Original Article

Effect of transforming growth factor-beta on activity of connective tissue growth factor gene promoter in mouse NIH/3T3 fibroblasts

Qing ZHAO, Nan CHEN, Wei-ming WANG, Jian LU, Bing-bing DAI

Abstract

AIM:
To investigate the regulatory mechanism of transforming growth factor-beta on activity of connective tissue growth factor promoter in mouse NIH/3T3 fibroblasts.
METHODS:
The regulation fragment of the 5' flanking region of the human CTGF gene was linked to pGL3-Basic vector, a firefly luciferase reporter construct without promoter. The recombinant plasmid pCTGF-luc was transiently transfected to NIH/3T3 fibroblasts. The activity of CTGF promoter after treatment of TGF-beta(1) and MAPK pathway inhibitors were assayed with luciferase reporter gene assay system.
RESULTS:
TGF-beta(1)-induced increase of CTGF promoter activity was concentration-dependent, with a plateau at 5 microg/L by 2.67-fold vs control (P<0.05). The TGF-beta(1) stimulation of CTGF promoter activity was time-dependent, too. After exposure to TGF-beta(1) (5 microg/L), the maximal level of luciferase activity was reached at 12 h and maintained to 24 h by 2.76- and 2.20-fold vs control, respectively (P<0.05). Blockade of MAPK pathway with PD98059 (10 micromol/L), the MAP kinase kinase 1 inhibitor, and SB203580 (10 micromol/L), the p38 MAP kinase inhibitor, decreased basal and TGF-beta (1)-induced activation of CTGF promoter. However, inhibition of c-Jun-N-terminal kinase/stress-activated protein kinase by SP600125 (20 micromol/L) was without effect.
CONCLUSION:
TGF-beta(1) stimulated the transcriptional activity of CTGF gene promoter in NIH/3T3 fibroblasts in a dose- and time-dependent manner. MAPK pathway may play a role in the regulation of TGF-beta(1)-induced CTGF expression.
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