Doxorubicin represses CARP gene transcription through the generation of oxidative stress in neonatal rat cardiac myocytes: possible role of serine/threonine kinase-dependent pathways.
Doxorubicin (Dox), an anthracyclin antineoplastic agent, causes dilated cardiomyopathy. CARP has been identified as a nuclear protein whose mRNA levels are exquisitely sensitive to Dox. In this study we investigated the molecular mechanisms underlying the repression of CARP expression by Dox in cultured neonatal rat cardiac myocytes. Dox (1 micromol/l)-mediated decrease in CARP mRNA levels was strongly correlated with BNP but not with ANP mRNA levels. Hydrogen peroxide scavenger catalase (1 mg/ml) but not hydroxyl radical scavengers dimethylthiourea (10 mmol/l) or mannitol (10 mmol/l) blunted the Dox-mediated decrease in CARP and BNP expression. Superoxide dismutase inhibitor diethyldithiocarbamic acid (10 mmol/l), which inhibits the generation of hydrogen peroxide from superoxide metabolism, attenuated the repression. PD98059 (MEK1 inhibitor, 50 micromol/l), SB203580 (p38 MAP kinase inhibitor, 10 micromol/l), calphostin C (protein kinase C (PKC) inhibitor, 1 micromol/l), non-selective protein tyrosine kinase inhibitors genistein (50 micromol/l) or herbimycin A (1 micromol/l) failed to abrogate the downregulation of CARP and BNP expression by Dox. In contrast, H7 (30 micromol/l), a potent inhibitor of serine/threonine kinase, significantly blocked Dox-mediated downregulation of CARP and BNP expression. Transient transfection of a series of 5'-deletion and site-specific mutation constructs revealed that M-CAT element located at -37 of the human CARP promoter mediates Dox-induced repression of CARP promoter activity. These results suggest that a genetic response to Dox is mediated through the generation of hydrogen peroxide, which is selectively linked to the activation of H7-sensitive serine/threonine kinase distinct from PKC and well characterized mitogen-activated protein (MAP) kinases (ERK and p38MAP kinase). Furthermore, our data implicated M-CAT element as a Dox-response element within the CARP promoter in cardiac myocytes.
Aihara
Y
Y
Second Department of Internal Medicine, Gunma University School of Medicine, Gunma, Japan.
Kurabayashi
M
M
Tanaka
T
T
Takeda
S I
SI
Tomaru
K
K
Sekiguchi
K I
KI
Ohyama
Y
Y
Nagai
R
R
D000818:Animals; D000831:Animals, Newborn; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D020097:Natriuretic Peptide, Brain / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
C491304:ANKRD1 protein, human; C491303:Ankrd1 protein, rat; D000903:Antibiotics, Antineoplastic; D000975:Antioxidants; D004791:Enzyme Inhibitors; D009124:Muscle Proteins; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D012097:Repressor Proteins; D020097:Natriuretic Peptide, Brain; D003609:Dactinomycin; D004317:Doxorubicin; D006861:Hydrogen Peroxide; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C