Catalytic activity of human and bovine adrenal cytochromes P-450 17 alpha, lyase expressed in Cos 1 cells.
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Animals↗
Biomedical subjects
J I Mason
Publications and source records attributed to J I Mason.
Developmental regulation of P-45017 alpha gene expression in fetal bovine adrenal.
Among the multifactorial aspects of regulation of steroid hydroxylase gene expression, it is the developmental process which leads to imprinting of expression of particular steroid hydroxylases in specific cell types. We have begun to investigate the ontogeny of steroidogenesis in fetal bovine tissues. Expression of most steroid hydroxylases and related enzymes is detectable in adrenals of the smallest fetuses studied and continues throughout fetal life. Like the other steroid hydroxylases, P-450(17)alpha is detectable in the earliest fetal adrenals studied. However, following an increase in expression, P-450(17)alpha disappears from the fetal adrenal by 100 days gestational age and remains absent until about 230 days gestational age. The absence of P-450(17)alpha is correlated with the absence of cortisol in the fetal adrenal and the absence of ACTH in fetal plasma. Thus expression of P-450(17)alpha in bovine fetal adrenal appears to be strictly dependent on cAMP while expression of other steroid hydroxylases appears to involve both cAMP-dependent and cAMP-independent mechanisms. Furthermore, P-450(17)alpha is expressed in fetal testis at gestational times when it is absent in fetal adrenal. We have begun to examine binding of nuclear proteins from adrenals of various gestational ages to the 5'-flanking region of the bovine P-450(17)alpha gene. Preliminary evidence indicates the presence of a protein in nuclei of fetal adrenals not expressing P-450(17)alpha that is not present in fetal adrenals of other gestational ages.
Adrenal Glands↗
Regulation of cholesterol side-chain cleavage and 17 alpha-hydroxylase/lyase activities in proliferating human theca interna cells in long term monolayer culture.
In this report we describe the development and characterization of a long term culture system to study regulation of the expression of 17 alpha-hydroxylase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase in human theca interna cells. Conditions have been established for the dispersal, growth, freezing, and storage of functional human theca interna cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Theca interna cells grown under these conditions have a doubling rate of 28-32 h and are morphologically distinct from human granulosa cells grown under the same conditions. Theca interna cells were grown, passed for successive passages, and transferred into serum-free medium containing forskolin, hCG, LH, or cAMP analogs. There was a time- and dose-dependent increase in 17 alpha-hydroxylase activity and progesterone synthesis from endogenous precursors. Added pregnenolone was converted to 17 alpha-hydroxypregnenolone, which was further converted primarily to dehydroepiandrosterone and, to a much lesser extent, androstenedione. Progesterone was converted to 17 alpha-hydroxyprogesterone and 16 alpha-hydroxyprogesterone. In studies using 17 alpha-hydroxyprogesterone as substrate, no metabolism to androstenedione or any other product was detectable. Similarly, 4-pregnen-20 alpha-ol-one (20 alpha-dihydroprogesterone) was not metabolized to any detectable products. Northern analysis performed on total RNA obtained from forskolin-stimulated theca interna cultures verified that the increase in 17 alpha-hydroxylase activity was associated with a corresponding increase in levels of mRNA specific for 17 alpha-hydroxylase cytochrome P-450. Message levels for cholesterol side-chain cleavage P-450 were similarly increased in cells treated with forskolin. No detectable mRNA encoding aromatase cytochrome P-450 was discerned. This procedure for the preparation and study of proliferating human theca internal cells provides an opportunity to study regulation of the expression of steroidogenic enzymes and other cellular processes unique to human ovarian cells.
Aldehyde-Lyases↗
Rat P450(17 alpha) from testis: characterization of a full-length cDNA encoding a unique steroid hydroxylase capable of catalyzing both delta 4- and delta 5-steroid-17,20-lyase reactions.
A cDNA clone encoding the complete rat 17 alpha-hydroxylase (P450(17 alpha] from testis has been identified and sequenced. The deduced amino acid sequence is found to have 69% similarity with human P450(17 alpha), 64% similarity with bovine P450(17 alpha), and 47% similarity with chicken P450(17 alpha). The protein contains 507 amino acids being one amino acid shorter than the human P450(17 alpha) as the result of a codon being absent at the position of amino acid 139 in the human sequence. The cDNA hybridizes to a single mRNA (approximately 2.0 kilobases) in rat testis RNA and Southern analysis indicates the presence of a single CYP17 gene in the rat genome. Expression of this cDNA in COS1 cells leads to production of a steroid hydroxylase which is capable of converting both 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone into C19 steroids, dehydroepiandrosterone, and androstenedione, respectively. This activity profile is distinct from that of either the human or bovine forms of P450(17 alpha) which are unable to catalyze 17,20-lyase conversion of delta 4-C21 steroids to delta 4-C19 steroids at significant rates.
Amino Acid Sequence↗
Immunolocalization of aromatase, 17 alpha-hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary.
Immunohistochemical localization of cholesterol side-chain-cleavage, 17 alpha-hydroxylase and aromtase cytochromes P-450 was performed in 35 morphologically normal human premenopausal ovaries by using specific antibodies against the enzymes. In well-developed ovarian follicles in the late stages of follicular growth, immunoreactivity of P-450AROM was only seen in granulosa cells while P-450(17 alpha) and P-450SCC activity was confined to theca interna cells, confirming that follicular oestrogen is produced in granulosa cells by the aromatization of androgens derived from the theca interna cells. In the corpus luteum, this functional differentiation is maintained, since immunoreactivity of P-450AROM was exclusively present in luteinized granulosa cells while that of P-450(17 alpha) was present in luteinized theca calls. Immunoreactivity of P-450SCC was present in both types of cells in the corpus luteum.
Animals↗
Ovine placental steroid 17 alpha-hydroxylase/C-17,20-lyase, aromatase and sulphatase in dexamethasone-induced and natural parturition.
Parturition in the sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to oestrogen production. This change is believed to be a consequence of the preparatum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17) alpha), steroid C-17,20-lyase, and possibly aromatase and steroid sulphatase. The activity levels have been determined of steroid 17 alpha-hydroxylase, aromatase and steroid sulphatase in placental microsomes in late pregnancy, dexamethasone-induced labour and in natural labour at term. Over the gestational period of 118-140 days, basal levels of placental aromatase were relatively constant (mean value (+/- S.E.M.) of 5.6 +/- 0.5 pmol/min per mg microsomal protein (n = 10]. Pregnenolone and progesterone 17 alpha-hydroxylase activities were undetectable (less than 0.5 pmol/min per mg microsomal protein (n = 7]. In six animals in labour induced with infusion of dexamethasone into the fetus, placental aromatase activity increased to a value of 14.0 +/- 1.0 pmol/min per mg protein; placental pregnenolone 17 alpha-hydroxylase, measured in four of the animals, also increased to 453 +/- 77 pmol/min per mg microsomal protein. In five animals in natural spontaneous labour with vaginal delivery, aromatase activity was 26.7 +/- 5.2 pmol/min per mg microsomal protein and pregnenolone 17 alpha-hydroxylase activity was 141 +/- 14 pmol/min per mg microsomal protein. Steroid sulphatase activity was barely detectable (less than 1.5 pmol/min per mg microsomal protein) during late pregnancy, dexamethasone-induced labour or natural parturition.(ABSTRACT TRUNCATED AT 250 WORDS)
Aldehyde-Lyases↗
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↗
The effects of alpha-human atrial natriuretic polypeptide on steroidogenesis by fetal zone cells of the human fetal adrenal gland.
The human fetal adrenal gland is primarily composed of fetal zone cells, which exhibit a high rate of steroidogenesis and a rapid growth rate during fetal life. alpha-Human atrial natriuretic polypeptide has been shown to inhibit basal and adrenocorticotropic hormone-stimulated steroidogenesis in the human adult and in some human adrenal adenoma cells. The purpose of this investigation was to determine the effect of atrial natriuretic polypeptide on steroidogenesis by fetal zone cells. Dispersed fetal zone cells were incubated in Krebs-Ringer's medium with adrenocorticotropic hormone, forskolin, 22R-hydroxycholesterol, or dibutyryl cyclic adenosine monophosphate and in the presence of atrial natriuretic polypeptide. The medium was analyzed for content of dehydroepiandrosterone sulfate and cortisol by radioimmunoassay. The addition of adrenocorticotropic hormone, forskolin, 22R-hydroxycholesterol, or dibutyryl cyclic adenosine monophosphate increased the secretion of dehydroepiandrosterone sulfate and cortisol twofold to threefold and twofold to sixfold, respectively, above basal rates. Atrial natriuretic polypeptide significantly inhibited basal dehydroepiandrosterone sulfate and cortisol secretion. When cells were incubated in the presence of adrenocorticotropic hormone, forskolin, 22R-hydroxycholesterol, or dibutyryl cyclic adenosine monophosphate plus atrial natriuretic polypeptide, cortisol secretion was inhibited 50% to 90% and dehydroepiandrosterone sulfate was inhibited 25% to 50%. Atrial natriuretic polypeptide had no effect on the metabolism of progesterone tagged with carbon 14 in fetal zone cells. In conclusion, atrial natriuretic polypeptide inhibited basal and adrenocorticotropic hormone-stimulated steroid secretion by fetal zone cells. Furthermore, these results suggested that the action of atrial natriuretic polypeptide was by inhibition of cholesterol side-chain cleavage or transfer of cholesterol to the mitochondrion.
Adrenal Glands↗
Inhibitors of cytochrome P-450-dependent arachidonic acid metabolism.
A new generation of heteroatom analogs of arachidonic acid are documented as powerful and selective inhibitors of the cytochrome P-450-dependent arachidonic acid oxygenase reaction (IC50, 5-10 microM) with little effect on either cyclooxygenase or soybean lipoxidase at 100 microM. The imidazole derivatives, ketoconazole and clotrimazole, are potent and selective inhibitors of the arachidonic acid epoxygenase and lipoxidase-like activities of phenobarbital-induced rat liver microsomal fractions (IC50, 2.0 and 0.3 microM, respectively). In contrast, the w/w-1 oxygenase activity of ciprofibrate-induced microsomal fractions was relatively resistant to inhibition by these compounds (IC50, 50 and 25 microM for ketoconazole and clotrimazole, respectively). Nordihydroguaiaretic acid (NDGA), eicosatetraynoic acid (ETYA), and indomethacin, extensively utilized inhibitors of the cyclooxygenase and lipoxygenase branches of the arachidonate cascade, also inhibit cytochrome P-450-dependent arachidonic acid metabolism. In decreasing order of potency, they were NDGA, ETYA, and indomethacin (IC50, 15, 40, and 70 microM, respectively).
5,8,11,14-Eicosatetraynoic Acid↗
Gerbil adrenal 11 beta- and 19-hydroxylating activities respond similarly to inhibitory or stimulatory agents: two activities of a single enzyme.
A high level of steroid 19-hydroxylation is exhibited by adrenal mitochondria of the gerbil, Meriones, unguiculatus, that accounts for the ability of that species to produce nearly equal amounts of corticosterone and 19-hydroxycorticosterone (Proc. Soc. exp. Biol. Med. 165 (1980) 69-74). Inhibitors of steroidogenesis and a polyclonal antibody against bovine cytochrome P-450(11 beta) were used to determine if the agents would effect differential or parallel suppression of 19- vs 11 beta-hydroxylation by gerbil adrenal mitochondria in vitro. The inhibitors (0.1-60 microM) tested (listed in order of decreasing effectiveness) were imazalil, metyrapone, miconazole and 4-hydroxyandrostenedione. With each inhibitor the degree of suppression of 11 beta-hydroxylation was accompanied by a parallel decline in 19-hydroxylation. The addition of the polyclonal antibody preparation also produced equivalent declines in the rates of the two hydroxylation reactions. The addition of ACTH 1 microM to primary cultures of gerbil adrenal cells brought about nearly equal increases in the secretion of 11 beta- and 19-hydroxylated steroids into the culture media. These results support the hypothesis that the 11 beta-hydroxylase of gerbil adrenal mitochondria has the capacity to carry out 11 beta- and 19-hydroxylations with nearly equal facility.
Adrenal Cortex↗
Effects of transforming growth factor beta on ovine adrenocortical cells.
Transforming growth factor beta (TGF beta) is a potent regulator of steroidogenic cell function. However, the mechanisms of the effects are not well understood. We studied the actions of TGF beta on primary cultures of ovine adrenocortical (OAC) cells. OAC cells had high affinity receptors for TGF beta (KD congruent to 7.6 +/- 1.5 X 16(-11) M). In addition, TGF beta inhibited the following markers of adrenocortical function: (1) ACTH, cholera toxin and forskolin acute stimulation of cAMP and steroid production; (2) the acute 8-bromo-cAMP stimulation of corticosteroid and pregnenolone production; and (3) the activity and amount of P-450 17 alpha-hydroxylase protein as well as activities of 11 beta- and 21-hydroxylases. The inhibitory effects of TGF beta on ACTH-induced cAMP and steroid production were time (half inhibition at 6 and 3 h respectively) and dose dependent (ID50 congruent to 10(-12) M). From these data we concluded that TGF beta acted rapidly on sites of OAC cell acute responses to stimulation by ACTH before and after the production of cAMP. Pregnenolone production in these cells was not inhibited by TGF beta when steroid production was stimulated on the addition of the readily permeable cholesterol derivative, 22 R-hydroxycholesterol. Thus, the rapid effect on OAC cells was manifest by TGF beta action on the utilization of cellular pools of cholesterol for the acute stimulation of steroid formation and not by direct action on the cholesterol side-chain cleavage enzyme. In addition, cells stimulated with ACTH in the absence or presence of lipoproteins (for up to 36 h) were susceptible to the inhibitory action of TGF beta. Taken together, these data amplify the pleiotropic actions of TGF beta on adrenocortical cell function and demonstrate that one acute action of TGF beta is on the utilization of endogenous supplies of cholesterol for steroid production.
8-Bromo Cyclic Adenosine Monophosphate↗
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↗
Simultaneous transfection of COS-1 cells with mitochondrial and microsomal steroid hydroxylases: incorporation of a steroidogenic pathway into nonsteroidogenic cells.
Transfected, nonsteroidogenic COS-1 cells derived from monkey kidney are found to be capable of supporting the initial and rate-limiting step common to all steroidogenic pathways, the side-chain cleavage of cholesterol to produce pregnenolone. Endogenous COS-1 kidney cell renodoxin reductase and renodoxin are able to sustain low levels of this activity catalyzed by bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) whose synthesis is directed by a transfected plasmid containing P450scc cDNA. Double transfection with both P450scc and adrenodoxin plasmids leads to greater pregnenolone production and indicates that adrenodoxin plays a role as a substrate for this reaction or that bovine adrenodoxin serves as a better electron donor than the endogenous iron-sulfur protein renodoxin. Also it is found that both the bovine adrenodoxin and P450scc precursor proteins are proteolytically processed upon their uptake by COS-1 cell mitochondria to forms having the same electrophoretic mobility as mature bovine adrenodoxin and P450scc. Following triple transfection of COS-1 cells with P450scc, adrenodoxin, and 17 alpha-hydroxylase cytochrome P450 plasmids, pregnenolone produced in mitochondria by the side-chain cleavage reaction can be further metabolized in the endoplasmic reticulum to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Although this functional steroidogenic pathway can be incorporated into this nonsteroidogenic cell type, it is found to be nonresponsive to cAMP, a potent activator of steroid hormone biosynthesis in adrenal cortex, testis, and ovary. Thus the cellular mechanisms necessary to support both microsomal and mitochondrial steroid hydroxylase activities appear not to be tissue specific, whereas the acute cAMP-dependent regulation of steroidogenesis is not present in transformed kidney (COS-1) cells.
Adrenodoxin↗
Protein kinase-C in the human fetal adrenal gland.
The fetal zone (FZ) of the human fetal adrenal gland undergoes rapid growth and exhibits a high rate of steroidogenesis throughout fetal life. In addition to cAMP-dependent processes regulating steroidogenesis and possibly growth of the FZ, evidence is accumulating that cAMP-independent mechanisms are also involved. The purpose of this study was to determine if the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase-C activity, stimulates steroidogenesis in FZ cells and to characterize protein kinase-C activity in FZ, neocortex zone, and anencephalic adrenal tissues. Adrenal glands were obtained from first and second trimester abortions and two anencephalic fetuses. The FZ was dissected from the neocortex. In some experiments, dispersed FZ cells were incubated in the presence and absence of ACTH and TPA for 3 h. TPA and ACTH stimulated steroidogenesis 2- and 5-fold, respectively. In other experiments, the separated zones and anencephalic adrenal tissues were homogenized, and the homogenates were subjected to DEAE-cellulose column chromatography. A single peak with phospholipid- and calcium-dependent activity was found. Subcellular distribution studies demonstrated greatest activity in the cytosolic fraction. The specific activity of protein kinase-C was significantly greater in FZ than neocortex zone, whether expressed per mg protein or per microgram DNA content. The activity in anencephalic tissue was low. In addition, protein kinase-C (80,000-dalton molecular size protein) was detected in adrenal tissues after electrophoresis and immunoblotting using an antibody directed against protein kinase-C. Greater amounts of protein kinase-C were detected in FZ tissue than in NC or anencephalic adrenal tissue. These results indicate that the lower activities of protein kinase-C in neocortex and anencephalic adrenal tissues were due to low amounts of enzyme rather than inactive enzyme. In summary, TPA-stimulated steroidogenesis in fetal zone cells and fetal zone cells contained greater activity and a greater amount of protein kinase-C than neocortex cells. Minimal activity and enzyme protein were found in anencephalic tissues. These results suggest that cAMP-independent mechanisms may play a role in fetal adrenal steroidogenesis.
Adrenal Cortex↗
The regulation of ovine placental steroid 17 alpha-hydroxylase and aromatase by glucocorticoid.
Parturition in the pregnant sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to estrogen production. This change is believed to be a consequence of the prepartum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha), steroid C-17,20-lyase, and possibly aromatase. We have investigated the activity levels of aromatase and 17 alpha-hydroxylase in placental microsomes in late pregnancy and dexamethasone-induced labor. Over the gestational period of 118-140 days basal levels of placental aromatase were relatively constant [mean value (+/- SD) of 5.6 +/- 1.6 pmol min-1 mg microsomal protein-1 (n = 10)]. Steroid 17 alpha-hydroxylase activity was undetectable [less than 0.5 pmol min-1 mg microsomal protein-1 (n = 7)]. In six animals in labor induced with infusion of dexamethasone into the fetus, placental aromatase activity had a mean value of 14.0 +/- 2.5 pmol min-1 mg protein-1; placental steroid 17 alpha-hydroxylase, measured in four of the animals, had a mean (+/- SD) activity of 319 +/- 58 pmol min-1 mg microsomal protein-1. Immunoblotting of placental microsomal preparations with specific antibodies to cytochrome P-450(17)alpha and NADPH-cytochrome P-450-reductase indicated that the glucocorticoid-induced activity of 17 alpha-hydroxylase was associated with increased content of cytochrome P-450(17)alpha. Northern blotting with a cDNA probe for cytochrome P-450(17)alpha showed that glucocorticoid increased the levels of mRNA for the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Regulation of steroid hydroxylase gene expression.
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Adrenal Cortex↗
Some enzymatic vagaries of a bovine adrenal microsomal cytochrome P-450 introduced and expressed in transformed monkey kidney cells.
The present studies illustrate the ability to carry out kinetic measurements of steroid hydroxylation using a cytochrome P-450 expressed in a tissue culture cell system. For these experiments a single species of cDNA, incorporated into a suitable expression vector, has been introduced via transfection. A number of interesting preliminary observations have been made on the function of the cytochrome P-450 associated with adrenocortical microsomes which catalyses the 17-hydroxylation of progesterone and pregnenolone. In confirmation of earlier reports the adrenal 17-OHase possesses both 17-hydroxylase as well as C17,20-lyase activities. However, the latter is only functional with 17-OH pregnenolone and not with 17-OH progesterone as substrate. This result differs from the numerous reports that a lyase activity for both substrates is associated with this P-450. The reason for this difference between a delta 4 and a delta 5 steroid remains unresolved although initial experiments indicate that the 5-alpha reduced progesterone is a suitable substrate for both the 17-OHase as well as lyase reactions. This result suggests an inhibitory effect of the delta 4 double bond preventing the carbon-carbon cleavage of the C-17,20 bond in 17-OH progesterone. Clearly more experiments will be required to resolve this question. Measurements of substrate affinity for the cytochrome P-450 expressed in COS cells appears to be influenced by a permeability barrier to the steroid effecting the transport of the steroid across the cell membrane into the cells. This conclusion is suggested by the presence of a time lag before the onset of metabolism as well as by the discrepancy in the concentration of substrate required to give half-maximal rates of metabolism, cf. the results obtained where the initial concentration of progesterone present in the reaction medium is altered versus those experiments measuring the kinetics of substrate depletion. The presence of such a barrier to the free movement of steroid across the membrane is interesting to contemplate when considering the build up of 17-OH pregnenolone required for the lyase reaction. Most unexpected where the results obtained when a comparable expression vector containing the cDNA for cytochrome b5 was cotransfected with pCD17 alpha 2. Cytochrome b5 has been postulated to be an electron transfer component participating in the cyclic function of some cytochromes P-450 (Hildebrandt and Estabrook, 1971).(ABSTRACT TRUNCATED AT 400 WORDS)
Adrenal Cortex↗
Regulation of the biosynthesis of steroidogenic enzymes.
Recombinant DNA technology can permit study of the regulation of steroid hydroxylase gene expression at three levels. The first of these is cAMP-regulated gene expression. In the adrenal, ACTH, via cAMP, increases the expression of the genes for all of the cytochrome P-450 species involved in the steroid biosynthetic pathway, as well as the iron-sulfur protein, adrenodoxin. This action of cAMP is inhibited by cycloheximide, suggestive of the involvement of a regulatory protein factor in mediating this action of cAMP. The second level is tissue-specific regulation of steroid hydroxylase gene expression. An example of this which we have studied is the expression of cholesterol side-chain cleavage cytochrome P-450 (P-450sec) and 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) in the bovine ovary. P-450sec is expressed at high levels in the corpus luteum but at low levels in follicles, whereas P-450(17)alpha is expressed in follicles, but is undetectable in the corpus luteum. The third level is fetal imprinting. A number of the cytochrome P-450 species involving in the steroidogenic pathway are expressed in the fetal adrenal at a time when exposure of the gland to ACTH is very low, suggestive that factor(s) other than pituitary ACTH mediate this expression in fetal life.
Adrenal Glands↗