Abstract
Purpose
Chronic low back pain has been associated with intervertebral disc (IVD) degeneration, which is characterized by the accumulation of extracellular matrix (ECM)-degrading proteases and inflammatory molecules in the degenerate tissue. IVD degeneration could be the outcome of natural organismal ageing and/or of the exposure of the disc to cumulative stressful environmental stimuli and is accompanied by an increased population of senescent cells in the tissue. On the other hand, senescent cells are known to secrete proteolytic enzymes and inflammatory molecules, which can contribute to ECM catabolism. The aim of this study was to investigate the transcriptional profile of selected metalloproteinases (MMPs) and inflammatory mediators in human nucleus pulposus IVD cells that became senescent using three different approaches: serial subculturing, exposure to ionizing radiation and p16INK4a overexpression.
Methods
Gene expression was assessed using quantitative RT-PCR and protein levels were determined by western blot analysis. The proliferative potential of the cells, as well as the percentage of senescent cells in the population were estimated by nuclear BrdU incorporation and by senescence-associated β galactosidase staining, respectively.
Results
All senescent cells showed a similar regulation of MMP-1, -2, -3, -9, interleukin (IL) 6, IL8 and interferon γ at the level of transcription, with only some quantitative differentiations observed in p16INK4a-overexpressing cells.
Conclusions
Data described here suggest that senescent cells may have similar functions in IVD homeostasis, irrespective of the origin of senescence induction.
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References
Vo NV, Hartman RA, Patil PR, Risbud MV, Kletsas D, Iatridis JC, Hoyland JA, Le Maitre CL, Sowa GA, Kang JD (2016) Molecular mechanisms of biological aging in intervertebral discs. J Orthop Res 34:1289–1306
Sivan SS, Hayes AJ, Wachtel E, Caterson B, Merkher Y, Maroudas A, Brown S, Roberts S (2014) Biochemical composition and turnover of the extracellular matrix of the normal and degenerate intervertebral disc. Eur Spine J 23(Suppl 3):S344–S353
Le Maitre CL, Freemont AJ, Hoyland JA (2004) Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc. J Pathol 204:47–54
Pockert AJ, Richardson SM, Le Maitre CL, Lyon M, Deakin JA, Buttle DJ, Freemont AJ, Hoyland JA (2009) Modified expression of the ADAMTS enzymes and tissue inhibitor of metalloproteinases 3 during human intervertebral disc degeneration. Arthritis Rheumatol 60:482–491
Bachmeier BE, Nerlich A, Mittermaier N, Weiler C, Lumenta C, Wuertz K, Boos N (2009) Matrix metalloproteinase expression levels suggest distinct enzyme roles during lumbar disc herniation and degeneration. Eur Spine J 18:1573–1586
Vo NV, Hartman RA, Yurube T, Jacobs LJ, Sowa GA, Kang JD (2013) Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration. Spine J 13:331–341
Wuertz K, Vo N, Kletsas D, Boos N (2012) Inflammatory and catabolic signalling in intervertebral discs: the roles of NF-kappaB and MAP kinases. Eur Cell Mater 23:103–119 (discussion 119–120)
Cuellar JM, Golish SR, Reuter MW, Cuellar VG, Angst MS, Carragee EJ, Yeomans DC, Scuderi GJ (2010) Cytokine evaluation in individuals with low back pain using discographic lavage. Spine J 10:212–218
Andrade P, Hoogland G, Garcia MA, Steinbusch HW, Daemen MA, Visser-Vandewalle V (2013) Elevated IL-1beta and IL-6 levels in lumbar herniated discs in patients with sciatic pain. Eur Spine J 22:714–720
Risbud MV, Shapiro IM (2014) Role of cytokines in intervertebral disc degeneration: pain and disc content. Nat Rev Rheumatol 10:44–56
Gruber HE, Ingram JA, Norton HJ, Hanley EN Jr (2007) Senescence in cells of the aging and degenerating intervertebral disc: immunolocalization of senescence-associated beta-galactosidase in human and sand rat discs. Spine 32:321–327 (Phila Pa 1976)
Le Maitre CL, Freemont AJ, Hoyland JA (2007) Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther 9:R45
Roberts S, Evans EH, Kletsas D, Jaffray DC, Eisenstein SM (2006) Senescence in human intervertebral discs. Eur Spine J 15(Suppl 3):S312–S316
Feng C, Liu H, Yang M, Zhang Y, Huang B, Zhou Y (2016) Disc cell senescence in intervertebral disc degeneration: causes and molecular pathways. Cell Cycle 15:1674–1684
Coppe JP, Patil CK, Rodier F, Sun Y, Munoz DP, Goldstein J, Nelson PS, Desprez PY, Campisi J (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6:2853–2868
Jeong SW, Lee JS, Kim KW (2014) In vitro lifespan and senescence mechanisms of human nucleus pulposus chondrocytes. Spine J 14:499–504
Kim KW, Chung HN, Ha KY, Lee JS, Kim YY (2009) Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs. Spine J 9:658–666
Johnson WE, Roberts S (2003) Human intervertebral disc cell morphology and cytoskeletal composition: a preliminary study of regional variations in health and disease. J Anat 203:605–612
Kletsas D (2009) Senescent cells in the intervertebral disc: numbers and mechanisms. Spine J 9:677–678
Dimozi A, Mavrogonatou E, Sklirou A, Kletsas D (2015) Oxidative stress inhibits the proliferation, induces premature senescence and promotes a catabolic phenotype in human nucleus pulposus intervertebral disc cells. Eur Cell Mater 30:89–102 (discussion 103)
Liakou E, Mavrogonatou E, Pratsinis H, Rizou S, Evangelou K, Panagiotou PN, Karamanos NK, Gorgoulis VG, Kletsas D (2016) Ionizing radiation-mediated premature senescence and paracrine interactions with cancer cells enhance the expression of syndecan 1 in human breast stromal fibroblasts: the role of TGF-beta. Aging (Albany NY) 8:1650–1669
Papadopoulou A, Kletsas D (2011) Human lung fibroblasts prematurely senescent after exposure to ionizing radiation enhance the growth of malignant lung epithelial cells in vitro and in vivo. Int J Oncol 39:989–999
Mavrogonatou E, Kletsas D (2009) High osmolality activates the G1 and G2 cell cycle checkpoints and affects the DNA integrity of nucleus pulposus intervertebral disc cells triggering an enhanced DNA repair response. DNA Repair (Amst) 8:930–943
Coppe JP, Rodier F, Patil CK, Freund A, Desprez PY, Campisi J (2011) Tumor suppressor and aging biomarker p16INK4a induces cellular senescence without the associated inflammatory secretory phenotype. J Biol Chem 286:36396–36403
Coppe JP, Desprez PY, Krtolica A, Campisi J (2010) The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol 5:99–118
Tasdemir N, Lowe SW (2013) Senescent cells spread the word: non-cell autonomous propagation of cellular senescence. EMBO J 32:1975–1976
Krishnamurthy J, Ramsey MR, Ligon KL, Torrice C, Koh A, Bonner-Weir S, Sharpless NE (2006) p16INK4a induces an age-dependent decline in islet regenerative potential. Nature 443:453–457
Rutges JP, Kummer JA, Oner FC, Verbout AJ, Castelein RJ, Roestenburg HJ, Dhert WJ, Creemers LB (2008) Increased MMP-2 activity during intervertebral disc degeneration is correlated to MMP-14 levels. J Pathol 214:523–530
Weiler C, Nerlich AG, Zipperer J, Bachmeier BE, Boos N (2002) 2002 SSE Award Competition in Basic Science: expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption. Eur Spine J 11:308–320
Purcell M, Kruger A, Tainsky MA (2014) Gene expression profiling of replicative and induced senescence. Cell Cycle 13:3927–3937
Philipot D, Guerit D, Platano D, Chuchana P, Olivotto E, Espinoza F, Dorandeu A, Pers YM, Piette J, Borzi RM, Jorgensen C, Noel D, Brondello JM (2014) p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis. Arthritis Res Ther 16:R58
Urban JP (2002) The role of the physicochemical environment in determining disc cell behaviour. Biochem Soc Trans 30:858–864
Acknowledgements
We would like to thank Dr. Lesley Probert for generously providing us plasmids pLenti7.3/V5-GW/lacZverA and pLenti6gw u6laminshrna verB.
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Vamvakas, SS., Mavrogonatou, E. & Kletsas, D. Human nucleus pulposus intervertebral disc cells becoming senescent using different treatments exhibit a similar transcriptional profile of catabolic and inflammatory genes. Eur Spine J 26, 2063–2071 (2017). https://doi.org/10.1007/s00586-017-5198-0
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DOI: https://doi.org/10.1007/s00586-017-5198-0