Open AccessA20 overexpression under control of mouse osteocalcin promoter in MC3T3‐E1 cells inhibited tumor necrosis factor‐alpha‐induced apoptosis 1
Author(s) -
QIN Yuejuan,
ZHANG Zhenlin,
YU Luyang,
HE Jinwei,
HOU Yanan,
LIU Tianjin,
WU Jiacai,
WU Songhua,
GUO Lihe
Publication year - 2006
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2006.00403.x
Subject(s) - apoptosis , tumor necrosis factor alpha , osteocalcin , alpha (finance) , necrosis , cancer research , microbiology and biotechnology , biology , chemistry , endocrinology , medicine , biochemistry , genetics , alkaline phosphatase , enzyme , construct validity , nursing , patient satisfaction
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.