Transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line.
H2O2, like other oxidants, is known to act as a mitogen at low concentrations in resting Balb/3T3 or mouse epidermal JB6 cells. We described previously that H2O2 induces some early response genes in Balb/3T3 cells. We extended these observations using another cell line, MC3T3 (mouse osteoblastic) cells by examination of transcriptional activity of these genes and by using inhibitors of protein kinases. H2O2 increased the expressions of c-fos, c-jun, egr-1 and JE genes which are known to be early response genes and are induced by mitogenic stimuli in many types of cells. Exogenous addition of H2O2 increased the mRNA levels of these genes, the kinetics of increase being similar to those of their inductions by a phorbol ester or serum. Nuclear run-on transcription showed that this induction occurred at the transcriptional level. H2O2 at 0.1-0.2 mM induced maximal expressions of c-fos and c-jun, whereas 0.3 mM H2O2 was required for induction of stress-induced heme oxygenase mRNA. The inductions of c-fos and c-jun were inhibited by 50 microM H7, a protein kinase inhibitor that is relatively specific for protein kinase C, but were not affected by H9, relatively specific for cAMP-dependent protein kinase. In cells pretreated with 12-O-tetradecanoylphorbol 13-acetate, however, in which protein kinase was supposed to be downregulated, H2O2 induced c-fos and heme oxygenase as efficiently as in untreated cells. H2O2 did not increase the phosphorylation of p80 protein, which is known to be a substrate for protein kinase C. Thus, H2O2 seemed to induce c-fos and c-jun by activating protein kinases distinct from protein kinase C. Activity of the chloramphenicol acetyltransferase gene under control of the serum-response element of human c-fos genes was increased by H2O2 treatment, whereas that under control of cAMP-response element was not affected. These results indicate that the inductions by H2O2 of c-fos and possibly other early response genes are mediated through activation of the serum-response element in their enhancer.
Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Japan.
K
K
Nose
M
M
Shibanuma
K
K
Kikuchi
H
H
Kageyama
S
S
Sakiyama
T
T
Kuroki
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D001483:Base Sequence; D001769:Blood; D002460:Cell Line; D016207:Cytokines / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D051766:Early Growth Response Protein 1; D015870:Gene Expression / Q000187:drug effects*; D016762:Genes, fos / Q000187:drug effects / Q000235:genetics; D016758:Genes, jun / Q000187:drug effects / Q000235:genetics; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D017874:Immediate-Early Proteins; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*
D016207:Cytokines; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D007546:Isoquinolines; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D014157:Transcription Factors; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D006861:Hydrogen Peroxide; D013755:Tetradecanoylphorbol Acetate