Abstract
Intervertebral disc cell apoptosis occurs through either death receptor or mitochondrial pathway, but whether disc cell apoptosis is also mediated by the endoplasmic reticulum (ER) pathway remains unclear. The objective of this study was to investigate whether ER and mitochondria are co-involved in disc cell apoptosis and intervertebral disc degeneration (IVDD) in rats. Forty-eight rats were used for in vivo experiments. IVDD was characterized by X-ray and histomorphology examination, disc cell apoptosis was detected by TUNEL staining, and the co-involvement of ER and mitochondria in apoptosis was determined by immunohistochemical staining for GRP78, GADD153, caspase-12, and cytochrome C. Additional eight rats were used for annular cell isolation and culture. After sodium nitroprusside treatment, annular cell apoptosis was observed morphologically and quantified by flow cytometry; the expression of biomarkers of ER stress and mitochondrial dysfunction were analyzed by reverse transcriptase PCR (RT-PCR), fluorescence double labeling, and Western blot; and mitochondrial membrane potential was detected by 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbo cyanine iodide (JC-1) staining. Finally, NS3694 and Z-ATAD-FMK were employed to inhibit the formation of apoptosome complex and the activation of caspase-12, respectively, and apoptotic incidence and caspase-9 activity were assayed. We found that IVDD, induced by unbalanced dynamic and static forces in the rats, was accompanied by increased disc cell apoptosis and enhanced expression of GRP78, GADD153, caspase-12, and cytochrome C. Annular cell apoptosis induced by sodium nitroprusside was confirmed by morphologic observation and flow cytometry. With increased apoptosis, the expression of GRP78, GADD153, and caspase-12 upregulated, mitochondrial membrane potential decreased, and accumulation of cytochrome C in the cytosol manifested. Furthermore, NS3694 and Z-ATAD-FMK dramatically suppress annular cell apoptosis and caspase-9 activity. In conclusion, disc cell apoptosis mediated simultaneously by ER and mitochondria plays a potent role in IVDD.
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References
Adams JM (2003) Ways of dying: multiple pathways to apoptosis. Genes Dev 17:2481–2495
Ariga K, Miyamoto S, Nakase T, Okuda S, Meng W, Yonenobu K et al (2001) The relationship between apoptosis of endplate chondrocytes and aging and degeneration of the intervertebral disc. Spine 26:2414–2420
Ariga K, Yonenobu K, Nakase T, Hosono N, Okuda S, Meng W et al (2003) Mechanical stress-induced apoptosis of endplate chondrocytes in organ-cultured mouse intervertebral discs: an ex vivo study. Spine 28:1528–1533
Battie MC, Videman T, Gibbons LE, Fisher LD, Manninen H, Gill K (1995) Determinants of lumbar disc degeneration: a study relating lifetime exposures and magnetic resonance imaging findings in identical twins. Spine 20:2601–2612
Benavides A, Pastor D, Santos P, Tranque P, Calvo S (2005) CHOP plays a pivotal role in the astrocyte death induced by oxygen and glucose deprivation. Glia 52:261–275
Court C, Colliou OK, Chin JR, Liebenberg E, Bradford DS, Lotz JC (2001) The effect of static in vivo bending on the murine intervertebral disc. Spine J 1:239–245
Ferreiro E, Costa R, Marques S, Cardoso SM, Oliveira CR, Pereira CM (2008) Involvement of mitochondria in endoplasmic reticulum stress-induced apoptotic cell death pathway triggered by the prion peptide PrP(106–126). J Neurochem 104:766–776
Fernandez-Segura E, Canizares FJ, Cubero MA, Warley A, Campos A (1999) Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis. Exp Cell Res 253:454–462
Gotoh T, Mori M (2006) Nitric oxide and endoplasmic reticulum stress. Arterioscler Thromb Vasc Biol 26:1439–1446
Gruber HE, Hanley EN Jr (1998) Analysis of aging and degeneration of the human intervertebral disc. Comparison of surgical specimens with normal controls. Spine 23:751–757
Gruber HE, Norton HJ, Hanley EN Jr (2000) Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro. Spine 25:2153–2157
Groenendyk J, Michalak M (2005) Endoplasmic reticulum quality control and apoptosis. Acta Biochim Pol 52:381–395
Kadowaki H, Nishitoh H, Ichijo H (2004) Survival and apoptosis signals in ER stress: the role of protein kinases. J Chem Neuroanat 28:93–100
Kohyama K, Saura R, Doita M, Mizuno K (2000) Intervertebral disc cell apoptosis by nitric oxide: biological understanding of intervertebral disc degeneration. Kobe J Med Sci 46:283–295
Kroeber MW, Unglaub F, Wang H, Schmid C, Thomsen M, Nerlich A et al (2002) New in vivo animal model to create intervertebral disc degeneration and to investigate the effects of therapeutic strategies to stimulate disc regeneration. Spine 27:2684–2690
Lademann U, Cain K, Gyrd-Hansen M, Brown D, Peters D, Jaattela M (2003) Diarylurea compounds inhibit caspase activation by preventing the formation of the active 700-kilodalton apoptosome complex. Mol Cell Biol 23:7829–7837
Liu N, Kuang X, Kim HT, Stoica G, Qiang W, Scofield VL et al (2004) Possible involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in MoMuLV-ts1-induced apoptosis in astrocytes. J Neurovirol 10:189–198
Lopez-Armada MJ, Carames B, Martin MA, Cillero-Pastor B, Lires-Dean M, Fuentes-Boquete I et al (2006) Mitochondrial activity is modulated by TNF-α and IL-1β in normal human chondrocyte cells. Osteoarthritis Cartilage 14:1011–1022
Lotz JC, Chin JR (2000) Intervertebral disc cell death is dependent on the magnitude and duration of spinal loading. Spine 25:1477–1483
Lotz JC, Colliou OK, Chin JR, Duncan NA, Liebenberg E (1998) Compression-induced degeneration of the intervertebral disc: an in vivo mouse model and finite-element study. Spine 23:2493–2506
Luo S, Mao C, Lee B, Lee AS (2006) GRP78/BiP is required for cell proliferation and protecting the inner cell mass from apoptosis during early mouse embryonic development. Mol Cell Biol 26:5688–5697
Luthra S, Dong J, Gramajo AL, Chwa M, Kim DW, Neekhra A et al (2008) 7-Ketocholesterol activates caspases-3/7, -8, and -12 in human microvascular endothelial cells in vitro. Microvasc Res 75:343–350
Nishimura K, Mochida J (1998) Percutaneous reinsertion of the nucleus pulposus. An experimental study. Spine 23:1531–1539
Masud A, Mohapatra A, Lakhani SA, Ferrandino A, Hakem R, Flavell RA (2007) Endoplasmic reticulum stress-induced death of mouse embryonic fibroblasts requires the intrinsic pathway of apoptosis. J Biol Chem 282:14132–14139
McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ (2001) Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol 21:1249–1259
Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y (2002) An endoplasmic reticulum stress-specific caspase cascade in apoptosis: cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem 277:34287–34294
Mulhern ML, Madson CJ, Danford A, Ikesugi K, Kador PF, Shinohara T (2006) The unfolded protein response in lens epithelial cells from galactosemic rat lenses. Invest Ophthalmol Vis Sci 47:3951–3959
Nagata T, Ilieva H, Murakami T, Shiote M, Narai H, Ohta Y et al (2007) Increased ER stress during motor neuron degeneration in a transgenic mouse model of amyotrophic lateral sclerosis. Neurol Res 29:767–771
Oliver BL, Cronin CG, Zhang-Benoit Y, Goldring MB, Tanzer ML (2005) Divergent stress responses to IL-1β, nitric oxide, and tunicamycin by chondrocytes. J Cell Physiol 204:45–50
Orrenius S (2004) Mitochondrial regulation of apoptotic cell death. Toxicol Lett 149:19–23
Park JB, Chang H, Kim KW (2001a) Expression of Fas ligand and apoptosis of disc cells in herniated lumbar disc tissue. Spine 26:618–621
Park JB, Kim KW, Han CW, Chang H (2001b) Expression of Fas receptor on disc cells in herniated lumbar disc tissue. Spine 26:142–146
Park JB, Lee JK, Park SJ, Kim KW, Riew KD (2005) Mitochondrial involvement in fas-mediated apoptosis of human lumbar disc cells. J Bone Joint Surg Am 87:1338–1342
Park JB, Park IC, Park SJ, Jin HO, Lee JK, Riew KD (2006) Anti-apoptotic effects of caspase inhibitors on rat intervertebral disc cells. J Bone Joint Surg Am 88:771–779
Rannou F, Lee TS, Zhou RH, Chin J, Lotz JC, Mayoux-Benhamou MA et al (2004) Intervertebral disc degeneration: the role of the mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload. Am J Pathol 164:915–924
Reddy RK, Mao C, Baumeister P, Austin RC, Kaufman RJ, Lee AS (2003) Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors: role of ATP binding site in suppression of caspase-7 activation. J Biol Chem 278:20915–20924
Sanges D, Marigo V (2006) Cross-talk between two apoptotic pathways activated by endoplasmic reticulum stress: differential contribution of caspase-12 and AIF. Apoptosis 11:1629–1641
Stokes IA, Iatridis JC (2004) Mechanical conditions that accelerate intervertebral disc degeneration: overload versus immobilization. Spine 29:2724–2732
Tan Y, Dourdin N, Wu C, De Veyra T, Elce JS, Greer PA (2006) Ubiquitous calpains promote caspase-12 and JNK activation during endoplasmic reticulum stress-induced apoptosis. J Biol Chem 281:16016–16024
Unterberger U, Hoftberger R, Gelpi E, Flicker H, Budka H, Voigtlander T (2006) Endoplasmic reticulum stress features are prominent in Alzheimer disease but not in prion diseases in vivo. J Neuropathol Exp Neurol 65:348–357
Walsh AJ, Lotz JC (2004) Biological response of the intervertebral disc to dynamic loading. J Biomech 37:329–337
Wang YJ, Shi Q, Lu WW, Cheung KC, Darowish M, Li TF et al (2006a) Cervical intervertebral disc degeneration induced by unbalanced dynamic and static forces: a novel in vivo rat model. Spine 31:1532–1538
Wang YJ, Shi Q, Sun P, Zhou Q, Darowish M, Li TF et al (2006b) Insulin-like growth factor-1 treatment prevents anti-Fas antibody-induced apoptosis in endplate chondrocytes. Spine 31:736–741
Williams BL, Lipkin WI (2006) Endoplasmic reticulum stress and neurodegeneration in rats neonatally infected with borna disease virus. J Virol 80:8613–8626
Wu GJ, Chen TG, Chang HC, Chiu WT, Chang CC, Chen RM (2007) Nitric oxide from both exogenous and endogenous sources activates mitochondria-dependent events and induces insults to human chondrocytes. J Cell Biochem 101:1520–1531
Xu H, Zhou Q, Liu X, Qi YP (2006) Co-involvement of the mitochondria and endoplasmic reticulum in cell death induced by the novel ER-targeted protein HAP. Cell Mol Biol Lett 11:249–255
Zhao CQ, Jiang LS, Dai LY (2006) Programmed cell death in intervertebral disc degeneration. Apoptosis 11:2079–2088
Zhao CQ, Liu D, Li H, Jiang LS, Dai LY (2007) Interleukin-1β enhances the effect of serum deprivation on rat annular cell apoptosis. Apoptosis 12:2155–2161
Yang L, Carlson SG, McBurney D, Horton WE Jr (2005) Multiple signals induce endoplasmic reticulum stress in both primary and immortalized chondrocytes resulting in loss of differentiation, impaired cell growth, and apoptosis. J Biol Chem 280:31156–31165
Acknowledgments
This study was supported by the National Natural Science Foundation of China (no. 30700851) and Science and Technology Commission of Shanghai Municipality (09411953000).
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C.-Q. Zhao and Y.-H. Zhang contributed equally to this paper.
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Zhao, CQ., Zhang, YH., Jiang, SD. et al. Both endoplasmic reticulum and mitochondria are involved in disc cell apoptosis and intervertebral disc degeneration in rats. AGE 32, 161–177 (2010). https://doi.org/10.1007/s11357-009-9121-4
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DOI: https://doi.org/10.1007/s11357-009-9121-4