CCAAT/Enhancer Binding Protein β, But Not Steroidogenic Factor-1, Modulates the Phthalate-Induced Dysregulation of Rat Fetal Testicular Steroidogenesis

Abstract

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 β (c/ebp β) and steroidogenic factor-1 (SF-1). Chromatin immunoprecipitation confirmed declines in DNA-protein interactions of c/ebp β 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 β, 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.

Phthalate-Induced Fetal Leydig Cell Dysfunction Mediates Male Reproductive Tract Anomalies

“…DBP inhibits NR5A1 binding in the promoters and introns of NR5A1-transactivated genes (such as Cyp11a1 , Cyp17a1 , and Star ) that are down-regulated by DBP (Plummer et al, 2013). The binding of NR5A1 to the NR5A1-transactivated gene promoter of Fshr is not affected phthalates (Kuhl et al, 2007; Plummer et al, 2007; Plummer et al, 2013), conforms to the unaltered Fshr expression after the treatment of phthalates (DEHP, DBP, and DINP) (Kuhl et al, 2007; Plummer et al, 2007; Lin et al, 2008a; Plummer et al, 2013; Li et al, 2014), therefore suggesting that the effects of phthalates on NR5A1 binding are indirect. The suppressive actions of phthalates on the binding of NR5A1 to the NR5A1-mediated steroidogenic genes correlate with profiles in the binding of PPARα, a subtype nuclear receptor of PPAR (Plummer et al, 2013).…”

“…Gene expression studies suggest that phthalates (e.g. DBP) affect steroidogenic gene expression via indirect suppression of NR5A1 action in fetal rat testes (Kuhl et al, 2007; Plummer et al, 2007). DBP inhibits NR5A1 binding in the promoters and introns of NR5A1-transactivated genes (such as Cyp11a1 , Cyp17a1 , and Star ) that are down-regulated by DBP (Plummer et al, 2013).…”

Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC1

“…Sertoli cells exposed to chromium(III) chloride have been reported to increase levels of Cebpa mRNA, which may influence germ cell development by altering downstream target gene expression [34]. Fetal exposure to dibutyl phthalate (DBP) inhibits steroidogenic genes by decreasing the DNA-binding activity of CEBPB [35]. These studies demonstrated the common participation of the CEBP family after toxicant-induced testicular injury.…”

Antagonistic Effects of Testosterone and the Endocrine Disruptor Mono-(2-Ethylhexyl) Phthalate on INSL3 Transcription in Leydig Cells

“…Activated PPAR can repress gene expression in a DNA-binding-independent manner through the recruitment of corepressors or by interfering with other DNA-associated transcription factors (65,66). Recently phthalates were also reported to act by decreasing recruitment of the transcription factor CCAAT/enhancerbinding protein-␤ to DNA but having no effect on SF1 (69). Although the exact mechanism remains to be identified, we found that the phthalate MEHP antagonizes AR action through a region of the Insl3 promoter known to bind transcriptional activators, which suggests that disruption of transcription factor interactions might be involved.…”