Biomedical subjects

C J Corbin

Publications and source records attributed to C J Corbin.

29 records · Page 2Linked to original sources

PubMed1993

Tissue-specific promoters regulate aromatase cytochrome P450 expression.

In the human, estrogen biosynthesis occurs in several tissue sites, including ovary, placenta, adipose, and brain. Recent work from our laboratory has indicated that tissue-specific expression of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, is determined, in part, by the use of tissue-specific promoters. Thus the expression of P450arom in human ovary appears to utilize a promoter proximal to the translation start-site. This promoter is not utilized in placenta but instead, the promoter used to drive aromatase expression in placenta is at least 40 kb upstream from the translational start-site. In addition, there is a minor promoter used in the expression of a small proportion of placental transcripts which is 9 kb upstream from the start of translation. Transcripts from these promoters are also expressed in other fetal tissues including placenta-related cells such as JEG-3 choriocarcinoma cells, hydatidiform moles, and other fetal tissues such as fetal liver. On the other hand, in adipose tissue expression of P450arom may be achieved by yet another, adipose-specific promoter. The various 5'-untranslated exons unique for expression driven by each of these promoters are spliced into a common intron/exon boundary upstream from the translational start-site. This means that the protein expressed in each of the various tissue-specific sites of estrogen biosynthesis is identical.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of a complementary deoxyribonucleic acid insert encoding bovine aromatase cytochrome P450.

Aromatase, an enzyme complex comprised of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) and the flavoprotein NADPH-cytochrome P450 reductase, catalyzes the conversion of androgens to estrogens. Three cDNA inserts encoding P450arom were isolated from a bovine placental cDNA library. These inserts were sequenced and found to correspond closely to human P450arom sequence from the internal EcoRI restriction site (exon III) through the termination codon (exon X) into the 3'-untranslated region. The rapid amplification of cDNA ends technique was used to generate the rest of the cDNA 5' of the internal EcoRI site, using mRNA obtained from bovine granulosa cells as a template. This insert was sequenced, and when aligned with the other inserts, an open reading frame was found which was predicted to encode a protein of 503 amino acid residues. The deduced polypeptide shares 84% identity with human P450arom and 79%, 76%, 71%, and 57% identity with mouse, rat, chicken, and trout P450arom, respectively. A full-length open reading frame was generated using the polymerase chain reaction and mRNA obtained from bovine granulosa cells as template. After this insert was ligated into the pCMV5 expression vector, it was transfected into COS-1 monkey kidney tumor cells. We were able to demonstrate aromatase activity by assaying the incorporation of tritium into [3H] water from [1 beta-3H]androstenedione. Northern analysis revealed a single transcript of approximately 6 kilobases in poly(A)+ RNA obtained from bovine placental tissue and granulosa cells. This indicated for the first time a correspondence between the pattern of estrogen biosynthesis throughout the bovine ovarian cycle and the levels of transcripts encoding P450arom. In addition, weak hybridization was noted to transcripts of the expected size, namely 3.4 and 2.9 kilobases, in poly(A)+ RNA obtained from human placental tissue. The large size of the bovine transcript is due to a long 3'-untranslated region, because, based on the rapid amplification of cDNA ends technique, there appeared to be approximately 150 basepairs 5' of the start site of translation, and we were never able to find a polyadenylation site, even in one clone that went well past the corresponding polyadenylation site in human P450arom.

Amino Acid Sequence↗

PubMed1993

Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis.

The relationship of function to structure of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) has been examined by means of sequence alignment and site-directed mutagenesis. Comparison has been made between the sequence of P450arom and the two soluble bacterial cytochrome P450 isoforms, whose three-dimensional structure has been determined (P450BM3 and P450cam). From this comparison, it appears that although there is a similarity of overall structure in cytochromes P450, there is enough significant difference in the regions involved in substrate recognition and substrate binding that residues believed to be involved, even in the known structures, must be tested. With this in mind, we have generated a detailed alignment of P450arom, including the definition of putative alpha-helices and beta-sheets based on comparison of the alignments of P450BM3 and P450cam, generated from their three-dimensional structure, and have made mutations in regions we believe to be involved in substrate recognition at the solvent surface and orientation in the heme pocket. We have mutated F116 and F134 to determine if they are present in the heme pocket, and Q225 and L228 to determine if they are a part of the substrate recognition loop. Although F116E is essentially inactive and may be a folding mutant or may inhibit reductase binding, F134E is more active than the wild type and may be located in the heme pocket facilitating the hydrogen abstraction from C2 of androstenedione. Mutations at Q225 and L228 also result in the anticipated changes in the apparent Km and maximum velocity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Tissue-specific promoters regulate aromatase cytochrome P450 expression.

In humans, estrogen biosynthesis occurs in several tissue sites, including ovary, placenta, adipose, and brain. Recent work from our laboratory indicates that tissue-specific expression of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, is determined, in part, by the use of tissue-specific promoters. Thus, the expression of P450arom in human ovary appears to utilize a promoter proximal to the translation start site. This promoter is not utilized in placenta; instead, the promoter used to drive aromatase expression in placenta is > or = 40 kb upstream from the translational start site. In addition, a minor promoter used in the expression of a small proportion of placental transcripts is 9 kb upstream from the start of translation. Transcripts from these promoters are also expressed in other fetal tissues, including placenta-related cells such as JEG-3 choriocarcinoma cells and hydatidiform moles and other fetal tissues such as fetal liver. In adipose tissue, on the other hand, expression of P450arom may be achieved by yet another, adipose-specific promoter. The various 5'-untranslated exons unique for expression driven by each of these promoters are spliced into a common intron/exon boundary upstream from the translational start site. This means that the protein expressed in each of the various tissue-specific sites of estrogen biosynthesis is identical.

Amino Acid Sequence↗

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

Structural analysis of the gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase.

The structural gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) was isolated from a human EMBL3 genomic library. The gene encompasses approximately 8 kilobases of DNA and is comprised of two large introns and three exons encoding amino acid residues 1-48, 49-103, and 104-373, respectively. The exonic sequence is identical to that of the cDNA that we previously isolated and expressed in COS 1 cells. DNA sequence analysis reveals a putative TATA (TATATAA) motif 26 basepairs up-stream of the beginning of exon I, as determined by S1 nuclease protection analysis. However, primer extension analysis using poly(A)+ RNA isolated from both placenta and corpora lutea indicates that the RNA initiates up-stream of the putative TATA motif, and that an additional 53-basepair exon, which is untranslated, is present 5' to the first coding exon. Southern hybridization analysis of genomic DNA using a single exon probe suggests that there may be more than one copy of the gene in the human genome. In addition, we confirm from Southern analysis of genomic DNA isolated from human x hamster somatic cell hybrids that the gene is located on human chromosome 1. These findings will provide a foundation for the characterization of apparent 3 beta HSD clinical deficiencies when these are due to a mutation in the structural gene.

Amino Acid Sequence↗

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↗

PubMed1988

Isolation of a full-length cDNA insert encoding human aromatase system cytochrome P-450 and its expression in nonsteroidogenic cells.

The isolation and cloning of a full-length cDNA insert complementary to mRNA encoding human aromatase system cytochrome P-450 is reported. The insert contains an open reading frame encoding a protein of 503 amino acids. This gene is clearly a member of the cytochrome P-450 gene superfamily, because the sequence contains regions of marked homology to those of other members, notably a putative membrane-spanning region, I helix, Ozols, and heme-binding regions. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental aromatase system cytochrome P-450, as detected by immunoblot analysis, and catalyzed the aromatization of androstenedione, testosterone, and 16 alpha-hydroxyandrostenedione. This activity was inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione and econazole. Thus the several steps involved in the aromatization reaction appear to be catalyzed by a single polypeptide chain, which can metabolize the three major physiological substrates.

Amino Acid Sequence↗

PubMed1987

Regulation of estrogen biosynthesis in human adipose stromal cells. Effects of dibutyryl cyclic AMP, epidermal growth factor, and phorbol esters on the synthesis of aromatase cytochrome P-450.

Using human adipose stromal cells in monolayer culture as a model system for study of the regulation of aromatase activity, as well as polyclonal antibodies raised in this laboratory against aromatase cytochrome P-450 (cytochrome P-450AROM), it was found that the rate of synthesis of cytochrome P-450AROM was stimulated by dibutyryl cyclic AMP. This stimulation was attenuated by epidermal growth factor and was potentiated by phorbol esters. These changes in cytochrome P-450AROM synthesis were associated with comparable changes in the levels of translatable cytochrome P-450AROM mRNA, as well as with changes in the activity of aromatase of these cells. By contrast, there was little change in the synthesis of the reductase component of the aromatase enzyme complex in response to these factors. The increase in mRNA was blocked by cycloheximide, indicative of a requirement for protein synthesis in mediating this inductive response. It is concluded that aromatase activity is regulated primarily by changes in the level of mRNA encoding cytochrome P-450AROM, and that such changes are likely a reflection of changes in the rate of transcription of the gene encoding this enzyme. Increases in the levels of cytochrome P-450AROM mRNA are apparently mediated by a regulatory protein(s), similar to that found for other steroidogenic forms of cytochrome P-450.

Adipose Tissue↗

PubMed1987

Sequencing of cDNA inserts encoding aromatase cytochrome P-450 (P-450AROM).

Two cDNA inserts complementary to mRNA encoding aromatase cytochrome P-450 (P-450AROM) have been isolated and characterized by restriction mapping and sequencing. The overlapping sequence encoded by these inserts is identical, and a putative heme-binding region has been identified. In addition, the open reading frame contains the sequences of all four cysteine-containing tryptic peptides isolated by Chen et al. (1986) from purified cytochrome P-450AROM. The inserts differ in the use of alternative poly A-addition signals, which is consistent with the presence of two major species of mRNA in human placenta, of 3.0 and 2.4 kb, which hybridize to these inserts. The identity of sequence between the two inserts and the likely presence of alternative poly A-addition signals, is suggestive that only one form of cytochrome P-450AROM is encoded by these mRNA species.

Amino Acid Sequence↗

PubMed1986

Growth factors suppress and phorbol esters potentiate the action of dibutyryl adenosine 3',5'-monophosphate to stimulate aromatase activity of human adipose stromal cells.

In previous studies, we observed that the stimulatory effect of (Bu)2cAMP on aromatase activity of human adipose stromal cells was markedly attenuated when fetal calf serum was present in the culture medium. To determine whether growth factors may be the inhibitors of (Bu)2cAMP-stimulated aromatase activity in serum, the effects of growth factors and phorbol esters on aromatase activity of human adipose stromal cells in monolayer culture were investigated. Epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) were all without effect on aromatase activity when added by themselves, but markedly inhibited aromatase activity stimulated by (Bu)2cAMP. On the other hand, nerve growth factor, multiplication-stimulating activity, relaxin, and insulin had no effect on aromatase activity, either by themselves or in the presence of (Bu)2cAMP. Thus, EGF, PDGF, and FGF can mimic the inhibitory action of fetal calf serum on (Bu)2cAMP-stimulated aromatase activity of these cells. By contrast, none of these substances was capable of mimicking the effect of serum to facilitate the stimulatory action of dexamethasone on aromatase activity of these cells. The phorbol esters phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-diacetate were also capable of facilitating the action of (Bu)2cAMP to stimulate aromatase activity, but had little or no action on dexamethasone-stimulated aromatase activity or when added by themselves. It is concluded that aromatase is under multifactorial regulation in human adipose stromal cells. The activity is induced by glucocorticoids and by agents that stimulate cAMP-dependent protein kinase; the latter effect is potentiated by factors that stimulate protein kinase C, but is suppressed by growth factors such as EGF, FGF, and PDGF, whose actions are believed to be mediated by receptor-linked tyrosine kinase activity.

Adipose Tissue↗