Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development.

Early expression of estrogen receptors (esr) and their role in regulating early expression of cyp19a1b encoding brain aromatase were examined in the brain of zebrafish. Using in toto hybridization and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), a significant increase in the expression of esr1, esr2a, and esr2b was observed between 24 and 48 hours postfertilization (hpf). In toto hybridization demonstrated that esr2a and esr2b, but not esr1, are found in the hypothalamus. Using real-time RT-PCR, an increase in cyp19a1b mRNAs occurs between 24 and 48 hpf, indicating that expression of cyp19a1b is temporally correlated with that of esr. This increase is blocked by the pure anti-estrogen ICI182,780. Furthermore, E2 treatment of cyp19a1b-GFP (green fluorescent protein) transgenic embryos results in appearance of GFP expression in the brain as early as 25 hpf. These results indicate that basal expression of cyp19a1b expression in the brain of developing zebrafish most likely relies upon expression of esr that are fully functional before 25 hpf.

Mutations of P450c21 (steroid 21-hydroxylase) at Cys428, Val281, and Ser268 result in complete, partial, or no loss of enzymatic activity, respectively.

Steroid 21-hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH), a common genetic disease. To define the relationship between gene mutations and enzyme deficiency, we generated missense mutations of the 21-hydroxylase cDNA at three different sites and characterized the mutant proteins after expressing them in cultured mammalian and yeast cells. Among them, Ser268 and Val281 have been found to be mutated in CAH patients, whereas Cys428 has been implicated as the heme ligand. Our results show mutations at these sites result in complete, partial, or no loss of the enzymatic activity. All the Cys428 mutants had neither enzymatic activity nor P450 absorption, thus supporting the notion that Cys428 is the heme ligand. All the 268-mutants exhibited the same activity as normal 21-hydroxylase, demonstrating that the clinically observed Ser268----Thr change represents a polymorphism rather than the cause of the enzyme deficiency. The 281-mutants had normal Km but greatly reduced Vmax values that also paralleled the reduction in the heme content, in the order Val281 (normal, 100%) greater than Ile281 (50%) greater than Leu281 (20%) greater than Thr281 (10%). Our findings suggest that the methyl group at the beta-carbon of Val281 is required for heme incorporation and consequently enzymatic activity.

Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli.

P450c21 catalyzes an important step in steroid synthesis. Its deficiency leads to symptoms of steroid imbalance. To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli. The 5'-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification. Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells. One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity. The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by Km were normal. The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra. The D380 mutant protein had 3-fold reduction in peak heights in both spectra. This reduced heme binding resulted in 3-fold lower enzymatic activity.

Ethanol modulates the expression of GABA(B) receptor mRNAs in the prenatal rat brain in an age and area dependent manner.

Prenatal ethanol exposure has various deleterious effects on neuronal development. As GABA(B) receptor is known to play an important role during the development of the CNS, we now focused on its mRNA expression pattern in the rat brain during the late gestational days (GD) from 15.5 to GD 21.5. Ethanol's effect was also observed from GD 11.5 to GD 21.5. GABA(B1) receptor mRNA showed a high expression level in GD 15.5 and 19.5, while GABA(B2) receptor mRNA did in GD 15.5 and 21.5. The mRNAs levels depended on age and area during development. Ethanol exposure decreased GABA(B1) receptor from GD 11.5 to GD 19.5 with slight increases in GD 21.5. The decreasing effects were area dependent, with the highest effects in the forebrain including cortex, whereas slight effects were observed in the midbrain and hindbrain. The present results suggest an important role of GABA(B) receptor in the effects of ethanol on prenatal brain developmental processes.

Difference in transcriptional activity of two homologous CYP21A genes.

Steroid 21-hydroxylase (CYP21) deficiency is the major cause of congenital adrenal hyperplasia, a common genetic disease due to steroid imbalance. The main cause for the mutation of the CYP21A2 (c21B) gene is conversion of its nucleotide sequence to the neighboring homologous but nonfunctional c21A gene. In this report the transcriptional activities of the c21A and c21B genes have been analyzed. Transient transfection assays showed that transcription derived from the c21A gene was about 5-fold lower in strength than that of the c21B gene, although both sequences responded to cAMP normally in two adrenocortical cell lines. The normal response to cAMP could probably be attributed to equal activation of both genes by a transcription factor Nur77. The lower transcriptional activity of the c21A gene was attributed to sequence changes within 167 base pairs of the 5'-flanking region, which differs from the c21B gene by only four nucleotides at positions around -100. These four nucleotide changes render the c21A sequence to bind proteins less tightly than the -100 region of the c21B sequence, which binds proteins such as transcription factor Sp1 in electrophoretic mobility shift assays. The reduced transcription due to nucleotide changes at the regulatory region of the c21A gene, in combination with other mutations in the coding region, could play important roles in 21-hydroxylase deficiency.

Expression of human 21-hydroxylase (P450c21) in bacterial and mammalian cells: a system to characterize normal and mutant enzymes.

Cytochrome P450c21 (steroid 21-hydroxylase) is a key enzyme in the synthesis of cortisol, whose deficiency is the cause of a common genetic disease, congenital adrenal hyperplasia. We have expressed P450c21 (steroid 21-hydroxylase) in E. coli and mammalian cells. In E. coli, P450c21 cDNA was cloned into a T7 expression vector to produce a large amount of P450c21 fusion protein, which enabled antiserum production. In mammalian cells, a plasmid containing full-length P450c21 cDNA (phc21) was constructed and transfected into COS-1 cells to produce active P450c21, which was detected by immunoblotting and 21-hydroxylase activity assay. This system was used to assay mutations involved in the disease. Ile172 of phc21 corresponding to the site of mutation in some cases of the disease was mutagenized to become Asn, Leu, His, or Gln. Mutant as well as normal P450c21 was produced when their cDNAs were transfected into COS-1 cells. The mutant proteins, however, had greatly reduced 21-hydroxylase activities. Therefore, missense mutation at Ile172 resulted in inactivation of the enzyme, but not in repression of enzyme synthesis. The Leu for Ile substitution at amino acid 172 did not result in partial restoration of enzymatic activity, indicating that hydrophobicity at this residue may not play a role in its function.

Variegated expression of a mouse steroid 21-hydroxylase/beta- galactosidase transgene suggests centripetal migration of adrenocortical cells.

5'-Flanking sequences (6.4 kb) of the mouse steroid 21-hyrodxylase (21-OHase) A gene linked to a LacZ reporter gene directed appropriate cell-specific expression in cultured Y1 adrenocortical tumor cells and in the adrenal cortex of transgenic mice. The transgene expression initiated at the same stage of adrenal development as the endogenous 21-OHase gene (embryonic day 11.5). Although the endogenous 21-OHase gene is expressed throughout the adrenal cortex, the 21-OHase/beta-gal transgene showed a strikingly variegated pattern of adrenocortical expression in all 10 transgene-expressing mouse lines examined. This presents as radial stripes of beta-gal staining transcending the classical zonal structure of the adrenal cortex but paralleling the columnar arrangement of cells of the zona fasciculata on the centripetal organization of the adrenocortical blood supply. To the extent that the variegated pattern of 21-OHase/beta-gal transgene expression depicts adrenocortical cell lineage, these results suggest that all cells within an individual stripe have a common clonal origin; the radial pattern of clonally derived cells argues that cellular migration maintains the adult adrenocortical cell population. Adrenal glands of developing embryos also exhibited a variegated pattern of 21-OHase/beta-gal transgene expression. However, this presented as islands of beta-gal reporter staining within the developing gland, suggesting that the rapid embryonic adrenal growth phase, which precedes the establishment of the classic adrenocortical zonal structure, may be governed by cellular mechanisms distinct from those responsible for maintenance of the adult adrenocortical cell population.

Transcription of the human ferredoxin gene through a single promoter which contains the 3',5'-cyclic adenosine monophosphate-responsive sequence and Sp 1-binding site.

We have investigated the functional elements involved in cAMP-stimulated transcription of the human ferredoxin gene. Unlike the bovine gene, the human gene lacked a second upstream RNA initiation site as demonstrated by sequence analysis of the exon boundary, lack of upstream RNA, and analysis of the promoter. The presence of a single promoter was determined by testing the ability of various gene segments to drive the expression of the chloramphenicol acetyltransferase gene after transfection into a mouse adrenal cell line Y1. Full promoter activity was conferred by a DNA fragment spanning -209 to +55, although the -94 to +55 fragment already provided some promoter activity. Transcription from the -94 to +55 segment was stimulated by 2-fold when 8-bromo-cAMP was added to the cell. Footprinting analyses showed two GC boxes at -50 to -70 and -87 to -108 were protected by proteins from both Y1 and HeLa cells. Competition experiments showed that a protein with a recognition sequence indistinguishable from Sp1 bound to these sites. When connected to a heterologous TATA box, the sequence at -76 to -42, which contained the proximal GC box, was able to confer a high level of basal transcription and cAMP stimulation. This sequence does not show sequence homology with the known cAMP-responsive element. Mutations or deletion of the Sp 1-binding site showed diminished basal transcription and defined the cAMP responsive sequence to be from -76 to -62. Therefore the cAMP-responsive sequence of the human ferredoxin gene was located at -76 to -62, which was adjacent to the Sp 1-binding site.

Function of steroidogenic factor 1 domains in nuclear localization, transactivation, and interaction with transcription factor TFIIB and c-Jun.

Normal endocrine development and function require nuclear hormone receptor SF-1 (steroidogenic factor 1). To understand the molecular mechanism of SF-1 action, we have investigated its domain function by mutagenesis and functional analyses. Our mutant studies show that the putative AF2 (activation function 2) helix located at the C-terminal end is indispensable for gene activation. SF-1 does not have an N-terminal AF1 domain. Instead, it contains a unique FP region, composed of the Ftz-F1 box and the proline cluster, after the zinc finger motif. The FP region interacts with transcription factor IIB (TFIIB) in vitro. This interaction requires residues 178-201 of TFIIB, a domain capable of binding several transcription factors. The FP region also mediates physical interaction with c-Jun, and this interaction greatly enhances SF-1 activity. The putative SF-1 ligand, 25-hydroxycholesterol, has no effects on these bindings. In addition, the Ftz-F1 box contains a bipartite nuclear localization signal (NLS). Removing the basic residues at either end of the key nuclear localization sequence NLS2.2 abolishes the nuclear transport. Expression of mutants containing only the FP region or lacking the AF2 domain blocks wild-type SF-1 activity in cells. By contrast, the mutant having a truncated nuclear localization signal lacks this dominant negative effect. These results delineate the importance of the FP and AF2 regions in nuclear localization, protein-protein interaction, and transcriptional activation.

DNA-PK/Chk2 induces centrosome amplification during prolonged replication stress.

The antineoplastic drug hydroxyurea (HU), when used at subtoxic doses, induces prolonged replication stress and centrosome amplification. This causes genomic instability and increases the malignancy of the recurring tumor. The mechanism of centrosome amplification induced by prolonged replication stress, however, is still unclear. Here, we examined the involvement of ataxia telangiectasia, mutated (ATM), ataxia telangiectasia, mutated and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) and found that HU-induced centrosome amplification was inhibited by the depletion of DNA-PKcs, but not ATM and ATR. Inactivation of ATM/ATR in U2OS cells instead caused aneuploidy and cell death. We found DNA-PKcs depletion also abrogated ATM phosphorylation, indicating that ATM activation during prolonged replication stress depends on DNA-PK. Depletion of DNA-PK abrogated checkpoint kinase (Chk)2 activation and partially reduced Chk1 activation. Chk2 depletion blocked HU-induced centrosome amplification, indicating a function of Chk2 in centrosome amplification. We further found that Chk2 was phosphorylated at Thr68 on the mother centriole at late G2 and mitosis when unstressed and on all amplified centrioles induced by HU. In summary, we have elucidated that DNA-PK/Chk2 signaling induces centrosome amplification upon long-term HU treatment, therefore increasing our insight into tumor recurrence after initial chemotherapy.

Biochemical roles of testosterone and epitestosterone to 5 alpha-reductase as indicators of male-pattern baldness.

In establishing a theory to predict male-pattern baldness, we investigated the correlation of testosterone, epitestosterone, and dihydrotestosterone with 5alpha-reductase in hair using gas chromatography-mass spectrometry. One hundred milligram hair samples were obtained from a group of balding subjects and their sons, as well as from a corresponding aged-matched, nonbalding group. The ratio of testosterone to epitestosterone was significantly greater (mean 46.41, p < 0.001; mean 35.83, p < 0.001, respectively) in the hair of balding fathers (n = 19, age 28-50 y) and their sons (n = 16, age 8-16 y) than in the hair of the nonbalding control subjects (mean 9.17 and 10.47, respectively). These findings demonstrate that analysis of terminal hair may not only provide a basis for predicting baldness when the subject is still young, but also for preventing and treating male-pattern baldness by controlling the steroid hormone balance.

Study of cholesterol side-chain cleavage (20,22 desmolase) deficiency causing congenital lipoid adrenal hyperplasia using bovine-sequence P450scc oligodeoxyribonucleotide probes.

Conversion of cholesterol to pregnenolone is mediated by the cholesterol side-chain cleavage (SCC) enzyme, P450scc. Deficient SCC activity causes congenital lipoid adrenal hyperplasia (also known as 20,22 desmolase deficiency), a potentially lethal defect in the synthesis of all steroid hormones. To probe for possible genetic defects causing this disease we synthesized four oligodeoxyribonucleotides containing 63 to 72 bases corresponding to portions of the bovine complementary DNA (cDNA) sequence for P450scc. The bovine oligonucleotides were labeled and used directly to probe Southern blots of normal human genomic DNA, revealing a pattern indicating there is a single P450scc gene in the human genome. Hybridization to Northern blots of normal human and bovine adrenal messenger RNA indicates that P450scc messenger RNA is about 2.0 kilobases long in both species. Hybridizations of the oligonucleotides to genomic DNA from three unrelated patients with SCC deficiency did not detect a deletion in the human P450scc gene. The bovine sequence oligonucleotides were then used to isolate a human P450scc cDNA clone. The isolated P450scc cDNA fragment contains 818 bases encoding 239 amino acids of the protein, the translation termination signal, and 98 bases of the 3' untranslated region. The sequence of this carboxy-terminal half of the human P450scc protein is 72% homologous with the bovine sequence and contains an additional amino acid not found in bovine P450scc; the human and bovine nucleotide sequences are 81% homologous. Repetition of the genomic DNA blotting studies with the cDNA probe gave the same results obtained with the bovine-sequence oligonucleotide probes, confirming that SCC deficiency is not due to a deletion in the regions of the P450scc hybridizing with the probes. Long, chemically synthesized heterologous sequence oligonucleotides containing unknown numbers of base mismatches with human sequences may thus be used to study human genes so that access to a cDNA is not necessary for such studies.

Tissue-specific, hormonal, and developmental regulation of SCC-LacZ expression in transgenic mice leads to adrenocortical zone characterization.

We report here the study of the human CYP11A1 promoter in driving tissue-specific, developmentally and hormonally regulated reporter gene expression. A 4.4-kb fragment containing all known regulatory elements is more efficient than a short basal promoter fused to an upstream adrenal enhancer in driving reporter LacZ gene expression both in cell culture and in transgenic mice. The LacZ gene controlled by the 4.4- and 2.3-kb promoters was expressed in the adrenal cortex, testicular Leydig cells, ovarian corpora lutea, and granulosa cells. Transgene expression in the adrenals was stimulated by ACTH, indicating the presence of ACTH-responsive sequence. Beta-galactosidase activity was first detected in the adrenal primordia at 11.5 days postcoitum. Its expression continued throughout all stages of adrenal development in a pattern similar to that of the endogenous CYP11A1, which was expressed in all zones of the adrenal cortex, but was strongest in the X zone. The X zone grew before puberty but regressed afterward, as did the levels of CYP11A1 and LacZ gene expression in the X zone. Our study of the CYP11A1 promoter in transgenic mice led to characterization of the adrenocortical zones.

Assignment of the gene for adrenal P450c17 (steroid 17 alpha-hydroxylase/17,20 lyase) to human chromosome 10.

P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20 lyase activities. We identified several human P450c17 cDNA clones in a human adrenal cDNA library we constructed in lambda gt10. A short clone containing the 3'-terminal 650 bases of the full-length sequence was used to examine Southern blots of DNA from normal persons and from a panel of mouse/human somatic cell hybrid lines. The pattern of hybridization of this cDNA to normal human DNA cut with 8 restriction endonucleases suggests the human genome has two (or more) P450c17 genes. The pattern of hybridization to the somatic cell hybrid cell lines, each containing a limited, known number of human chromosomes, indicates the human adrenal P450c17 gene lies on chromosome 10. The chromosomal locations of the other P450c17 genes could not be determined.

Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17 alpha-hydroxylase/17,20-lyase) in cultured human granulosa cells.

Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to 32P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0-100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02-2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490% and 240% of control values, respectively, with hCG, to 166% and 168% with FSH, and to 495% and 380% with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10(-6) M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin-specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription. Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with 32P-labeled human P450c17 cDNA (P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450c17 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis.

Carrier analysis and prenatal diagnosis of congenital adrenal hyperplasia caused by 21-hydroxylase deficiency in Chinese.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We screened 1,000 healthy people, using a previously developed differential PCR method combined with single-strand conformation polymorphism and amplification-created restriction site methods for the carrier detection of the CYP21 gene deficiency. Our results indicated that the rate of occurrence of the heterozygous CAH carrier was about 12 in 1,000, with a gene frequency of 0.0060 and an incidence frequency of 1 in 28,000 in the Chinese population. In addition, 9 cases of CAH families were performed with prenatal diagnosis. Among them, 3 cases were diagnosed as the severe form, 4 cases carried the heterozygous mutation, and 2 were normal. This is the first report of carrier frequency analysis and prenatal diagnosis of 21-hydroxylase deficiency in Chinese.

Altered hydroxylation of estrogen in patients with postmenopausal osteopenia.

To study the possible contributions of the differences in estrogen metabolism to bone mass in postmenopausal osteopenia, spinal and femoral bone mineral densities (BMD) were measured, and 18 urinary metabolites of estrogen were analyzed by a gas chromatography-mass spectrometry assay system in 59 postmenopausal women (5-10 yr after menopause). The BMD of the spine and femoral neck showed positive correlations with body weight, height, and body mass index as we expected. Compared to nonosteopenic subjects, there were no significant differences in serum estrone (E1) and estradiol (E2) levels in patients with osteopenia. However, the urinary 16 alpha-hydroxyestrone [16 alpha-(OH)E1] level was significantly lower in patients with spinal osteopenia (P < 0.001). Among the 18 urinary metabolites of estrogen, the 16 alpha-(OH)E1 and 16 alpha-(OH)E1/2-hydroxyestrone [2-(OH)E1) ratio showed positive correlations with spinal BMD (P < 0.05), whereas 2-(OH)E2 showed a negative correlation with femoral neck BMD (P < 0.05). The urinary 16 alpha-(OH)E1 level also revealed a positive correlation with the age-matched z score of BMD in the spine (P < 0.05). In multiple stepwise regression analysis, weight, 16 alpha-(OH)E1, interaction between 16 alpha-(OH)E1 and 2-(OH)E2, 2-(OH)E2, and years after menopause were statistically significant for spinal BMD (r2 = 0.4968). For femoral neck BMD and weight, 16 alpha-(OH)E1 and 2-(OH)E2 were the independent determinants (r2 = 0.3369). In conclusion, the activity of estrogen 16 alpha-hydroxylase was decreased and/or the activity of estrogen 2-hydroxylase was enhanced in post-menopausal osteopenia. We speculated that these derangements may serve as contributing factors for the acceleration of bone loss in post-menopausal osteoporosis.

Characterization of Alu repeats surrounding the human ferredoxin-encoding gene.

Three Alu sequences have been identified surrounding the human ferredoxin-encoding genes. Among them, one is located about 1000 bp upstream from the active gene, whereas two others flank the ferredoxin pseudogene, psi FDX3. All these Alu sequences contain poly(A) tails and are flanked by direct repeats, indicating that they arose by RNA-mediated transposition events.

Estrogens and polyamines in breast cancer: their profiles and values in disease staging.

The urinary concentrations of 16 estrogens and 11 polyamines were quantitatively determined by gas chromatography-mass spectrometry and gas chromatography with nitrogen-phosphorus detection. Samples from patients with stages I-IV of breast cancer (35 cases, aged 27-65 years) as well as from age-matched normal female subjects (25 cases, aged 22-61 years) were tested. Also, the ratios of precursor to product metabolite including 16alpha-OH E1 to 2-OH E1, which are linked to estrogen and polyamine biosynthetic pathways, were determined to explore enzyme involvement in breast cancer and to evaluate the potential usefulness of these ratios and concentrations as disease staging markers. It was confirmed that major estrogens and 16a-OH E1 were positively associated with breast cancer and catechol estrogens including 2-OH E1 were inversely associated with breast cancer. The ratios of N1-acSp/Spd and 16alpha-OH E1/2-OH E1 might be a useful dual marker for staging of breast cancer. From the variation of the relative ratios of polyamines, it is suggested that alteration in polyamine oxidase (PAO) activity may play an important role in the development of breast cancer.

Polyamine profiles in the urine of patients with leukemia.

Eleven urinary polyamine levels were determined in controls (32 cases) and 43 patients with varying stages of leukemia including initial, relapse and complete remission, using gas chromatography nitrogen-phosphorus detection. Also, to indirectly evaluate the possible involvement of enzymes, precursor to product concentration ratios were compared between controls and patients with each stage of leukemia. As a result, it is confirmed that the ratio of N1-acSpd/N8-acSpd could be used as a diagnostic marker and the level of N1,N12-diacetylspermine could be used for determining disease stage and as a malignancy marker for leukemia. An altered metabolic pathway related to leukemia is also proposed in which N1,N12-diacetylspermine can be produced directly from spermine and N1,N12-diacetylspermine is a major source of N1-acetylspermidine.