The Roles of MAPKs in Rabbit Nucleus Pulposus Cell Apoptosis Induced by High Osmolality
Authors: Wang D. , Li Fanghui , Liu Ruilong , Wei Jianwei , Zhou Chuanli 
Published: May 2014

Introduction

Earlier work has suggested that the p38 MAPK, JNK1/2, and ERK1/2 signal pathway existed in nucleus pulposus cells and the cell growth, differentiation, and apoptosis were regulated by them. Because osmotic fluctuations are inevitable in the physicochemical environment of intervertebral disc cells, high osmolality could activate p38 MAPK, JNK1/2, and ERK1/2 signal pathway. The effects of high osmolality on the catabolic program and proliferation of nucleus pulposus cells are still not clear.

Materials and Methods

Rabbit nucleus pulposus cells were cultured and divided into different group at random. The cells were pretreated with inhibitor for p38 MAPK, JNK1/2, and ERK1/2 signal pathway, respectively. In next step, the cells were cultured in different osmolality environment for different time at 37°C in 5% carbon dioxide incubator. After treatments, ratio of apoptosis was measured by flow cytometry, and Western blotting was performed to quantify the expression of the activated forms of p38 MAPK, JNK1/2, and ERK1/2. Furthermore, immunofluorescence analysis with confocal microscopy was performed to confirm the hyperosmolality effects on activation of p38 MAPK, JNK1/2, and ERK1/2 signal pathways in nucleus pulposus cells.

Results

Our results show that in 500 and 600 mOsm/kg medium, rabbit nucleus pulposus cell apoptosis increased, and a persistent phosphorylation of p38 MAPK, JNK1/2, and ERK1/2 proteins were observed. In the same condition, the apoptotic cells death remarkably decreased when the p38 MAPK and JNK1/2 signal pathways were blocked by their inhibitors SB203580 and SP600125, respectively. On the other side, the apoptotic cells death rate reraised greatly when the ERK1/2 signal pathways were blocked by its inhibitor PD98059.

Conclusion

High osmolality activated p38MAPK, JNK1/2, and ERK1/2 in rabbit nucleus pulposus cell, and the activated p38 MAPK and JNK1/2 induced cell apoptosis, on the contrary, the activated ERK1/2 made the cell survived. Disclosure of Interest None declared References Freemont AJ. The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford) 2009;48(1):5–10 Urban JP, McMullin JF. Swelling pressure of the inervertebral disc: influence of proteoglycan and collagen contents. Biorheology 1985;22(2):145–157 Wilke HJ, Neef P, Caimi M, Hoogland T, Claes LE. New in vivo measurements of pressures in the intervertebral disc in daily life. Spine 1999;24(8):755–762 Takeno K, Kobayashi S, Negoro K, et al. Physical limitations to tissue engineering of intervertebral disc cells: effect of extracellular osmotic change on glycosaminoglycan production and cell metabolism. Laboratory investigation. J Neurosurg Spine 2007;7(6):637–644 Boyd LM, Richardson WJ, Chen J, Kraus VB, Tewari A, Setton LA. Osmolarity regulates gene expression in intervertebral disc cells determined by gene array and real-time quantitative RT-PCR. Ann Biomed Eng 2005;33(8):1071–1077 Wuertz K, Urban JP, Klasen J, et al. Influence of extracellular osmolarity and mechanical stimulation on gene expression of intervertebral disc cells. J Orthop Res 2007;25(11):1513–1522 Johnson NL, Gardner AM, Diener KM, et al. Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death. J Biol Chem 1996;271(6):3229–3237 Hiyama A, Gogate SS, Gajghate S, Mochida J, Shapiro IM, Risbud MV. BMP-2 and TGF-beta stimulate expression of beta1,3-glucuronosyl transferase 1 (GlcAT-1) in nucleus pulposus cells through AP1, TonEBP, and Sp1: role of MAPKs. J Bone Miner Res 2010;25(5):1179–1190 Raucci A, Laplantine E, Mansukhani A, Basilico C. Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes. J Biol Chem 2004;279(3):1747–1756 Borghetti P, Della Salda L, De Angelis E, et al. Adaptive cellular response to osmotic stress in pig articular chondrocytes. Tissue Cell 1995;27(2):173–183 Gasz B, Rácz B, Roth E, et al. Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis. Peptides 2006;27(1):87–94 Tsai TT, Guttapalli A, Agrawal A, Albert TJ, Shapiro IM, Risbud MV. MEK/ERK signaling controls osmoregulation of nucleus pulposus cells of the intervertebral disc by transactivation of TonEBP/OREBP. J Bone Miner Res 2007;22(7):965–974 Pritchard S, Erickson GR, Guilak F. Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton. Biophys J 2002;83(5):2502–2510 Mavrogonatou E, Kletsas D. 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) 2009;8(8):930–943 Uchiyama Y, Cheng CC, Danielson KG, et al. Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling. J Bone Miner Res 2007;22(12):1996–2006 Mavrogonatou E, Kletsas D. Effect of varying osmotic conditions on the response of bovine nucleus pulposus cells to growth factors and the activation of the ERK and Akt pathways. J Orthop Res 2010;28(10):1276–1282 Han J. The Sun (Baltim, Md) 2007;8:364-3 Hui L, Bakiri L, Stepniak E, Wagner EF. p38alpha: a suppressor of cell proliferation and tumorigenesis. Cell Cycle 2007;6(20):2429–2433 Mishra S, Mishra JP, Kumar A. Activation of JNK‐dependent pathway is required for HIV viral protein R‐induced apoptosis in human monocytic cells: involvement of antiapoptotic BCL2 and c‐IAP1 genes. J Biol Chem 2007;282(7):4288–4300 Ono K, Han J. The p38 signal transduction pathway: activation and function. Cell Signal 2000;12(1):1–13 Chen XL, Xia ZF, Yu YX, Wei D, Wang CR, Ben DF. p38 mitogen-activated protein kinase inhibition attenuates burn-induced liver injury in rats. Burns 2005;31(3):320–330 Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell 2PO.010;103(2):239–252 Guan QH, Pei DS, Zhang QG, Hao ZB, Xu TL, Zhang GY. The neuroprotective action of SP600125, a new inhibitor of JNK, on transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 via nuclear and non-nuclear pathways. Brain Res 2005;1035(1):51–59 Muniyappa H, Das KC. Activation of c-Jun N-terminal kinase (JNK) by widely used specific p38 MAPK inhibitors SB202190 and SB203580: a MLK-3-MKK7-dependent mechanism. Cell Signal 2008;20(4):675–683 Studer RK, Aboka AM, Gilbertson LG, et al. p38 MAPK inhibition in nucleus pulposus cells: a potential target for treating intervertebral disc degeneration. Spine 2007;32(25):2827–2833 Risbud MV, Fertala J, Vresilovic EJ, Albert TJ, Shapiro IM. Nucleus pulposus cells upregulate PI3K/Akt and MEK/ERK signaling pathways under hypoxic conditions and resist apoptosis induced by serum withdrawal. Spine 2005;30(8):882–889 Risbud MV, Guttapalli A, Albert TJ, Shapiro IM. Hypoxia activates MAPK activity in rat nucleus pulposus cells: regulation of integrin expression and cell survival. Spine 2005;30(22):2503–2509 Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 1995;270(46):27489–27494