Overexpression of BNIP3 in rat intervertebral disk cells triggers autophagy and apoptosis
Keywords:apoptosis, autophagy, BNIP3, intervertebral disc cell, intervertebral disc degeneration
- Apoptosis of intervertebral disc cells is the primary cause of intervertebral disc degeneration (IDD). Bcl-2 and adenovirus E1B 19 kDa interacting protein 3 (BNIP3) is involved in cell autophagy and apoptosis.
- We explored the effect of BNIP3 on the autophagy and apoptosis in the isolated rat intervertebral disc cells by overexpression and RNA interference.
- BNIP3 overexpression in the rat intervertebral disc cells significantly upregulated the expression of the autophagy-related and pro-apoptotic proteins, the effects of which were substantially reversed by BNIP3 interference.
- BNIP3 enhanced autophagy and apoptosis of intervertebral disc cells in vitro, which might promote IDD development.
Abstract: Excessive apoptosis of intervertebral disk cells and intervertebral disk degeneration (IDD) is the prime cause of low back pain. B-cell lymphoma 2 (Bcl-2) and adenovirus E1B 19 kDa interacting protein 3 (BNIP3), a member of the Bcl-2 family, are involved in cell autophagy and apoptosis. The roles and mechanisms of BNIP3 in intervertebral disk cell autophagy and apoptosis are unclear. In this study, primary rat intervertebral disk cells were prepared to study the effect of BNIP3 overexpression on their autophagy and apoptosis. The cell counting kit (CCK)-8 assay showed that BNIP3 overexpression decreased cell viability. Real-time PCR and Western blotting showed that BNIP3 overexpression significantly upregulated the expression of autophagy-related proteins and pro-apoptotic proteins, including hypoxia-inducible factor-1α, apoptotic protease activating factor 1, caspase 3 and cleaved caspase 3, microtubule-associated proteins 1A/1B light chain 3 (LC3) and Beclin-1 while downregulating the expression of anti-apoptotic protein Bcl-2. Cell staining detection of autophagy and apoptosis showed that BNIP3 overexpression increased the autophagy and apoptosis of rat intervertebral disk cells. BNIP3 RNA interference revealed that the effects of BNIP3 overexpression can be reversed. These findings suggested that BNIP3 enhanced the autophagy and apoptosis in the rat intervertebral disk cells in vitro, which might promote IDD development.
Edward J, Carreon LY, Williams MV, Glassman S, Li J. The importance and impact of patients' health literacy on low back pain management: a systematic review of literature. Spine J. 2018;18(2):370-6. https://doi.org/10.1016/j.spinee.2017.09.005
Le Maitre CL, Binch AL, Thorpe AA, Hughes SP. Degeneration of the intervertebral disc with new approaches for treating low back pain. J Neurosurg Sci. 2015;59(1):47-61.
Petit A, Roquelaure Y. Low back pain, intervertebral disc and occupational diseases. Int J Occup Saf Ergon. 2015;21(1):15-9. https://doi.org/10.1080/10803548.2015.1017940
Xu TT, Liao F, Jin HT, Tong PJ, Xiao LW, Wu CL. [Research advance on intervertebral disc degeneration and cell death]. Zhongguo Gu Shang. 2015;28(7):673-8. Chinese.
Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol. 2007;8(9):741-52. https://doi.org/10.1038/nrm2239
Yurube T, Ito M, Kakiuchi Y, Kuroda R, Kakutani K. Autophagy and mTOR signaling during intervertebral disc aging and degeneration. JOR Spine. 2020;3(1):e1082. https://doi.org/10.1002/jsp2.1082
Zhang SJ, Yang W, Wang C, He WS, Deng HY, Yan YG, Zhang J, Xiang YX, Wang WJ. Autophagy: A double-edged sword in intervertebral disk degeneration. Clin Chim Acta. 2016;457:27-35. https://doi.org/10.1016/j.cca.2016.03.016
Azad MB, Chen Y, Henson ES, Cizeau J, McMillan-Ward E, Israels SJ, Gibson SB. Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3. Autophagy. 2008;4(2):195-204. https://doi.org/10.4161/auto.5278
Kubasiak LA, Hernandez OM, Bishopric NH, Webster KA. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3. Proc Natl Acad Sci U S A. 2002;99(20):12825-30. https://doi.org/10.1073/pnas.202474099
Hamacher-Brady A, Brady NR, Logue SE, Sayen MR, Jinno M, Kirshenbaum LA, Gottlieb RA, Gustafsson AB. Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy. Cell Death Differ. 2007;14(1):146-57. https://doi.org/10.1038/sj.cdd.4401936
Sowter HM, Ratcliffe PJ, Watson P, Greenberg AH, Harris AL. HIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors. Cancer Res. 2001;61(18):6669-73.
Zhang T, Xue L, Li L, Tang C, Wan Z, Wang R, Tan J, Tan Y, Han H, Tian R, Billiar TR, Tao WA, Zhang Z. BNIP3 Protein suppresses PINK1 kinase proteolytic cleavage to promote mitophagy. J Biol Chem. 2016;291(41):21616-29. https://doi.org/10.1074/jbc.M116.733410
Choi H, Merceron C, Mangiavini L, Seifert EL, Schipani E, Shapiro IM, Risbud MV. Hypoxia promotes noncanonical autophagy in nucleus pulposus cells independent of MTOR and HIF1A signaling. Autophagy. 2016;12(9):1631-46. https://doi.org/10.1080/15548627.2016.1192753
Vasagiri N, Kutala VK. Structure, function, and epigenetic regulation of BNIP3: a pathophysiological relevance. Mol Biol Rep. 2014;41(11):7705-14. https://doi.org/10.1007/s11033-014-3664-x
Ney PA. Mitochondrial autophagy: Origins, significance, and role of BNIP3 and NIX. Biochim Biophys Acta. 2015;1853(10 Pt B):2775-83. https://doi.org/10.1016/j.bbamcr.2015.02.022
Liu J, Peng N, Lu Y, Wang J. Preliminary research for correlation between BNIP3 expression and disc degeneration. Chin J Spine Spinal Cord. 2012;22(1):65-71. Chinese.
Liu J, Peng N, Wang J. Immunohistochemistry research on BNIP3 expressions of degenerated intervertebral disc in rabbit. J Reg Anat Oper Surg. 2012;21(1):3-5. Chinese.
Zhang F, Zhao X, Shen H, Zhang C. Molecular mechanisms of cell death in intervertebral disc degeneration (Review). Int J Mol Med. 2016 Jun;37(6):1439-48. https://doi.org/10.3892/ijmm.2016.2573
Shen X, Jiang H. Research progress on the relationship between autophagy and intervertebral disc degeneration. Chin J Trad Med Traum Orthop. 2017;25(8):77-82. Chinese.
Peng A, Yin C, Wu T, Huang W, Cheng X. Progress in association between autophagy and intervertebral disc degeneration. Chin J Cell Biol. 2016;38(11):1382-90. Chinese.
Chen D, Zhou K, Xu H. Research progress on the role of autophagy in intervertebral disc degeneration. J Med Res. 2016;45(12):9-12. Chinese.
Ding F, Shao ZW, Xiong LM. Cell death in intervertebral disc degeneration. Apoptosis. 2013;18(7):777-85. https://doi.org/10.1007/s10495-013-0839-1
Chen JW, Ni BB, Li B, Yang YH, Jiang SD, Jiang LS. The responses of autophagy and apoptosis to oxidative stress in nucleus pulposus cells: implications for disc degeneration. Cell Physiol Biochem. 2014;34(4):1175-89. https://doi.org/10.1159/000366330
Mizushima N. Autophagy: process and function. Genes Dev. 2007;21(22):2861-73. https://doi.org/10.1101/gad.1599207
Yorimitsu T, Klionsky DJ. Autophagy: molecular machinery for self-eating. Cell Death Differ. 2005;12 Suppl 2:1542-52. https://doi.org/10.1038/sj.cdd.4401765
Ye W, Xu K, Huang D, Liang A, Peng Y, Zhu W, Li C. Age-related increases of macroautophagy and chaperone-mediated autophagy in rat nucleus pulposus. Connect Tissue Res. 2011;52(6):472-8. https://doi.org/10.3109/03008207.2011.564336
Xu H, Xiong S, Wang H, Zhang M, Yu Y. The evidence and the possible significance of autophagy in degeneration model of human cervical end-plate cartilage. Exp Ther Med. 2014;7(3):537-42. https://doi.org/10.3892/etm.2013.1465
Gruber HE, Hoelscher GL, Ingram JA, Bethea S, Hanley EN Jr. Autophagy in the degenerating human intervertebral disc: in vivo molecular and morphological evidence, and induction of autophagy in cultured annulus cells exposed to proinflammatory cytokines-implications for disc degeneration. Spine (Phila Pa 1976). 2015;40(11):773-82. https://doi.org/10.1097/BRS.0000000000000865
Burton TR, Gibson SB. The role of Bcl-2 family member BNIP3 in cell death and disease: NIPping at the heels of cell death. Cell Death Differ. 2009;16(4):515-23. https://doi.org/10.1038/cdd.2008.185
Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ. 2009;16(7):939-46. https://doi.org/10.1038/cdd.2009.16
Liu J, Wang J, Zhou Y. Upregulation of BNIP3 and translocation to mitochondria in nutrition deprivation induced apoptosis in nucleus pulposus cells. Joint Bone Spine. 2012;79(2):186-91. https://doi.org/10.1016/j.jbspin.2011.04.011
Willis S, Day CL, Hinds MG, Huang DCS. The Bcl-2-regulated apoptotic pathway. J Cell Sci. 2003;116(20):4053-6. https://doi.org/10.1242/jcs.00754
Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011;18(4):571-80. https://doi.org/10.1038/cdd.2010.191
Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell. 2005;122(6):927-39. https://doi.org/10.1016/j.cell.2005.07.002
Decuypere JP, Parys JB, Bultynck G. Regulation of the autophagic bcl-2/beclin 1 interaction. Cells. 2012;1(3):284-312. https://doi.org/10.3390/cells1030284
Sheng R, Qin ZH. The divergent roles of autophagy in ischemia and preconditioning. Acta Pharmacol Sin. 2015;36(4):411-20. https://doi.org/10.1038/aps.2014.151
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