Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In vitro luteinization of bovine granulosa (LGC) and theca (LTC) cells was achieved by culturing cells with forskolin (10 microM) and insulin (2 micrograms/ml) for 9 days. This treatment induced the presence of cytochrome P450scc and adrenodoxin in both cell types, but to substantially higher levels in LGC than in LTC. Forskolin dose-dependently stimulated the secretion of progesterone and cAMP after 3 h of incubation in both cell types although LGC were less sensitive to this stimulation than were LTC. Only LTC were responsive to LH, in accordance with their higher LH/hCG binding capacity. Both prostaglandin F2 alpha (PGF2 alpha) and phorbol 12-myristate 13-acetate (TPA) increased progesterone production during 3 h incubation of LGC and LTC, and treatment with staurosporine (a protein kinase C inhibitor) reversed this effect. Neither TPA nor PGF2 alpha alone affected cAMP levels but each acted synergistically with forskolin to increase cAMP accumulation. These results indicate that 1) elevated progesterone output from LGC is related to steroidogenic enzyme level; 2) bovine LH (up to 100 ng/ml) does not provoke a response in LGC due to their low LH/hCG binding capacity; 3) cAMP-protein kinase A and protein kinase C pathways are both involved in progesterone production by LGC and LTC, possibly by enhancing cholesterol transport.
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PMID:Steroidogenic enzyme content and progesterone induction by cyclic adenosine 3',5'-monophosphate-generating agents and prostaglandin F2 alpha in bovine theca and granulosa cells luteinized in vitro. 131 23

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.
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PMID:Expression of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase in fetal human adrenocortical cells transfected with SV40 T antigen. 132 52

As pregnancy progresses in the cow, the secretory activity of the corpus luteum is markedly diminished. This reduced secretion is due to a decline in the number of viable luteal cells as well as reduction in the secretory activity and responsiveness of the cells to trophic agents. The principal extra-ovarian source of progesterone (P4) by mid-gestation therefore appears to be the placenta. Uniquely this P4 biosynthesis is cyclic-nucleotide independent, but the Ca+2 dependent. It therefore appears that the Ca+2 second messenger and protein kinase C systems are responsible for regulation of sterol biosynthesis in the cow placenta. Dispersed bovine caruncle cells from the first trimester of pregnancy in comparison to caruncle cells of older than 90 days of gestation produce little P4 and are refractory to agents which enhance placental steroidogenesis. In order to explain this refractoriness of the first trimester cells, we determine (1) the expression of P450scc and its mRNA and (2) the expression of adrenodoxin. It was found that P4 synthesis by bovine maternal caruncle cells was low or undetectable in the first trimester but increased more than 10-fold in the second trimester of gestation. Addition of 25-OH-cholesterol to second trimester maternal cells increased P5 production but no effect was observed in first trimester cells. Cytochrome P450scc and its mRNA and adrenodoxin content were determined using Western blot or dot-blot techniques. Both proteins and the mRNA were detected in maternal tissue of first and second trimesters of gestation. In conclusion low P4 levels synthesized by first trimester maternal cells are not due to the absence of either cytochrome P450scc or adrenodoxin protein or production of P450scc mRNA. The data suggest that the refractoriness of the maternal caruncle cells during the first trimester is the result of post-translational regulation.
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PMID:Regulation of steroidogenesis in the bovine placenta. 134 67

The regulated expression of the genes encoding the various steroidogenic enzymes is a crucial component in the control of steroid hormone biosynthesis. Tissue-specific transcription of each of the steroidogenic enzyme genes determines the array of enzymes present within a steroidogenic tissue, and therefore the types of steroid hormones the tissue produces. Transcriptional regulation also determines developmental changes in the steroid hormones synthesized by steroidogenic tissues and for the quantitative regulation of steroid hormones necessary for reproduction and for maintaining physiological homeostasis. The molecular mechanisms governing transcriptional regulation of steroidogenic enzyme genes is now being studied. The results so far indicate that, like most other genes, transcription of steroidogenic enzyme genes is regulated by cis-elements in the 5' flanking DNA of the genes that bind trans-acting proteins found in the nucleus. Several types of cis-elements have been identified: elements responsible for basal transcription, for induction by cAMP, and for both basal and cAMP induction. Some of the basal cis-elements identified may have a role in tissue-specific transcription of certain steroidogenic enzyme genes in steroidogenic tissues. We have also identified regions in both the human P450scc and human P450c17 promoters that repress transcription when activated by the Ca2+/protein kinase C intracellular second messenger system used by angiotensin II. This review summarizes our current understanding of transcriptional regulation of the steroidogenic enzyme genes.
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PMID:The role of transcriptional regulation in steroid hormone biosynthesis. 165 86

We have recently shown that granulosa cells from hen ovarian follicles, collected at a stage of development 2-3 wk prior to ovulation (e.g. 6-8 mm in diameter) are steroidogenically inactive. Therefore, the hypothesis tested in the present studies was that theca cells from follicles at this stage of development must contain sufficient levels of functional cytochrome P450 side-chain cleavage (P450scc) enzyme to produce the progestin precursor required for the synthesis of androgens and estrogens. Northern blot analysis of total theca RNA collected from 6-8-mm follicles indicated the presence of a single P450scc mRNA transcript of approximately 2 kb whose expression was increased following an 8-h preincubation with 200 ng/ml ovine LH (oLH) or 10 microM forskolin. Western blot analysis of crude mitochondrial protein revealed a band of immunoreactive P450scc protein of approximately 53 kDa that was determined to be capable of converting 25-hydroxycholesterol to pregnenolone in a cell-free system. In the second set of studies, conducted to examine the cellular regulation of steroidogenesis in isolated theca cells of 6-8-mm follicles, theca cells were found to produce measurable basal levels of cAMP, progesterone, androstenedione, and estradiol following a 3-h incubation of 5 x 10(5) cells. Furthermore, significant dose-dependent increases in steroidogenesis were observed in response to oLH (0.2-200 ng/ml), chicken FSH (cFSH; 20-200 ng/ml), cholera toxin (0.002-20 ng/ml), and 8-bromo-cAMP (0.1-3.33 mM). Phorbol 12-myristate 13-acetate (PMA; 10-167 nM) also stimulated dose-dependent increases in basal progesterone, androstenedione, and estradiol production. In addition, while PMA had no effect on oLH (200 ng/ml)-promoted cAMP accumulation, or on oLH (20 ng/ml)- or 8-bromo-cAMP (1 mM)-stimulated progesterone production, it attenuated oLH-induced and 8-bromo-cAMP-induced androstenedione and estradiol accumulation. We conclude that theca cells from 6-8-mm follicles possess mRNA and immunoreactive protein coding for functional P450scc. Furthermore, basal steroidogenesis is increased by both the protein kinase A and protein kinase C pathways, whereas evidence suggests that protein kinase C inhibits LH-induced androstenedione production at a site distal to cAMP and progesterone production, most likely by decreasing C17,20-lyase activity.
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PMID:Cytochrome P450 side-chain cleavage (P450scc) in the hen ovary. I. Regulation of P450scc messenger RNA levels and steroidogenesis in theca cells of developing follicles. 166 52

Previous studies have indicated that developing avian granulosa cells collected from follicles 2-3 wk prior to ovulation (e.g. 6-8-mm in diameter) are steroidogenically incompetent, apparently due to a lack of functional cytochrome P450 side-chain cleavage (P450scc) enzyme activity. The present studies were designed to test this hypothesis by determining the absence or presence of P450scc messenger RNA, immunoreactive protein, and enzyme activity in granulosa tissue of developing hen ovarian follicles. Additionally, the interactive roles of FSH, the adenylyl cyclase-cAMP system, and the protein kinase C pathway in granulosa cell differentiation were investigated. Granulosa cells collected from developing, 6-8-mm follicles were found to contain extremely low but detectable levels of a single, 2.0-kb P450scc mRNA transcript, as well as immunoreactive P450scc protein (53 kDa). However, this protein was apparently incapable of converting 25-hydroxycholesterol to pregnenolone in a cell-free system. Preincubation of granulosa cells with ovine FSH or forskolin for 24 h rendered the cells capable of converting cholesterol precursor to pregnenolone during a subsequent 3-h incubation. Inclusion of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), in the preincubation medium blocked the stimulatory actions of FSH and forskolin on the induction of P450scc activity; however, PMA-preincubation did not alter the ability of granulosa cells to convert exogenous pregnenolone to progesterone compared to vehicle-pretreated cells. These data suggest that steroidogenic incompetency in differentiating avian granulosa cells is primarily due to a lack of active P450scc enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cytochrome P450 side-chain cleavage (P450scc) in the hen ovary. II. P450scc messenger RNA, immunoreactive protein, and enzyme activity in developing granulosa cells. 166 53

Long-term regulation of mammalian steroid hormone synthesis occurs principally by transcriptional regulation of the gene for the rate-limiting cholesterol side-chain cleavage enzyme P450scc. Adrenal steroidogenesis is regulated primarily by two hormones: adrenocorticotropin, which works via cyclic AMP (cAMP) and protein kinase A, and angiotensin II, which works via Ca2+ and protein kinase C. Forskolin and 8-bromo-cAMP stimulated, while prolonged treatment with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]) and a calcium ionophore (A23187) additively suppressed accumulation of endogenous P450scc mRNA in transformed murine adrenal Y1 cells. In Y1 cells transfected with 2,327 base pairs of the human P450scc promoter fused to the bacterial gene for chloramphenicol acetyltransferase (CAT), forskolin increased CAT activity 900% while combined TPA plus A23187 reduced CAT activity to 15% of the control level. Forskolin induced the P450scc promoter as rapidly as a promoter containing two cAMP-responsive elements fused to a simian virus 40 promoter, a system known to respond directly to cAMP. Basal expression was increased by sequences between -89 and -152 and was increased further by sequences between -605 and -2327. This upstream region also conferred inducibility by cAMP. TPA plus A23187 transiently increased CAT activity before repressing it, reflecting the complex actions of angiotensin II in vivo. Repression by prolonged treatment with TPA plus A23187 was mediated by multiple elements between -89 and -343. Induction of CAT activity by forskolin was not diminished by treatment with TPA plus A23187, nor were the regions of the promoter responsible for regulation by the two pathways coisolated. Thus, the human gene for P450scc is repressed by TPA plus A23187 by mechanisms and sequences independent of those that mediate induction by cAMP.
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PMID:Human P450scc gene transcription is induced by cyclic AMP and repressed by 12-O-tetradecanoylphorbol-13-acetate and A23187 through independent cis elements. 170 Feb 77

Luteinizing hormone is the major regulator of Leydig cell differentiation and steroidogenic function. A number of hormones produced by the Leydig cell (e.g. estrogen, angiotensin, CRF, vasopressin) and the tubular compartment (inhibin, TGF beta), can influence both acute and long-term actions of LH. Conversely, hormones produced in the Leydig cells modulate tubular function (e.g. androgen, beta-endorphin, oxytocin). The LH stimulatory event can be negatively influenced by the action of angiotensin II through the guanyl nucleotide inhibitory unit of adenylate cyclase. We have recently discovered an action of corticotrophin releasing hormone through specific high-affinity low-capacity receptors in the Leydig cells which involves a pertussis toxin insensitive guanyl nucleotide regulatory unit with interaction between signalling pathways and resulting inhibition of LH induced cAMP generation and consequently of steroidogenesis. In contrast to other tissues the CRF receptor in the Leydig cells did not couple to Gs. CRF action is exerted through direct or indirect action of protein kinase C, at the level of the catalytic subunit of adenylate cyclase. Physiological increases in endogenous LH cause positive regulation of membrane receptors and steroidogenesis, while major elevations in circulating gonadotropin can induce down-regulation of LH receptors and desensitization of steroid responses in the adult cell. Gonadotropin-induced desensitization in adult rat tests include an estrogen mediated steroidogenic lesion of the microsomal enzymes 17 alpha-hydroxylase/17,20-desmolase. For further understanding of the regulation of this key enzyme of the androgen pathway the rat P450(17) alpha cDNA was cloned and sequenced. This cDNA expressed in COS-1 cells 17 alpha-hydroxylase/17,20-desmolase activities. From the deduced amino acid sequence, two transmembrane regions were identified, a signal peptide for insertion in the ER, and a 2nd transmembrane region separated from the first by 122 amino acids. The carboxy terminal non-transmembrane region possesses 4 hydrophobic clefts, of which cleft II would contain the putative steroid binding site for both hydroxylase and lyase activities. The rat cDNA was employed to evaluate the hormonal regulation of mRNA levels in adult and fetal Leydig cells. Low dose hCG treatment caused an early increase in mRNA levels followed by a return to control values at later times, while with higher desensitizing doses the initial increase in mRNA was followed by a marked reduction in mRNA at 24 h and a small recovery at 48 h. Fetal rat Leydig cells treated with E2 showed a 70% decrease in P450 mRNA levels, and testosterone production closely followed the changes in mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:LH action in the Leydig cell: modulation by angiotensin II and corticotropin releasing hormone, and regulation of P450(17) alpha mRNA. 269 45

In earlier studies in cultures of porcine granulosa cells prepared from small antral follicles, steroidogenesis-related loci were inhibited by treatment for 48 h with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC). In the present investigation, cells were incubated in serum-free medium for 48 h, with various agents present during the last 2-24 h. With TPA at 30 ng/ml, the FSH-stimulated cAMP accumulation was markedly enhanced at all time points. FSH increased the concentration of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA throughout the 24-h incubation. At 4 and 8 h, TPA increased the accumulation of P450scc mRNA, having an additive effect with FSH. However, at 24 h, TPA markedly suppressed the FSH-induced increased in P450scc mRNA. Pretreatment of cells with FSH did not shorten the time required for TPA to become inhibitory. The stimulatory effect of 8-bromo-cAMP on P450scc mRNA also was augmented by TPA at 4 h, but significant inhibition was not observed at 24 h. The concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA, intended to be used for correction of gel loading, was stably increased by both cAMP and TPA. These effects of TPA suggest multiple actions of PKC(s) on the regulation of P450scc expression and other endpoints in ovarian granulosa cells.
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PMID:Dual actions of phorbol ester on cytochrome P450 cholesterol side-chain cleavage messenger ribonucleic acid accumulation in porcine granulosa cells. 762 23

The rate-limiting step in luteal biosynthesis of progesterone consists of cleavage of the side chain of cholesterol by mitochondrial cytochrome P450 side-chain cleavage enzyme (P450scc) to form pregnenolone. Luteal mRNA encoding P450scc, quantitated on selected days of the 16-day ovine estrous cycle, was similar on days 3 and 6, increased by 2-fold on day 9 (P < 0.05) and remained elevated on day 15. Levels of P450scc mRNA on day 15 of pregnancy were not different from those found on any day of the cycle (P < 0.05). To determine whether levels of mRNA encoding P450scc are hormonally regulated, ewes on day 10 of the estrous cycle were injected with hCG or prostaglandin F2 alpha (PGF2 alpha). P450scc mRNA was not increased for up to 36 h after injection of hCG, nor decreased within 8 h after injection of PGF2 alpha (P < 0.05). An assay for P450scc activity was developed which utilized ovine small and large luteal cells in the presence of 22R-hydroxycholesterol and ovine high density lipoprotein. Enzyme activity was quantitated by measurement of progesterone production. In small luteal cells activation of the protein kinase A (PKA) second-messenger system by treatment with LH resulted in 910% increase in progesterone production without altering activity of P450scc. Activation of the protein kinase C (PKC) second-messenger system with phorbol 12-myristate 13-acetate caused a 51% reduction in progesterone secretion from large luteal cells but did not alter activity of P450scc. These findings suggest that in mature luteal tissue steady state levels of mRNA encoding P450scc, and enzyme activity are independent of acute regulation by activation of PKA or PKC second-messenger systems.
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PMID:Regulation of cytochrome P450scc synthesis and activity in the ovine corpus luteum. 782 90


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