Estrogen receptor α (ERα) is a ligand-inducible transcription factor that mediates the biological effects of estrogens and antiestrogens. Many point mutations in the human ERα gene have been reported to be associated with breast cancer, endometrial cancer, and psychiatric diseases. However, functional analyses for most mutants with amino acid changes are still lacking. In the present study, to investigate the effects of point mutations on the function, gel-shift assays and luciferase assays were performed for eight kinds of mutated ERα proteins, including a single nucleotide change of C207G (N69K), G478T (G160C), T887C (L296P), A908G (K303R), C926T (S309F), A1058T (E353V), A1186G (M396V) and G1231deletion (411fsX7). The mutated ERα expression plasmids were constructed by sitedirected mutagenesis. With gel-shift assays using in vitro translated ERα proteins, binding to the consensus estrogen response element (ERE) was observed for the mutated ERα proteins except ERα (G160C) and ERα (411fsX7), the binding of which was comparable with that of the wild type. Western blot analyses showed that ERα (G160C) could not be efficiently translated with the in vitro transcription/translation system and that ERα (411fsX7) produced a truncated protein. To investigate the transactivation potency, wild-type or mutated ERα expression plasmids were cotransfected with pGL3-3EREc38 reporter plasmid into human breast adenocarcinoma MDA-MB-435 cells. The concentration-response curves (10 pM -100 nM E2) of the mutant ERα proteins except ERα (E353V) and ERα (411fsX7) were similar to that of wild-type ERα. However, at a low level of E2 (100 pM), the mutants ERα (N69K), ERα (L296P), ERα (S309F), and ERα (M396V) showed a significant decrease of transactivation compared with that of the wild-type ERα. The mutants ERα (E353V) and ERα (411fsX7) did not show responsiveness to E2 and antiestrogens, 4-hydroxytamoxifen (4OHT) and ICI 182,780. The mutant ERα (S309F) showed decreased responsiveness for the antiestrogenicity of 4OHT. In conclusion, we found that some of the naturally occurring human ERα mutants with amino acid changes may have an altered responsiveness to estrogen and antiestrogens.

ABSTRACT:Human CYP2A6, which is predominantly expressed in liver, is a key enzyme responsible for the metabolism of nicotine, coumarin, and some pharmaceutical drugs. CYP2A6 is also expressed in sex steroid-responsive tissues such as breast, ovary, uterus, testis, and adrenal grand. In this study, we examined the regulation of CYP2A6 gene by estrogen. Reverse transcription-polymerase chain reaction (RT-PCR) assays revealed that CYP2A6 mRNA was induced by estradiol in estrogen receptor (ER)-positive MCF-7 (2.9-fold) and HepG2 (1.3-fold) cells, but not in ER-negative MDA-MB-435 cells. Real-time RT-PCR assays revealed the CYP2A6 induction by estradiol in human hepatocytes (1.2-to 1.5-fold). Computer-assisted homology search identified a putative estrogen response element (ERE) at ؊2436 on the CYP2A6 gene. Electrophoretic mobility shift assays demonstrated specific binding of ER␣ to this element. Luciferase assays using MCF-7 cells revealed that the transcriptional activity of the CYP2A6 promoter was significantly activated by estradiol in an ER␣-dependent manner, in which ERE was responsible for the activation. Chromatin immunoprecipitation assays verified the in vivo association of ER␣ with the ERE on the CYP2A6 gene. Immunohistochemical analyses using human endometrial tissues indicated that the CYP2A6 protein level in glandular cells was significantly higher in the proliferative phase than in the secretory phase, concomitant with local estrogen secretion during the menstrual cycle. These findings clearly demonstrated that CYP2A6 is directly induced by estrogen in an ER␣-dependent manner, implying a biological role of CYP2A6 in estrogen-responsive tissues. Furthermore, this mechanism can also explain clinical aspects of increased nicotine metabolism under estrogen-rich environments.

CYP2A6 plays important roles in the metabolism of nicotine and some clinically used drugs. Interindividual variability in the CYP2A6 expression level in human liver might be caused by an inducible property, but the molecular mechanism of induction is unclear. Rifampicin, phenobarbital, -(3,4-dichlorobenzyl)oxime, which are activators of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), induced CYP2A6 mRNA in human hepatocytes. We identified three direct repeat separated by four nucleotides (DR4)-like elements at Ϫ6698, Ϫ5476, and Ϫ4618 in the CYP2A6 gene, to which PXR and CAR could bind after dimerization with retinoid X receptor (RXR)-␣. In luciferase assays, overexpression of PXR or CAR could not activate the transcriptional activity of CYP2A6 promoter constructs (Ϫ6754 to Ϫ1) in HepG2 cells. Cotransfection of hepatocyte nuclear factor-4␣ did not affect the transcriptional activities in the absence or presence of PXR or CAR. Interestingly, cotransfection of peroxisome proliferator-activated receptor-␥ coactivator 1␣ (PGC-1␣) as well as PXR significantly enhanced the transcriptional activity (3.9-fold of control). By the deletion of a possible suppresser region (Ϫ4533 to Ϫ185), the effects of PXR/PGC-1␣ on the transcriptional activity were increased (6.9-fold of control). Deletion or mutation analyses revealed that two DR4-like elements at Ϫ5476 and Ϫ4618 are essential for transactivation by PXR/PGC-1␣. Chromatin immunoprecipitation assay revealed that PXR and PGC-1␣ bind to CYP2A6 chromatin. In conclusion, we found that CYP2A6 is induced via PXR and PGC-1␣ through the DR4-like element at the distal response region. This is the first study to report the molecular mechanism of the induction of CYP2A6.

ABSTRACT:Genetic polymorphisms of CYP2A6 gene are known as a causal factor of the interindividual differences in nicotine metabolism. We found three novel CYP2A6 alleles. The CYP2A6*18A allele has a single nucleotide polymorphism (SNP) of A5668T (A1175T, Y392F) in exon 8. The CYP2A6*18B allele has synonymous SNPs of G51A (G51A), T5684C (T1191C), and T5702C (T1209C) in addition to A5668T (A1175T, Y392F). The CYP2A6*19 allele has the SNPs of A5668T (A1175T, Y392F), T6354C (intron 8), and T6558C (T1412C, I471T) as well as the conversion with the CYP2A7 sequence in the 3-untranslated region, in which the latter two changes correspond to CYP2A6*7. Ethnic differences in the frequencies of these alleles were observed between whites, African-Americans, Japanese, and Koreans. Wild or variant CYP2A6 (CYP2A6*18, CYP2A6*19, and CYP2A6*7) were expressed in Escherichia coli. For coumarin 7-hydroxylation and 5-fluorouracil formation from tegafur, the K m values were increased, and V max values were decreased in CYP2A6.18 compared with those in CYP2A6.1, resulting in decreased clearance to 50 and 35% of that of the wild type, respectively. The K m and V max values for nicotine C-oxidation were both increased, resulting in no change of clearance. In CYP2A6.19, the effects on the coumarin 7-hydroxylation and 5-fluorouracil formation (increased K m and decreased V max ) were prominent, resulting in decreased clearance to 8% of those of the wild type. For nicotine C-oxidation, the K m and V max values were both decreased, resulting in decreased clearance to 30% of that of the wild type. The changes of the kinetics in CYP2A6.19 were similar to those in CYP2A6.7. In vivo nicotine metabolism was evaluated in whites (n ‫؍‬ 56) and Koreans (n ‫؍‬ 40). Although the CYP2A6*18 and CYP2A6*19 alleles were found only heterozygously, a subject with CYP2A6*7/CYP2A6*19 showed a lower cotinine/nicotine ratio of the plasma concentration compared with homozygotes of the CYP2A6*1A, supporting the in vitro results that the CYP2A6*19 allele leads to decreased enzymatic activity.

UGT2B7 is transcriptionally regulated by Nrf2, but the mechanism is hindered by polymorphisms in the promoter region of UGT2B7*2. The allele-specific mechanism may cause variability of the glucuronidation in response to oxidative stress.

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Human CYP2A6 is responsible for the metabolism of nicotine and coumarin as well as the metabolic activation of tobacco-related nitrosamines. Earlier studies revealed that CYP2A6 activity was increased by dietary cadmium or cruciferous vegetables, but the underlying mechanisms remain to be clarified. In the present study, we investigated the possibility that Nrf2 might be involved in the regulation of CYP2A6. Real-time RT-PCR analysis revealed that the CYP2A6 mRNA level in human hepatocytes was significantly (P < 0.01, 1.4 fold) induced by 10 µM sulforaphane (SFN), a typical activator of Nrf2. A computer-based search identified three putative antioxidant response elements (AREs) in the 5'-flanking region of the CYP2A6 gene at positions -1212, -2444, and -3441, termed ARE1, ARE2, and ARE3, respectively. Electrophoretic mobility shift assays demonstrated that Nrf2 bound only to ARE1. Luciferase assays using HepG2 cells revealed that the overexpression of Nrf2 significantly increased the reporter activities of the constructs containing a 30-bp fragment that included ARE1. However, the activity of the construct containing the intact 5'-flanking region (-1 to -1395) including ARE1 was not increased by the overexpression of Nrf2. In contrast, when the reporter construct was injected into mice via the tail vein, the reporter activity in the liver was significantly (P < 0.05, 1.9 fold) increased by SFN (1 mg/head) administration. In conclusion, we found that human CYP2A6 is regulated via Nrf2, suggesting that CYP2A6 is induced under oxidative stress.

Differentiation of stem cells to hepatocytes provides an unlimited supply of human hepatocytes and therefore has been vigorously studied. However, to date, the stem cell-derived hepatocytes were suggested to be of immature features. To obtain matured hepatocytes from stem cells, we tested the effect of culturing human-induced pluripotent stem (hiPS) cell-derived endoderm cells on collagen vitrigel membrane and compared with our previous reported nanofiber matrix. We cultured hiPS cell-derived endoderm cells on a collagen vitrigel membrane and examined the expression profiles, and tested the activity of metabolic enzymes. Gene expression profile analysis of hepatocytic differentiation markers revealed that upon culture on collagen vitrigel membrane, immature markers of AFP decreased, with a concomitant increase in the expression of mature hepatocyte transcription factors and mature hepatocyte markers such as ALB , ASGR1 . Mature markers involved in liver functions, such as transporters, cytochrome P450 enzymes and phase II metabolic enzymes were also upregulated. We observed the upregulation of the liver markers for at least 2 weeks. Gene array profiling analysis revealed that hiPS cell-derived hepatocyte-like cells (hiPS-hep) resemble those of the primary hepatocytes. Functions of the CYP enzyme activities were tested in multi-institution and all revealed high CYP1A, CYP2C19, CYP2D6, CYP3A activity, which could be maintained for at least 2 weeks in culture. Taken together, the present approach identified that collagen vitrigel membrane provides a suitable environment for the generation of hepatocytes from hiPS cells that resemble many characteristics of primary human hepatocytes.