A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis.
OBJECTIVE
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 fibroblast 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.


CONCLUSIONS
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.
Qin
Yue-juan
YJ
Center for Preventing and Treating Osteoporosis, Osteoporosis Research Unit, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, China.
Zhang
Zhen-lin
ZL
Yu
Lu-yang
LY
He
Jin-wei
JW
Hou
Ya-nan
YN
Liu
Tian-jin
TJ
Wu
Jia-cai
JC
Wu
Song-hua
SH
Guo
Li-he
LH
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D005822:Genetic Vectors; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D015675:Osteocalcin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D015675:Osteocalcin; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3