemia. Sci Transl Med. 2019;11:eaav4881.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Han X, Yoshizaki K, Miyazaki K, Arai C, Funada K, Yuta T, et al. The transcription factor NKX2-3 mediates p21 expression and ectodysplasin-A signaling in the enamel knot for cusp formation in tooth development. J Biol Chem. 2018;293:14572–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Yang Liu for technical support in flow cytometry, Liting Wang for technical support in immunofluorescence microscopy, and Quanfang Wei for technical assistance in transmission electron microscopy. This work was supported by grants from the National Natural Science Foundation of China (No. 81930090, 82203974, 81725019), Project funded by China Postdoctoral Science Foundation (No. 2022M723867), Postdoctoral Innovative Talent Support Program of China (No. BX20220398), Postdoctoral Innovative Talent Support Program of Chongqing (No. CQBX2021017) and the Scientific Research Project of PLA (No. BWS21J022).

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  1. These authors contributed equally: Mengjia Hu, Naicheng Chen, Mo Chen.

Authors and Affiliations

  1. State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing, 400038, China

    Mengjia Hu, Naicheng Chen, Mo Chen, Fang Chen, Yukai Lu, Yang Xu, Lijing Yang, Hao Zeng, Mingqiang Shen, Shilei Chen, Fengchao Wang, Song Wang & Junping Wang

  2. Chinese PLA Center for Disease Control and Prevention, Beijing, 100071, China

    Mengjia Hu

  3. Department of Obstetrics and Gynecology, Liangping District Maternal and Child Health Care Hospital, Chongqing, 405200, China

    Xuehong Chen

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  1. Mengjia Hu
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Contributions

MH, NC and MC designed the study, performed experiments, analyzed data and wrote the paper. FC, YL and YX performed experiments and analyzed data. LY and HZ participated in some animal experiments. MS and XC participated in some in vitro experiments. SC and FW participated in the initial experimental design and discussed the manuscript. JW and SW conceived and supervised the study, and revised the manuscript.

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Correspondence to Song Wang or Junping Wang.

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Hu, M., Chen, N., Chen, M. et al. Transcription factor Nkx2-3 maintains the self-renewal of hematopoietic stem cells by regulating mitophagy. Leukemia 37, 1361–1374 (2023). https://doi.org/10.1038/s41375-023-01907-y

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