Acid-sensing ion channel 1a regulates the survival of nucleus pulposus cells in the acidic environment of degenerated intervertebral discs

Document Type : Original Article

Authors

The Department of Orthopedics, Zhongda Hospital, Medical School of Southeast University, Nanjing, Jiangsu 210009, China

Abstract

Objective(s): Activation of acid-sensing ion channel 1a (ASIC1a) is responsible for tissue injury caused by acidosis in nervous systems. But its physiological and pathological roles in nucleus pulposus cells (NPCs) are unclear. The aim of this study is to investigate whether ASIC1a regulates the survival of NPCs in the acidic environment of degenerated discs.
Materials and Methods: NPCs were isolated and cultured followed by immunofluorescent staining and Western-blot analysis for ASIC1a. Intracellular calcium ([Ca2+]i) was determined by Ca2+-imaging using Fura-2-AM. Cell necrosis, apoptosis, and senescence following acid exposure were determined using lactate dehydrogenase (LDH) release assay, annexin V-fluorescein isothiocyanate/propidium iodide dual-staining and cell cycle analysis, respectively, followed by Western-blot analysis for apoptosis-related proteins (Bax, Bcl-2, and caspase-3) and senescence-related proteins (p53, p21, and p16). Effects of treatment with psalmotoxin-1 (PcTX1, blocker of ASIC1a) on [Ca2+]i and cell survival were investigated.
Results:ASIC1a was detected in healthy NPCs, and its expression was significantly higher in degenerated cells. When NPCs were treated with PcTX1, acid-induced increases in [Ca2+]i were significantly inhibited. PcTX1 treatment also resulted in decreased LDH release, cell apoptosis and cell cycle arrest in acid condition. Acid exposure decreased the expression of Bcl-2 and increased the expression of Bax, cleaved caspase-3 and senescence-related proteins (p53, p21, and p16), which was inhibited by PcTX1.
Conclusion: The present findings suggest that further understanding of ASIC1a functionality may provide not only a novel insight into intervertebral disc biology but also a novel therapeutic target for intervertebral disc degeneration.

Keywords


1.   Martin BI, Deyo RA, Mirza SK, Turner JA, Comstock BA, Hollingworth W, et al. Expenditures and health status among adults with back and neck problems. JAMA 2008; 299:656-664.
2.   Dagenais S, Caro J, Haldeman S. A systematic review of low back pain cost of illness studies in the United States and internationally. Spine J 2008; 8:8-20.
3.   Sakai D, Grad S. Advancing the cellular and molecular therapy for intervertebral disc disease. Adv Drug Deliv Rev 2015; 84:159-171.
4.   Leung VY, Chan D, Cheung KM. Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J 2006; 15:S406-413.
5. Wuertz K, Godburn K, Iatridis JC. MSC response to pH levels found in degenerating intervertebral discs. Biochem Biophys Res Commun 2009; 379:824-829.
6.   Stairmand JW, Holm S, Urban JP. Factors influencing oxygen concentration gradients in the intervertebral disc. A theoretical analysis. Spine 1991; 16:444-449.
7.   Ishihara H, Urban JP. Effects of low oxygen concentrations and metabolic inhibitors on proteoglycan and protein synthesis rates in the intervertebral disc. J Orthop Res 1999; 17:829-835.
8.   Li H, Liang C, Tao Y, Zhou X, Li F, Chen G, et al. Acidic pH conditions mimicking degenerative intervertebral discs impair the survival and biological behavior of human adipose-derived mesenchymal stem cells. Exp Biol Med 2012; 237:845-852.
9.   Razaq S, Wilkins RJ, Urban JP. The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus. Eur Spine J 2003; 12:341-349.
10. Gray ML, Pizzanelli AM, Grodzinsky AJ, Lee RC. Mechanical and physiochemical determinants of the chondrocyte biosynthetic response. J Orthop Res 1988; 6:777-792.
11. Olmarker K, Larsson K. Tumor necrosis factor alpha and nucleus-pulposus-induced nerve root injury. Spine 1998; 23:2538-2544.
12. Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Evans CH. Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2. Spine (Phila Pa 1976) 1996; 21:271-277.
13. Stefanovic-Racic M, Stadler J, Georgescu HI, Evans CH. Nitric oxide and energy production in articular chondrocytes. J Cell Physiol 1994; 159:274-280.
14. Ichimura K, Tsuji H, Matsui H, Makiyama N. Cell culture of the intervertebral disc of rats: factors influencing culture, proteoglycan, collagen, and deoxyribonucleic acid synthesis. J Spinal Disord 1991; 4:428-436.
15. Diamant B, Karlsson J, Nachemson A. Correlation between lactate levels and pH in discs of patients with lumbar rhizopathies. Experientia 1968; 24:1195-1196.
16. Kitano T, Zerwekh JE, Usui Y, Edwards ML, Flicker PL, Mooney V. Biochemical changes associated with the symptomatic human intervertebral disk. Clin Orthop Relat Res 1993; 372-377.
17. Yermolaieva O, Leonard AS, Schnizler MK, Abboud FM, Welsh MJ. Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a. Proc Natl Acad Sci U S A 2004; 101:6752-6757.
18. Cuesta A, Del Valle ME, Garcia-Suarez O, Vina E, Cabo R, Vazquez G, et al. Acid-sensing ion channels in healthy and degenerated human intervertebral disc. Connect Tissue Res 2014; 55:197-204.
19. Wang YZ, Xu TL. Acidosis, acid-sensing ion channels, and neuronal cell death. Mol Neurobiol 2011; 44:350-358.
20. Li X, Wu FR, Xu RS, Hu W, Jiang DL, Ji C, et al. Acid-sensing ion channel 1a-mediated calcium influx regulates apoptosis of endplate chondrocytes in intervertebral discs. Expert Opin Ther Targets 2014; 18:1-14.
21. Ye JH, Gao J, Wu YN, Hu YJ, Zhang CP, Xu TL. Identification of acid-sensing ion channels in adenoid cystic carcinomas. Biochem Biophys Res Commun 2007; 355:986-992.
22. Berridge MJ, Bootman MD, Lipp P. Calcium--a life and death signal. Nature 1998; 395:645-648.
23. Pfirrmann CW, Metzdorf A, Zanetti M, Hodler J, Boos N. Magnetic resonance classification of lumbar intervertebral disc degeneration. Spine (Phila Pa 1976) 2001; 26:1873-1878.
24. Xiong ZG, Zhu XM, Chu XP, Minami M, Hey J, Wei WL, et al. Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels. Cell 2004; 118:687-698.
25. Ciapetti G, Granchi D, Devescovi V, Leonardi E, Greggi T, Di Silvestre M, et al. Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs. Eur Spine J 2012; 21:S10-19.
26. Mamet J, Baron A, Lazdunski M, Voilley N. Proinflammatory mediators, stimulators of sensory neuron excitability via the expression of acid-sensing ion channels. J Neurosci 2002; 22:10662-10670.
27. Yingjun G, Xun Q. Acid-sensing ion channels under hypoxia. Channels (Austin) 2013; 7:231-237.
28. Yuan FL, Chen FH, Lu WG, Li X, Wu FR, Li JP, et al. Acid-sensing ion channel 1a mediates acid-induced increases in intracellular calcium in rat articular chondrocytes. Mol Cell Biochem 2010; 340:153-159.
29. MacDonald G, Shi L, Vande Velde C, Lieberman J, Greenberg AH. Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis. J Exp Med 1999; 189:131-144.
30. Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, et al. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 1999; 284:339-343.
31. Egnatchik RA, Leamy AK, Jacobson DA, Shiota M, Young JD. ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload. Mol Metabol 2014; 3:544-553.
32. Passos JF, Saretzki G, Ahmed S, Nelson G, Richter T, Peters H, et al. Mitochondrial dysfunction accounts for the stochastic heterogeneity in telomere-dependent senescence. PLoS Biol 2007; 5:e110.
33. Balaban RS, Nemoto S, Finkel T. Mitochondria, oxidants, and aging. Cell 2005; 120:483-495.
34. Kim JS, Kim EJ, Kim HJ, Yang JY, Hwang GS, Kim CW. Proteomic and metabolomic analysis of H2O2-induced premature senescent human mesenchymal stem cells. Exp Gerontol 2011; 46:500-510.
35. Zhao CQ, Wang LM, Jiang LS, Dai LY. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev 2007; 6:247-261.
36. Gruber HE, Ingram JA, Norton HJ, Hanley EN Jr. Senescence in cells of the aging and degenerating intervertebral disc: immunolocalization of senescence-associated beta-galactosidase in human and sand rat discs. Spine (Phila Pa 1976) 2007; 32:321-327.