WIP1基因调控骨髓造血干细胞的维持和功能
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摘要
WIP1(Wild-type p53-induced phosphatase)是一癌基因,在众多癌症细胞中高表达,同时是p53负反馈调节环路的重要组成成分,参与调控细胞的稳态平衡。为了研究WIP1基因与骨髓造血干细胞(Haematopoietic stem cells,HSCs)的维持和再生的关系,我们通过WIP1基因敲除小鼠的体外HSC单克隆培养、体内竞争性移植等实验研究WIP1在HSC自我更新能力和多系分化能力中的作用。
实验结果显示WIP1基因缺失小鼠的骨髓B细胞比例下降,胸腺T细胞发育障碍；在干细胞和祖细胞水平,WIP1基因缺失小鼠的长期/短期造血干细胞增多,多潜能祖细胞及淋系共同祖细胞比例下降；体外单克隆培养实验显示,WIP1基因缺失HSC形成克隆能力下降；全骨髓及干细胞竞争性移植实验和连续移植实验显示,WIP1基因缺失小鼠骨髓长期造血干细胞造血重建能力明显低于野生型；5-FU处理实验显示,WIP1基因缺失的HSC对5-FU敏感性增强；在半致死剂量辐射损伤情况下,WIP1基因缺失的小鼠长期造血干细胞的比例进一步增加；8.5Gy放射线照射损伤实验显示,WIP1基因缺失的HSC对放射线敏感性增强；移植实验显示WIP1基因缺失小鼠HSC数目增多及功能下降的表型不依赖于HSC微环境及整体环境；WIP1缺失小鼠HSC功能下降不依赖于p38及p38介导的氧化应激信号通路。这样研究结果显示,无论在正常情况下还是应激反应中,WIP1基因缺失均导致HSC数目增加但是再生能力下降,说明WIP1基因在维持HSC稳态方面起重要作用。
WIP1 (or PPM ID), Wild-type p53-induced phosphatase 1, is recognized as an oncogene, displaying high expression level in cells of multiple cancer types. Meanwhile, it is also known as a very important component in p53 negative feedback pathway, hence plays a vital role in regulating homeostasis of cells. In this study, we aimed to examine the effects of WIP1 gene on self-renewal and differentiation of hematopoietic stem cells (HSCs) in both normal and stress situations. To this end, HSCs of WIP1 gene knockout mouse were evaluated by in vitro clonal assay and in vivo tranplantation expriment..
Our data showed that the total number of B cell in bone marrow was decreased although the proportion of different subsets in B cell development was normal; in thymus development, the proportion of CD8/CD4 double negative cells was increased while the CD8/CD4 double positive cells was decreased. We also found an increased cell number in the stem cell compartment but decreased cell numbers at the multipotent progenitors and common lymphoid progenitor level in Wipl gene knockout mice bone marrow,. The in vitro single-cell clonal assay and the in vivo bone marrow transplantation experiment showed that Wipl gene knockout murine HSC has defect in forming big size colony and reconstituting the recipient hematopoietic system. Under replicative stress and the irradiation stress conditions, Wipl(?) murine HSC frequencies were further increased compared to the littermate controls. However, the Wipl(?) HSCs showed increased sensitivity to myelotoxic stress and high dose irradiation. Further expriment showed that these phenotype was due to HSC cell intrinsic defect but not dependent on the defect of HSC microenvironment. Moreover, the defect of WIPl(?) HSCs cannot be rescued by blocking p38 signal pathway or antioxidative treatment. Thus, our results demonstrate that Wipl plays a pivotal role in regulating the HSC maintenance and function.
实验结果显示WIP1基因缺失小鼠的骨髓B细胞比例下降,胸腺T细胞发育障碍；在干细胞和祖细胞水平,WIP1基因缺失小鼠的长期/短期造血干细胞增多,多潜能祖细胞及淋系共同祖细胞比例下降；体外单克隆培养实验显示,WIP1基因缺失HSC形成克隆能力下降；全骨髓及干细胞竞争性移植实验和连续移植实验显示,WIP1基因缺失小鼠骨髓长期造血干细胞造血重建能力明显低于野生型；5-FU处理实验显示,WIP1基因缺失的HSC对5-FU敏感性增强；在半致死剂量辐射损伤情况下,WIP1基因缺失的小鼠长期造血干细胞的比例进一步增加；8.5Gy放射线照射损伤实验显示,WIP1基因缺失的HSC对放射线敏感性增强；移植实验显示WIP1基因缺失小鼠HSC数目增多及功能下降的表型不依赖于HSC微环境及整体环境；WIP1缺失小鼠HSC功能下降不依赖于p38及p38介导的氧化应激信号通路。这样研究结果显示,无论在正常情况下还是应激反应中,WIP1基因缺失均导致HSC数目增加但是再生能力下降,说明WIP1基因在维持HSC稳态方面起重要作用。
WIP1 (or PPM ID), Wild-type p53-induced phosphatase 1, is recognized as an oncogene, displaying high expression level in cells of multiple cancer types. Meanwhile, it is also known as a very important component in p53 negative feedback pathway, hence plays a vital role in regulating homeostasis of cells. In this study, we aimed to examine the effects of WIP1 gene on self-renewal and differentiation of hematopoietic stem cells (HSCs) in both normal and stress situations. To this end, HSCs of WIP1 gene knockout mouse were evaluated by in vitro clonal assay and in vivo tranplantation expriment..
Our data showed that the total number of B cell in bone marrow was decreased although the proportion of different subsets in B cell development was normal; in thymus development, the proportion of CD8/CD4 double negative cells was increased while the CD8/CD4 double positive cells was decreased. We also found an increased cell number in the stem cell compartment but decreased cell numbers at the multipotent progenitors and common lymphoid progenitor level in Wipl gene knockout mice bone marrow,. The in vitro single-cell clonal assay and the in vivo bone marrow transplantation experiment showed that Wipl gene knockout murine HSC has defect in forming big size colony and reconstituting the recipient hematopoietic system. Under replicative stress and the irradiation stress conditions, Wipl(?) murine HSC frequencies were further increased compared to the littermate controls. However, the Wipl(?) HSCs showed increased sensitivity to myelotoxic stress and high dose irradiation. Further expriment showed that these phenotype was due to HSC cell intrinsic defect but not dependent on the defect of HSC microenvironment. Moreover, the defect of WIPl(?) HSCs cannot be rescued by blocking p38 signal pathway or antioxidative treatment. Thus, our results demonstrate that Wipl plays a pivotal role in regulating the HSC maintenance and function.
引文
1. Udayakumar, T., et al., The E2F1/Rb and p53/MDM2 pathways in DNA repair and apoptosis:understanding the crosstalk to develop novel strategies for prostate cancer radiotherapy. Semin Radiat Oncol,2010. 20(4):p.258-66.
2. Viale, A., et al., Cell-cycle restriction limits DNA damage and maintains self-renewal of leukaemia stem cells. Nature,2009. 457(7225):p.51-6.
3. Akala,O.O., et al., Long-term haematopoietic reconstitution by Trp53-/-p16Ink4a-/-p19Arf-/-multipotent progenitors. Nature,2008. 453(7192):p.228-32.
4. Harrison, D. E. and C.M. Astle, Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure. J Exp Med,1982.156(6):p.1767-79.
5. Beerman, I., et al., Stem cells and the aging hematopoietic system. Curr Opin Immunol,2010.22(4):p.500-6.
6. Cheng, T., et al., Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science,2000.287(5459):p.1804-8.
7. Lu, X., et al., The type 2C phosphatase Wip1:an oncogenic regulator of tumor suppressor and DNA damage response pa thways. Cancer Metastasis Rev,2008.27(2):p.123-35.
8. Cha, H., et al., Wip1 directly dephosphorylates gamma-H2AX and attenuates the DNA damage response. Cancer Res,2010.70(10):p. 4112-22.
9. Moon, S. H., et al., Dephosphorylation of gammaH2AX by WIP1:An important homeostatic regulatory event in DNA repair and cell cycle control. Cell Cycle,2010.9(11).
10. Choi, J., et al., Mice deficient for the wild-type p53-induced phosphatase gene (Wip1) exhibit defects in reproductive organs, immune function, and cell cycle control. Mol Cell Biol,2002.22(4): p.1094-105.
11. Harrison, M., et al., Wip1-deficient mice are resistant to common cancer genes. Trends Mol Med,2004.10(8):p.359-61.
12. Demidov,O. N., et al., Wip1 phosphatase regulates p53-dependent apoptosis of stem cells and tumorigenesis in the mouse intestine. Cell Stem Cell,2007.1(2):p.180-90.
13. Zhu, Y. H., et al., Wip1 regulates the generation of new neural cells in the adult olfactory bulb through p53-dependent cell cycle control. Stem Cells,2009.27(6):p.1433-42.
14. Lee, J. S., et al., Senescent growth arrest in mesenchymal stem cells is bypassed by Wip1-mediated downregulation of intrinsic stress signaling pathways. Stem Cells,2009.27(8):p.1963-75.
15. Wong, E. S., et al., p38MAPK controls expression of multiple cell cycle inhibi tors and isle t prolifera tion wi th advancing age. Dev Ce 11, 2009.17(1):p.142-9.
16. Schito, M. L., et al., Wipl phosphatase-deficient mice exhibit defective Tcell maturation due to sustainedp53 activation. J Immunol, 2006.176(8):p.4818-25.
17. Morita, Y., H. Ema, and H. Nakauchi, Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment. J Exp Med,2010.207(6):p.1173-82.
18. Ema, H., et al., Adult mouse hematopoietic stem cells: purification and single-cell assays. Nat Protoc,2006.1(6):p. 2979-87.
19. Essers, M. A., et al., IFNalpha activates dormant haematopoietic stem cells in vivo. Nature,2009.458(7240):p.904-8.
20. Min, H., E. Montecino-Rodriguez, and K. Dorshkind, Effects of aging on the common lymphoid progenitor to pro-B cell transition. J Immunol,2006.176(2):p.1007-12.
21. Miyamoto, K., et al., Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell Stem Cell,2007.1(1):p.101-12.
22. Takano, H., et al., Asymmetric division and lineage commitment at the level of hematopoietic stem cells:inference from differentiation in daughter cell and granddaughter cell pairs. J Exp Med,2004.199(3):p.295-302.
23. Ema, H., et al., In vitro self-renewal division of hematopoietic stem cells. J Exp Med,2000.192(9):p.1281-8.
24. Abkowitz, J. L., et al., Evidence that the number of hematopoietic stem cells per animal is conserved in mammals. Blood,2002.100(7): p.2665-7.
25. Song, Z., et al., Alterations of the systemic environment are the primary cause of impaired B and T lymphopoiesis in telomere-dysfunctional mice. Blood,2010.115(8):p.1481-9.
26. Liu, Y., et al., The p53 tumor suppressor protein is a critical regulator of hematopoietic stem cell behavior. Cell Cycle,2009.8(19): p.3120-4.
27. Dumble, M., et al., The impact of altered p53 dosage on hematopoietic stem cell dynamics during aging. Blood,2007.109(4): p.1736-42.
28. Sahin, E. and R. A. Depinho, Linking functional decline of telomeres, mitochondria and stem cells during ageing. Nature,2010. 464(7288):p.520-8.
29. Sahin, E., et al., Telomere dysfunction induces metabolic and mitochondrial compromise. Nature,2011.470(7334):p.359-65.
30. I to, K., et al., Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat Med,2006.12(4): p.446-51.
31. Rudolph, K. L., et al., Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell,1999.96(5):p.701-12.
32. Ju, Z., et al., Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment. Nat Med,2007.13(6):p.742-7.
33. Chambers, S. M., et al., Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation. PLoS Biol,2007. 5(8):p. e201.
2. Viale, A., et al., Cell-cycle restriction limits DNA damage and maintains self-renewal of leukaemia stem cells. Nature,2009. 457(7225):p.51-6.
3. Akala,O.O., et al., Long-term haematopoietic reconstitution by Trp53-/-p16Ink4a-/-p19Arf-/-multipotent progenitors. Nature,2008. 453(7192):p.228-32.
4. Harrison, D. E. and C.M. Astle, Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure. J Exp Med,1982.156(6):p.1767-79.
5. Beerman, I., et al., Stem cells and the aging hematopoietic system. Curr Opin Immunol,2010.22(4):p.500-6.
6. Cheng, T., et al., Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science,2000.287(5459):p.1804-8.
7. Lu, X., et al., The type 2C phosphatase Wip1:an oncogenic regulator of tumor suppressor and DNA damage response pa thways. Cancer Metastasis Rev,2008.27(2):p.123-35.
8. Cha, H., et al., Wip1 directly dephosphorylates gamma-H2AX and attenuates the DNA damage response. Cancer Res,2010.70(10):p. 4112-22.
9. Moon, S. H., et al., Dephosphorylation of gammaH2AX by WIP1:An important homeostatic regulatory event in DNA repair and cell cycle control. Cell Cycle,2010.9(11).
10. Choi, J., et al., Mice deficient for the wild-type p53-induced phosphatase gene (Wip1) exhibit defects in reproductive organs, immune function, and cell cycle control. Mol Cell Biol,2002.22(4): p.1094-105.
11. Harrison, M., et al., Wip1-deficient mice are resistant to common cancer genes. Trends Mol Med,2004.10(8):p.359-61.
12. Demidov,O. N., et al., Wip1 phosphatase regulates p53-dependent apoptosis of stem cells and tumorigenesis in the mouse intestine. Cell Stem Cell,2007.1(2):p.180-90.
13. Zhu, Y. H., et al., Wip1 regulates the generation of new neural cells in the adult olfactory bulb through p53-dependent cell cycle control. Stem Cells,2009.27(6):p.1433-42.
14. Lee, J. S., et al., Senescent growth arrest in mesenchymal stem cells is bypassed by Wip1-mediated downregulation of intrinsic stress signaling pathways. Stem Cells,2009.27(8):p.1963-75.
15. Wong, E. S., et al., p38MAPK controls expression of multiple cell cycle inhibi tors and isle t prolifera tion wi th advancing age. Dev Ce 11, 2009.17(1):p.142-9.
16. Schito, M. L., et al., Wipl phosphatase-deficient mice exhibit defective Tcell maturation due to sustainedp53 activation. J Immunol, 2006.176(8):p.4818-25.
17. Morita, Y., H. Ema, and H. Nakauchi, Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment. J Exp Med,2010.207(6):p.1173-82.
18. Ema, H., et al., Adult mouse hematopoietic stem cells: purification and single-cell assays. Nat Protoc,2006.1(6):p. 2979-87.
19. Essers, M. A., et al., IFNalpha activates dormant haematopoietic stem cells in vivo. Nature,2009.458(7240):p.904-8.
20. Min, H., E. Montecino-Rodriguez, and K. Dorshkind, Effects of aging on the common lymphoid progenitor to pro-B cell transition. J Immunol,2006.176(2):p.1007-12.
21. Miyamoto, K., et al., Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell Stem Cell,2007.1(1):p.101-12.
22. Takano, H., et al., Asymmetric division and lineage commitment at the level of hematopoietic stem cells:inference from differentiation in daughter cell and granddaughter cell pairs. J Exp Med,2004.199(3):p.295-302.
23. Ema, H., et al., In vitro self-renewal division of hematopoietic stem cells. J Exp Med,2000.192(9):p.1281-8.
24. Abkowitz, J. L., et al., Evidence that the number of hematopoietic stem cells per animal is conserved in mammals. Blood,2002.100(7): p.2665-7.
25. Song, Z., et al., Alterations of the systemic environment are the primary cause of impaired B and T lymphopoiesis in telomere-dysfunctional mice. Blood,2010.115(8):p.1481-9.
26. Liu, Y., et al., The p53 tumor suppressor protein is a critical regulator of hematopoietic stem cell behavior. Cell Cycle,2009.8(19): p.3120-4.
27. Dumble, M., et al., The impact of altered p53 dosage on hematopoietic stem cell dynamics during aging. Blood,2007.109(4): p.1736-42.
28. Sahin, E. and R. A. Depinho, Linking functional decline of telomeres, mitochondria and stem cells during ageing. Nature,2010. 464(7288):p.520-8.
29. Sahin, E., et al., Telomere dysfunction induces metabolic and mitochondrial compromise. Nature,2011.470(7334):p.359-65.
30. I to, K., et al., Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat Med,2006.12(4): p.446-51.
31. Rudolph, K. L., et al., Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell,1999.96(5):p.701-12.
32. Ju, Z., et al., Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment. Nat Med,2007.13(6):p.742-7.
33. Chambers, S. M., et al., Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation. PLoS Biol,2007. 5(8):p. e201.