Progressive development of insulin resistance phenotype in male mice with complete aromatase (CYP19) deficiency.

Aromatase (CYP19) is a cytochrome P450 enzyme that catalyzes the formation of aromatic C18 estrogens from C19 androgens. It is expressed in various tissues and contributes to sex-specific differences in cellular metabolism. We have generated aromatase-knockout (ArKO) mice in order to study the role of estrogen in the regulation of glucose metabolism. The mean body weights of male ArKO (-/-) mice (n=7) and wild-type littermates (+/+) (n=7) at 10 and 12 weeks of age were 26.7+/-1.9 g vs 26.1+/-0.8 g and 28.8+/-1.4 g vs 26.9+/-1.0 g respectively. The body weights of the ArKO and wild-type mice diverged between 10 and 12 weeks of age with the ArKO males weighing significantly more than their wild-type littermates (P<0.05). The ArKO males showed significantly higher blood glucose levels during an intraperitoneal glucose tolerance test compared with wild-type littermates beginning at 18 weeks of age. By 24 weeks of age, they had higher fasting blood glucose levels compared with wild-type littermates (133.8+/-22.8 mg/dl vs 87.8+/-20.3 mg/dl respectively; P<0.01). An intraperitoneal injection of insulin (0.75 mU insulin/g) caused a continuous decline in blood glucose levels in wild-type mice whereas ArKO males at 18 weeks and older exhibited a rebound increase in glucose levels 30 min after insulin injection. Thus, ArKO male mice appear to develop glucose intolerance and insulin resistance in an age-dependent manner. There was no difference in fasting serum triglyceride and total cholesterol levels between ArKO male mice and wild-type littermates at 13 and 25 weeks of age. However, serum triglyceride and cholesterol levels were significantly elevated following a meal in ArKO mice at 36 weeks of age. Serum testosterone levels in ArKO male mice were continuously higher compared with wild-type littermates. Treatment of ArKO males with 17beta-estradiol improved the glucose response as measured by intraperitoneal glucose and insulin tolerance tests. Treatment with fibrates and thiazolidinediones also led to an improvement in insulin resistance and reduced androgen levels. As complete aromatase deficiency in man is associated with insulin resistance, obesity and hyperlipidemia, the ArKO mouse may be a useful animal model for examining the role of estrogens in the control of glucose and lipid homeostasis.

Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol.

Aromatase P450 (CYP19) is an enzyme catalysing the conversion of androgens into oestrogens. We generated mice lacking aromatase activity (ArKO) by targeted disruption of Cyp19 and report the characteristic features of the ArKO ovaries and uteri as revealed by histological and biochemical analyses. ArKO females were totally infertile but there were as many developing follicles in their ovaries at 8 weeks of age as in wild-type ovaries. Nevertheless, no typical corpus luteum was observed in the ArKO ovaries. Electron microscopy revealed the presence of well-developed smooth endoplasmic reticulum, few lipid droplets and mitochondria with less organized tubular structures in the ArKO luteinized interstitial cells. These ultrastructural features were different from those of the wild-type interstitial cells, where there are many lipid droplets and mitochondria with well-developed tubular structures, characteristic of steroid-producing cells. When ArKO mice were supplemented with 17beta-oestradiol (E(2); 15 microg/mouse) every fourth day from 4 weeks of age for 1 month, increased numbers of follicles were observed in the ovaries as compared with those of untreated ArKO mice, although no typical corpus luteum was detectable. Ultrastructural analysis revealed the disappearance of the accumulated smooth endoplasmic reticulum in the luteinized interstitial cells after E(2 )supplementation. Transcripts of pro-apoptotic genes such as p53 and Bax genes were markedly elevated in the ArKO ovaries as compared with those of wild-type mice. Although E(2) supplementation did not cause suppression of the elevated expression of p53 and Bax mRNAs, it caused marked enhancement of expression levels of lactoferrin and progesterone receptor mRNAs in the uteri as well as increases in uterine wet weight. At 8 months of age, ArKO mice developed haemorrhages in the ovaries, in which follicles were nearly depleted, while age-matched wild-type females still had many ovarian follicles. Furthermore, macrophage-like cells were occasionally observed in the ArKO ovarian follicles. These results suggested that targeted disruption of Cyp19 caused anovulation and precocious depletion of ovarian follicles. Additionally, analysis of mice supplemented with E(2) demonstrated that E(2) apparently supports development of ovarian follicles, although it did not restore the defect in ovulation.

Oestrogen at the neonatal stage is critical for the reproductive ability of male mice as revealed by supplementation with 17beta-oestradiol to aromatase gene (Cyp19) knockout mice.

Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.

A loss of aggressive behaviour and its reinstatement by oestrogen in mice lacking the aromatase gene (Cyp19).

Aromatase P450 (CYP19) is an enzyme responsible for conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeting disruption of the CYP19 gene and observed that the ArKO males exhibited a complete loss of aggressive behaviour against intruder mice when examined using a resident-intruder paradigm. The defect in the behaviour of ArKO males was reinstated when the mice received supplements of 17beta-oestradiol soon after birth. Nevertheless, the cumulative duration of the behaviour displayed by the treated mice during the test period of 15 min was 19+/-10 s, which was much shorter than that displayed by wild-type males, 90+/-17 s. When the supplementation was started at 7 days after birth, the defect was not restored. These findings illustrate an absolute requirement for oestrogen during the neonatal stage of a male's life for the development of the potential for aggression observed in adulthood. Furthermore, the present study demonstrates that ArKO males are a useful model in which to investigate the neural mechanisms by which aggressive behaviour is controlled.

Sex- and age-related response to aromatase deficiency in bone.

Deficiency of sex steroids causes osteoporosis, but the relationship between estrogen and androgen is not clear because androgen is converted into estrogen by aromatase. In this study, we characterized bone metabolism in the aromatase-deficient (ArKO) mouse. At 9 weeks old, a marked loss of cancellous bone due to increased bone resorption was observed not only in female ArKO mice but also in males. The degree of bone loss in ArKO males was similar to that in females, and treatment with 17beta-estradiol completely restored the bone mass in both sexes. At 32 weeks old, female ArKO mice showed severe loss of cancellous and cortical bone. Male ArKO mice of this age also showed reduced bone mass, but the degree of bone loss in females was more marked than that in males. Here, we report sex- and age-related responses to aromatase deficiency in bone.

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator.

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid beta-oxidation. The impairment was reversed to the wild-type levels by treatment with 17beta-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid beta-oxidation and in maintaining lipid homeostasis.

Altered expression of fatty acid-metabolizing enzymes in aromatase-deficient mice.

Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid beta-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid beta-oxidation. In vitro assays of fatty acid beta-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid beta-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17beta-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid beta-oxidation and in maintaining hepatic lipid homeostasis.

Nitric oxide inhibits HIV tat-induced NF-kappaB activation.

To evaluate the roles of nitric oxide (NO) on human immunodeficiency virus (HIV) Tat-induced transactivation of HIV long terminal repeat (HIV-LTR), we examined the effect of NO in the regulation of nuclear factor (NF)-kappaB, a key transcription factor involved in HIV gene expression and viral replication. In the present study, we demonstrate that HIV Tat activates NF-kappaB and that this activation can be attenuated by endogenous or exogenous NO. Inhibition of endogenous NO production with the NO synthase (NOS) inhibitor L-NMMA causes a significant increase in Tat-induced NF-kappaB activity. In addition, NO attenuates signal-initiated degradation of IkappaBalpha, an intracellular inhibitor of NF-kappaB, and blocks the DNA binding activity of the NF-kappaB p50/p50 homodimer and p50/p65 heterodimer. To determine how NO is induced by HIV Tat, reverse transcription polymerase chain reaction was used to demonstrate the induction of NOS-2 and NOS-3 mRNA by Tat. Although a putative NF-kappaB binding site was identified in the -74 GGAGAGCCCCC -64 region of the NOS-3 gene promoter, gel mobility shift assays and site-directed mutation analyses suggest that the putative NF-kappaB site is not of primary importance. Rather, several Sp-1 sites adjoining the putative NF-kappaB binding site in the promoter region of NOS-3 gene are required for the induction of NOS-3 gene expression by Tat.

Tissue factor pathway inhibitor-2 is a novel mitogen for vascular smooth muscle cells.

A mitogen for growth-arrested cultured bovine aortic smooth muscle cells was purified to homogeneity from the supernatant of cultured human umbilical vein endothelial cells by heparin affinity chromatography and reverse-phase high performance liquid chromatography. This mitogen was revealed to be tissue factor pathway inhibitor-2 (TFPI-2), which is a Kunitz-type serine protease inhibitor. TFPI-2 was expressed in baby hamster kidney cells using a mammalian expression vector. Recombinant TFPI-2 (rTFPI-2) stimulated DNA synthesis and cell proliferation in a dose-dependent manner (1-500 nM). rTFPI-2 activated mitogen-activated protein kinase (MAPK) activity and stimulated early proto-oncogene c-fos mRNA expression in smooth muscle cells. MAPK, c-fos expression and the mitogenic activity were inhibited by a specific inhibitor of MAPK kinase, PD098059. Thus, the mitogenic function of rTFPI-2 is considered to be mediated through MAPK pathway. TFPI has been reported to exhibit antiproliferative action after vascular smooth muscle injury in addition to the ability to inhibit activation of the extrinsic coagulation cascade. However, structurally similar TFPI-2 was found to have a mitogenic activity for the smooth muscle cell.

Effects of irradiation on telomerase activity in human lymphoma and myeloma cell lines.

The effect of high-dose irradiation on telomerase activity was examined in some human lymphoma (DL40, DL95, DL110) and myeloma (U266) cell lines. The survival rate was reduced in DL40, DL110 and U266 by irradiation. Irradiation, however, showed no effect on the rate of DL95. Telomerase activity was detected in non-irradiated samples of all cell lines, as measured by PCR-based telomeric repeat amplification protocol assay. The telomerase activity increased 2-6.5 fold by irradiation. Especially in DL110, the activation increased in a dose-dependent manner. In the early phase after irradiation, we observed no correlation between telomerase activity and cell viability, suggesting that telomerase-mediated chromosome healing might not be a major cause and/or not sufficiently effective to protect the cells from irradiation.

Induction of DNA fragmentation by total-body irradiation in murine liver.

Total-body irradiation (TBI) is an accepted modality to treat patients with disseminated tumors. The influence of the treatment on normal tissues is evaluated using mice by measuring the rate of the induction and distribution of apoptosis, as well as DNA fragmentation which occurs in the murine liver within hours of irradiation. Unanesthetized female C3H/He mice were exposed to gamma-ray TBI of 2, 7, and 20 gray (Gy) delivered from 60Co at a dose rate of 114 cGy/min. Frozen sections of livers which were excised from the animals at various times after irradiation were stained by hematoxylin-eosin (H-E) to count numbers of apoptotic cells, or were examined to detect DNA fragmentation. The percentages of apoptotic cells and length of the period during which the maximum levels of the percentages were exhibited showed a dose-dependent increase in the sections stained with H-E. No positive cells for 3'-OH ends of fragmented DNA were found in the liver before TBI, whereas positive cells were observed immediately after irradiation without dose-dependency, these positive cells returned to nearly basal levels after several hours. Positive cells were observed prior to showing apoptosis, suggesting that DNA fragmentation occurs immediately after TBI independent of apoptosis. The difference in the time courses between induction of DNA fragmentation and of apoptosis was not observed in other organs or in the samples treated with the detergent. These results suggested that the 3'-OH ends newly generated by TBI were masked by a detergent-soluble DNA-binding molecule which might be preferentially present in the murine liver.

CMO I deficiency caused by a point mutation in exon 8 of the human CYP11B2 gene encoding steroid 18-hydroxylase (P450C18).

Corticosterone methyloxidase I (CMO I) deficiency is an autosomal recessive disorder of aldosterone biosynthesis. To determine further the molecular genetic basis of CMO I deficiency, a patient of Turkish origin that suffered from CMO I deficiency was studied. Nucleotide sequencing of the PCR-amplified exons from the genomic DNA of this patient revealed a single point mutation CTG (leucine) CCG (proline) at codon 461 in exon 8 of CYP11B2, which is involved in the putative heme binding site of steroid 18-hydroxylase (P450(C18)). The expression study using a cDNA introducing the point mutation revealed that the amino acid substitution totally abolishes the P450(C18)p3 enzyme activities required for conversion of 11-deoxycorticosterone to aldosterone, even though the mutant product was detected in the mitochondrial fraction of the transfected cells. These results suggest that this point mutation causes CMO I deficiency.

Cooperative regulation of the human aromatase cytochrome P450 gene transcription by placenta-specific cis-acting elements.

Aromatase cytochrome P450 catalyses the reaction to convert androgens to estrogens by coupling with NADPH-cytochrome P450 reductase in the endoplasmic reticulum. The human aromatase cytochrome P450 gene (CYP19) is expressed in a variety of tissues under regulation of tissue-specific promoters. Previously, we localized a cell-type specific transcriptional enhancer element between -242 and -166 relative to the major cap site of the gene, by transient expression analysis in human BeWo choriocarcinoma cells. In the present study, we demonstrate that the enhancer element consists of two subelements, element I (located between -238 and -200), and element II (located between -196 and -176) as analysed by DNase I footprinting using the nuclear extracts of BeWo cells. The gel mobility shift assay shows that each of these subelements binds specific nuclear factor(s). The transient expression of the bacterial chloramphenicol acetyltransferase gene constructs involving the subelements in BeWo cells reveals that the elements activate reporter gene expression synergistically when present together, nevertheless each of the elements by itself also has an enhancer activity. The transient expression analysis further shows that element I is responsible for the transcriptional synergism with the binding site of a nuclear factor-interleukin-6 (NF-IL-6) (also known as CCAAT enhancer/binding protein beta), which is located between -2141 and -2115 relative to the major cap site of the gene. These results suggest that the enhancer element plays important roles in sustaining the high levels of CYP19 expression in placental cells in cooperation with other cis-acting transcritional regulatory elements.

Mutation of cytochrome P-45017 alpha gene (CYP17) in a Japanese patient previously reported as having glucocorticoid-responsive hyperaldosteronism: with a review of Japanese patients with mutations of CYP17.

A 17-yr-old female Japanese patient, who was reported in 1968 as having glucocorticoid-responsive hyperaldosteronism but was presumed to have a defect of 17 alpha-hydroxylation mainly in the adrenal glands as the etiology of her illness, was followed. The relationship between clinical manifestations and molecular abnormalities in cytochrome P-45017 alpha gene (CYP17) was also reviewed based on the literature on Japanese patients with 17 alpha-hydroxylase deficiency. She has been treated with dexamethasone, resulting in normal blood pressure and normokalemia for 28 yr. She had almost normal gonadal function with regular menstruation on her first admission. Because of sustained genital bleeding, however, she underwent total hysterectomy with an ovarian biopsy at the age of 42 yr. No follicles or corpus luteum were detected in the ovarian specimen. At the age of 45 yr, the basal levels of sex steroids were decreased, while those of gonadotropins were increased. A genetic study on CYP17 revealed a homozygous deletion of phenylalanine (Phe) codon (TTC) at either amino acid position 53 or 54 in exon 1. A review of the literature revealed 4 patients with this type of CYP17 mutation, including the present patient, out of a total of 11 young adult Japanese patients. The clinical manifestations caused by congenitally deficient gonadal function were not marked in any of these 4 patients, but were marked in 5 of the 7 patients with different mutations of CYP17. The remaining 2 female patients had irregular menstruation. The pretreatment urine/plasma values of aldosterone were variable, normal to high, in individual patients, regardless of the structural abnormalities of CYP17. The following conclusions were suggested: 1) this type of CYP17 mutation is associated with well preserved gonadal function in young adult patients, but it likely causes early reduction of gonadal function with increasing age in these patients; 2) the prevalence of this type of CYP17 mutation is quite high in Japanese patients; and 3) the pretreatment hyperaldosteronism observed in the present patient seems not to be related to the mutation of CYP17.

Overexpression of int-5/aromatase in mammary glands of transgenic mice results in the induction of hyperplasia and nuclear abnormalities.

Our recent studies have shown that the cellular gene at the mouse mammary tumor virus integration site in the int-5 locus is aromatase. To study the role of int-5/aromatase in normal mammary development and mammary neoplasia, we have generated transgenic mice that overexpress int-5/aromatase under the control of mouse mammary tumor virus enhancer/promoter. All the transgenic virgin (n = 10) and postlactational (n = 15) females that overexpress int-5/aromatase show various histological abnormalities. Overexpression of int-5/aromatase in mammary glands of virgin females leads to the enlargement of 40% of ducts, of which 65% had hyperplastic lesions, 20% had dysplastic lesions, and 15% had fibroadenoma lesions. Overexpression of int-5/aromatase in involuted mammary glands of transgenic females induces hyperplasia in 75-80% of ducts and glands that exhibit a range of morphological abnormalities, including formation of hyperplastic alveolar nodule (10%), ductal and glandular hyperplasia (70-80%), ductal and lobular dysplasia (15%), and nuclear abnormalities (2-5%) such as multinucleation and karyomegaly, which are all indicative of preneoplastic changes. Our results show that early exposure of mammary epithelium to in situ estrogen and continued exposure to in situ estrogen as a result of overexpression of int-5/aromatase appears to predispose mammary tissue to preneoplastic changes, which may, in turn, increase the risk of developing neoplasia and increase susceptibility to environmental carcinogens. These findings support clinical and experimental data that suggest that early estrogen exposure increases breast cancer risk.

Identification and characterization of transcriptional regulatory elements of the human aromatase cytochrome P450 gene (CYP19).

Aromatase cytochrome P450, a member of the cytochrome P450 gene super family, catalyzes conversion of androgens to estrogens in a form of an enzyme-complex with NADPH-cytochrome P450 reductase. Transcription of the aromatase cytochrome P450 gene (CYP19) is regulated in part by tissue-specific promoters coupled with alternative splicing mechanisms. The transcription in human placenta is governed by a promoter activity of the 5' flanking region of exon I.1, which is mapped more than 40 kb upstream from the translational start codon observed in exon II. Transient expression analyses with chimeric constructs containing the 5' flanking sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene in human BeWo choriocarcinoma cells localized a cell-type specific enhancer element between -242 and -166 relative to the major cap site. DNase I footprinting and transient expression analyses of the enhancer element indicate that it consists of two sub-elements and that both sub-elements are necessary for the maximum enhancement of the transcription. In addition to the enhancer element, a cis-acting element important for transcriptional enhancement of the gene in response to 12-O-tetradecanoylphorbol 13-acetate in BeWo cells is localized between -2141 and -2115. A nuclear factor binding to the element is identified as NF-IL6 (also termed as LAP and C/EBP beta). Transient expression analyses using the CAT constructs containing the NF-IL6 binding sites involvement of the factor in transcriptional regulation of CYP19.

A SP1 binding site in the GC-rich region is essential for a core promoter activity of the human endothelial nitric oxide synthase gene.

Endothelial nitric oxide synthase (eNOS) is an important oxygenase which catalyzes the conversion of L-arginine to L-citrulline to form nitric oxide (NO), a potent important factor for vasodilation and inhibition of platelet aggregation. We have analyzed characteristics of the promoter region of the human eNOS gene using the transient expression in human endothelial cells of CAT constructs with a series of 5'-deletion mutants. The 5'-flanking region between -116 and -98, which contains a putative consensus sequence for binding of transcription factor Sp1, is essential to direct a basal promoter activity. Gel mobility shift analysis involving anti-Sp1 antibody and competitor DNAs disrupted at the binding site for Sp1 reveals that Sp1 or its closely related protein(s) binds to the consensus sequence located between -104 and -96. These results indicate that the Sp1 site is essential for a core promoter activity of the human eNOS gene.

Identification of a transcriptional regulatory factor for human aromatase cytochrome P450 gene expression as nuclear factor interleukin-6 (NF-IL6), a member of the CCAAT/enhancer-binding protein family.

Human aromatase cytochrome P450 catalyzes the ultimate reaction in the estrogen biosynthetic pathway by coupling with another enzyme, NADPH-cytochrome P450 reductase, in the endoplasmic reticulum. The expression of the gene encoding the enzyme (CYP19) is regulated, in part, by tissue-specific promoters through the use of alternative-splicing mechanisms. Recently, we have localized a transcriptional activating element at positions -2141 to -2115 relative to the major cap site of the gene, by transient expression analyses in human BeWo choriocarcinoma cells using the bacterial chloramphenicol acetytransferase reporter gene ligated with CYP19 promoter sequences which regulate expression in this tissue. Here, we report the isolation of a cDNA encoding a DNA-binding protein which binds specifically to the regulatory element. The deduced amino-acid sequence of the insert is identical to that corresponding to the DNA-binding domain and the dimerization domain of a transcription factor, nuclear factor interleukin-6 (NF-IL6), a member of the CCAAT/enhancer-binding protein (C/EBP) family. Studies using specific antibodies against members of the C/EBP family demonstrate that NF-IL6 is the major nuclear factor binding to the regulatory element in BeWo cells; nevertheless. C/EBP alpha also seems to be involved. Disruption of the NF-IL6-binding site within the regulatory element resulted in the disappearance of the transcriptional enhancing activity of the element, indicating that NF-IL6 is at least one of the nuclear factor(s) which enhances transcription through binding to the cis-acting element. These results indicate the intrinsic importance of NF-IL6 in the transcriptional regulation of CYP19 expression.

Transcriptional regulation of the human aromatase cytochrome P450 gene expression in human placental cells.

The human aromatase cytochrome P450 gene, CYP 19, spans more than 75 kb in the human genome. Recently, it is proposed that the expression of the CYP 19 gene is regulated in part by tissue-specific promoters through the use of mechanisms involving alternative splicing of a number of untranslated exons. In this study, we have characterized cis-acting elements involved in the transcriptional regulation of the gene in human placental cells, where the majority of the transcripts contain the 5'-untranslated sequence encoded by exon I.1. By transient expression analyses in human BeWo choriocarcinoma cells using the bacterial chloramphenicol acetyltransferase gene as a reporter gene, we localized an enhancer element in the region between -242 and -166 relative to the major cap site of the gene. Furthermore, we demonstrate that the element between -2141 and -2115 participates in the 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated enhancement of gene expression. By screening a human placental cDNA expression library, we have isolated a cDNA clone (lambda 1-2) encoding a peptide which binds specifically to the element between -2141 and -2115. Sequence analysis of the clone revealed that the insert of lambda 1-2 encodes a part of the amino acid sequence of NF-IL6 (also termed as LAP and C/EBP beta). Northern blot analysis reveals expression of the NF-IL6 gene in BeWo cells and human placenta. These results indicate that NF-IL6 is one of the nuclear factors which participate in TPA-mediated transcriptional enhancement of CYP 19 gene expression.

Inborn errors of aldosterone biosynthesis in humans.

Corticosterone methyl oxidase (CMO) type I and type II deficiencies are inborn errors at the penultimate and ultimate steps in the biosynthesis of aldosterone in humans. Recently, steroid 18-hydroxylase (P450C18), or aldosterone synthase (P450aldo), was shown to be a multifunctional enzyme catalyzing these two steps of aldosterone biosynthesis, i.e., the conversion of corticosterone to 18-hydroxycorticosterone and the subsequent conversion of 18-hydroxycorticosterone to aldosterone. This observation suggests that CMO I and CMO II deficiencies are derived from two different mutations in the P450C18 gene (CYP11B2). To elucidate whether or not this is the case, we performed molecular genetic studies on CYP11B2 of both types of patients. Nucleotide sequence analysis has indicated that the gene of CMO I deficient patients is completely inactivated by a frameshift to form a stop codon due to a 5-bp nucleotide deletion in exon 1. Sequence analysis of CYP11B2 of CMO II deficient patients has revealed two point mutations, CGG-->TGG (Arg181-->Trp) in exon 3 and GTG-->GCG (Val386-->Ala) in exon 7. CYP11B1, the gene for steroid 11 beta-hydroxylase (P45011 beta) which was previously postulated to be the target for CMO II deficiency, is not impaired in these two types of patients. Expression studies using the corresponding mutant cDNAs have shown that CMO I deficient patients are null mutants with a complete lack of P450C18 whereas CMO II deficient patients are leaky mutants with an altered P450C18 activity.(ABSTRACT TRUNCATED AT 250 WORDS)