Cholesterol Side-chain Cleavage Enzyme Complex Research Articles

Biosynthesis and metabolism of steroid hormones by human adrenal carcinomas.

Over a 15-year period, our university-based laboratory obtained 125 adrenal tumors, of which 15 (12%) were adrenal cortical carcinomas. Of these, 6 (40% of the carcinomas) occurred in patients with clear clinical manifestations of steroid hormone excess. Adrenal cortical carcinoma cells derived from the surgically resected tumors in 4 of these patients were isolated and established in primary culture. Radiotracer steroid interconversion studies were carried out with these cultures and also on mitochondria isolated from homogenized tissues. Large tumors had the lowest steroidogenic activities per weight, whereas small tumors had more moderately depressed enzyme activities relative to cells from normal glands. In incubations with pregnenolone as substrate, 1 mM metyrapone blocked the synthesis of corticosterone and cortisol and also the formation of aldosterone. Metyrapone inhibition was associated with a concomitant increase in the formation of androgens (androstenedione and testosterone) from pregnenolone. Administration of metyrapone in vivo before surgery in one patient resulted in a similar increase in plasma androstenedione, though plasma testosterone levels were not significantly affected. In cultures of two of four tumors examined, dibutyryl cAMP stimulated 11ss-hydroxylase activity modestly; ACTH also had a significant stimulatory effect in one of these tumors. Unlike results obtained with normal or adenomatous adrenal cortical tissues, mitochondria from carcinomatous cells showed a lack of support of either cholesterol side-chain cleavage enzyme complex or steroid 11ss-hydroxylase activity by Krebs cycle intermediates (10 mM isocitrate, succinate or malate). This finding is consistent with the concept that these carcinomas may tend to function predominantly in an anaerobic manner, rather than through the oxidation of Krebs cycle intermediates.

Influence of season and low-level oestradiol immunoneutralization on episodic LH and testosterone secretion and testicular steroidogenic enzymes and steroidogenic acute regulatory protein in the adult ram

The regulation of LH-dependent and -independent increases in testosterone secretion by key proteins in the testes of adult rams was investigated. Serial blood samples were collected from groups of four control and passively immunized (oestradiol antiserum for 3 weeks) rams and the animals were gonadectomized in either the non-breeding season (April) or the breeding season (September). LH pulse frequency and basal (interpulse) concentrations were several times greater (P < 0.01) in the breeding season than in the non-breeding season. Neither of these parameters nor LH pulse amplitude were affected by oestradiol immunization. Parameters of testosterone episodic secretion and response to an injection (i.v.) of 15 micrograms NIH-LH-S25 were also greater (P < 0.05) in the breeding season and, with the exception of pulse frequency, in immunized rams versus controls. Substrate utilization established that testosterone biosynthesis was predominantly via the 5-ene pathway. Increases in blood testosterone concentration in the breeding season were associated with a fivefold higher (P < 0.01) activity of cytochrome P450 17alpha-hydroxylase/C-17,20 lyase (P450(17alpha)) and a 65% higher (P < 0.05) relative amount of mRNA for cytochrome P450 cholesterol side-chain cleavage enzyme complex (P450scc) in the testis. Of the steroidogenic enzyme activities examined, only that for 17beta-hydroxysteroid dehydrogenase (17beta-HSD) tended to be increased by oestradiol immunization. Blood concentrations of cholesterol lipoproteins and expression of the testicular low density lipoprotein receptor were not affected by season or immunization. The amount of steroidogenic acute regulatory protein (StAR) mRNA was 65% higher (P < 0.01) in the breeding season and 20% higher (P < 0.01) in immunized rams versus controls. These results indicate that greater LH stimulation may increase testosterone biosynthesis in the breeding season by increasing StAR mRNA (and presumably delivery of cholesterol to P450scc) and the activity of P450(17alpha), and possibly that of P450scc (activity not measured). More moderate increases in StAR mRNA and 17beta-HSD activity may explain, in part, the increases in testosterone secretion with oestradiol immunization.

Bursal antisteroidogenic peptide alters the activity of steroidogenic enzymes in chicken granulosa cells

We have previously reported that a peptide from chicken bursa of Fabricius, bursal antisteroidogenic peptide (BASP), inhibits luteinizing hormone-stimulated progesterone biosynthesis by chicken ovarian granulosa cells. The objective of this study was to determine the site(s) of BASP inhibition within the steroidogenic pathway of chicken granulosa cells. The effects of BASP on key steroidogenic enzymes, including adenylyl cyclase (AC), phosphodiesterase, the cholesterol side-chain cleavage enzyme complex and 3ß-hydroxysteroid dehydrogenase were determined. Luteinizing hormone (10 ng/tube) stimulated a fivefold increase in granulosa cell progesterone production that was inhibited by BASP (0.06, 0.12 or 0.25 bursal equivalents) in a dose-dependent manner. Luteinizing hormone stimulated a sixfold increase in cyclic 3′,5′-adenosine monophosphate (cAMP) formation, and this increase was potentiated by BASP in a dose-dependent manner. In addition, BASP stimulated cAMP formation in the absence of luteinizing hormone without affecting progesterone production. The AC activator forskolin (0.1 mM) stimulated a 4.5-fold increase in progesterone synthesis, which was inhibited by BASP. In the presence of forskolin. BASP increased cAMP formation in a dose-dependent manner. A fivefold increase in progesterone synthesis induced by the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (1.0 mM) was inhibited by BASP. In the presence of 3-isobutyl-l-methylxanthine, BASP increased cAMP formation in a dose-dependent manner. Finally, 22(R)-hydroxycholesterol (250, 500, 1,000, or 2,500 ng/tube) or pregnenolone (50, 100, 200, or 500 ng/tube) resulted in up to 15- or 10-fold increases in progesterone production, respectively. Increasing concentrations of BASP caused a dose-dependent suppression of the conversion of 22(R)-hydroxycholesterol, but not pregnenolone, to progesterone. The inhibition of steroidogenesis by BASP is not associated with reduced cAMP levels, and BASP appears to strongly stimulate AC activity. In addition, these findings suggest that BASP may limit the availability of progesterone precursors by inhibiting the activity of the cholesterol side-chain cleavage enzyme complex.

Follicle-stimulating hormone-regulated Sertoli cell proteins SCc1 and SCc2 are phosphorylated and mitochondrially associated

Sertoli cell intracellular protein 1 (SCc1) and 2 (SCc2) are polypeptides found in rat Sertoli cell cultures incubated with either FSH or (Bu)2cAMP. They were first identified in [35S]methionine-labeled Sertoli cell lysates using two-dimensional gel electrophoresis. Here we extend these observations by showing that SCc1 and SCc2 are present in rat seminiferous tubules, ovaries, and granulosa cells incubated with either FSH or (Bu)2cAMP and in testicular peritubular cells incubated with (Bu)2cAMP. Peritubular cells do not, however, respond to FSH with the production of SCc1 and SCc2. Peptide mapping with N-chlorosuccinimide revealed that SCc1 and SCc2 have similar cleavage patterns, suggesting a common primary amino acid sequence that is modified posttranslationally. Metabolic labeling with [32P]orthophosphate provided direct evidence that SCc1 and SCc2 are phosphoproteins. A shift in mobility of SCc1 and SCc2 toward the basic region of the gel to positions designated SCc1' and SCc2' occurred when cell lysates were treated with alkaline phosphatase before electrophoresis, providing additional evidence that SCc1 and SCc2 are phosphoproteins. SCc1 and SCc2 are also shown to be mitochondrially-associated in the Sertoli cell. Peptide maps of SCc1, SCc2, SCc1', and SCc2' obtained by treatment with alpha-chymotrypsin, are identical to proteolytic maps of proteins pp30', p30, and pp30 from adrenocortical cells. SCc1, SCc2, SCc1', and SCc2' are homologous with regard to their regulated expression, electrophoretic mobility, and mitochondrial localization to the adrenal proteins pp30' and pp30 as well as a series of 30 kilodalton proteins from MA-10 Leydig tumor cells. Both the adrenal cell proteins and the Leydig tumor cell proteins are thought to participate in cholesterol transport to the inner mitochondrial membrane, providing substrate for the cholesterol side-chain cleavage enzyme complex, an activity which the Sertoli cell does not perform, suggesting that alternative functions must be sought for SCc1 and SCc2 in Sertoli cells.

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.

Direct hypothalamic control of vasoactive intestinal peptide (VIP) levels in the developing rat ovary

We have previously identified the neurotransmitter vasoactive intestinal peptide (VIP) in nerve fibers of the immature rat ovary and showed that it stimulates steroid release by a mechanism involving increased synthesis of the components of the cholesterol side-chain cleavage enzyme complex. The present experiments were undertaken to study the ontogeny of ovarian VIP levels and to determine if they change in relation to the initiation of puberty. VIP was already detected in 2-day-old ovaries; levels remained constant at approximately 4.5 pg/mg ovary until the end of juvenile development (day 30). Thereafter, and preceding the peripubertal activation of the ovary, VIP levels increased two-fold, decreased gradually towards the first proestrus, and returned to juvenile values after the first ovulation. Transection of the ovarian nerves eliminated radioimmunoassayable VIP levels in both intact and hypophysectomized rats, indicating that ovarian VIP derives mostly from the extrinsic innervation of the gland. Treatment of hypophysectomized immature female rats with human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), growth hormone (GH), prolactin (Prl), estradiol, or their combination, failed to reproduce the peripubertal increase in VIP. In contrast, unilateral direct anodal current lesions of the left preoptic-anterior hypothalamic area (POA-AHA) of hypophysectomized juvenile rats led to a significant increase in VIP in the ipsilateral ovary. Surprisingly, both bilateral lesions and lesions of the right POA-AHA also increased VIP levels in the left ovary suggesting the existence of a marked asymmetry in the hypothalamic control of ovarian VIP. Lesions of the ventromedial nucleus, dorsal AHA or small lesions of the AHA were ineffective. The results suggest that: a) in prepubertal rats ovarian VIP levels are controlled by a pituitary-independent, neurally-mediated, mechanism which involves the POA-AHA region of the hypothalamus; b) this control is markedly asymmetric; and c) the lesion-induced increase in ovarian VIP levels may not result from the loss of a discrete inhibitory center, but rather may be the consequence of compensatory reactivity of the areas surrounding the lesion.

Tetradecanoyl phorbol acetate suppresses follicle-stimulating hormone-induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells.

The effect of tetradecanoylphorbol acetate (TPA) on follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studied in rat ovarian granulosa cells cultured for 48 h in serum-free medium. Cell proteins were radiolabeled with [35S]methionine, followed by immunoprecipitation of cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) as well as the iron-sulfur protein adrenodoxin. Polyacrylamide gel electrophoresis and fluorography of the immunoprecipitates showed that TPA, when added in combination with FSH (50 ng/ml) or dibutyryl cAMP (Bt2cAMP; 1 mM), suppressed the stimulatory effects of these compounds on the synthesis of the SCC components in a concentration-dependent fashion. The effect of TPA was accompanied by decreased progesterone formation and decreased cAMP accumulation. The structural analog of TPA, phorbol-4 alpha-didecanoate, which does not activate protein kinase C (Ca2+/phospholipid-dependent enzyme), had no effect on the FSH- or Bt2cAMP-stimulated synthesis of SCC and progesterone or on cAMP formation. In addition to inhibiting the synthesis of these proteins, TPA greatly reduced the FSH- and Bt2cAMP-induced increase in levels of mRNA encoding the precursor form of P-450SCC. It is concluded that the effect of the phorbol ester TPA to inhibit FSH-stimulated progesterone formation in cultured ovarian granulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levels of mRNA encoding the precursor forms of these proteins. The results are indicative that TPA not only inhibits FSH-mediated stimulation of cAMP formation but also may block cAMP-mediated induction of SCC synthesis. It is postulated that the effects of TPA may reflect the physiological role of protein kinase C in the regulation of ovarian steroidogenesis.

Effects of epidermal growth factor on the synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells in primary culture

The effect of epidermal growth factor (EGF) on the synthesis of the components of the cholesterol side-chain cleavage enzyme complex (SCC) was studied in rat ovarian granulosa cells. The cells were cultured for 48 h in the presence or absence of EGF (15 ng/nil) and/or FSH (50 ng/ml) after which proteins were radiolabeled by incubation with [ 35S]methionine followed by immunoprecipitation of newly synthesized P-450 scc or adrenodoxin (ISP) with polyclonal antibodies directed against the corresponding proteins from bovine adrenal cortex. In addition the action of EGF on the level of translatable RNA for P-450 scc was evaluated using a cell-free translation system programmed with RNA isolated from treated and untreated cells, followed by immunoisolation of newly synthesized proteins. Immunoisolated proteins were separated by polyacrylamide-gel electrophoresis, visualized by fluorography and quantified by densitometry. EGF stimulated progesterone formation by the cells 3-fold and potentiated the FSH-induced stimulation of progesterone formation, but had no effect on cAMP accumulation. EGF also stimulated the synthesis of P-450 scc and ISP, and enhanced the FSH-induced synthesis of P-450 scc and ISP in a concentration-dependent fashion with a maximal stimulation attained at concentrations ranging from 1.0 to 100 ng/ml. No appreciable changes in the induction pattern were observed when EGF and dibutyryl cyclic AMP (Bt 2cAMP) were added together, as compared to when Bt 2cAMP was added alone. Neither treatment affected the synthesis of the constitutive mitochondrial enzyme, F 1~ATPase. Immunoisolation of P-450 scc from the proteins synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from EGF- and/or FSH-treated cells, revealed that EGF enhanced the FSH-stimulated synthesis of the precursor form of P-450 scc It is concluded that EGF stimulates the synthesis of the SCC components, either alone or together with FSH, through increasing levels of translatability of the mRNA encoding the precursor form of P-450 scc , independently of cAMP formation.

Vasoactive intestinal peptide induces the synthesis of the cholesterol side-chain cleavage enzyme complex in cultured rat ovarian granulosa cells.

Vasoactive intestinal peptide (VIP) has been identified in ovarian nerves and stimulates steroid secretion from immature ovaries. To gain insight into its mechanism of action, the effect of VIP on the synthesis of the cholesterol side-chain cleavage enzyme complex was studied in ovarian granulosa cells from immature estrogen-primed rats. The cells were cultured for 48 hr in serum-free medium; the proteins were labeled with [35S]methionine; and the synthesis of cytochrome P-450, iron-sulfur protein, and NADPH:iron-sulfur protein reductase was evaluated by electrophoretic analysis after immunoisolation with polyclonal antibodies directed against the bovine adrenal enzymes. VIP at concentrations ranging from 0.001 to 1 microM stimulated 3- to 5-fold the synthesis of cytochrome P-450 and iron-sulfur protein. Peptide NH2-terminal histidine, COOH-terminal isoleucine, which has greater than 50% sequence homology of VIP, stimulated the synthesis of both proteins at approximately 50% of VIP effectiveness. Secretin, another member of the glucagon-secretin family of peptides, which has only 30% sequence homology to VIP, was without effect. Similar results were observed with the NADPH:iron-sulfur protein reductase. VIP-induced synthesis of the cholesterol side-chain cleavage enzyme complex was accompanied by a dose-related increase in cAMP accumulation and progestin formation. It is concluded that VIP regulates the synthesis of the ovarian cholesterol side-chain cleavage enzyme complex, which catalyzes the rate-limiting reaction in progesterone biosynthesis, and that the VIP effect is at least partially mediated through cAMP. It is suggested that a stimulatory action of VIP on the synthesis of ovarian progesterone may contribute to regulating the functional development of the ovary.

Clomiphene and Tamoxifen Inhibit Progesterone Synthesis in Granulosa Cells: Comparison with Estradiol

The actions of clomiphene, tamoxifen, and 17 beta-estradiol were studied in an isolated avian granulosa cell system during short term incubations. A dose-dependent inhibition of ovine LH-enhanced progesterone production was observed for all three compounds, with an IC50 of 3 X 10(-6) M and maximal inhibition (95%) at 5 X 10(-5) M. A similar inhibition was found when cells were stimulated by either 8-bromo-cAMP or forskolin. Neither clomiphene nor tamoxifen had an inhibitory effect on the conversion of pregnenolone to progesterone, a step that was blocked by 17 beta-estradiol and isoxazole , a known inhibitor of 3 beta-hydroxysteroid dehydrogenase. On the other hand, cells exposed to clomiphene failed to use [6-3H]25-hydroxycholesterol, while estradiol significantly increased the conversion of labeled substrate to pregnenolone at the expense of progesterone (30% vs. 7% of the total radioactivity). By comparison, in control cells, progesterone represented the major metabolite with 36% of the total radioactivity; pregnenolone had only 2.8%. The results demonstrate that these triphenylethylene compounds, which are used clinically as antiestrogens, inhibit steroidogenesis at a step before pregnenolone formation, probably at the site of the cholesterol side-chain cleavage enzyme complex, while 17 beta-estradiol inhibits 3 beta-hydroxysteroid dehydrogenase.

Regulation of cholesterol metabolism in rat adrenal mitochondria

Addition of cholesterol to rat adrenal mitochondria resulted in a stimulation of pregnenolone synthesis. The slow step of the mitochondrial cholesterol side-chain cleavage reaction could be the interaction of the sterol with cytochrome P-450. The rate of cholesterol binding to this enzyme as observed spectroscopically correlated with the equilibration period (20 min) of the mitochondria and exogenous cholesterol required for maximal rates of pregnenolone synthesis. It is suggested that translocation of cholesterol between different sterol pools occurs within the mitochondria. Potential intracellular effectors that could be of importance in the movement or regulation of mitochondrial cholesterol include bivalent metallic ions, prostaglandins, cyclic nucleotides, polyamines and polylysine. Of the effectors studied, only calcium ions and polylysine markedly stimulated pregnenolone synthesis. These effectors might stimulate steroidogenesis by lateral displacement of cholesterol in the mitochondrial membrane into a compartment easily accessible to the cholesterol side-chain cleavage enzyme complex.

Gonadotrophin Stimulation of Progesterone Synthesis by Midpregnancy Mouse Ovarian Cellsin Vitro

The ability of cultured midpregnancy mouse ovarian cells to synthesize progesterone de novo and from oxogenous pregnenolone has been assessed. The conversion of pregnenolone to progesterone is almost completely blocked by cyanoketone, a known inhibitor of delta5,3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity, but is unaffected by aminoglutethimide, which inhibits the cholesterol side-chain cleavage enzyme complex (desmolase). Since there is little metabolism of the formed progesterone, the ability of ovarian cells to convert exogenous pregnenolone to progesterone in vitro reflects the activity of 3beta-HSD in these cells. Cultured ovarian cells are also capable of endogenous progesterone production in the absence of added pregnenolone, although the absolute amount of progesterone produced is considerably less than that produced from exogenous pregnenolone. Since endogenous progesterone accumulation is almost completely blocked by the addition of aminoglutethimide to the culture medium, it is concluded that this response does represent de novo progesterone synthesis. Neither bovine luteinizing hormone (LH) nor human chorionic gonadotrophin (hCG) affects 3beta-HSD activity in cultured ovarian cells. The ability of the cells to secrete or to further metabolize the progesterone formed is also unaffected. However, both LH and hCG stimulate endogenous progesterone production within one hour of their addition to the culture medium. The stimulation, 2-10 fold in several experiments, can be maintained for at least six days of culture, and is not a result of an increase in the growth rate of the ovarian cells. As would be expected, the stimulation is blocked by the addition of aminoglutethimide to the culture medium. Taken together, these facts suggest that gonadotrophic hormones stimulate progesterone production by ovarian cells specifically by their action at steps prior to the conversion of pregnenolone to progesterone.