Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells.
TGF-beta 1 controls the expression of numerous genes, including early response and cellular matrix genes. However, the signal-transducing mechanism underlying this regulation of gene expression is not fully understood. In this study, we investigated whether redox regulation plays a role in the TGF-beta 1 signal transduction in the mouse osteoblastic cell line (MC3T3-E1). The overall intracellular oxidized state of the cells, when measured using 2',7'-dichlorofluorescin diacetate by laser-scanning confocal microscopy, was increased transiently after the addition of TGF-beta 1. This increase was abolished by the addition of oxygen radical scavengers such as catalase and N-acetylcysteine. In a variant cell line lacking the TGF-beta 1 receptor, the intracellular oxidized state was not modulated by treatment with TGF-beta 1. We then examined the expression of early growth response-1 (egr-1) gene, which is inducible by TGF-beta 1 and H2O2. Radical scavengers inhibited the induction of egr-1 by TGF-beta 1, but not that by 12-O-tetradecanoylphorbol-13 acetate. A nuclear run-on assay indicated that this inhibition was at the transcriptional level. From transient expression experiments using chloramphenicol acetyltransferase gene linked to serially deleted egr-1 gene 5'-upstream region, the CArG element in the 5' flanking region of egr-1 was identified as an essential sequence in the transcriptional activation for both TGF-beta 1 and H2O2 stimulation. These findings suggest that H2O2 acts as a mediator for the TGF-beta 1-induced transcription of egr-1 gene.
Ohba
M
M
Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Japan.
Shibanuma
M
M
Kuroki
T
T
Nose
K
K
D016475:3T3 Cells; D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002374:Catalase / Q000494:pharmacology; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D051766:Early Growth Response Protein 1; D016166:Free Radical Scavengers; D015870:Gene Expression; D011905:Genes, ras; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D017874:Immediate-Early Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000096:biosynthesis*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D016166:Free Radical Scavengers; D017874:Immediate-Early Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D006861:Hydrogen Peroxide; D002374:Catalase; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate; D000111:Acetylcysteine