CCAAT/enhancer binding protein beta, but not steroidogenic factor-1, modulates the phthalate-induced dysregulation of rat fetal testicular steroidogenesis.
Prolonged in utero exposure of fetal male rats to dibutyl phthalate (DBP) can result in a feminized phenotype characterized by malformed epididymides, hypospadias, cryptorchidism, and retained thoracic nipples, among others. These symptoms likely result, in part, from decreased expression of steroidogenic enzymes and, therefore, reduced testosterone biosynthesis. However, the molecular mechanisms involved in these changes in gene expression profiles are unknown. To understand these mechanisms in rats, in vivo DNase footprinting was adapted to provide a semiquantitative map of changes in DNA-protein interactions in the promoter region of steroidogenic genes, including steroidogenic acute regulatory, scavenger receptor B-1, cytochrome P450 side chain cleavage, and cytochrome P450 17A1, that are down-regulated after an in utero DBP exposure. Regions with altered DNase protection were coordinated with a specific DNA binding protein event by EMSA, and binding activity confirmed with chromatin immunoprecipitation. Results demonstrated altered DNase protection at regions mapping to CCAAT/enhancer binding protein beta (c/ebp beta) and steroidogenic factor-1 (SF-1). Chromatin immunoprecipitation confirmed declines in DNA-protein interactions of c/ebp beta in DBP treated animals, whereas SF-1 was reduced in both diethyl phthalate (nontoxic) and DBP (toxic) treatments. These results suggest that inhibition of c/ebp beta, and not SF-1, is critical in DBP induced inhibition of steroidogenic genes. In addition, these observations suggest a pathway redundancy in the regulation of steroidogenesis in fetal testis. In conclusion, this study presents a snapshot of changes in the structure of transcriptional machinery and proposes a mechanism of action resulting from DBP exposure.
Kuhl
Adam J
AJ
The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709-2137, USA. akuhl@thehamner.org
Ross
Susan M
SM
Gaido
Kevin W
KW
D000818:Animals; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D047369:Chromatin Immunoprecipitation; D003850:Deoxyribonuclease I / Q000378:metabolism; D003993:Dibutyl Phthalate / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D008297:Male; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010795:Phthalic Acids / Q000494:pharmacology*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051122:Scavenger Receptors, Class B / Q000235:genetics / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D054339:Steroidogenic Factor 1 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D013256:Steroids / Q000378:metabolism*; D013737:Testis / Q000187:drug effects* / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D000098682:Steroidogenic Acute Regulatory Protein
D022782:CCAAT-Enhancer-Binding Protein-beta; D050991:GATA4 Transcription Factor; C084365:Gata4 protein, rat; D010750:Phosphoproteins; D010795:Phthalic Acids; C495125:Scarb1 protein, rat; D051122:Scavenger Receptors, Class B; D054339:Steroidogenic Factor 1; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D003993:Dibutyl Phthalate; D013739:Testosterone; C032279:phthalic acid; D013254:Steroid 17-alpha-Hydroxylase; D003850:Deoxyribonuclease I