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


CONCLUSIONS
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.
Zhao
Qing
Q
Department of Nephrology, Ruijin Hospital, Shanghai 200025, China.
Chen
Nan
N
Wang
Wei-ming
WM
Lu
Jian
J
Dai
Bing-bing
BB
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D055513:Connective Tissue Growth Factor; D004305:Dose-Response Relationship, Drug; D005419:Flavonoids / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000008:administration & dosage / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases
D000894:Anti-Inflammatory Agents, Non-Steroidal; C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; D005419:Flavonoids; D007093:Imidazoles; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D011725:Pyridines; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D008156:Luciferases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one