Fetal liver CYP3A7 plays an important role in placental estriol synthesis during pregnancy, yet little is known concerning the extent or consequences of variability in expression. The purpose of this investigation was to characterize the variability in CYP3A7 expression using several phenotypic measures in a panel of 54 fetal livers ranging in age from 76 days to 32 weeks gestation. CYP3A7 mRNA expression was measured using quantitative polymerase chain reaction, whereas immunoreactive CYP3A7 was determined using an affinity-purified antipeptide antibody. Variability in catalytic activity was evaluated using testosterone and dehydroepiandrosterone (DHEA) as substrates. Across the entire panel, CYP3A7 was the most abundant CYP3A mRNA species present and varied 634-fold from 151 to 95,700 transcripts/ng total RNA, corrected for 18S ribosomal RNA. CYP3A4 expression was minimal based on mRNA expression (1000-fold lower than CYP3A7) and the ratio of testosterone 2␣-(T2␣H) to 6␤-(T6␤H) hydroxylation. T2␣H and T6␤H were highly correlated (r 2 ϭ 0.859), and the correlation increased (r 2 ϭ 0.974) in livers with CYP3A5*3/*3 genotypes implying that the same enzyme (CYP3A7) generated both products. Overall, T2␣H and DHEA16␣H activities varied 175-and 250-fold, respectively. A subset of five samples had extremely low mRNA, protein, and catalytic activity, possibly due to pathology affecting fetal viability (anencephaly, porencephaly). In the remaining samples, T2␣H activity varied 6.7-fold (358 Ϯ 142, range 97 to 643 pmol/min/mg) and DHEA16␣H activity varied 6.2-fold (8.07 Ϯ 2.87, range 2.41 to 14.9 nmol/ min/mg). Observed variability in CYP3A7 activity was not related to CYP3A7*2, and alternative regulatory mechanisms require further investigation.

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Treatment of HEC1A endometrial cancer cells with 10 nM 17␤-estradiol (E2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA expression, and a similar response was observed using a construct, pVEGF1, containing a VEGF gene promoter insert from ؊2018 to ؉50. In HEC1A cells transiently transfected with pVEGF1 and a series of deletion plasmids, it was shown that E2-dependent down-regulation was dependent on wild-type estrogen receptor ␣ (ER␣) and reversed by the anti-estrogen ICI 182,780, and this response was not affected by progestins. Deletion analysis of the VEGF gene promoter identified an overlapping G/GCrich site between ؊66 to ؊47 that was required for decreased transactivation by E2. Protein-DNA binding studies using electrophoretic mobility shift and DNA footprinting assays showed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter. Coimmunoprecipitation and pull-down assays demonstrated that Sp3 and ER␣ proteins physically interact, and the interacting domains of both proteins are different from those previously observed for interactions between Sp1 and ER␣ proteins. Using a dominant negative form of Sp3 and transcriptional activation assays in Schneider SL-2 insect cells, it was confirmed that ER␣-Sp3 interactions define a pathway for E2-mediated inhibition of gene expression, and this represents a new mechanism for decreased gene expression by E2.

ABSTRACT:The mechanisms underlying interindividual variation and developmental changes in cytochrome P450 3A (CYP3A) expression and activity are not fully understood. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods were used to detect, during human fetal and pediatric development, mRNA expression of nuclear receptors involved in the regulation of CYP3A genes. Quantitative RT-PCR was conducted on RNA extracted from prenatal (n ‫؍‬ 60, 76 days to 32 weeks estimated gestational age) and pediatric (n ‫؍‬ 20, 4 days to 18 years of age) liver tissue with primers for nuclear receptors implicated in regulating CYP3A gene expression. Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) were expressed at low (and highly variable) levels in pre-and neonatal liver relative to liver tissue derived from older children. CAR was expressed at higher levels relative to In conclusion, nuclear receptor mRNA expression demonstrates considerable interindividual variability in human fetal and pediatric liver and is significantly correlated with CYP3A expression.

-Estradiol (E2) induces transforming growth factor(TGF ) gene expression in MCF-7 cells and previous studies have identified a 53 bp ( 252 to 200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGF gene promoter in this study identified a second upstream region of the promoter ( 623 to 549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and transacting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor (ER )/Sp1 complex interacting with an Sp1(N) 30 ERE half-site (½) motif in which both ER and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ER or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N) x ERE½ motif identified in the TGF gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.