Maternal cocaine administration causes an epigenetic modification of protein kinase Cepsilon gene expression in fetal rat heart.
Protein kinase Cepsilon (PKCepsilon) plays a pivotal role in cardioprotection during cardiac ischemia and reperfusion injury. Recent studies demonstrated that prenatal cocaine exposure caused a decrease in PKCepsilon expression and increased heart susceptibility to ischemic injury in adult offspring, suggesting an in utero programming of PKCepsilon gene expression pattern in the heart. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for cocaine-mediated repression of the PKCepsilon gene in the heart. Pregnant rats were administered either saline or cocaine intraperitoneally (15 mg/kg) twice daily from days 15 to 20 of gestational age, and term fetal hearts were studied. Cocaine treatment significantly decreased PKCepsilon mRNA and protein levels in the heart. CpG dinucleotides found in cAMP response element-binding protein (CREB), CREB/c-Jun1, and CREB/c-Jun2 binding sites at the proximal promoter region of the PKCepsilon gene were densely methylated and were not affected by cocaine. In contrast, methylation of CpGs in the activator protein 1 (AP-1) binding sites was low but was significantly increased by cocaine. Reporter gene assays showed that the AP-1 binding site played a strong stimulatory role of PKCepsilon gene transcription. Methylation of the AP-1 binding sites significantly decreased AP-1 binding to the PKCepsilon promoter. Supershift analyses implicated c-Jun homodimers binding to the AP-1 binding sites. Cocaine did not affect nuclear c-Jun levels or the binding of c-Jun to the unmethylated AP-1 binding sites. The results indicate a role for DNA methylation in cocaine-mediated PKCepsilon gene repression in the developing heart and suggest an epigenetic mechanism affecting this gene linked with vulnerability of ischemic injury in the heart of adult offspring.
Zhang
Haitao
H
Center for Perinatal Biology, Department of Pharmacology and Physiology, Loma Linda University School of Medicine, Loma Linda, CA 92350, USA.
Darwanto
Agus
A
Linkhart
Thomas A
TA
Sowers
Lawrence C
LC
Zhang
Lubo
L
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D003042:Cocaine / Q000008:administration & dosage / Q000494:pharmacology*; D019175:DNA Methylation / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005260:Female; D005318:Fetal Heart / Q000187:drug effects* / Q000201:enzymology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D018811:Maternal Exposure; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051744:Protein Kinase C-epsilon / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000201:enzymology; D018808:Transcription Factor AP-1 / Q000378:metabolism
D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D051744:Protein Kinase C-epsilon; D003042:Cocaine