高渗应激下ERK信号转导通路在兔髓核细胞凋亡中的作用
|
摘要
目的：观察高渗透压对体外培养的兔髓核细胞凋亡的影响及细胞外信号调节激酶(ERK1/2)信号转导通路在此过程中的作用。
方法：用不同的渗透压梯度以及ERK1/2特异性抑制剂PD98059(50μM)分不同作用时间处理髓核细胞；流式细胞仪检测髓核细胞在不同处理组的凋亡情况,蛋白质免疫印迹技术检测各组ERK1/2和p-ERK1/2蛋白的表达情况；免疫荧光标记技术检测p-ERK1/2蛋白在髓核细胞内的分布情况。
结果：高渗透压(600mOsm)培养基中兔髓核细胞凋亡显著增加,与对照组相比差异有统计学意义(P=0.013),并呈时间依赖性；PD98059预处理组与相应时间段同渗透压组相比,细胞凋亡进一步显著地增加(P=0.035)；400mOsm组及其抑制组在各时间段细胞凋亡均不明显；600mOsm高渗透压显著激活p-ERK1/2的表达,并且在3h组表达水平最高,与对照组比较差异显著(P=0.027),而PD98059几乎阻断p-ERK1/2的表达；免疫荧光标记检测到p-ERK1/2在600mOsm高渗组的细胞胞浆和胞核中均有分布。
结论：体外培养的兔髓核细胞能适应轻度增加的渗透压(400mOsm)环境,而高渗透压(600mOsm)应激下可显著诱导其凋亡,PD98059特异性抑制高渗透压应激诱导的短暂的ERKl/2激活并进一步明显诱导兔髓核细胞的凋亡,高渗透压应激诱导激活的ERKl/2信号转导通路具有抑制兔髓核细胞凋亡的作用。
Objective:To observe the apoptosis of primary culture rabbit nucleus pulposus cells in hypertonic environment and the effect of extracellular signal-regulated kinase/ERK signal pathways in this process.
Methods:After treatment with different osmotic pressure gradients and time, Flow cytometer/FCM was used to detect the apoptosis, Western blotting technology was used to analyse the expression of phospho-ERK1/2, and Immunofluorescence was done to observe the distribution of phospho-ERK1/2in the nucleus pulposus cells treated with hypertonic mediums.
Results:600mOsm hypertonic mediums powerfully induced nucleus pulposus cells apoptosis compared with control groups (P=0.013), and that appeared time dependence. However, pretreatment groups with PD98059(50μM) further increased apoptosis (P=0.035). Apoptosis was not obvious in400mOsm hypertonic mediums with or without PD98059.600mOsm hypertonic mediums markedly increased the expression of phospho-ERK1/2compared with control groups (P=0.027), and the expression level was highest after3hours. While PD98059(50μM) almost blocked expression of phospho-ERK1/2. The results of immunofluorescence showed that phospho-ERK1/2dispersed throughout cytoplasm and nucleus.
Conclusion:In hypertonic mediums (600mOsm) nucleus pulposus cells apoptosis obviously increased and anti-apoptosis of nucleus pulposus cells was related to activation of ERK1/2, moreover nucleus pulposus cells could adapt low-grade hypertonic environment (400mOsm).
方法：用不同的渗透压梯度以及ERK1/2特异性抑制剂PD98059(50μM)分不同作用时间处理髓核细胞；流式细胞仪检测髓核细胞在不同处理组的凋亡情况,蛋白质免疫印迹技术检测各组ERK1/2和p-ERK1/2蛋白的表达情况；免疫荧光标记技术检测p-ERK1/2蛋白在髓核细胞内的分布情况。
结果：高渗透压(600mOsm)培养基中兔髓核细胞凋亡显著增加,与对照组相比差异有统计学意义(P=0.013),并呈时间依赖性；PD98059预处理组与相应时间段同渗透压组相比,细胞凋亡进一步显著地增加(P=0.035)；400mOsm组及其抑制组在各时间段细胞凋亡均不明显；600mOsm高渗透压显著激活p-ERK1/2的表达,并且在3h组表达水平最高,与对照组比较差异显著(P=0.027),而PD98059几乎阻断p-ERK1/2的表达；免疫荧光标记检测到p-ERK1/2在600mOsm高渗组的细胞胞浆和胞核中均有分布。
结论：体外培养的兔髓核细胞能适应轻度增加的渗透压(400mOsm)环境,而高渗透压(600mOsm)应激下可显著诱导其凋亡,PD98059特异性抑制高渗透压应激诱导的短暂的ERKl/2激活并进一步明显诱导兔髓核细胞的凋亡,高渗透压应激诱导激活的ERKl/2信号转导通路具有抑制兔髓核细胞凋亡的作用。
Objective:To observe the apoptosis of primary culture rabbit nucleus pulposus cells in hypertonic environment and the effect of extracellular signal-regulated kinase/ERK signal pathways in this process.
Methods:After treatment with different osmotic pressure gradients and time, Flow cytometer/FCM was used to detect the apoptosis, Western blotting technology was used to analyse the expression of phospho-ERK1/2, and Immunofluorescence was done to observe the distribution of phospho-ERK1/2in the nucleus pulposus cells treated with hypertonic mediums.
Results:600mOsm hypertonic mediums powerfully induced nucleus pulposus cells apoptosis compared with control groups (P=0.013), and that appeared time dependence. However, pretreatment groups with PD98059(50μM) further increased apoptosis (P=0.035). Apoptosis was not obvious in400mOsm hypertonic mediums with or without PD98059.600mOsm hypertonic mediums markedly increased the expression of phospho-ERK1/2compared with control groups (P=0.027), and the expression level was highest after3hours. While PD98059(50μM) almost blocked expression of phospho-ERK1/2. The results of immunofluorescence showed that phospho-ERK1/2dispersed throughout cytoplasm and nucleus.
Conclusion:In hypertonic mediums (600mOsm) nucleus pulposus cells apoptosis obviously increased and anti-apoptosis of nucleus pulposus cells was related to activation of ERK1/2, moreover nucleus pulposus cells could adapt low-grade hypertonic environment (400mOsm).
引文
1. Wilke HJ, Neef P, Caimi M. et al. New In Vivo Measurements of Pressures in the Intervertebral Disc in Daily Life [J]. Spine 1999,24(8):755-762.
2. Walsh AJ, Lotz JC. Biological response of the intervertebral disc to dynamic loading [J].Biomech,2004,37 (3):329-337.
3. Tsai TT, Danielson KG, Guttapalli A et al. TonEBP/OREBP Is a Regulator of Nucleus Pulposus Cell Function and Survival in the Intervertebral Disc [J].Biological Chemistry,2006,281(35):25416-25424.
4. Paul H, PHilip ER. The strain-dependent osmotic pressure and stiffness of the bovine nucleus pulposus apportioned into ionic and non-ionic contributors [J]. Biomechanics 200841:2411-2416.
5. Urban J.P. The role of the pHysicochemical environment in determining disc cell behaviour [J]. Biochemical Society transactions 2002,30(6):858-864.
6. Pritchard S, Erickson GR, Guilak F. Hyperosmotically Induced Volume Change and Calcium Signaling in Intervertebral Disk Cells:The Role of the Actin Cytoskeleton [J]. BiopHysical Journal 2002,83:2502-2510.
7. 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):637-644.
8. Tsai TT, Guttapalli A, Agrawal A. et al. MEK/ERK signaling controls osmoregulation of nucleus pulposus cells of the intervertebral disc by transactivation of TonEBP/OREBP [J]. Bone Miner Res 2007 (7):965-974.
9. Junger H, Edelman DB, Junger WG. et al. Hypertonicity promotes survival of corticospinal motoneurons via mitogen-activated protein kinase p38 signaling [J]. Mol Neurosci,2003,21(2):111-120.
10. Racz B, Reglodi D, Fodor B. et al. Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes [J]. Bone 2007,40 1536-1543.
11. Gentile LB, Piva B, Capizzani BC. et al. Hypertonic environment elicits cyclooxygenase-2-driven prostaglandin E2 generation by colon cancer cells:role of cytosolic phospholipase A2-alpha and kinase signaling pathways [J]. Prostaglandins Leukot Essent Fatty Acids.2010;82(2-3):131-139.
12. Paleari L, Catassi A, Ciarlo M. et al. Role of a7-nicotinic acetylcholine receptor in human non-small cell lung cancer proliferation [J]. Cell Prolif,2008 (41):936-959.
13. Alessi DR, Cuenda A, Cohen P. et al. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo [J]. Biological Chemistry 1995,17,270 (46):27489-27494.
14. Risbud MV, Fertala J, Vresilovic EJ et al. Nucleus pulposus cells upregulate PI3K/Akt and MEK/ERK signaling pathways under hypoxic conditions and resist apoptosis induced by serum withdrawal [J]. Spine 2005,30(8):882-889.
15. Risbud MV, Guttapalli A, Albert TJ. et al. Hypoxia Activates MAPK Activity in Rat Nucleus Pulposus Cells:Regulation of Integrin Expression and Cell Survival [J]. Spine 2005,30:2503-2509.
16. Ishihara H, Warensjo K, Roberts S. et al. Proteoglycan synthesis in the intervertebral disk nucleus:the role of extracellular osmolality [J]. Am. J. Physiol.272 (41) 1997: C1499-C1506.
17. 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.
18. Lee JS, Lee JJ, Seo JS. HSP70 Deficiency Results in Activation of c-Jun N-terminal Kinase, Extracellular Signal-regulated Kinase, and Caspase-3 in Hyperosmolarity-induced Apoptosis [J]. BIOLOGICAL CHEMISTRY, Vol.280, No. 8,2005:6634-6641.
19. Maruyama T, Kadowaki H, Okamoto N.et al. CHIP-dependent termination of MEKK2 regulates temporal ERK activation required for proper hyperosmotic response [J]. The EMBO Journal,2010 (29):2501-2514.
20. 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.
21. Chen J, Baer AE, Paik PY et al. Matrix protein gene expression in intervertebral disc cells subjected to altered osmolarity [J]. Biochemical and Biophysical Research Communications 2002,293:932-938.
22. Boyd LM, Richardson WJ, Chen J et al. Osmolarity Regulates Gene Expression in Intervertebral Disc Cells Determined by Gene Array and Real-Time Quantitative RT-PCR [J]. Annals of Biomedical Engineering 2005,33(8):1071-1077.
23. Gao J, Li T, Lu L et al. Functional role of CCCTC binding factor in insulin-stimulatedcell proliferation [J]. Cell Prolif,2007 (40):795-808.
24. Zhang L, Niu T, Yang SY et al. The Occurrence and Regional Distribution of DR4 on Herniated Disc Cells-A Potential Apoptosis Pathway in Lumbar Intervertebral Disc [J]. Spine 2008,33:422-427.
25. Zhao CQ, Wang LM, Jiang LS et al. The cell biology of intervertebral disc aging and degeneration [J]. Ageing Research Reviews 2007,6(3):247-261.
26. Neidlinger WC, Wiirtz K, Urban JP et al. Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure [J]. Spine 2006,15(3):S372-S378.
1. Caffrey DR, O'Neill LA, Shields DC.The evolution of the MAP kinase pathways: coduplication of interacting proteins leads to new signaling cascades [J] Mol Evol. 1999,49(5):567-582.
2. Widmann C, Gibson S, Jarpe MB et al. Mitogen-activated protein kinase:cones-rvation of a three-kinase module from yeast to human. [J] Physiol Rev. 1999,79(1):143-180.
3. Seger R, Krebs EG. The MAPK signaling cascade [J] FASEB J.1995,9 (9):726-735.
4. Waskiewicz AJ, Cooper JA. Mitogen and stress response pathways:MAP kinase cascades and phosphatase regulation in mammals and yeast [J]Curr Opin Cell Biol. 1995,7(6):798-805.
5. Tibbles LA, Woodgett JR. The stress-activated protein kinase pathways [J]Cell Mol Life Sci.1999,55(10):1230-1254.
6. Davis RJ. Signal transduction by the JNK group of MAP kinases [J] Cell. 2000,103(2):239-252.
7. Chang L, Karin M. Mammalian MAP kinase signalling cascades [J] Nature. 2001,410(6824):37-40.
8. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases [J] Science.2002,298(5600):1911-1912.
9. Werlen G, Hausmann B, Naeher D et al. Signaling life and death in the thymus: timing is everything [J] Science.2003,299(5614):1859-1863.
10. Zhou. G, Bao ZQ, Dixon JE. Components of a new human protein kinase signal transduction pathway [J] Biol Chem.1995,270(21):12665-12669.
11. Abe MK, Kuo WL, Hershenson MB et al. Extracellular signal- regulated kinase 7 (ERK7), a novel ERK with a C-terminal domain that regulates its activity, its cellular localization, and cell growth [J] Mol Cell Biol.1999,19 (2):1301-1312.
12. Abe MK, Saelzler MP, Espinosa R et al. ERK8, a new member of the mitogen-activated protein kinase family [J] Biol Chem.2002,277(19):16733-16743.
13. Crews CM, Alessandrini A, Erikson RL. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product [J] Science,1992,258(50 81):478-480.
14. Alessi DR, Saito Y, Campbell DG et al. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1 [J] EMBO J.1994,13(7):1610-1619.
15. Davis RJ. The mitogen-activated protein kinase signal transduction pathway [J] Biological Chemistry,1993,268 (20):14553-14556.
16. Janknecht R, Ernst WH, Pingoud V et al. Activation of ternary complex factor Elk-1 by MAP kinases [J] EMBO J.1993,12(13):5097-5104.
17. Treisman R. Regulation of transcription by MAP kinase cascades [J] Curr Opin Cell Biol.1996,8(2):205-215.
18. Shaharabany M, Holtzman EJ, Mayan H et al. Distinct pathways for the involvement of WNK4 in the signaling of hypertonicity and EGF [J] FEBS 2008,275(8): 1631-1642.
19. Favata MF, Horiuchi KY, Manos EJ et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase [J] Biol Chem.1998,273(29):18623-18632.
20. Alessi DR, Cuenda A, Cohen P et al. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo [J] Biological Chemistry.1995,270(46):27489-27494.
21. Kojima R, Taniguchi H, Tsuzuki A et al. Hypertonicity-induced expression of monocyte chemoattractant protein-1 through a novel cis-acting element and MAPK signaling pathways [J] Immunol.2010,184(9):5253-5262.
22. Junger H, Edelman DB, Junger WG. Hypertonicity promotes survival of corticospinal motoneurons via mitogen-activated protein kinase p38 signaling [J] Mol Neurosci. 2003;21(2):111-120.
23. Cuschieri J, Gourlay D, Garcia I et al. Hypertonic precondi-tioning inhibits macrophage responsiveness to endotoxin [J] Immunol.2002,168(3):1389-1396.
24. Chen Y, Hashiguchi N, Yip L et al. Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors [J] Cell Physiology. 2006,290(4):C1051-C1059.
25. Shaharabany M, Holtzman EJ, Mayan H et al. Distinct pathways for the involvement of WNK4 in the signaling of hypertonicity and EGF [J] FEBS,2008,275 (8):1631-1642.
26. Luo L, Li DQ, Corrales RM et al. Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface [J] Eye Contact Lens.2005,31(5):186-193.
27. Umenishi F, Schrier RW. Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene [J] Biol Chem.2003,278(18):15765-15770.
28. Gillis D, Shrode LD, Krump E et al. Osmotic stimulation of the Na+/H+exchanger NHE1:relationship to the activation of three MAPK pathways [J] Membranous Biology.2001,181(3):205-214.
29. Luo L, Li DQ, Pflugfelder SC. Hyperosmolarity-induced apoptosis in human corneal epithelial cells is mediated by cytochrome c and MAPK pathways [J] Cornea. 2007,26(4):452-460.
30. Zhuang S, Hirai SI, Ohno S. Hyperosmolality induces activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells [J] Cell Physiology. 2000,278(1):C102-C109.
31. Roger F, Martin PY, Rousselot M et al. Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle's loop. Requirement of p38 kinase for the regulatory volume increase response [J] Biological Chemistry.1999,274 (48):34103-34110.
32. Urban JP. The role of the physicochemical environment in determining disc cell behaviour [J]. Biochemical Society Transactions,2002,30:858-864.
33. Tsai TT, Danielson KG, Guttapalli A et al. TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc [J] Biological Chemistry. 2006,281 (35):25416-25424.
34. Tsai TT, Guttapalli A, Agrawal A et al. 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.
35. Li L, Xu L, Qu X et al. Cbl-regulated Akt and ERK signals are involved in β-elemene-induced cell apoptosis in lung cancer cells [J] Mol Med Report. 2011,4(6):1243-1246.
36. Liu HZ, Yu C, Yang Z et al. Tubeimoside I sensitizes cisplatin in cisplatin-resistant human ovarian cancer cells (A2780/DDP) through down-regulation of ERK and up-regulation of p38 signaling pathways [J] Mol Med Report.2011,4(5):985-992.
37. Xu C, Gui Q, Chen W et al. Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo [J] Exp Clin Cancer Res. 2011,30:63.
38. Xiao CL, Tao ZH, Guo L et al. Isomalto oligosaccharide sulfate inhibits tumor growth and metastasis of hepatocellular carcinoma in nude mice [J] BMC Cancer. 2011,11:150.
39. Wang S, Huang X, Li Y et al. RN181 suppresses hepatocellular carcinoma growth by inhibition of the ERK/MAPK pathway [J] Hepatology.2011,53(6):1932-1942.
40. Xu ZW, Wang FM, Gao MJ et al. Cardiotonic steroids attenuate ERK phosphorylation and generate cell cycle arrest to block human hepatoma cell growth [J] Steroid Biochem Mol Biol.2011,125(3-5):181-91.
41. Zheng JF, Sun LC, Liu H et al. EBP50 exerts tumor suppressor activity by promoting cell apoptosis and retarding extracellular signal-regulated kinase activity [J] Amino Acids.2010,38(4):1261-1268.
42. Wu FS, Zheng SS, Wu LJ et al. Calcitriol inhibits the growth of MHCC97 heptocellular cell lines by down-modulating c-met and ERK expressions [J] Liver Int. 2007,(5):700-707.
43. Li D, Wu LJ, Tashiro S et al. Oridonin-induced A431 cell apoptosis partially through blockage of the Ras/Raf/ERK signal pathway [J] Pharmacol Sci.2007,103(l):56-66.
44. Liu L, Cao Y, Chen C et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5 [J] Cancer Res.2006,66(24):11851-11858.
45. Sutter AP, Maaser K, Gerst B et al. Enhancement of peripheral benzodiazepine receptor ligand-induced apoptosis and cell cycle arrest of esophageal cancer cells by simultaneous inhibition of MAPK/ERK kinase [J] Biochem Pharmacol.2004, 67(9):1701-1710.
46. Fan Y, Chen H, Qiao B et al. Opposing effects of ERK and p38 MAP kinases on HeLa cell apoptosis induced by dipyrithione [J] Mol Cells.2007,23(1):30-38.
47. Gentile LB, Piva B, Capizzani BC et al. Hypertonic environment elicits cyclooxygenase-2-driven prostaglandin E2 generation by colon cancer cells:role of cytosolic phospholipase A2-alpha and kinase signaling pathways [J] Prostaglandins Leukot Essent Fatty Acids.2010,82(2-3):131-139.
48. Wang J, Kuiatse I, Lee AV et al. Sustained c-Jun-NH2-kinase activity promotes epithelial-mesenchymal transition, invasion, and survival of breast cancer cells by regulating extracellular signal-regulated kinase activation [J] Mol Cancer Res.2010, 8(2):266-277.
49. Belyanskaya LL, Ziogas A, Hopkins DS et al. TRAIL-induced survival and proliferation of SCLC cells is mediated by ERK and dependent on TRAIL-R2/DR5 expression in the absence of caspase-8 [J] Lung Cancer.2008,60(3):355-365.
50. Prakash GJ, Suman P, Prieto DM et al. Leukaemia inhibitory factor mediated proliferation of HTR-8/SVneo trophoblast cells is dependent on activation of extracellular signal-regulated kinase 1/2 [J] Reprod Fertil Dev.2011,23(5):714-724.
51. Du K, Zheng Q, Zhou M et al. Chlamydial antiapoptotic activity involves activation of the Raf/MEK/ERK survival pathway [J] Curr Microbiol.2011,63(4):341-346.
52. Malki A, Youssef A. Antidiabetic drug metformin induces apoptosis in human MCF breast cancer via targeting ERK signaling [J] Oncol Res.2011,19(6):275-285.
53. Tong JS, Zhang QH, Huang X et al. Icaritin causes sustained ERK1/2 activation and induces apoptosis. in human endometrial cancer cells [J] PLoS One.2011, 6(3):e16781.
54. Guo Y, Zhang X, Meng J et al. An anticancer agent icaritin induces sustained activation of the extracellular signal-regulated kinase (ERK) pathway and inhibits growth of breast cancer cells [J] Eur J Pharmacol.2011,658(2-3):114-122.
55. Racz B, Reglodi D, Fodor B et al. Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes [J] Bone.2007,40(6):1536-1543.
56. Lee SM, Lee YJ, Kang DG et al. Effect of Poria cocos on hypertonic stress-induced water channel expression and apoptosis in renal collecting duct cells [J] Ethnopharmacol.2012,6.
57. Han Q, Zhang X, Xue R et al. AMPK potentiates hypertonicity-induced apoptosis by suppressing NFκB/COX-2 in medullary interstitial cells [J] Soc Nephrol.2011,22 (10):1897-1911.
58. Schenk LK, Rinschen MM, Klokkers J et al. Cyclosporin-A induced toxicity in rat renal collecting duct cells:interference with enhanced hypertonicity induced apoptosis [J] Cell Physiol Biochem.2010;26(6):887-900.
59. Nishimura T, Sai Y, Fujii J et al. Roles of TauT and system A in cytoprotection of rat syncytiotrophoblast cell line exposed to hypertonic stress [J] Placenta.2010,31 (11):1003-1009.
60. Bevilacqua E, Wang X, Majumder M et al. eIF2alpha phosphory-lation tips the balance to apoptosis during osmotic stress [J] Biol Chem.2010,285(22): 17098-17111.
61. Racz B, Reglodi D, Fodor B et al. Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes [J] Bone.2007,40(6):1536-1543.
2. Walsh AJ, Lotz JC. Biological response of the intervertebral disc to dynamic loading [J].Biomech,2004,37 (3):329-337.
3. Tsai TT, Danielson KG, Guttapalli A et al. TonEBP/OREBP Is a Regulator of Nucleus Pulposus Cell Function and Survival in the Intervertebral Disc [J].Biological Chemistry,2006,281(35):25416-25424.
4. Paul H, PHilip ER. The strain-dependent osmotic pressure and stiffness of the bovine nucleus pulposus apportioned into ionic and non-ionic contributors [J]. Biomechanics 200841:2411-2416.
5. Urban J.P. The role of the pHysicochemical environment in determining disc cell behaviour [J]. Biochemical Society transactions 2002,30(6):858-864.
6. Pritchard S, Erickson GR, Guilak F. Hyperosmotically Induced Volume Change and Calcium Signaling in Intervertebral Disk Cells:The Role of the Actin Cytoskeleton [J]. BiopHysical Journal 2002,83:2502-2510.
7. 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):637-644.
8. Tsai TT, Guttapalli A, Agrawal A. et al. MEK/ERK signaling controls osmoregulation of nucleus pulposus cells of the intervertebral disc by transactivation of TonEBP/OREBP [J]. Bone Miner Res 2007 (7):965-974.
9. Junger H, Edelman DB, Junger WG. et al. Hypertonicity promotes survival of corticospinal motoneurons via mitogen-activated protein kinase p38 signaling [J]. Mol Neurosci,2003,21(2):111-120.
10. Racz B, Reglodi D, Fodor B. et al. Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes [J]. Bone 2007,40 1536-1543.
11. Gentile LB, Piva B, Capizzani BC. et al. Hypertonic environment elicits cyclooxygenase-2-driven prostaglandin E2 generation by colon cancer cells:role of cytosolic phospholipase A2-alpha and kinase signaling pathways [J]. Prostaglandins Leukot Essent Fatty Acids.2010;82(2-3):131-139.
12. Paleari L, Catassi A, Ciarlo M. et al. Role of a7-nicotinic acetylcholine receptor in human non-small cell lung cancer proliferation [J]. Cell Prolif,2008 (41):936-959.
13. Alessi DR, Cuenda A, Cohen P. et al. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo [J]. Biological Chemistry 1995,17,270 (46):27489-27494.
14. Risbud MV, Fertala J, Vresilovic EJ et al. Nucleus pulposus cells upregulate PI3K/Akt and MEK/ERK signaling pathways under hypoxic conditions and resist apoptosis induced by serum withdrawal [J]. Spine 2005,30(8):882-889.
15. Risbud MV, Guttapalli A, Albert TJ. et al. Hypoxia Activates MAPK Activity in Rat Nucleus Pulposus Cells:Regulation of Integrin Expression and Cell Survival [J]. Spine 2005,30:2503-2509.
16. Ishihara H, Warensjo K, Roberts S. et al. Proteoglycan synthesis in the intervertebral disk nucleus:the role of extracellular osmolality [J]. Am. J. Physiol.272 (41) 1997: C1499-C1506.
17. 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.
18. Lee JS, Lee JJ, Seo JS. HSP70 Deficiency Results in Activation of c-Jun N-terminal Kinase, Extracellular Signal-regulated Kinase, and Caspase-3 in Hyperosmolarity-induced Apoptosis [J]. BIOLOGICAL CHEMISTRY, Vol.280, No. 8,2005:6634-6641.
19. Maruyama T, Kadowaki H, Okamoto N.et al. CHIP-dependent termination of MEKK2 regulates temporal ERK activation required for proper hyperosmotic response [J]. The EMBO Journal,2010 (29):2501-2514.
20. 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.
21. Chen J, Baer AE, Paik PY et al. Matrix protein gene expression in intervertebral disc cells subjected to altered osmolarity [J]. Biochemical and Biophysical Research Communications 2002,293:932-938.
22. Boyd LM, Richardson WJ, Chen J et al. Osmolarity Regulates Gene Expression in Intervertebral Disc Cells Determined by Gene Array and Real-Time Quantitative RT-PCR [J]. Annals of Biomedical Engineering 2005,33(8):1071-1077.
23. Gao J, Li T, Lu L et al. Functional role of CCCTC binding factor in insulin-stimulatedcell proliferation [J]. Cell Prolif,2007 (40):795-808.
24. Zhang L, Niu T, Yang SY et al. The Occurrence and Regional Distribution of DR4 on Herniated Disc Cells-A Potential Apoptosis Pathway in Lumbar Intervertebral Disc [J]. Spine 2008,33:422-427.
25. Zhao CQ, Wang LM, Jiang LS et al. The cell biology of intervertebral disc aging and degeneration [J]. Ageing Research Reviews 2007,6(3):247-261.
26. Neidlinger WC, Wiirtz K, Urban JP et al. Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure [J]. Spine 2006,15(3):S372-S378.
1. Caffrey DR, O'Neill LA, Shields DC.The evolution of the MAP kinase pathways: coduplication of interacting proteins leads to new signaling cascades [J] Mol Evol. 1999,49(5):567-582.
2. Widmann C, Gibson S, Jarpe MB et al. Mitogen-activated protein kinase:cones-rvation of a three-kinase module from yeast to human. [J] Physiol Rev. 1999,79(1):143-180.
3. Seger R, Krebs EG. The MAPK signaling cascade [J] FASEB J.1995,9 (9):726-735.
4. Waskiewicz AJ, Cooper JA. Mitogen and stress response pathways:MAP kinase cascades and phosphatase regulation in mammals and yeast [J]Curr Opin Cell Biol. 1995,7(6):798-805.
5. Tibbles LA, Woodgett JR. The stress-activated protein kinase pathways [J]Cell Mol Life Sci.1999,55(10):1230-1254.
6. Davis RJ. Signal transduction by the JNK group of MAP kinases [J] Cell. 2000,103(2):239-252.
7. Chang L, Karin M. Mammalian MAP kinase signalling cascades [J] Nature. 2001,410(6824):37-40.
8. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases [J] Science.2002,298(5600):1911-1912.
9. Werlen G, Hausmann B, Naeher D et al. Signaling life and death in the thymus: timing is everything [J] Science.2003,299(5614):1859-1863.
10. Zhou. G, Bao ZQ, Dixon JE. Components of a new human protein kinase signal transduction pathway [J] Biol Chem.1995,270(21):12665-12669.
11. Abe MK, Kuo WL, Hershenson MB et al. Extracellular signal- regulated kinase 7 (ERK7), a novel ERK with a C-terminal domain that regulates its activity, its cellular localization, and cell growth [J] Mol Cell Biol.1999,19 (2):1301-1312.
12. Abe MK, Saelzler MP, Espinosa R et al. ERK8, a new member of the mitogen-activated protein kinase family [J] Biol Chem.2002,277(19):16733-16743.
13. Crews CM, Alessandrini A, Erikson RL. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product [J] Science,1992,258(50 81):478-480.
14. Alessi DR, Saito Y, Campbell DG et al. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1 [J] EMBO J.1994,13(7):1610-1619.
15. Davis RJ. The mitogen-activated protein kinase signal transduction pathway [J] Biological Chemistry,1993,268 (20):14553-14556.
16. Janknecht R, Ernst WH, Pingoud V et al. Activation of ternary complex factor Elk-1 by MAP kinases [J] EMBO J.1993,12(13):5097-5104.
17. Treisman R. Regulation of transcription by MAP kinase cascades [J] Curr Opin Cell Biol.1996,8(2):205-215.
18. Shaharabany M, Holtzman EJ, Mayan H et al. Distinct pathways for the involvement of WNK4 in the signaling of hypertonicity and EGF [J] FEBS 2008,275(8): 1631-1642.
19. Favata MF, Horiuchi KY, Manos EJ et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase [J] Biol Chem.1998,273(29):18623-18632.
20. Alessi DR, Cuenda A, Cohen P et al. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo [J] Biological Chemistry.1995,270(46):27489-27494.
21. Kojima R, Taniguchi H, Tsuzuki A et al. Hypertonicity-induced expression of monocyte chemoattractant protein-1 through a novel cis-acting element and MAPK signaling pathways [J] Immunol.2010,184(9):5253-5262.
22. Junger H, Edelman DB, Junger WG. Hypertonicity promotes survival of corticospinal motoneurons via mitogen-activated protein kinase p38 signaling [J] Mol Neurosci. 2003;21(2):111-120.
23. Cuschieri J, Gourlay D, Garcia I et al. Hypertonic precondi-tioning inhibits macrophage responsiveness to endotoxin [J] Immunol.2002,168(3):1389-1396.
24. Chen Y, Hashiguchi N, Yip L et al. Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors [J] Cell Physiology. 2006,290(4):C1051-C1059.
25. Shaharabany M, Holtzman EJ, Mayan H et al. Distinct pathways for the involvement of WNK4 in the signaling of hypertonicity and EGF [J] FEBS,2008,275 (8):1631-1642.
26. Luo L, Li DQ, Corrales RM et al. Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface [J] Eye Contact Lens.2005,31(5):186-193.
27. Umenishi F, Schrier RW. Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene [J] Biol Chem.2003,278(18):15765-15770.
28. Gillis D, Shrode LD, Krump E et al. Osmotic stimulation of the Na+/H+exchanger NHE1:relationship to the activation of three MAPK pathways [J] Membranous Biology.2001,181(3):205-214.
29. Luo L, Li DQ, Pflugfelder SC. Hyperosmolarity-induced apoptosis in human corneal epithelial cells is mediated by cytochrome c and MAPK pathways [J] Cornea. 2007,26(4):452-460.
30. Zhuang S, Hirai SI, Ohno S. Hyperosmolality induces activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells [J] Cell Physiology. 2000,278(1):C102-C109.
31. Roger F, Martin PY, Rousselot M et al. Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle's loop. Requirement of p38 kinase for the regulatory volume increase response [J] Biological Chemistry.1999,274 (48):34103-34110.
32. Urban JP. The role of the physicochemical environment in determining disc cell behaviour [J]. Biochemical Society Transactions,2002,30:858-864.
33. Tsai TT, Danielson KG, Guttapalli A et al. TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc [J] Biological Chemistry. 2006,281 (35):25416-25424.
34. Tsai TT, Guttapalli A, Agrawal A et al. 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.
35. Li L, Xu L, Qu X et al. Cbl-regulated Akt and ERK signals are involved in β-elemene-induced cell apoptosis in lung cancer cells [J] Mol Med Report. 2011,4(6):1243-1246.
36. Liu HZ, Yu C, Yang Z et al. Tubeimoside I sensitizes cisplatin in cisplatin-resistant human ovarian cancer cells (A2780/DDP) through down-regulation of ERK and up-regulation of p38 signaling pathways [J] Mol Med Report.2011,4(5):985-992.
37. Xu C, Gui Q, Chen W et al. Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo [J] Exp Clin Cancer Res. 2011,30:63.
38. Xiao CL, Tao ZH, Guo L et al. Isomalto oligosaccharide sulfate inhibits tumor growth and metastasis of hepatocellular carcinoma in nude mice [J] BMC Cancer. 2011,11:150.
39. Wang S, Huang X, Li Y et al. RN181 suppresses hepatocellular carcinoma growth by inhibition of the ERK/MAPK pathway [J] Hepatology.2011,53(6):1932-1942.
40. Xu ZW, Wang FM, Gao MJ et al. Cardiotonic steroids attenuate ERK phosphorylation and generate cell cycle arrest to block human hepatoma cell growth [J] Steroid Biochem Mol Biol.2011,125(3-5):181-91.
41. Zheng JF, Sun LC, Liu H et al. EBP50 exerts tumor suppressor activity by promoting cell apoptosis and retarding extracellular signal-regulated kinase activity [J] Amino Acids.2010,38(4):1261-1268.
42. Wu FS, Zheng SS, Wu LJ et al. Calcitriol inhibits the growth of MHCC97 heptocellular cell lines by down-modulating c-met and ERK expressions [J] Liver Int. 2007,(5):700-707.
43. Li D, Wu LJ, Tashiro S et al. Oridonin-induced A431 cell apoptosis partially through blockage of the Ras/Raf/ERK signal pathway [J] Pharmacol Sci.2007,103(l):56-66.
44. Liu L, Cao Y, Chen C et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5 [J] Cancer Res.2006,66(24):11851-11858.
45. Sutter AP, Maaser K, Gerst B et al. Enhancement of peripheral benzodiazepine receptor ligand-induced apoptosis and cell cycle arrest of esophageal cancer cells by simultaneous inhibition of MAPK/ERK kinase [J] Biochem Pharmacol.2004, 67(9):1701-1710.
46. Fan Y, Chen H, Qiao B et al. Opposing effects of ERK and p38 MAP kinases on HeLa cell apoptosis induced by dipyrithione [J] Mol Cells.2007,23(1):30-38.
47. Gentile LB, Piva B, Capizzani BC et al. Hypertonic environment elicits cyclooxygenase-2-driven prostaglandin E2 generation by colon cancer cells:role of cytosolic phospholipase A2-alpha and kinase signaling pathways [J] Prostaglandins Leukot Essent Fatty Acids.2010,82(2-3):131-139.
48. Wang J, Kuiatse I, Lee AV et al. Sustained c-Jun-NH2-kinase activity promotes epithelial-mesenchymal transition, invasion, and survival of breast cancer cells by regulating extracellular signal-regulated kinase activation [J] Mol Cancer Res.2010, 8(2):266-277.
49. Belyanskaya LL, Ziogas A, Hopkins DS et al. TRAIL-induced survival and proliferation of SCLC cells is mediated by ERK and dependent on TRAIL-R2/DR5 expression in the absence of caspase-8 [J] Lung Cancer.2008,60(3):355-365.
50. Prakash GJ, Suman P, Prieto DM et al. Leukaemia inhibitory factor mediated proliferation of HTR-8/SVneo trophoblast cells is dependent on activation of extracellular signal-regulated kinase 1/2 [J] Reprod Fertil Dev.2011,23(5):714-724.
51. Du K, Zheng Q, Zhou M et al. Chlamydial antiapoptotic activity involves activation of the Raf/MEK/ERK survival pathway [J] Curr Microbiol.2011,63(4):341-346.
52. Malki A, Youssef A. Antidiabetic drug metformin induces apoptosis in human MCF breast cancer via targeting ERK signaling [J] Oncol Res.2011,19(6):275-285.
53. Tong JS, Zhang QH, Huang X et al. Icaritin causes sustained ERK1/2 activation and induces apoptosis. in human endometrial cancer cells [J] PLoS One.2011, 6(3):e16781.
54. Guo Y, Zhang X, Meng J et al. An anticancer agent icaritin induces sustained activation of the extracellular signal-regulated kinase (ERK) pathway and inhibits growth of breast cancer cells [J] Eur J Pharmacol.2011,658(2-3):114-122.
55. Racz B, Reglodi D, Fodor B et al. Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes [J] Bone.2007,40(6):1536-1543.
56. Lee SM, Lee YJ, Kang DG et al. Effect of Poria cocos on hypertonic stress-induced water channel expression and apoptosis in renal collecting duct cells [J] Ethnopharmacol.2012,6.
57. Han Q, Zhang X, Xue R et al. AMPK potentiates hypertonicity-induced apoptosis by suppressing NFκB/COX-2 in medullary interstitial cells [J] Soc Nephrol.2011,22 (10):1897-1911.
58. Schenk LK, Rinschen MM, Klokkers J et al. Cyclosporin-A induced toxicity in rat renal collecting duct cells:interference with enhanced hypertonicity induced apoptosis [J] Cell Physiol Biochem.2010;26(6):887-900.
59. Nishimura T, Sai Y, Fujii J et al. Roles of TauT and system A in cytoprotection of rat syncytiotrophoblast cell line exposed to hypertonic stress [J] Placenta.2010,31 (11):1003-1009.
60. Bevilacqua E, Wang X, Majumder M et al. eIF2alpha phosphory-lation tips the balance to apoptosis during osmotic stress [J] Biol Chem.2010,285(22): 17098-17111.
61. Racz B, Reglodi D, Fodor B et al. Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes [J] Bone.2007,40(6):1536-1543.