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At least 19 recordsLinked to original sources
PubMed1995

Transcriptional regulation of the genes encoding the cytochrome P-450 steroid hydroxylases.

Steroid hormone biosynthesis requires the concerted action of a related group of cytochrome P-450 steroid hydroxylases. In recent years considerable effort has been directed toward defining the molecular basis for the cell-selective expression of these genes and their transcriptional regulation by trophic hormones. The orphan nuclear receptor SF-1, acting through a conserved element found in the proximal promoter regions of all steroid hydroxylase genes, seems to be a major, but not exclusive, determinant of cell-selective gene expression. In contrast, the coordinate responses of the steroid hydroxylases to trophic hormones apparently involves an interplay of multiple proteins that collectively lead to a synchronous induction of gene expression. In some instances these interactions apparently involve transcription factors that also contribute to the cell-selective expression of these genes.

Animals↗
PubMed2000

Allelic variants of human cytochrome P450 1A1 (CYP1A1): effect of T461N and I462V substitutions on steroid hydroxylase specificity.

Steroid hydroxylation specificities were determined for the wild-type and the two allelic variants of the polymorphic human cytochrome P450 1A1 (CYP1A1) that were associated with amino acid exchanges near the active site of the enzyme. All three variants were expressed in insect cells using recombinant baculoviruses. Each variant protein was spectrally and enzymatically active, as judged by the ability of the prepared microsomes to catalyse O-dealkylation of ethoxyresorufin and pentoxyresorufin in cumene hydroperoxide-mediated reactions. With progesterone and testosterone as substrate, all variants of CYP1A1 exhibited high, but different steroid hydroxylation activities (8-40 pmol hydroxysteroid/min/pmol CYP1A1, i.e. approximately 800-4000 pmol/min/mg microsomal protein). All three variants exclusively catalysed 6beta-hydroxylation of both steroids. In addition, towards progesterone as substrate, all variants also catalysed 16alpha-hydroxylations with approximately half of the rate of 6beta-hydroxylation activity. With progesterone as substrate for hydroxylation in 6beta position, CYP1A1 T461N had the lowest catalytic efficiency (Vmax/Km) followed by the CYP1A1 I462V variant and the wild-type enzyme. For 16alpha-hydroxylation of progesterone, the catalytic efficiencies of the three variants are not statistically significantly different. With testosterone as substrate the CYP1A1 1462V variant catalysed 6beta-hydroxylation with an efficiency considered not significantly different compared to the wild-type, although both the apparent Km and Vmax were significantly decreased. In contrast, the CYP1A1 T461N variant exhibited significantly decreased catalytic efficiencies compared to both the 1462V variant and the wild-type enzyme. These results indicate that all three naturally occurring allelic variants of human CYP1A1 hydroxylate steroid hormones with varying efficiencies in a stereo- and regioselective manner, whereby the CYP1A1 T461N variant exhibited the lowest catalytic efficiency.

Alleles↗
PubMed2006

CYP17- and CYP11B-dependent steroid hydroxylases as drug development targets.

Steroid hormone biosynthesis is catalyzed by the action of a series of cytochrome P450 enzymes as well as reductases. Defects in steroid hydroxylating P450s are the cause of several severe defects such as the adrenogenital syndrome (AGS), corticosterone methyl oxidase (CMO) I or II deficiencies, or pseudohermaphroditism. In contrast, overproduction of steroid hormones can be involved in breast or prostate cancer, in hypertension, and heart fibrosis. Besides inhibiting the action of the steroid hormones on the level of steroid hormone receptors by using antihormones, which often is connected with severe side effects, more recently the steroid hydroxylases themselves turned out to be promising new targets for drug development. Since the 3-dimensional structures of steroid hydroxylases are not yet available, computer models of the corresponding CYPs may help to develop new inhibitors of these enzymes. During the past years, the necessary test systems have been developed and new compounds have been synthesized, which displayed selective and specific inhibition of CYP17, CYP11B2, and CYP11B1. With some of these potential new drugs, clinical trials are under way. It can be expected that in the near future some of these compounds will contribute to our arsenal of new and selective drugs.

Animals↗
PubMed1993

Regulation of steroid hydroxylase gene expression: importance to physiology and disease.

Steroid hydroxylase gene expression is multifactorial in nature, being regulated by tissue-specific, developmental, constitutive and signal transduction systems. The biochemistry of this complex pattern of regulation is not yet clearly elucidated, but studies in several laboratories have led to an understanding of specific aspects of regulation, particularly that involving signal transduction. The complexity of regulation appears to be necessary for normal human physiology because of the wide variety of steroid hormones produced by these enzymes. Genetic diseases associated with the steroid hydroxylases provide examples of how aberrant physiology can result from alterations in the multifactorial regulation of steroid hydroxylase gene expression.

Animals↗
PubMed1996

Mechanisms of ACTH(cAMP)-dependent transcription of adrenal steroid hydroxylases.

The action of peptide hormones from the anterior pituitary regulates transcription of a large number of genes located in most, if not all, tissues. This action is mediated through regulation of steroid hormone production in the steroidogenic factories (adrenals, gonads). These steroid hormones are transported through the circulation to the peripheral tissues where they serve as ligands for the family of zinc-finger nuclear receptor transcription factors. The mechanisms by which peptide hormones regulate steroid hormone production include a chronic response mediated by elevated levels of cAMP resulting from the binding of peptide hormones to their cell surface receptors which enhances transcription of the genes encoding steroid hydroxylases required for steroid hormone biosynthesis. The action of ACTH in the adrenal cortex has been studied in greatest detail leading to identification of unique cAMP-response sequences (CRS) in the different bovine steroid hydroxylase genes. Most likely FSH and LH mediate steroid hydroxylase gene expression in the gonads via the same response elements. Unlike developmental/tissue-specific transcription of these genes which is regulated by a common transcription factor (SF-1), cAMP-dependent transcription of each steroid hydroxylase gene requires a different transcription factor.

Adrenal Cortex↗
PubMed1989

Regulation of steroid hydroxylase gene expression is multifactorial in nature.

In summary, regulation of steroid hydroxylase gene expression is complex and multifactorial, involving cAMP-dependent and -independent mechanisms required for maintenance of optimal steroidogenic capacity, tissue-specific mechanisms which lead to different steroidogenic pathways in different tissues, and developmental mechanisms which lead to fetal imprinting of steroid hydroxylase expression and which probably overlap with both maintenance and tissue-specific mechanisms. Future studies will involve identification of the trans-acting factors associated with each of these aspects of the multifactorial regulation and characterization of the cis-regulatory elements to which they bind. Such studies will inevitably lead to the identification of genes encoding these trans-acting factors and investigation of their regulation. In this way, it will be possible to work outward from the steroid hydroxylase genes toward the cell surface receptors in order to elucidate the series of events which lead to cAMP-dependent and -independent regulation of steroid hydroxylase gene expression.

Adrenal Cortex↗
PubMed1978

Topological studies of the steroid hydroxylase complexes in bovine adrenocortical mitochondria.

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria was studied by using nonpenetrating artificial electron acceptors and the impermeable protein reagent diazobenzenesulfonate. Inhibition of steroid hydroxylase activity by ferricyanide and dichlorophenolindophenol sulfonate was only observed in mitochondria which had been damaged by various techniques. Intact mitochondria were not inhibited by these reagents. The reaction was monitored by oxygen uptake due to hydroxylation of deoxycorticosterone, as well as P-450 reduction and corticosterone formation. The results obtained were similar regardless of how the activity was measured. Labeling of the mitochondria with the nonpenetrating protein reagent diazobenzenesulfonate also inhibited P-450 reduction and corticosterone formation in mitochondria which had been damaged prior to addition of this reagent. Intact mitochondria which were labeled with this reagent showed very little inhibition of both activities. These results strongly suggest that all protein components of the steroid 11beta-hydroxylase system are located on the matrix side of the mitochondrial inner membrane. The inability of ferricyanide, dichlorophenolindophenol sulfonate, and diazobenzenesulfonate to inhibit the malate-dependent reduction of P-450 in intact mitochondria implies that all the P-450-dependent mitochondrial steroid hydroxylase systems are located on the matrix side of the inner mitochondrial membrane.

Adrenal Cortex↗
PubMed1993

Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression.

The cytochrome P450 steroid hydroxylases are coordinately regulated by steroidogenic factor 1 (SF-1), a protein expressed selectively in steroidogenic cells. Based on its expression in steroidogenic tissues and DNA-binding specificity, we isolated a putative SF-1 cDNA from an adrenocortical cDNA library. As evidence that this cDNA encodes SF-1, we now show that it is selectively expressed in steroidogenic cells, that an antiserum against its protein product specifically abolishes the SF-1-related gel-shift complex, and that its coexpression increases promoter activity of the 21-hydroxylase 5'-flanking region in transfection experiments. Sequence analyses of the SF-1 cDNA revealed that it is the mouse homolog of fushi tarazu factor I (FTZ-F1), a nuclear receptor that regulates the fushi tarazu homeobox gene in Drosophila. A second FTZ-F1 homolog, embryonal long terminal repeat-binding protein (ELP), was recently isolated from embryonal carcinoma cells. SF-1 and ELP cDNAs are virtually identical for 1017 base pairs, including putative DNA-binding domains, but diverge at their 5'- and 3'-ends. One genomic clone contained both SF-1- and ELP-specific sequences, confirming their origin from a single gene. Characterization of this gene defined shared exons encoding common regions and alternative promoters and 3'-exons leading to differences between the two FTZ-F1 transcripts. We used in situ hybridization with transcript-specific probes to study the ontogeny of SF-1 and ELP expression. ELP transcripts were not detected from embryonic day 8 to adult, consistent with its previous isolation from embryonal carcinoma cells and its postulated role in early embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Neoplasms↗
PubMed2003

ACTH modulation of transcription factors responsible for steroid hydroxylase gene expression in the adrenal cortex.

Steroid hormone biosynthesis in the adrenal cortex and gonads involves the coordinated transcription of the genes encoding the steroid hydroxylases, 3beta-hydroxysteroid dehydrogenase (3betaHSD), the steroidogenic acute regulatory protein (StAR), and adrenodoxin (Adx). Transcriptional regulation of steroidogenic genes is multifactorial, entailing developmental, tissue-specific, constitutive, and cAMP-dependent mechanisms. Optimal steroidogenic capacity is achieved by the actions of ACTH which exerts transcriptional pressure on all steroidogenic genes. The actions of ACTH in the adrenal cortex have been studied in great detail and is mediated by cAMP and protein kinase A (PKA) via two temporally distinct pathways. The acute response leads to mobilization of cholesterol, the initial substrate for all steroidogenic pathways, from cellular stores to the inner mitochondrial membrane where cholesterol sidechain cleavage cytochrome P450 (P45011A1) resides. The slower, chronic response of ACTH in the adrenal cortex directs transcription of the genes encoding the steroidogenic enzymes. Although steroidogenic gene transcription in response to ACTH is cAMP-dependent, the consensus cAMP response pathway (CRE/CREB) is not involved. Instead, each steroidogenic gene utilizes unique cAMP-responsive sequences (CRS) found in the promoters of each gene, which bind a diverse array of transcription factors. Moreover, once specific transcription factors are bound to the promoters of the steroidogenic genes, increased gene expression requires posttranslational modification (phosphorylation/dephosphorylation) of the transcription factors and binding of coactivator proteins. This review provides a general view (with emphasis on the human) of the important factors involved in regulating steroidogenic gene expression and ultimately steroid hormone biosynthesis.

Adrenal Cortex↗
PubMed1979

Competitive inhibitors of rabbit hepatic microsomal 12 alpha-steroid hydroxylase.

Rabbit hepatic microsomal 12 alpha-steroid hydroxylase which is stable to storage at -70 degrees C in the pellet form was assayed for activity with [5 alpha,6 alpha-3H2]cholestane-3 alpha,7 alpha-diol solubilized with Tween 80 since methanol was incapable of maintaining the sterol in aqueous solution. Under optimized conditions in phosphate buffer, pH 7.4, containing nicotinamide, magnesium chloride, and NADPH, the enzyme conversion appeared linear for the initial 10 min. The rate of hydroxylation was proportional to protein concentration up to 4 mg/ml. Apparent Km and Vmax were 71 microM and 323 pmol of product/mg of protein/min. Based on the known structural requirements of the enzyme system, competitive inhibitors were prepared with the C-12 position derivatized as an alkene, hydroxyl, or oxo functional group. A Dixon plot revealed that 5 alpha-cholest-11-ene-3 alpha,7 alpha,26-triol was the best inhibitor with an apparent Ki of 26 microM.

Animals↗
PubMed1991

Regulation of steroid hydroxylases in normal and SV40 T antigen-transfected bovine adrenocortical cells in long-term culture.

Over long periods of growth in culture, bovine adrenocortical cells lose the ability to express the steroid hydroxylase genes. For 17 alpha-hydroxylase, cells show a stochastic pattern of phenotypic switching from a state in which they express this gene in response to cyclic AMP to a state in which the gene is no longer inducible. Introducing SV40 T antigen into bovine adrenocortical cells greatly increases their replicative potential; steroid hydroxylase expression in these clones resembles that of the precursor cells before transfection. The other steroid hydroxylases (21-hydroxylase and 11 beta-hydroxylase) appear to undergo phenotypic switching like 17 alpha-hydroxylase. The loss of expression of these genes appears to be more rapid, but there are differences in the requirements of 21-hydroxylase and 11 beta-hydroxylase versus 17 alpha-hydroxylase for induction by cyclic AMP; additionally, growth of cells in extracellular matrix Matrigel was required for expression of 21-hydroxylase and 11 beta-hydroxylase in long-term cultures of either normal or SV40 T antigen-transfected cells. Understanding the molecular basis for the phenotypic switching of steroid hydroxylases that occurs in bovine adrenocortical cells may elucidate mechanisms for cellular senescence and for maintenance of tissue-specific functions during long-term growth in culture.

Adrenal Cortex↗
PubMed1995

A cell-specific nuclear receptor regulates the steroid hydroxylases.

Recent studies of the gene regulation of the cytochrome P450 steroid hydroxylases have established a key role for an orphan nuclear receptor, designated steroidogenic factor 1 (SF-1). SF-1 binds to shared promoter elements upstream of the steroid hydroxylases to mediate their coordinate expression in steroidogenic cells. Analyses of SF-1 expression during mouse embryonic development showed that SF-1 is expressed from the earliest stages of organogenesis of the steroidogenic tissues, suggesting an intimate link between SF-1 and steroidogenic cell differentiation. Finally, in gene disruption experiments, the gene encoding SF-1 was shown to be essential for development of the adrenal glands and gonads. These results establish the essential role of this orphan nuclear receptor in the development and function of the primary steroidogenic tissues.

Adrenal Glands↗
PubMed1992

Effect of placement of dexamethasone adjacent to the ovine fetal paraventricular nucleus on adrenocortical steroid hydroxylase messenger ribonucleic acid.

The preterm rise in the concentration of ovine fetal plasma cortisol that initiates the events of parturition in sheep commences at approximately 125 days of gestational age (dGA; term = approximately 147 dGA). Concurrent with the rise in fetal plasma cortisol, adrenocortical steroid hydroxylase enzyme activity increases. The purpose of this study was 1) to quantitate changes in levels of mRNA for the steroid hydroxylases, 17-hydroxylase cytochrome P450 (P450(17)alpha), side-chain cleavage cytochrome P450 (P450scc), 11 beta-hydroxylase cytochrome P450 (P450(11)beta), and C21-hydroxylase cytochrome P450 (P450C21); and 2) examine the role of the fetal paraventricular nucleus (PVN) in the onset of adrenocortical steroid hydroxylase mRNA expression. Unperturbed fetuses were collected by cesarian section under halothane anesthesia at 105 (n = 4), 120 (n = 4), 126-128 (n = 4), and 136 dGA (n = 3); neonatal animals were collected within 2 h after birth. To examine the role of the fetal PVN in regulation of adrenocortical steroid hydroxylase mRNA expression, ovine fetuses were stereotaxically implanted bilaterally 2 mm lateral to the fetal PVN at 105-107 dGA with either cholesterol (n = 4) or dexamethasone (DEX; n = 3). Implanted fetuses were collected by cesarian section under halothane anesthesia at 126-128 dGA. mRNA for both cytochrome P450(17)alpha and P450scc declined 3-fold from 105 to 120 dGA (P less than or equal to 0.05), and then increased by 126-128 dGA compared to that on 120 dGA (P less than or equal to 0.05) and continued to increase through term. Cytochrome P450C21 increased at 126-128 dGA compared to that on 105 and 120 dGA (P less than or equal to 0.05) and remained elevated through term. Three distinct transcripts [approximately 6.2, 4.2, and 2.5 kilobases (kb)] were observed for cytochrome P450(11)beta; the 4.2-kb transcript was predominant. While total message for P450(11)beta declined over increasing gestational age, no differences were noted for the 4.2-kb transcript until after birth, when levels significantly declined (P less than or equal to 0.025). Placement of DEX adjacent to the fetal PVN prevented reemergence of expression of mRNA for P450(17)alpha and P450scc at 126-128 dGA, but had no effect on mRNA for P450C21 or P45011 beta. We conclude that mRNA for P450(17)alpha and P450scc undergoes a decline in expression concurrent with the previously described period of adrenal hyporesponsiveness from 105-126 dGA, followed by an increase in mRNA that accompanies the preterm rise in fetal plasma cortisol.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Cortex↗
PubMed1996

Adrenocortical-specific transgene expression directed by steroid hydroxylase gene promoters.

The 5'-flanking regions of genes for three mouse adrenal steroid hydroxylases were analyzed for their ability to direct adrenal cortex-specific beta-galactosidase (beta-gal) reporter expression both in cell culture and transgenic mice. The 5'-flanking regions chosen were from the genes for steroid 21-hydroxylase (21-OHase), expressed throughout the adrenal cortex and mediating both glucocorticoid and mineralocorticoid synthesis, and aldosterone synthetase (AS) and steroid 11 beta-hydroxylase (11 beta-OHase), which catalyze respectively the terminal steps of mineralocorticoid synthesis in the zona glomerulosa and glucocorticoid synthesis in the zona fasciculata/reticularis. While 5.0 kb of 11 beta-OHase gene 5'-flanking region and 5.4 kb of the AS gene 5'-flanking region mediated respectively moderate and low levels of beta-gal reporter expression in Y1 adrenocortical tumor cells, neither of these 5'-flanking regions was able to direct reporter expression to the appropriate adrenocortical zone of transgenic mice. This suggests that additional regulatory elements, lying outside these 5'-flanking regions, are required for 11 beta-OHase and AS gene expression in the intact mouse. In contrast, 6.4 kb of the mouse 21-OHase A gene 5' flanking region was able to direct specific beta-galactosidase reporter expression, in both Y1 cells and transgenic mice, indicating that elements directing adrenal cortex-specific gene expression in vivo are located not more than 6.4 kb 5' of the 21-OHase gene transcription start site.

Adrenal Cortex↗
PubMed1988

Developmental expression of bovine adrenocortical steroid hydroxylases. Regulation of P-450(17 alpha) expression leads to episodic fetal cortisol production.

The developmental expression of adrenocortical steroid hydroxylases was studied in bovine fetuses from 40 to 280 days gestational age. The expression of P-450(17 alpha) is first detected at a gestational age of 50 days and reaches a maximum at 60-70 days. The expression of P-450(17 alpha) then declines and is nondetectable at a gestational age of 100 days. P-450(17 alpha) is not expressed again until about 240 days, i.e. shortly before birth (approximately 280 days). P-450scc, P-450c21, P-450(11 beta) and adrenodoxin were present in fetal adrenals throughout gestation. This "on-off-on" pattern of P-450(17 alpha) expression during fetal development was associated with a corresponding episodic production of cortisol. Immunoreactive corticotropin (ACTH) levels in fetal plasma were elevated in small fetuses (corresponding to less than or equal to 100 days) and in near-term fetuses (corresponding to greater than 250 days) compared with those in mid-gestation fetuses. In primary culture, adrenal cells from mid-gestation fetuses contained no detectable P-450(17 alpha) but rapidly responded to ACTH with an increase in P-450(17 alpha) protein and mRNA. The tissue specificity of the developmental patterns is emphasized by the fact that both P-450(17 alpha) and P-450scc were detectable throughout the development of the fetal testes, whereas only P-450scc was detectable in fetal bovine ovary prior to 200 days. Thus, in fetal bovine adrenal it appears that ACTH is the major regulatory factor effecting the intermittent presence of P-450(17 alpha), whereas the presence of the other steroid hydroxylases is either regulated by additional factors or shows a much different sensitivity to ACTH.

Adrenal Cortex↗
PubMed2006

Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases.

The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.

Adrenodoxin↗