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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adrenocorticotropin (ACTH) is known to exert an acute effect on adrenal steroidogenesis as well as long-term effects by regulation of gene expression. In order to further study the long-term action of ACTH, guinea pig fasciculata-glomerulosa (FG) cells in primary culture were treated for up to 72 h with ACTH. The effects of this treatment on steroid secretion, enzyme activity and mRNA levels for steroid enzymes were measured. While the rate of 17-deoxy C-21 steroid secretion decreased over the 72-h period of incubation with ACTH, the 17-hydroxy C-21 steroid secretion rate remained constant for the first 24 h of incubation and declined thereafter; the rate of 4-ene C-19 steroid secretion increased over the 72-h incubation period. ACTH treatment increased 17-hydroxylase and 17,20-lyase activities and the maximal stimulation was reached after 48 h. In contrast, the activity of 21-hydroxylase (P450c21) steadily declined over the 72-h incubation period. ACTH also caused an increase in mRNA levels for P450c21, 17-hydroxylase and 17,20-lyase (P450c17), 3 beta-hydroxysteroid dehydrogenase 4-ene-5-ene-isomerase (3 beta-HSD) and cholesterol side-chain cleavage enzyme (P450scc). The maximal stimulation for the four mRNAs was observed after 18 h of incubation with ACTH, decreasing afterwards except for P450c17 mRNA levels which remained elevated over the 72-h incubation period. Despite the increase in mRNA levels for 3 beta-HSD and P450c21, no increase in their respective enzyme activities was observed and 21-hydroxylase activity even declined over the 72-h incubation period with ACTH, thus suggesting that mechanism(s) other than gene expression alone regulate steroid secretion in FG cells. In conclusion ACTH caused major changes in steroid distribution due to increased 17-hydroxylase and 17,20-lyase activities and decreased 21-hydroxylase activity in FG cells in culture. Moreover, our data revealed major differences in the induction of mRNAs for steroidogenic enzymes and their activities following ACTH treatment.
J Steroid Biochem Mol Biol 1992 Jan
PMID:Effect of ACTH on steroidogenic enzymes in guinea pig fasciculata-glomerulosa cells: changes in activity and mRNA levels. 131 Apr 15

We report here the effects of a 7-day treatment of guinea-pigs with ACTH on adrenal mRNA levels for steroid-transforming enzymes. Adrenal 3 beta-hydroxysteroid dehydrogenase 4-ene-5-ene-isomerase (3 beta-HSD), 17-hydroxylase, 17,20-lyase, 21-hydroxylase and 11-hydroxylase activities were also examined as well as plasma and adrenal steroid levels. Our data reveal that chronic ACTH-treatment stimulated all post-pregnenolone enzyme activities in glomerulosa-fasciculata cells. Plasma steroid levels increased 8 h after the last injection of ACTH and returned to the control levels 24 h later whereas, in the adrenal, the content in steroids in the group sacrificed 8 h after the last injection of ACTH were similar to the values of the control group and decreased markedly 24 h later. It is suggested that the steroid turn-over in the adrenal may be affected by the chronic ACTH-treatment. On the other hand, despite the significant stimulation in steroid-transforming enzyme activities, our data reveal that chronic ACTH administration caused a decrease in mRNA levels for P450c21 and P450c17 while P450scc, 3 beta-HSD and P450c11 remained unchanged. Taken together, these results suggest that in vivo chronic ACTH-treatment of guinea-pigs increases adrenal steroidogenic capacity by increasing steroid secretion and steroid enzyme activity. Moreover, the chronic treatment with ACTH may have a post-transcriptional effect on steroidogenic enzymes gene expression by affecting the half-life of their mRNAs.
J Steroid Biochem Mol Biol 1992 Jan
PMID:Effect of chronic ACTH treatment on guinea-pig adrenal steroidogenesis: steroid plasma levels, steroid adrenal levels, activity of steroidogenic enzymes and their steady-state mRNA levels. 131 Apr 16

We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17 alpha-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11 beta-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.
J Steroid Biochem Mol Biol 1992 May
PMID:Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex. 131 35

Primary fetal human adrenocortical cells of definitive zone origin were transfected by electroporation with pSV3neo, a plasmid coding for SV40 T antigen and neo, which confers resistance to the antibiotic G418. The clones obtained proliferated for 30 to 40 population doublings after isolation when grown under standard medium conditions, and then entered 'crisis'. When early-passage clones were incubated with cyclic AMP (1:1 N6-monobutyryl and 8-bromo analogues), cell rounding was observed, as in primary cultures of human adrenocortical cells. As previously shown in bovine adrenocortical cells, rounding was inhibited with a monoclonal antibody against urokinase plasminogen activator but not with a monoclonal antibody against tissue plasminogen activator. The regulation of the steroidogenic pathway in clones was investigated. The effects of cyclic AMP and activation of protein kinase C were examined in cells maintained in defined medium or in the presence of serum. 17 alpha-Hydroxylase was strongly induced by cyclic AMP, as evidenced by Northern blotting and by the conversion of progesterone or 25-hydroxy-[1,2-3H]cholesterol, this induction being blocked by low concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA). Cholesterol side-chain cleavage enzyme was strongly induced by cyclic AMP, and clones also showed low activities of 21-hydroxylase and 11 beta-hydroxylase. Under all circumstances levels of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), as assessed by Northern blotting or by conversion of 25-hydroxycholesterol, were very low. 3 beta-HSD was not induced by cyclic AMP or TPA alone, but was induced by the combination of the two agents. The regulation of 17 alpha-hydroxylase and 3 beta-HSD resembles that previously described in primary cultures of human fetal adrenocortical cells. Thus, transfection with SV40 T antigen resulted in the production of clones which preserve the unique characteristics of the human adrenal cortex.
J Mol Endocrinol 1992 Aug
PMID:Expression of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase in fetal human adrenocortical cells transfected with SV40 T antigen. 132 52

The adjacent C4 and P450c21 genes encode the fourth component of serum complement and steroid 21-hydroxylase respectively, and are tandemly duplicated in the human, murine, and bovine genomes. We recently cloned a cDNA for another duplicated gene, operationally termed X, which overlaps the 3' end of human P450c21 and has the opposite transcriptional orientation. Thus, the organization of the locus is 5'-C4A-21A-XA-C4B-21B-XB-3' (Y. Morel, J. Bristow, S. E. Gitelman, and W. L. Miller, Proc. Natl. Acad. Sci. USA 86:6582-6586, 1989). To determine how this locus was duplicated, we sequenced the DNA at the duplication boundaries and the 7 kb between P450c21A and C4B comprising the XA locus. The sequences located the duplication boundaries precisely and indicate that the duplication occurred by nonhomologous recombination. The boundaries are substantially different from those of the corresponding duplication in the mouse genome, suggesting that similar gene duplications may have occurred independently in ancestors of rodents and primates after mammalian speciation. Compared with XB, the XA gene is truncated at its 5' end and bears a 121-bp intragenic deletion causing a frameshift and premature translational stop signal. Nevertheless, XA is transcribed into a stable 2.6-kb polyadenylated RNA that is expressed uniquely in the adrenal gland.
Mol Cell Biol 1992 May
PMID:Mechanism and consequences of the duplication of the human C4/P450c21/gene X locus. 162 Jan 34

We proposed that a cell-selective regulatory protein coordinately regulates the expression of three enzymes that are required for the biosynthesis of corticosteroids: cholesterol side chain cleavage enzyme, steroid 21-hydroxylase, and the aldosterone synthase isozyme of steroid 11 beta-hydroxylase. In this report, we identify a 53-kilodalton protein, termed steroidogenic factor 1 (SF-1), that interacts with the related promoter elements from these steroidogenic enzymes, and we isolate and characterize a cDNA that very likely encodes this protein. We first showed that nuclear extracts from bovine adrenal glands interact with the mouse steroidogenic regulatory elements, forming complexes indistinguishable from those produced by nuclear extracts from mouse Y1 adrenocortical cells. These bovine adrenal extracts were subjected to sequential ion exchange and affinity chromatography to yield a highly enriched preparation of SF-1. The predominant protein in the affinity-purified preparation comigrated with shift activity and had a mol wt of 53,000; UV cross-linking experiments demonstrated directly that this 53-kilodalton protein interacted with the steroidogenic regulatory element. Even with this marked enrichment, affinity-purified SF-1 bound six steroidogenic regulatory elements. These results support strongly the model that a steroidogenic cell-selective protein interacts with related promoter elements from three steroidogenic enzymes to regulate their coordinate expression. The recognition sequence of SF-1 closely resembles those of nuclear hormone receptor family members, suggesting that SF-1 may belong to this supergene family. By screening a Y1 cell cDNA library with the DNA-binding region of the H-2RIIBP nuclear hormone receptor cDNA, we isolated a cDNA that is selectively expressed in steroidogenic cells. When expressed as a glutathione S-transferase fusion protein in Escherichia. coli, the protein encoded by this cDNA interacts with all six related steroidogenic regulatory elements with a binding specificity indistinguishable from that of SF-1. Surprisingly, the sequence of the putative DNA-binding domain of this cDNA matches exactly the corresponding sequence of the mouse homolog of the Drosophila transcription factor fushi tarazu-factor I. The demonstration that a member of the nuclear hormone receptor family interacts with the steroidogenic regulatory elements provides intriguing insights into possible mechanisms by which these essential genes are regulated.
Mol Endocrinol 1992 Aug
PMID:Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I. 140 3

Stimulation of aldosterone synthesis by angiotensin II (AII) is associated with depolarization of the cell membrane. Since the potential difference of adrenocortical cells is dependent on membrane permeability to potassium ions, the effects of agents which hyperpolarize the cell (by increasing permeability to K+) on the control of aldosterone synthesis were investigated further. Basal and AII-stimulated aldosterone synthesis was increased by 20-70% in cells incubated with 1 or 10 nM of the potassium ionophore valinomycin; higher concentrations markedly inhibited AII-stimulated synthesis. Cromakalim, a potential antihypertensive drug which facilitates the opening of K+ channels in smooth muscle cells, stimulated basal aldosterone synthesis at 2 microM but had no effect at 40 microM. AII-stimulated aldosterone synthesis was not affected by cromakalim except at 40 microM, which was inhibitory. The inhibitory effects of cromakalim, unlike those of valinomycin, were not reversible. Aldosterone synthesis from added hydroxycholesterol and pregnenolone (but not from deoxycorticosterone and corticosterone) was significantly inhibited by 40 microM cromakalim. Potassium efflux from cells preloaded with 43K was unaffected by low concentrations of valinomycin, but was markedly increased by concentrations which inhibited AII-stimulated aldosterone production. Small decreases and increases in 43K efflux, caused by 1 and 40 microM cromakalim respectively, corresponded with increases and decreases in basal aldosterone production; cromakalim did not affect 43K efflux from AII-stimulated cells. We suggest that increasing adrenocortical cell membrane permeability to K+ reduces steroidogenesis, but that valinomycin and cromakalim have other actions which complicate the relationship between 43K efflux and aldosterone production. Cromakalim appears to inhibit 21-hydroxylase activity in the biosynthetic pathway and may also affect 3 beta-hydroxysteroid dehydrogenase activity.
J Mol Endocrinol 1992 Oct
PMID:Membrane permeability to K+ and the control of aldosterone synthesis: effects of valinomycin and cromakalim in bovine adrenocortical cells. 141 87

Progesterone 21-hydroxylation in hepatic microsomes from adult male sheep is a quantitatively important metabolic pathway (0.27 +/- 0.08 nmol deoxycorticosterone formed/min/mg protein; representing 13-25% of total progesterone conversion). This study was undertaken to determine whether the ovine hepatic progesterone 21-hydroxylase may be another member of the P450 2C subfamily, normally associated with progesterone 21-hydroxylation in rodent liver. An IgG preparation raised in rabbits against purified rat liver microsomal cytochrome P450 2C6 was found to recognize a single antigen (MW 52 kDa) in sheep liver microsomes. This protein was present in sheep liver (apparent concentration 16 +/- 4 ng/micrograms microsomal protein) representing approx. 28% of the corresponding content of P450 2C6 in untreated rat liver. Preincubation of the anti-P450 2C6 IgG with hepatic microsomes was found to decrease the rate of progesterone 21-hydroxylation to 50-80% of uninhibited control. Taken together, from these findings it is apparent that a P450 enzyme, most likely from the 2C subfamily, catalyses deoxycorticosterone formation from progesterone in sheep liver and that this is a quantitatively important pathway of progesterone hydroxylation in these fractions.
J Steroid Biochem Mol Biol 1992 Nov
PMID:Participation of a cytochrome P450 enzyme from the 2C subfamily in progesterone 21-hydroxylation in sheep liver. 141 95

Regulation of cytochromes P-450 21-hydroxylase (P-450C21) and P-450 17 alpha-hydroxylase/C17,20-lyase (P-450(17) alpha,lyase) activities and impairment of this regulation by Aroclor 1254 was studied in guinea-pig adrenal microsomes. In a membrane depleted system, a decrease in the normally predominant, P-450C21 activity and an increase in P-450(17) alpha,lyase activities was observed. The same deviations were observed in intact microsomes with increase in the reaction temperature (0-40 degrees C). Breaks in Arrhenius plots for activities of P-450C21 and P-450(17) alpha,lyase correlate with transition temperatures reported for the microsomal membrane. These results point to: (1) preference of a gel state membrane for catalytic expression of P-450C21 suggesting a clustered organization of this P-450 species with reductase; (2) preference of a fluid membrane for lyase activity suggesting a random collision mechanism for reduction of P-450(17) alpha,lyase. Aroclor 1254 introduced to reaction mixtures containing intact microsomes elicited basically the same changes as caused by depletion of the microsomal membrane or by increase in the incubation temperature. Lack of effect of Aroclor 1254 on P-450C21 and P-450(17) alpha,lyase activities in the membrane depleted system demonstrates that its interference with monooxygenase activities is mediated by the microsomal membrane. The similarities between altered cytochrome P-450 mediated activities in the presence of Aroclor 1254 and the deviations observed in the membrane depleted system or upon increase in the incubation temperature may suggest that this chemical exerts its impacts by influencing membrane fluidity.
J Steroid Biochem Mol Biol 1992 Mar
PMID:The interference of polychlorinated biphenyls (Aroclor 1254) with membrane regulation of the activities of cytochromes P-450C21 and P-450(17) alpha,lyase in guinea-pig adrenal microsomes. 155 19

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.
J Steroid Biochem Mol Biol 1992 Mar
PMID:The role of cytochrome b5 in adrenal microsomal steroidogenesis. 155 20


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