Abstract
Aim:
To construct an A20 expression vector under the control of mouse osteocalcin promoter (OC-A20), and investigate osteoblastic MC3T3-E1 cell line, which stably overexpresses A20 protein prevented tumor necrosis factor (TNF)-alpha-induced apoptosis.
Methods:
OC-A20 vector was constructed by fusing a fragment of the mouse osteocalcin gene-2 promoter with human A20 complementary DNA. Then the mouse MC3T3-E1 cell line, stably transfected by A20, was established. The expression of A20 mRNA and A20 protein in the cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To determine the specificity of A20 expression in osteoblast, the mouse osteoblastic MC3T3-E1 cell line and mouse embryo fibro-blast NIH3T3 cell line were transiently transfected with OC-A20. The anti-apoptotic role of A20 in MC3T3-E1 cells was determined by Flow cytometric analysis (FACS), terminal dUTP nick endo-labeling (TUNEL) and DNA gel electrophoresis analysis (DNA Ladder), respectively.
Results:
Weak A20 expression was found in MC3T3-E1 cells with the primers of mouse A20. A20 mRNA and A20 protein expression were identified in MC3T3-E1 cells transfected with OC-A20 using RT-PCR and Western blot analysis. Only A20 mRNA expression was found in MC3T3-E1 cell after MC3T3-E1 cells and NIH3T3 cells were transient transfected with OC-A20. A decrease obviously occurred in the rate of apoptosis in the OC-A20 group compared with the empty vector (pcDNA3) group by FACS (P<0.001). A significant increase in TUNEL positive staining was found in the pcDNA group compared with OC-A20 group (P<0.001). Simultaneously, similar effects were demonstrated in DNA gel electrophoresis analysis.
Conclusion:
We constructed an osteoblast-specific expression vector that expressed A20 protein in MC3T3-E1 cells and confirmed that A20 protects osteoblast against TNF-alpha-induced apoptosis.
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Manolagas SC, Jilka RL . Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N Engl J Med 1995; 332: 305–11.
Jilka RL, Weinstein RS, Bellido T, Parfitt AM, Manolagas SC . Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Miner Res 1998; 13: 793–802.
Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC . Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest 1998; 102: 274–82.
Plotkin LI, Weinstein RS, Parfitt AM, Roberson RK, Manolagas SC, Bellido T . Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 1999; 104: 1363–74.
Daniel S, Arvelo MB, Patel VI, Longo CR, Shrikhande G, Shukri T, et al. A20 protects endothelial cells from TNF-, Fas-, and NK-mediated cell death by inhibiting caspase 8 activation. Blood 2004; 104: 2376–84.
Heyninck K, Beyaert R . A20 inhibits NF-kappaB activation by dual ubiquitin-editing function. Trends Biochem Sci 2005; 30: 1–4.
Storz P, Doppler H, Ferran C, Grey ST, Toker A . Functional dichotomy of A20 apoptotic and necrotic cell death. Biochem J 2005; 387: 47–55.
Wertz IE, O'Rourke KM, Zhou H, Eby M, Aravind L, Seshagiri S, et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signaling. Nature 2004; 430: 694–9.
Gon Y, Asai Y, Hashimoto S, Mizumura K, Jibiki I, Machino T, et al. A20 inhibits toll-like receptor 2- and 4-mediated interleukin-8 synthesis in airway epithelial cells. Am J Respir Cell Mol Biol 2004; 31: 330–6.
Longo CR, Patel VI, Shrikhande GV, Scali ST, Csizmadia E, Daniel S, et al. A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21 waf1-dependent mechanism. Hepatology 2005; 42: 156–64.
Janicke RU, Lee FH, Porter AG . Nuclear c-Myc plays an important role in the cytotoxicity of tumor necrosis factor a in tumor cells. Mol Cell Biol 1994; 14: 5661–70.
Jaattela M, Mouritzen H, Elling F, Bastholm L . A20 zinc finger protein inhibits TNF and IL-1 signaling. J Immunol 1996; 156: 1166–73.
Lee EG, Boone DL, Chai S, Libby SL, Chien M, Lodolce JP, et al. Failure to regulate TNF-induced NF-kB and cell death responses in A20-deficient mice. Science 2000; 289: 2350–4.
Boone DL, Turer EE, Lee EG, Ahmad RC, Wheeler MT, Tsui C, et al. The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses. Nat Immunol 2004; 5: 1052–60.
Yu LY, Lin B, Zhang ZL, Guo LH . Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes. Acta Pharmacol Sin 2004; 25: 721–6.
Miao HS, Yu LY, Hui GZ, Guo LH . Antiapoptotic effect both in vivo and in vitro of A20 gene when transfected into rat hippocampal neurons. Acta Pharmacol Sin 2005; 26: 33–8.
Zhang ZL, Lin B, Yu LY, Shen SX, Zhu LH, Wang WP, et al. Gene therapy of experimental autoimmune thyroidits mice by in vivo administration of plasmid DNA coding for human interleukin-10. Acta Pharmacol Sin 2003; 24: 885–90.
Opipari AW, Boguski MS, Dixit VM . The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein. J Biol Chem 1990; 265: 14705–8.
Grey ST, Arvelo MB, Hasenkamp W, Bach FH, Ferran C . A20 inhibits cytokine-induced apoptosis and nuclear factor kB-de-pendent gene activation in islets. J Exp Med 1999; 190: 1135–46.
Laybutt DR, Kaneto H, Hasenkamp W, Grey S, Jonas JC, Sgroi DC, et al. Increased expression of antioxidant and anti-apoptotic genes in islets that may contribute to beta-cell survival during chronic hyperglycemia. Diabetes 2002; 51: 413–23.
Zhang SQ, Kovalenko A, Cantarella G, Wallach D . Recruitment of the IKK signalosome to the p55 TNF receptor: RIP and A20 bind to NEMO (IKKgamma) upon receptor stimulation. Immunity 2000; 12: 301–11.
Ducy P, Karsenty G . Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Mol Cell Biol 1995; 15: 1858–69.
Bach FH, Hancock WW, Ferran C . Protective genes expressed in endothelial cells: a regulatory response to injury. Immunol Today 1997; 18: 483–6.
He KL, Ting AT . A20 inhibits tumor necrosis factor (TNF) alpha-induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1 complex in Jurkat T cells. Mol Cell Biol 2002; 20: 6034–45.
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Project supported by the Natural Science Foundation of Shanghai (No 03ZR14058) and in part by National Natural Science Foundation of China (No 30570891).
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Qin, Yj., Zhang, Zl., Yu, Ly. et al. A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis. Acta Pharmacol Sin 27, 1231–1237 (2006). https://doi.org/10.1111/j.1745-7254.2006.00403.x
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DOI: https://doi.org/10.1111/j.1745-7254.2006.00403.x
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