UPREGULATION OF Bcl-2/ADENOVIRUS E1B 19-kDa-INTERACTING PROTEIN 3 AND TRANSLOCATION TOMITOCHONDRIA IN NUCLEUS PULPOSUS CELLS INDUCED BY NUTRITION DEPRIVATION_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIU Jie ^1,2 , PENG Na ³ , ZENG Shunfu ¹ , LU Yan ⁴ , WANG Jian ¹ , ZHOU Yue. ¹

1Department of Orthopaedics, 3Department of Operating Room, XinqiaoHospital, the Third Military Medical University, Chongqing, 400037, P.R.China;;
2 Health Department of 93197 Unit of Chinese PLA;;
4Department of Orthopaedics, the 187th Hospital of Chinese PLA.;

Corresponding author：

Keywords：

Nucleus pulposus cells; Bcl-2/adenovirus E1B; 19-kDa-interacting protein 3; Mitochondria; Nutrition deprivation; Rat

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【Abstract】 Objective To detect the expression of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3)
in cell death induced by nutrition deprivation in nucleus pulposus cells so as to further understand the mechanism of death
in nucleus pulposus cells. Methods Two adult Sprague Dawley rats, male or female, weighing 150-200 g, were involved
in this experiment. The cells isolated from rat caudal disc were cultured under the condition of L-DMEM culture media,
10%FBS, and 21%O2 (control group) and under the condition of DMEM-free glucose culture media, no serum, and 1% O2
(experimental group). The expressions of BNIP3 gene and protein were detected by real-time fluorescent quantitative PCR,
immunofluorescence staining, and Western blot. The cell apoptosis rate and mitochondrial membrane potential were measured
by flow cytometry at 24, 48, and 72 hours after culture. Results The expression of BNIP3 decreased in the control group;
the expressions of BNIP3 showed an increasing tendency with time in the experimental group, and BNIP3 combined with
mitochondria. Significant differences were observed in the expressions of BNIP3 gene and protein between 2 groups at the other
time (P lt; 0.05) except that no significant difference was observed in the expression of BNIP3 gene at 24 hours (P gt; 0.05). The
cell apoptosis rate and mitochondrial membrane potential were significantly lower in the experimental group than those in the
control group (P lt; 0.05). Conclusion Upregulation of BNIP3 and translocation to mitochondria may be involved in nucleus
pulposus cell death in nutrition deprivation.

Citation： LIU Jie,PENG Na,ZENG Shunfu,LU Yan,WANG Jian,ZHOU Yue.. UPREGULATION OF Bcl-2/ADENOVIRUS E1B 19-kDa-INTERACTING PROTEIN 3 AND TRANSLOCATION TOMITOCHONDRIA IN NUCLEUS PULPOSUS CELLS INDUCED BY NUTRITION DEPRIVATION. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(2): 166-171. doi: Copy

1.	Crock HV, Goldwasser M, Yoshizawa H. Vascular anatomy related to the intervertebral disc//Ghosh P. The biology of the intervertebral disc. Boca Raton (FL): CRC Press, 1988: 109-133.
2.	Urban JP, Holm S, Maroudas A, et al. Nutrition of the intervertebral disk. An in vivo study of solute transport. Clin Orthop Relat Res, 1977, (129): 101-114.
3.	Holm S, Maroudas A, Urban JP, et al. Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res, 1981, 8(2): 101-119.
4.	Stairmand J, Holm S, Urban J. Factors influencing oxygen concentration gradients in the intervertebral disc: a theoretical analysis. Spine (Phila Pa 1976), 1991, 16(4): 444-449.
5.	Thompson JP, Pearce RH, Schechter MT, et al. Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine (Phila Pa 1976), 1990, 15(5): 411-415.
6.	Yasuma T, Koh S, Okamura T, et al. Histological changes in aging lumbar intervertebral discs. Their role in protrusions and prolapses. J Bone Joint Surg (Am), 1990, 72(2): 220-229.
7.	Taylor JR, Twomey LT. The role of the notochord and blood vessels in vertebral column development and in the aetiology of Schmorl’s nodes//Grieve J. Modern manual therapy. Edinburgh: Churchill Livingstone, 1986: 21-29.
8.	Battié MC, Videman T, Gill K, et al. Smoking and lumbar intervertebral disc degeneration: an MRI study of identical twins. Spine (Phila Pa 1976), 1991, 16(9): 1015-1021.
9.	Kauppila LI, Penttilä A, Karhunen PJ, et al. Lumbar disc degeneration and atherosclerosis of the abdominal aorta. Spine (Phila Pa 1976), 1994, 19(8): 923-929.
10.	McFadden KD, Taylor JR. End-plate lesions of the lumbar spine. Spine (Phila Pa 1976), 1989, 14(8): 867-869.
11.	Roberts S, Menage J, Eisenstein SM. The cartilage end-plate and intervertebral disc in scoliosis: calcification and other sequelae. J Orthop Res, 1993, 11(5): 747-757.
12.	Aoki J, Yamamoto I, Kitamura N, et al. End plate of the discovertebral joint: degenerative change in the elderly adult. Radiology, 1987, 164(2): 411-414.
13.	Brown MD, Tsaltas T. Studies on the permeability of the intervertebral disc during skeletal maturation. Spine (Phila Pa 1976), 1986, 1: 240-244.
14.	Hassler O. The human intervertebral disc. A micro-angiographical study of its vascular supply at various ages. Acta Orthop Scand, 1970, 40(6): 765-772.
15.	Roberts S, Urban JPG, Evans H, et al. Transport properties of the human cartilage endplate in relation to its composition and calcification. Spine (Phila Pa 1976), 1996, 21(4): 415-420.
16.	Urban MR, Fairbank JC, Etherington PJ, et al. Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer. Spine (Phila Pa 1976), 2001, 26(8): 984-990.
17.	Nachemson A, Lewin T, Maroudas A, et al. In vitro diffusion of dye through the end-plates and annulus fibrosus of human lumbar inter-vertebral discs. Acta Orthop Scand, 1970, 41(6): 589-607.
18.	Lee H, Paik SG. Regulation of BNIP3 in normal and cancer cells. Mol Cells, 2006, 21(2): 1-6.
19.	Vande Velde C, Cizeau J, Dubik D, et al. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol Cell Biol, 2000, 20(15): 5454-5468.
20.	Ray R, Chen G, Vande Velde C, et al. BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology3 (BH3) domain at both mitochondrial and nonmitochondrial sites. J Biol Chem, 2000, 275(2): 1439-1448.
21.	Chen G, Ray R, Dubik D, et al. The E1B 19K/Bcl-2 binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. J Exp Med, 1997, 186(12): 1975-1983.
22.	Yan J, Yun H, Yang Y, et al. Upregulation of BNIP3 promotes apoptosis of lung cancer cells that were induced by p53. Biochem Biophys Res Commun, 2006, 346(2): 501-507.
23.	Twomey LT, Taylor JR. Age changes in lumbar vertebrae and intervertebral discs. Clin Orthop Relat Res, 1987, (224): 97-104.
24.	Roberts S, Menage J, Urban JP. Biochemical and structural properties of the cartilage end-plate and its relation to the intervertebral disc. Spine (Phila Pa 1976), 1989, 14(2): 166-174.
25.	Maroudas A, Stockwell RA, Nachemson A, et al. Factors involved in the nutrition of the intervertebral disc: cellularity and diffusion of glucose in vitro. J Anat, 1975, 120(Pt 1): 113-130.
26.	Trout JJ, Buckwalter JA, Moore KC. Ultrastructure of the human intervertebral disc: II. Cells of the nucleus pulposus. Anat Rec, 1982, 204(4): 307-314.
27.	Gruber HE, Hanley EN. Analysis of aging and degeneration of the human intervertebral disc—comparison of surgical specimens with normal controls. Spine (Phila Pa 1976), 1998, 23(7): 751-757.
28.	Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
29.	Webster KA, Graham RM, Bishopric NH. BNIP3 and signalspecific programmed death in the heart. J Mol Cell Cardiol, 2005, 38(1): 35-45.
30.	Kubasiak LA, Hernandez OM, Bishopric NH, et al. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNip3. Proc Natl Acad Sci U S A, 2002, 99(20): 12825-12830.
31.	Yasuda M, Han JW, Dionne CA, et al. BNIP3alpha: a human homolog of mitochondrial proapoptotic protein BNIP3. Cancer Res, 1999, 59(3): 533-537.
32.	Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res, 2002, 91(3): 226-231.
33.	Zhang Z, Yang X, Zhang S, et al. BNIP3 upregulation and EndoG translocation in delayed neuronal death in stroke and in hypoxia. Stroke, 2007, 38(5): 1606-1613.
34.	Goping IS, Gross A, Lavoie JN, et al. Regulated targeting of BAX to mitochondria. J Cell Biol, 1998, 143(1): 207-215.
35.	Shimizu S, Konishi A, Kodama T, et al. BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc Natl Acad Sci U S A, 2000, 97(7): 3100-3105.
36.	Zhang L, Li L, Liu H, et al. HIF-1alpha activation by a redox-sensitive pathway mediates cyanide-induced BNIP3 upregulation and mitochondrial-dependent cell death. Free Radic Biol Med, 2007, 43(1): 117-127.

1. Crock HV, Goldwasser M, Yoshizawa H. Vascular anatomy related to the intervertebral disc//Ghosh P. The biology of the intervertebral disc. Boca Raton (FL): CRC Press, 1988: 109-133.
2. Urban JP, Holm S, Maroudas A, et al. Nutrition of the intervertebral disk. An in vivo study of solute transport. Clin Orthop Relat Res, 1977, (129): 101-114.
3. Holm S, Maroudas A, Urban JP, et al. Nutrition of the intervertebral disc: solute transport and metabolism. Connect Tissue Res, 1981, 8(2): 101-119.
4. Stairmand J, Holm S, Urban J. Factors influencing oxygen concentration gradients in the intervertebral disc: a theoretical analysis. Spine (Phila Pa 1976), 1991, 16(4): 444-449.
5. Thompson JP, Pearce RH, Schechter MT, et al. Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine (Phila Pa 1976), 1990, 15(5): 411-415.
6. Yasuma T, Koh S, Okamura T, et al. Histological changes in aging lumbar intervertebral discs. Their role in protrusions and prolapses. J Bone Joint Surg (Am), 1990, 72(2): 220-229.
7. Taylor JR, Twomey LT. The role of the notochord and blood vessels in vertebral column development and in the aetiology of Schmorl’s nodes//Grieve J. Modern manual therapy. Edinburgh: Churchill Livingstone, 1986: 21-29.
8. Battié MC, Videman T, Gill K, et al. Smoking and lumbar intervertebral disc degeneration: an MRI study of identical twins. Spine (Phila Pa 1976), 1991, 16(9): 1015-1021.
9. Kauppila LI, Penttilä A, Karhunen PJ, et al. Lumbar disc degeneration and atherosclerosis of the abdominal aorta. Spine (Phila Pa 1976), 1994, 19(8): 923-929.
10. McFadden KD, Taylor JR. End-plate lesions of the lumbar spine. Spine (Phila Pa 1976), 1989, 14(8): 867-869.
11. Roberts S, Menage J, Eisenstein SM. The cartilage end-plate and intervertebral disc in scoliosis: calcification and other sequelae. J Orthop Res, 1993, 11(5): 747-757.
12. Aoki J, Yamamoto I, Kitamura N, et al. End plate of the discovertebral joint: degenerative change in the elderly adult. Radiology, 1987, 164(2): 411-414.
13. Brown MD, Tsaltas T. Studies on the permeability of the intervertebral disc during skeletal maturation. Spine (Phila Pa 1976), 1986, 1: 240-244.
14. Hassler O. The human intervertebral disc. A micro-angiographical study of its vascular supply at various ages. Acta Orthop Scand, 1970, 40(6): 765-772.
15. Roberts S, Urban JPG, Evans H, et al. Transport properties of the human cartilage endplate in relation to its composition and calcification. Spine (Phila Pa 1976), 1996, 21(4): 415-420.
16. Urban MR, Fairbank JC, Etherington PJ, et al. Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer. Spine (Phila Pa 1976), 2001, 26(8): 984-990.
17. Nachemson A, Lewin T, Maroudas A, et al. In vitro diffusion of dye through the end-plates and annulus fibrosus of human lumbar inter-vertebral discs. Acta Orthop Scand, 1970, 41(6): 589-607.
18. Lee H, Paik SG. Regulation of BNIP3 in normal and cancer cells. Mol Cells, 2006, 21(2): 1-6.
19. Vande Velde C, Cizeau J, Dubik D, et al. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore. Mol Cell Biol, 2000, 20(15): 5454-5468.
20. Ray R, Chen G, Vande Velde C, et al. BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology3 (BH3) domain at both mitochondrial and nonmitochondrial sites. J Biol Chem, 2000, 275(2): 1439-1448.
21. Chen G, Ray R, Dubik D, et al. The E1B 19K/Bcl-2 binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis. J Exp Med, 1997, 186(12): 1975-1983.
22. Yan J, Yun H, Yang Y, et al. Upregulation of BNIP3 promotes apoptosis of lung cancer cells that were induced by p53. Biochem Biophys Res Commun, 2006, 346(2): 501-507.
23. Twomey LT, Taylor JR. Age changes in lumbar vertebrae and intervertebral discs. Clin Orthop Relat Res, 1987, (224): 97-104.
24. Roberts S, Menage J, Urban JP. Biochemical and structural properties of the cartilage end-plate and its relation to the intervertebral disc. Spine (Phila Pa 1976), 1989, 14(2): 166-174.
25. Maroudas A, Stockwell RA, Nachemson A, et al. Factors involved in the nutrition of the intervertebral disc: cellularity and diffusion of glucose in vitro. J Anat, 1975, 120(Pt 1): 113-130.
26. Trout JJ, Buckwalter JA, Moore KC. Ultrastructure of the human intervertebral disc: II. Cells of the nucleus pulposus. Anat Rec, 1982, 204(4): 307-314.
27. Gruber HE, Hanley EN. Analysis of aging and degeneration of the human intervertebral disc—comparison of surgical specimens with normal controls. Spine (Phila Pa 1976), 1998, 23(7): 751-757.
28. Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
29. Webster KA, Graham RM, Bishopric NH. BNIP3 and signalspecific programmed death in the heart. J Mol Cell Cardiol, 2005, 38(1): 35-45.
30. Kubasiak LA, Hernandez OM, Bishopric NH, et al. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNip3. Proc Natl Acad Sci U S A, 2002, 99(20): 12825-12830.
31. Yasuda M, Han JW, Dionne CA, et al. BNIP3alpha: a human homolog of mitochondrial proapoptotic protein BNIP3. Cancer Res, 1999, 59(3): 533-537.
32. Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ Res, 2002, 91(3): 226-231.
33. Zhang Z, Yang X, Zhang S, et al. BNIP3 upregulation and EndoG translocation in delayed neuronal death in stroke and in hypoxia. Stroke, 2007, 38(5): 1606-1613.
34. Goping IS, Gross A, Lavoie JN, et al. Regulated targeting of BAX to mitochondria. J Cell Biol, 1998, 143(1): 207-215.
35. Shimizu S, Konishi A, Kodama T, et al. BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc Natl Acad Sci U S A, 2000, 97(7): 3100-3105.
36. Zhang L, Li L, Liu H, et al. HIF-1alpha activation by a redox-sensitive pathway mediates cyanide-induced BNIP3 upregulation and mitochondrial-dependent cell death. Free Radic Biol Med, 2007, 43(1): 117-127.

Chinese Journal of Reparative and Reconstructive Surgery

UPREGULATION OF Bcl-2/ADENOVIRUS E1B 19-kDa-INTERACTING PROTEIN 3 AND TRANSLOCATION TOMITOCHONDRIA IN NUCLEUS PULPOSUS CELLS INDUCED BY NUTRITION DEPRIVATION

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