Transcriptional regulation of aromatase in placenta and ovary

Our goal is to define the cellular and molecular mechanisms for tissue- and cell-specific, developmental and hormonal regulation of the human CYP19 (aromatase P450/P450arom) gene in estrogen-producing cells. In this article, we review studies using transgenic mice and transfected cells to identify genomic regions and response elements that mediate CYP19 expression in placenta and ovary, as well as to define the molecular mechanisms for O₂ regulation of differentiation and CYP19 gene expression in human trophoblast cells in culture. We also highlight recent findings regarding LRH-1 versus SF-1 mRNA expression and cellular localization in the mouse ovary during the estrous cycle and various stages of pregnancy. Spatial and temporal expression patterns of mRNAs encoding these orphan nuclear receptors in comparison to those of P450arom and 17-hydroxylase/17,20-lyase mRNAs, suggest an important role of LRH-1 together with SF-1 in ovarian steroidogenesis.     

Mechanisms in the regulation of aromatase in developing ovary and placenta

During human gestation, the placental syncytiotrophoblast develops the capacity to synthesize large amounts of estrogen from C₁₉-steroids secreted by the fetal adrenals. The conversion of C₁₉-steroids to estrogens is catalyzed by aromatase P450 (P450arom), product of the CYP19 gene. The placenta-specific promoter of the hCYP19 gene lies 100,000 bp upstream of the translation initiation site in exon II. In studies using transgenic mice and transfected human trophoblast cells we have defined a 246-bp region upstream of placenta-specific exon I.1 that mediates placental cell-specific expression. Using transgenic mice, we also observed that as little as 278 bp of DNA flanking the 5′-end of ovary-specific hCYP19 exon IIa was sufficient to target ovary-specific expression. This ovary-specific promoter contains response elements that bind cAMP-response element-binding protein (CREB) and the orphan nuclear receptors SF-1 and LRH-1, which are required for cAMP-mediated stimulation of CYP19 expression in granulosa and luteal cells during the estrous cycle and pregnancy. In this article, we review our studies to define genomic regions and response elements that mediate placenta-specific expression of the hCYP19 gene. The temporal and spatial expression of LRH-1 versus SF-1 in the developing gonad during mouse embryogenesis and in the postnatal ovary also will be considered.     

Association between metabolic gene polymorphisms and susceptibility to peripheral nerve damage in workers exposed to n-hexane: A preliminary study

Chronic exposure to n-hexane may result in peripheral neuropathy. 2,5-Hexanedione (2,5-HD) has been identified as a toxic metabolite of n-hexane. The CYP2E1, CYP1A1 and GST genes are involved in the formation of 2,5-hexanedione from n-hexane as well as the elimination of 2,5-HD-formed electrophile, and these genes are highly polymorphic in the general population. A nested case-control study in an industrial cohort was conducted to evaluate the associations between polymorphisms in these metabolic genes and n-hexane-induced peripheral nerve damage. The study subjects included 22 cases, who worked in a printing factory with symptoms of peripheral nerve damage, and 163 controls, who came from the same factory of cases. DNA was extracted from blood samples and genotyping was conducted for CYP2E1 Pst, CYP2E1 Dra, CYP2E1 Ins96, CYP1A1 Msp, GSTT1 null, GSTM1 null and GSTP1 105V. Unconditional logistic regression was applied to estimate the odds ratio and 95% confidence intervals. There were no significant differences between the two groups regarding age, sex, smoking and alcohol status. A significant association between Dra polymorphism and peripheral nerve damage was found. The frequency of CYP2E1 Dra homozygous mutation in the case group (18.2%) was higher than that in the control group (3.7%, p=0.015). Individuals with homozygote genotype (CC) of CYP2E1 Dra had a significantly higher risk of peripheral nerve damage compared with those with DD genotype (adjusted OR = 5.58, 95% CI = 1.32-23.65) after n-hexane exposure duration, sex, age, smoking and alcohol status were adjusted. No significant association was found that CYP2E1 Pst, CYP2E1 Ins96, CYP1A1 Msp, GSTT1, GSTM1, GSTP gene polymorphisms associated with the susceptibility of peripheral nerve damage. These findings suggested that CYP2E1 gene might increase the susceptibility to n-hexane-induced peripheral damage.     

Activation of the SCPx promoter in mouse adrenocortical Y1 cells

Sterol carrier protein X (SCPx) is a peroxisomal protein with both lipid transfer and thiolase activity. Treatment of mouse adrenal Y1 cells with cAMP for 24h caused a significant induction of SCPx mRNA levels. Reporter gene studies demonstrated that treatment with cAMP and SF-1 was able to activate the SCPx promoter. Sequence analysis revealed the presence of three putative steroidogenic factor-1 (SF-1) binding motifs (designated SFB1, SFB2, and SFB3) and one CRE. Only SFB1 and SFB3 were able to bind recombinant SF-1 protein in electrophoretic mobility shift assays. The CRE was able to form a DNA/protein complex in the presence of Y1 nuclear extracts. Mutational analysis studies demonstrated that SFB3 is required for full activation of the SCPx promoter by cAMP treatment. Regulation of the SCPx gene by SF-1 and cAMP is similar to the regulatory mechanisms observed for other steroidogenic genes.     

CYP1A1, GSTM1 and GSTT1 polymorphisms and lung cancer: a pooled analysis of gene-gene interactions

Gene-environment interactions have been extensively studied in lung cancer. It is likely that several genetic polymorphisms cooperate in increasing the individual risk. Therefore, the study of gene-gene interactions might be important to identify high-susceptibility subgroups. GSEC is an initiative aimed at collecting available data sets on metabolic polymorphisms and the risks of cancer at several sites and performing pooled analyses of the original data. Authors of published papers have provided original data sets. The present paper refers to gene-gene interactions in lung cancer and considers three polymorphisms in three metabolic genes: CYP1A1, GSTM1 and GSTT1. The present analyses compare the gene-gene interactions of the CYP1A1*2A, GSTM1 and GSTT1 polymorphisms from studies on lung cancer conducted in Europe and the USA between 1991 and 2000. Only Caucasians have been included. The data set includes 1466 cases and 1488 controls. The only clear-cut association was found with CYP1A1*2A. This association remained unchanged after stratification by polymorphisms in other genes (with an odds ratio [OR] of approximately 2.5), except when interaction with GSTM1 was considered. When the OR for CYP1A1*2A was stratified according to the GSTM1 genotype, the OR was increased only among the subjects who had the null (homozygous deletion) GSTM1 genotype (OR=2.8, 95% CI=0.9-8.4). The odds ratio for the interactive term (CYP1A1*2A by GSTM1) in logistic regression was 2.7 (95% CI=0.5-15.3). An association between lung cancer and the homozygous CYP1A1*2A genotype is confirmed. An apparent and biologically plausible interaction is suggested between this genotype and GSTM1.     

Polymorphism of metabolic genes and susceptibility to occupational chronic manganism

In this study we investigated genetic polymorphisms of five metabolizing genes and their association with occupational chronic manganism. We recruited 49 patients with chronic manganism and 50 unrelated healthy control subjects who were welders and ferromanganese smelters and occupationally exposed to manganese dust and fume in the same workshops from three metallurgical industries. The controls were matched to the cases by sex, age, cigarette and alcohol intake, as well as the manganese exposure duration. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the cytochrome P450 2D6L gene (CYP2D6L) and the NAD(P)H:quinone oxidoreductase gene (NQO1). Allele-specific PCR was used to detect the cytochrome P450 1A1 gene (CYP1A1), and the glutathione-S-transferase mu and theta genes (GSTM and GSTT). The frequency of polymorphic alleles, a mutation of CYP2D6L, was significantly lower in patients with chronic manganism (16.3%) than in controls (29.0%). Individuals with the homozygote polymorphism (L/L) of CYP2D6 had a 90% decreased risk of chronic manganism compared with the wild-type (Wt/Wt) (odds ratio =0.10, 95% confidence interval = 0.01-0.82). A significant association between the CYP2D6 genotype subgroup and the latency of chronic manganese poisoning was also found. Patients who had homozygous (L/L) or heterozygous (Wt/L) mutant alleles developed manganism an average of 10 years later than those who were homozygous wildtype (Wt/Wt). However, the allele and genotype frequencies of CYP1A1 and NQO1 genes were distributed similarly in cases and controls. In addition, no difference in the frequencies of GSTM1 and GSTT1 null genotypes were observed between cases and controls. The results suggest that CYP2D6L gene polymorphism might influence susceptibility to manganese-induced neurotoxicity. However, because of limited sample size, our results should be validated in large-scale studies.     

Evidence that functional interactions of CREB and SF-1 mediate hormone regulated expression of the aromatase gene in granulosa cells and constitutive expression in R2C cells

The proximal promoter of the rat aromatase CYP19 gene contains two functional domains that can confer hormone/cAMP inducibility in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Region A contains a hexameric sequence that binds steroidogenic factor-1 (SF-1). Region B contains a CRE-like sequence that binds CREB and two other factors, X and Y. To determine if CRE binding factors X and Y had overlapping functions with CREB, and to determine if the CREB and SF-1 binding sites exhibited functional interactions in the context of the intact promoter, mutations within the CRE and hexameric SF-1 binding site were generated. Mutations within the CRE showed that CREB but not factors X and Y mediated cAMP-dependent activity of chimeric transgenes in primary granulosa cell cultures. Granulosa cells transfected with constructs that bound CREB but not SF-1 (or the converse) resulted in a loss of approximately 50% cAMP-dependent CAT activity. Transgenes that did not bind CREB or SF-1 exhibited no cAMP-dependent CAT activity. When these same constructs where transfected into R2C Leydig cells, mutation of either the CREB or SF-1 binding sites resulted in a greater than 90% loss of CAT activity. Western blot and immunocytochemistry analyses revealed that the amount of phosphorylated CREB increased in response to hormone/cAMP in granulosa cells and was high in R2C Leydig cells, coinciding with expression of the transgenes and endogenous aromatase mRNA in each cell type. Therefore, in both cell types the aromatase promoter is dependent upon a functional CRE and the presence of phosphoCREB. The CREB and SF-1 binding sites interact in an additive manner to mediate cAMP transactivation in granulosa cells, whereas they interact synergistically to confer high basal transactivation in R2C Leydig cells. Taken together, the results indicated that the molecular mechanisms or pathways that activate CREB, SF-1 or their interaction are different in granulosa cells and R2C cells.     

The orphan nuclear receptor SF-1 is involved in the effect of PCBs,DDT, and DDE on the secretion of steroid hormones and oxytocin from bovine luteal cells during the estrous cycle in vitro

The orphan receptor steroidogenic factor-1 (SF-1) is involved in the regulation of ovarian steroidogenesis in cows. It is hypothesized that estrogen-like chlorinated compounds might affect SF-1, and thus impair the function of the ovary. Bovine luteal cells from the estrous cycle (Days: 1–5, 6–10, 11–15, and 16–19) were treated for 50 hours with DDT, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene, 3,3′4,4′-tetrachlorobiphenyl or 2′2′4,4′,5,5′-hexachlorobiphenyl (each at a dose of 10 ng/mL). Luteal cells were also treated with 4-(heptyloxy)phenol (1 × 10⁻⁷ M), an SF-1 agonist, and F0160 (1 × 10⁻⁶ M), an SF-1 blocker, jointly or separately. The secretion of progesterone and oxytocin and the expression of oxytocin precursor (NP-I/OT) messenger RNA were increased (P < 0.05) by all studied xenobiotics and 4-(heptyloxy)phenol, although they were inhibited (P < 0.05) by F0160. However, the xenobiotics did not affect (P > 0.05) SF-1 messenger RNA expression. In summary, SF-1 is involved in the adverse effect of chlorinated xenobiotics on the regulation of the bovine CL.     

Aromatase and cyclooxygenases: enzymes in breast cancer

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C₁₉ androgens to C₁₈ estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE₂ increases intracellular cAMP levels and stimulates estrogen biosynthesis, and previous studies in our laboratories have shown a strong linear association between aromatase (CYP19) expression and expression of the cyclooxygenases (COX-1 and COX-2) in breast cancer specimens. To further investigate the pathways regulating COX and CYP19 gene expression, studies were performed in normal breast stromal cells, in breast cancer cells from patients, and in breast cancer cell lines using selective pharmacological agents. Enhanced COX enzyme levels results in increased production of prostaglandins, such as PGE₂. This prostaglandin increased aromatase activity in breast stromal cells, and studies with selective agonists and antagonists showed that this regulation of signaling pathways occurs through the EP₁ and EP₂ receptor subtypes. COX-2 gene expression was enhanced in breast cancer cell lines by ligands for the various peroxisome proliferator-activated receptors (PPARs), and differential regulation was observed between hormone-dependent and -independent breast cancer cells. Thus, the regulation of both enzymes in breast cancer involves complex paracrine interactions, resulting in significant consequences on the pathogenesis of breast cancer.     

牦牛B细胞淋巴瘤2相关蛋白A1的原核表达及体外活性

为研究牦牛(Bos grunniens) B细胞淋巴瘤2相关蛋白A1(B cell lymphoma 2 related protein A1,BCL2A1)的原核表达及体外活性。采用原核表达载体构建、细胞划痕实验、CCK-8法、透射电镜和实时荧光定量PCR等方法。结果显示,成功构建pET-32a-BCL2A1重组质粒,表达获得约33kDa的BCL2A1。终浓度为0.02μg/mL、0.2μg/mL、2.0μg/mL的BCL2A1均能使HepG2细胞活性显著降低(P < 0.05),并在一定程度上抑制细胞的迁移。2.0μg/mL BCL2A1导致HepG2细胞核固缩,胞质中高密度物质聚集,溶酶体吞噬形成致密的凋亡小体等。此外,细胞凋亡相关基因CASP9的mRNA水平在2.0 μg/mL组中显著上调(P < 0.05),CASP8的mRNA水平在0.2 μg/mL和2.0 μg/mL组中显著上调(P < 0.05),而CASP3和Cyt c的mRNA水平在3个浓度处理组均显著上调(P < 0.05)。这提示BCL2A1可能通过凋亡途径影响HepG2细胞活性,为进一步探索牦牛BCL2A1基因功能积累资料。     

Benzo(a)pyrene diolepoxide adducts to albumin in workers exposed to polycyclic aromatic hydrocarbons: association with specific CYP1A1, GSTM1, GSTP1 and EHPX genotypes

We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg^-1 SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg^-1 SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034).

Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.     

Regulation of the CYP27B1 5'-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.