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E R Simpson

Publications and source records attributed to E R Simpson.

At least 145 records · Page 8Linked to original sources
PubMed1990

Transcriptional regulation of the bovine CYP17 (P-450(17)alpha) gene. Identification of two cAMP regulatory regions lacking the consensus cAMP-responsive element (CRE).

Regions within the 5'-flanking sequence of the bovine CYP17 (P-450(17)alpha) gene which are required for cAMP-dependent regulation of transcription have been localized by transient transfection of chimeric reporter gene constructs into mouse adrenal tumor Y1 cells. Two sequences have been found which individually confer cAMP responsiveness to reporter genes; they are located at -243/-225 and -80/-40 base pairs (bp). Obvious sequence homology between these two regions is not apparent. Gel shift competition analysis indicates that nuclear protein(s) binding to the -243/-225-bp region can be competed for by the addition of a double-stranded oligonucleotide containing a consensus cAMP-responsive element (CRE) from the human chorionic gonadotropin alpha gene, whereas addition of this CRE does not abolish protein-DNA complexes formed with fragments containing the -80/-40-bp sequence. Gel shift and Southwestern analysis indicate that the -243/-225-bp region of the P-450(17)alpha gene and the CRE both bind a 47-kDa protein and that the CRE binds additional proteins (43 and 68 kDa) not apparently recognized by the -243/-225-bp sequence. Thus cAMP-dependent regulation of the bovine P-450(17)alpha gene appears to involve two independent cis-regulatory regions, neither of which contains a consensus CRE. Based on protein binding analysis, one of these regions (that including -80/-40 bp) is distinct from the consensus CRE while the other (that containing -243/-225 bp) may be related to the consensus CRE.

Adrenal Gland Neoplasms↗
PubMed1990

Characterization of the promoter/regulatory region of the bovine CYP11A (P-450scc) gene. Basal and cAMP-dependent expression.

The promoter/regulatory region of the bovine CYP11A (P-450scc) gene was cloned from a bovine genomic library. One major start site of transcription was identified by primer extension analysis with a minor start site four nucleotides further upstream. A putative TATA box is located at position -31, and at position -68 resides a putative binding site for the transcription factor Sp1. Transient transfection of chimeric reporter gene constructs into mouse adrenal tumor Y1 cells was used to locate regions within the P-450scc 5'-flanking sequences that are important for basal and cAMP-dependent transcription of the reporter genes. While cAMP-dependent accumulation of mRNA derived from expression of the endogenous bovine P-450scc gene can be inhibited by protein synthesis inhibitors, transcription of reporter gene constructs containing the promoter/regulatory region of the P-450scc gene is not affected by cycloheximide following transient transfection of Y1 cells or primary bovine adrenocortical cells. Basal expression of these constructs as well as cAMP responsiveness is reduced upon deletion of sequences between -186 and -101, further deletion to -50 leading to loss of virtually all the remaining cAMP responsiveness. The sequence between -183 and -83 alone will direct both basal and cAMP-enhanced transcription when fused to a heterologous promoter and is equally active in either the correct or reverse orientation. No homology to the consensus cAMP-responsive element (CRE) or AP-2 binding site is found in this region whereas an activator protein 1-like sequence is found at position -116. It is concluded that the cAMP responsiveness of P-450scc gene expression is mediated by sequences different from canonical consensus regulatory elements. Whether or not there are sequences conferring cAMP responsiveness which are common both to P-450scc and the other steroidogenic P-450 genes remains to be established.

Adrenal Gland Neoplasms↗
PubMed1990

Angiotensin II inhibits luteinizing hormone-stimulated cholesterol side chain cleavage expression and stimulates basic fibroblast growth factor expression in bovine luteal cells in primary culture.

Angiotensin II has been identified immunohistochemically in the ovaries of both rats and humans. Here we present evidence that angiotensin II (an extremely vasoactive agent in a wide range of tissues) may be involved in the regulation of the major steroidogenic enzyme in the ovary, cholesterol side chain cleavage cytochrome P-450 (P-450scc), as well as of basic fibroblast growth factor (bFGF), which has been implicated as an angiogenic factor in the bovine corpus luteum. We have used primary cultures of bovine luteal cells to examine the effect of angiotensin II and its receptor antagonist, saralasin, on expression of mRNA encoding bFGF as well as on progesterone production and the expression of mRNA encoding cholesterol side chain cleavage cytochrome P-450 (P-450scc). Neither angiotensin II nor saralasin when added alone to the culture medium had any effect on basal progesterone production. Luteinizing hormone (LH) caused a 15-fold increase in progesterone accumulation after 24 h of exposure which was reduced to 5-fold in the presence of angiotensin II. This appeared to be receptor-mediated in that although saralasin alone had no effect on LH-stimulated progesterone accumulation, it significantly reversed the inhibition by angiotensin II. This pattern was mirrored by the levels of mRNA encoding P-450scc, i.c., LH induced the highest levels of expression of this message, these levels were reduced by angiotensin II, and saralasin partially overcame this reduction. Levels of mRNA encoding bFGF were elevated by both LH and angiotensin II. Treatment with saralasin, however, resulted in complete inhibition of bFGF mRNA expression in the presence of both LH and angiotensin II. These results suggest a role for angiotensin II to mediate the action of LH as a regulator of bFGF expression and hence, potentially, angiogenesis. Local production of angiotensin II might also contribute to the refractoriness of luteal progesterone secretion to LH at the time of luteal regression.

Angiotensin II↗
PubMed1990

Use of molecular probes to study regulation of aromatase cytochrome P-450.

Aromatase, an enzyme complex localized in the endoplasmic reticulum of estrogen-producing cells, is composed of NADPH-cytochrome P-450 reductase, and aromatase cytochrome P-450 (cytochrome P-450AROM). To define the molecular mechanisms involved in the multifactorial regulation of cytochrome P-450AROM in estrogen-producing cells, we have isolated a cDNA specific for human cytochrome P-450AROM and have used this cDNA to isolate the human cytochrome P-450AROM gene. The cDNA sequence encodes a polypeptide of 503 amino acids and contains--near the carboxy-terminus, a region of high homology with the putative heme-binding regions of other P-450 cytochromes. COS1 cells transfected with an expression plasmid containing the cytochrome P-450AROM cDNA had the capacity to aromatize testosterone, androstenedione and 16 alpha-hydroxyandrostenedione, suggesting that a single polypeptide catalyzes all steps of the aromatization reaction using either of the three major C19-substrates. The human cytochrome P-450AROM gene is greater than 52 kb in size and consists of 10 exons and 9 introns. Hormonally induced changes in aromatase activity of human ovarian granulosa and adipose stromal cells are associated with comparable changes in cytochrome P-450AROM gene expression and synthesis, whereas the reductase component is only modestly affected. Studies are in progress to define the molecular mechanisms involved in the regulation of cytochrome P-450AROM gene expression in estrogen-producing cells.

Adipose Tissue↗
PubMed1990

Expression of neural 5 alpha-reductase messenger ribonucleic acid: comparison to 5 alpha-reductase activity during prenatal development in the rat.

The present study was designed to characterize the developmental pattern of 5 alpha-reductase messenger RNA (mRNA) levels and enzyme activity in intact medial basal hypothalamic (MBH) and preoptic area (POA) tissue fragments of rats during fetal development. 5 alpha-Reductase activity was determined using [3H]testosterone as the substrate and quantifying, by TLC, the radiolabeled 5 alpha-reduced metabolites (dihydrotestosterone [DHT] and 5 alpha-androstane-3 alpha,17 beta-diol). Confirmation of the identity of the [3H]5 alpha-DHT formed was demonstrated by recrystallization of the derivatized DHT to constant specific activity. Under saturating substrate conditions (1.7 microM testosterone), there were no significant sex differences (male vs. female) detected in neural 5 alpha-reductase during prenatal, perinatal, or neonatal development. 5 alpha-Reductase activity was low but detectable at gestational day (GD) 15 (approximately 10 pmol/h.mg protein), increased over 3-fold to peak levels at GD 18, and then declined to moderate levels at GD 22 (approximately 22 pmol/h.mg protein) and 4 days after birth (approximately 17 pmol/h.mg protein). In addition, the developmental pattern of 5 alpha-reductase mRNA expression in fetal MBH-POA tissue was determined, by RNA blot hybridization, using a complementary DNA encoding the rat 5 alpha-reductase enzyme. A single mRNA species was detected at 2.5 kilobase in MBH-POA tissue, which is similar in size to adult rat liver and prostate 5 alpha-reductase mRNA. At GD 15, 5 alpha-reductase mRNA was clearly detectable; the abundance increased on GD 17, remained at maximal levels at GDs 18, 19, and 20, and then decreased to moderate levels on GD 22. A correlation calculated between the normalized 5 alpha-reductase mRNA levels and enzyme activities revealed a strong correspondence between mRNA abundance and enzyme activity values during prenatal development, r = 0.80, P = 0.056. These findings indicate that neural 5 alpha-reductase activity may be developmentally regulated, and that the changes in 5 alpha-reductase activity in MBH-POA tissue during late fetal development are regulated, in part, by the levels of mRNA encoding the 5 alpha-reductase enzyme.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1990

Expression of messenger ribonucleic acid species encoding steroidogenic enzymes in human follicles and corpora lutea throughout the menstrual cycle.

The levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450(17 alpha], aromatase cytochrome P-450 (P-450AROM), and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were examined in human follicles and corpora lutea (CL) throughout the menstrual cycle. Tissues were obtained from women undergoing hysterectomy and oophorectomy. The largest follicle or the CL was dissected from the ovary depending on whether the surgery was performed in the follicular or luteal phase. The day of the cycle was determined by onset of last menstrual period and was confirmed by endometrial histology. Total RNA was examined by Northern blot analysis, using as probes specific 32P-labeled cDNA inserts encoding each human enzyme. Early follicles demonstrated detectable mRNA for both P450scc and P450(17 alpha), but not for P450AROM or 3 beta HSD. P450AROM was detectable late in the follicular phase and appeared markedly induced in the CL. 3 beta HSD was detectable only in the CL. Levels of P450(17 alpha) mRNA remained relatively unchanged throughout the cycle, whereas P450scc mRNA levels were greatly increased in the CL. The presence of P450(17 alpha) mRNA in the human CL is of interest, since it is absent from the bovine CL, and this is consistent with the ability of the human, but not the bovine, CL to synthesize 17 alpha-hydroxyprogesterone and estrogens. The fact that P450AROM expression is highest in CL is surprising, since plasma estrogen levels are highest during the late follicular phase of the cycle, and may suggest that CL estrogen biosynthesis is limited by 17 alpha-hydroxylase or 17,20-lyase activities.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Combined 17 alpha-hydroxylase/17,20-lyase deficiency due to a 7-basepair duplication in the N-terminal region of the cytochrome P45017 alpha (CYP17) gene.

17 alpha-Hydroxylase deficiency is characterized by defects in either or both of the 17 alpha-hydroxylase/17,20-lyase activities. We have elucidated the molecular basis of the combined deficiency of these activities in a Japanese female who is genotypically male and the child of a consanguineous marriage. The complete exonic sequence of the patient's CYP17 (P45017 alpha) gene revealed a seven-basepair duplication (GCGCACA) in exon 2 which leads to a frame shift and, subsequently, a premature stop codon. Because this stop codon occurs N-terminal to the heme-binding sequence, the presence of this mutation leads to the absence of a functional P45017 alpha-protein in adrenal cortex and testis. This, in turn, leads to an absence of sex steroids and excessive secretion of steroids with mineralocorticoid activity and, consequently, female external genitalia and hypertension in this 46XY patient.

Adolescent↗
PubMed1990

Proliferating human granulosa-lutein cells in long term monolayer culture: expression of aromatase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase.

The development of long term culture conditions with which to study the regulation of expression of aromatase, cholesterol side-chain cleavage enzyme, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human granulosa-lutein cells is described in this report. Conditions have been established for the dispersal, growth, freezing, and storage of functional human granulosa cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Optimal growth conditions for human granulosa-lutein cells were determined by plating cells at a low density and testing the capacity of a variety of culture conditions to support growth. A combination of fetal bovine serum (FBS), horse serum, and the serum substitute UltroSer G was found to increase cell number to maximal levels, 8- to 10-fold higher than with sera alone. Human granulosa-lutein cells grown under these conditions had a doubling rate of 36-40 h and were morphologically distinct from human theca interna cells grown under similar conditions. Human granulosa-lutein cells treated with forskolin retracted and rounded up, whereas cultures of human ovarian theca interna cells or human fibroblasts treated similarly did not retract. Human granulosa-lutein cells were grown for successive passages and transferred to serum-free medium containing forskolin, LH, hCG, or cholera toxin. Addition of these agents resulted in a time- and dose-dependent increase in aromatase activity and progesterone secretion. In these studies FSH treatment was found not to increase aromatase activity. In a study of the time course of 3 beta HSD activity in the absence of forskolin under serum-free conditions, it was found that 3 beta HSD activity increased 3-fold during the 72-h treatment period. Forskolin-stimulated 3 beta HSD activity also increased in a time-dependent manner, with levels in treated cells 3-fold higher than those in control cells. Northern analysis performed on total RNA obtained from forskolin- or hCG-stimulated granulosa-lutein cells confirmed that the increase in aromatase activity was associated with a corresponding increase in levels of mRNA specific for aromatase cytochrome P-450. Levels of mRNA encoding cholesterol side-chain cleavage cytochrome P-450 were similarly increased in cells treated with forskolin compared with unstimulated values at each of the time points investigated. Under serum-free conditions in the absence of stimulation, the 3.4-kilobase band of aromatase cytochrome P-450 mRNA was detectable.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

The aromatase enzyme: from cloning to cancer.

Estrogen production by adipose tissue has been implicated in the etiology of such human cancers as endometrial and breast cancer. Estrogen production by adipose cells is subject to complex multifactorial regulation by a number of growth factors and cytokines, including those produced by breast cancer cells. In order to understand the mechanisms responsible for aromatase regulation, the structural gene encoding aromatase cytochrome P-450 (P-450AROM) was isolated from human genomic DNA. The gene spans at least 70 kb and is comprised of 10 exons, the first of which is untranslated. DNA sequence analysis indicates that the gene has a putative TATA (ATAAAA) sequence at -23 bp and putative CAAT binding sequences beginning at -41, -67, and -83 bp, that constitute a promoter region responsible for expression in placenta. However, this promoter does not appear to be responsible for expression in adipose, which may therefore be under the control of another, tissue-specific, promoter. Use of Polymerase Chain Reaction (PCR) technology has allowed for determination of expression of P-450AROM in samples of breast adipose. Preliminary results indicate that expression is highest in the upper lateral region, similar to the site of most frequent localization of tumors.

Adipose Tissue↗
PubMed1989

Structural analysis of the gene encoding human aromatase cytochrome P-450, the enzyme responsible for estrogen biosynthesis.

The structural gene encoding aromatase cytochrome P-450 (P-450AROM) was isolated from human genomic DNA. The gene spans at least 52 kilobases and is composed of 10 exons, the first of which is untranslated. Analysis of the transcription initiation site of human P-450AROM mRNA reveals the differential use of 1 of 3 consecutive G residues at the cap site. DNA sequence analysis indicates that the gene has a putative TATA (ATAAAA) sequence at -23 base pairs (bp) and putative CAAT binding sequences beginning at -41, -67, and -83 bp. The 5'-flanking region contains sequences similar to consensus sequences of cis-acting elements defined as regulators of aromatase gene expression. These putative sequences include a cAMP regulatory element at -211 bp, an AP1 (protein kinase C) site at -54 bp, and glucocorticoid regulatory elements at -352 bp and within the first intron at +346 bp. There appears to be only one gene encoding P-450AROM in the human genome. Two major species of human P-450AROM mRNA (3.4 and 2.9 kilobases) are derived from the use of two polyadenylation signals.

Amino Acid Sequence↗
PubMed1989

Deletion of a phenylalanine in the N-terminal region of human cytochrome P-450(17 alpha) results in partial combined 17 alpha-hydroxylase/17,20-lyase deficiency.

Steroid 17 alpha-hydroxylase and 17,20-lyase activities reside within the same polypeptide chain (cytochrome P-450(17 alpha)), and consequently human 17 alpha-hydroxylase deficiencies are characterized by defects in either or both of these activities. Human mutants having these deficiencies represent an excellent source of material for investigation of P-450(17 alpha) structure-function relationships. The CYP17 gene from an individual having partial combined 17 alpha-hydroxylase/17,20-lyase deficiency has been characterized structurally and the homozygous mutation found to be the deletion of the phenylalanine codon (TTC) at either amino acid position 53 or 54 in exon 1. Reconstruction of this mutation into a human P-450(17 alpha) cDNA followed by expression in COS 1 cells led to production of the same amount of immunodetectable P-450(17 alpha) protein as found with expression of the normal human P-450(17 alpha) cDNA. However, 17 alpha-hydroxylase activity of this mutant protein measured in intact cells was less than 37% of that observed upon expression of the wild-type enzyme, whereas 17,20-lyase activity of the mutant was less than 8% of that observed with the normal enzyme. When estimated in intact cells, the Km for 17 alpha-hydroxylation of progesterone was increased by a factor of 2 in the mutant enzyme, whereas the Vmax was reduced by a factor of 3. In order to estimate the kinetic parameters for the 17,20-lyase reaction, microsomes were isolated from transfected COS 1 cells to enrich for this activity. Surprisingly, the specific activity of the mutant 17 alpha-hydroxylase in microsomes was 3-fold less than that observed in intact cells, indicating that the structure of mutant P-450(17 alpha) was dramatically altered upon disruption of COS 1 cells. Apparently the deletion of a single phenylalanine in the N-terminal region of P-450(17 alpha) alters its folding in such a way that both enzymatic activities are dramatically decreased, leading to the partial combined deficiency observed in this individual.

Adrenal Hyperplasia, Congenital↗
PubMed1989

Effect of ACTH on the stability of mRNAs encoding bovine adrenocortical P-450scc, P-45011 beta, P-45017 alpha, P-450C21 and adrenodoxin.

ACTH treatment of bovine adrenocortical cells in primary culture causes increased accumulation of mRNAs encoding cytochromes P-450SCC, P-450(11)beta, P-450(17) alpha, P-450C21 and adrenodoxin as well as increased transcriptional activity of their respective genes. In this study we have shown that ACTH does not greatly affect the half-life of mRNAs encoding P-450(11)beta, P-450(17)alpha, P-450C21 and adrenodoxin. However, in the case of P-450SCC mRNA, ACTH causes a five-fold increase in the half-life leading to a significant stabilization of P-450SCC mRNA. Thus it appears that the levels of mRNAs encoding P-450(11)beta, P-450(17)alpha, P-450C21 and adrenodoxin are regulated by ACTH primarily at the transcriptional level, while that for P-450SCC is regulated at both the transcriptional and post-transcriptional levels.

Adrenal Cortex↗
PubMed1989

Glucocorticoid regulation of the rat cytochrome P450c (P450IA1) gene: receptor binding within intron I.

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the expression of cytochrome P450c were investigated using cultured fetal rat hepatocytes. Cytochrome P450c mRNA content, determined by Northern blot analysis, was induced in cells treated with 1,2-benzanthracene from levels undetectable in untreated cells. When dexamethasone was included in the culture medium together with 1,2-benzanthracene there was a further 2-fold increase in the induction of cytochrome P450c mRNA. The concentration of dexamethasone required for a half-maximal increase in cytochrome P450c mRNA content was approximately 10(-9) M. By nuclear run-on transcription assays, treatment with 1,2-benzanthracene induced cytochrome P450c transcription 5.3-fold over untreated cells. In the presence of dexamethasone and 1,2-benzanthracene, there was a further 2-fold increase in cytochrome P450c transcription. Southwestern blotting and exonuclease footprinting methods have identified binding interactions of a purified glucocorticoid receptor fraction with portions of the cytochrome P450c gene within the first intron. Using a chimeric plasmid containing the first intron, the first exon, and 824 bp of 5'-flanking region of the cytochrome P450c gene, chloramphenicol acetyltransferase activity was induced in transfected HepG2 hepatoma cells by the addition of 1,2-benzanthracene. The addition of dexamethasone induced a further 2.2-fold increase in activity. Deletion of the first intron within the chimeric plasmid abolished responsiveness to dexamethasone. It is concluded that glucocorticoids act together with polycyclic aromatic hydrocarbons to increase the levels of cytochrome P450c expressed in the fetal rat hepatocyte, and that this action is mediated by the glucocorticoid receptor. A glucocorticoid responsive element, which binds the glucocorticoid receptor, has been identified within the first intron of the cytochrome P450c gene. These results suggest that glucocorticoids play a significant role in the response of the hepatic cytochrome P450c gene to xenobiotics.

Actins↗
PubMed1989

Identification of a common molecular basis for combined 17 alpha-hydroxylase/17,20-lyase deficiency in two Mennonite families.

During the course of studies to characterize mutations of the CYP17 gene that cause the 17 alpha-hydroxylase-deficient form of congenital adrenal hyperplasia we have discovered two ostensibly unrelated Mennonite families in which affected individuals are homozygous for the same mutation. The defect is a four-base duplication in exon 8 of the CYP17 gene, which alters the reading frame encoding the C-terminal 26 amino acids of cytochrome P450(17 alpha).

Adrenal Hyperplasia, Congenital↗