Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The control of androgen production by the Leydig cell is dependent upon the episodic secretion of hormone (LH), which is released from the anterior pituitary gland in pulses of high biological activity. This mode of episodic LH secretion supports steroidogenic enzyme activity in the testis through interaction with LH receptors and stimulation of the adenylate cyclase/protein kinase sequence, leading to phosphorylation of key intermediates in the steroid biosynthetic pathway. The plasma membrane events that are rapidly activated by the specific interaction of LH or hCG with Leydig cell receptors include increased binding of guanyl nucleotide, and stimulation of cAMP-independent, Ca2+-dependent phosphorylation of a 44,500 Mr protein, with the characteristics of the adenylate cyclase nucleotide regulatory unit. Hormonal activation of adenylate cyclase is affected by Ca2+ with the same concentration-dependence, suggesting that nucleotide-induced phosphorylation is related to activation of the catalytic cyclase unit. In addition to the characteristic increases in pregnenolone synthesis and androgen production, gonadotropin-stimulated Leydig cells show prominent changes in LH receptor content and steroidogenic activity that modify their subsequent responses to hormonal signals. Thus, after exposure to increased LH and hCG levels in vivo and in vitro, LH receptors show an initial transient increase (up-regulation) followed by a marked decrease (down-regulation) and a prolonged depletion of LH receptor sites. Large doses of hCG cause "early" (prior to pregnenolone) and "late" steroidogenic lesions (17 alpha-hydroxylase, 17-20 desmolase) that are independent of receptor loss. The early lesion is partly due to reduced activity of HMG CoA reductase, and is mainly attributable to the increased activity of an inhibitory protein factor that modulates the activity of cholesterol side chain cleavage enzyme in Leydig cell mitochondria. In contrast, the late steroidogenic lesion is related to the nuclear actions of E2 produced during hormonal action. After hCG stimulation, an increase in nuclear E2 binding was accompanied by an early rise of RNA polymerase activities within 45 min coincident with the maximal increases in circulating testosterone and estradiol levels. These events were followed by the emergence of an E2-induced protein of Mr 27,000 at 3-6 h, and by reduction in the activity of 17 alpha-hydroxylase/17-20 desmolase, and a decrease in microsomal cytochrome P-450.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Hormonal regulation of androgen production by the Leydig cell. 632 62

Mouse Leydig MA-10 tumor cells are a good model of testicular steroidogenesis. The endogenous murine P450scc mRNA in these cells accumulated in response to 8-bromo-cAMP, forskolin, cholera toxin, and 1-methyl-3-isobutylxanthine, but not in response to 1,9-dideoxyforskolin, indicating that this accumulation was stimulated by the protein kinase-A pathway. Inhibiting transcription with actinomycin-D showed that the half-life of cytochrome P450scc mRNA in these cells was not altered by cAMP, consistent with earlier nuclear run-on data showing that the effect of cAMP on P450scc is at the transcriptional level. A series of 17 fragments of 5'-flanking DNA from the human P450scc gene were fused to the gene for firefly luciferase and transiently transfected into MA-10 cells. The longest construct, containing 2327 basepairs of 5'-flanking DNA, responded 4-fold to forskolin and, hence, was used to optimize the forskolin dose response, showing that 30 microM forskolin elicited a 90% maximal effect. Examination of the activity of the deletion constructs located basal and cAMP-responsive sequences. Constructions containing 79 basepairs of 5'-flanking DNA had basal activity; adding sequences between -79 and -110 had minimal effect, but adding sequences between -110 and -127 increased basal activity 3-fold. Adding sequences beyond -127 did not increase basal transcription further, indicating the presence of a basal transcription element between -110 and -127. These serial deletion mutants were used similarly to locate cAMP responsiveness between -1620 and -1676, indicating the presence of a cAMP response element in this region. The locations of these basal and cAMP-responsive sequences correspond well with those previously identified when human P450scc promoter/reporter constructions were transfected into mouse adrenocortical Y-1 cells, but differ from those identified when such constructions were transfected into human JEG-3 choriocarcinoma cells.
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PMID:Basal transcriptional activity and cyclic adenosine 3',5'-monophosphate responsiveness of the human cytochrome P450scc promoter transfected into MA-10 Leydig cells. 767 94

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

FSH induces the expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) in rat ovarian granulosa cells. The present study reveals that the tyrphostin AG18, a member of novel protein tyrosine kinase inhibitors, can arrest the FSH-induced synthesis of P450scc with an apparent IC50 of 30 microM. Total inhibition of P450scc expression was achieved at 80 microM AG18. AG18-mediated inhibition of P450scc was also observed when the enzyme was induced by prostaglandin E2, forskolin, or 8-bromo-cAMP. Studies examining functional LH receptors showed that the tyrphostin inhibits the expression of FSH-induced LH receptors. The drug did not affect FSH-induced cAMP accumulation, suggesting that it may interfere with the flow of FSH signal transduction at a site distal intracellular accumulation of cAMP. Control experiments demonstrated that the inhibitory action of AG18 was reversible, did not hamper total protein synthesis in the cells, and did not change the adenine nucleotide (ATP:ADP:AMP) ratio or their levels in the treated cells. A cell-free assay of cAMP-dependent protein kinase showed that the tyrphostin AG18 does not affect this enzyme activity up to concentrations above 200 microM. These results suggest that a putative tyrosine kinase activity is involved in the gonadotropin signal transduction pathway leading to expression of functional genes in ovarian cells.
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PMID:Tyrphostins inhibit follicle-stimulating hormone-mediated functions in cultured rat ovarian granulosa cells. 838 Mar 82

NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.
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PMID:Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal. 838 59

This study examines the transcriptional regulation of the bovine CYP11A (P450scc) gene by activators of protein kinase A and protein kinase C in bovine ovarian luteal cells. Cells were transfected with reporter gene constructs containing deletion mutations of the 5'-flanking region of the bovine CYP11A gene linked to the minimal beta-globin gene. A construct containing -118/-101 base pairs of CYP11A sequence retains the same degree of stimulation by forskolin and inhibition by co-treatment with phorbol 12-myristate 13-acetate as larger constructs. This sequence contains two putative binding sites for nuclear proteins, an AP1-like sequence and an overlapping GA box element. Gel shift analysis using nuclear extracts of bovine ovarian luteal cells demonstrated that both the wild-type -118/-101-base pair sequence and a consensus GC box bound Sp1 or Sp1-like proteins. Mutation of the GA box element completely suppressed stimulation by forskolin. Absence of binding using the same mutated sequence correlated with the reporter gene transcription results. Mutation of the AP1-like site had little effect on forskolin induction of phorbol 12-myristate 13-acetate inhibition. These results indicate that both stimulation by forskolin and inhibition by phorbol esters are mediated by the same GA box element, which binds Sp1 or an Sp1-like protein.
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PMID:Regulation of expression of the CYP11A (P450scc) gene in bovine ovarian luteal cells by forskolin and phorbol esters. 839 39

The conversion of cholesterol to pregnenolone, the rate-limiting step in steroid hormone synthesis, occurs on mitochondrial cytochrome P450scc, which catalyzes this reaction by receiving electrons from NADPH via a flavoprotein [adrenodoxin reductase (AdRed)] and an iron sulfur protein [adrenodoxin (Adx)]. The behavior of the genes and mRNAs encoding these proteins has been studied in several systems, but little is known about the behavior of the human proteins. Using cloned cDNAs for human P450scc and AdRed, we constructed bacterial expression vectors to make milligram quantities of the corresponding proteins. These, plus purified human Adx similarly prepared by Dr. L. Vickery, were injected into rabbits to raise antiserum to each of the proteins. Each antiserum was highly specific and did not cross-react with other mitochondrial proteins detectable by Western blotting. Human JEG-3 choriocarcinoma cells and mouse Y-1 adrenocortical carcinoma cells were then incubated for 0-24 h with 1 mM 8-bromo-cAMP (8Br-cAMP) or 30 nM phorbol 12-myristate 13-acetate (PMA; phorbol ester) plus 1 microM A23187 (calcium ionophore) to activate the protein kinase-A and -C pathways, respectively. In JEG-3 cells, 8Br-cAMP increased and PMA/A23187 slightly decreased the abundance of P450scc and Adx, but neither treatment had a detectable effect on AdRed. The production of pregnenolone by these cells increased 3-fold in response to 8Br-cAMP and fell to one third in response to PMA/A23187. In Y-1 cells, 8Br-cAMP increased the abundance of all three proteins, while PMA/A23187 decreased the abundance of P450scc and Adx. The production of pregnenolone by these cells increased 9-fold in response to 8Br-cAMP and was unaffected by TPA/A23187. These studies show that the three proteins of the cholesterol side-chain cleavage system behave in response to 8Br-cAMP and PMA/A23187 as predicted from the study of their genes and mRNAs, indicating that the chronic regulation of steroidogenesis in these cell systems is regulated principally at the level of mRNA abundance.
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PMID:Regulation of proteins in the cholesterol side-chain cleavage system in JEG-3 and Y-1 cells. 842 75

We tested the hypothesis that low density lipoprotein (LDL) metabolism and cellular concentrations of gene transcripts of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc mRNA) are sites of significant protein kinase-C (PKC) action in the long term (48-h) inhibitory modulation of steroid hormone biosynthesis in ovarian granulosa cells. To this end, we used 12-O-tetradecanoylphorbol-13-acetate (TPA) as an activator of PKC and a monolayer culture system of immature swine granulosa cells responsive to insulin and lipoprotein under serum-free conditions. Insulin-regulated LDL metabolism was identified as a major site of TPA-mediated inhibition of steroidogenesis in granulosa cells. Treatment with TPA (30 ng/ml), but not inactive phorbol base, effectively decreased insulin-stimulated [125I]iodo-LDL binding by 75%, internalization by 90%, and degradation by 75%, as well as delivery and utilization of the [3H]cholesterol moiety of LDL in progesterone biosynthesis by intact granulosa cells. Cellular concentrations of P450scc mRNA, as measured by Northern blot hybridization with a 32P-labeled 1-kilobase porcine cDNA clone, were significantly increased by insulin. This insulin effect was virtually abolished by cotreatment with TPA (30 ng/ml). In contrast, accumulation of mRNA transcripts of a non-steroidogenic gene, 3-phosphoglyceraldehyde dehydrogenase, but not 18S ribosomal RNA, was enhanced by TPA. In summary, major inhibitory actions of PKC activation on granulosa cell steroidogenesis are expressed at specific loci of LDL metabolism, including LDL receptor number, internalization, and degradation, as well as the delivery and utilization of the [3H]cholesterol moiety of LDL to intact granulosa cells. Moreover, a PKC activator suppresses the intracellular accumulation of insulin-stimulated P450scc mRNA, but not that of phosphoglyceraldehyde dehydrogenase or 18S ribosomal RNA. The results obtained in this in vitro study suggest that the inhibition by TPA at these different sites along the steroidogenic pathway may be similar to that which occurs via hormones that work through the PKC system, such as prostaglandin F2 alpha.
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PMID:Sites of inhibition of steroidogenesis by activation of protein kinase-C in swine ovarian (granulosa) cells. 847 49

Lipoprotein lipase (LPL), an enzyme which hydrolyzes triglycerides and participates in the catabolism of remnant lipoproteins, plays a crucial role in energy and lipid metabolism. The goal of this study was to analyze the expression and regulation of the LPL gene in human adrenals. Reverse transcriptase-polymerase chain reaction amplification and sequence analysis demonstrated the presence of LPL mRNA in fetal and adult human adrenal cortex. Furthermore, the human adrenocortical carcinoma cell line, NCI-H295, expresses LPL mRNA and protein, which is localized to the outer cellular membrane as demonstrated by immunofluorescence confocal microscopy and can be released in the medium by heparin addition. To asses whether the LPL gene is regulated by agents regulating adrenal steroidogenesis, NCI-H295 cells were treated with activators of second messenger systems. Whereas the calcium-ionophore A23187 did not affect LPL gene expression, treatment with phorbol 12-myristate 13-acetate decreased LPL mRNA levels in a time- and dose-dependent manner. This decrease after phorbol 12-myristate 13-acetate was associated with diminished heparin-releasable LPL mass and activity in the culture medium. Addition of the cAMP analog 8-Br-cAMP to NCI-H295 cells resulted in a rapid, but transient dose-dependent induction of LPL mRNA. Treatment with the protein synthesis inhibitor cycloheximide gradually induced, whereas simultaneous addition of cAMP and cycloheximide superinduced LPL mRNA levels. Nuclear run-on analysis indicated that the effects of cAMP and cycloheximide occurred at the transcriptional and post-transcriptional level, respectively. Transient co-transfection assays demonstrated that the first 230 base pairs of the proximal LPL promoter contain a cAMP-responsive element activated by protein kinase A and transcription factors belonging to the CREB/CREM family. These data indicate that LPL is expressed in human adrenal cortex and regulated in NCI-H295 adrenocortical carcinoma cells by activators of the protein kinase A and protein kinase C second messenger pathways in a manner comparable to P450scc, which catalyzes the first step in adrenal steroidogenesis. These observations suggest a role for LPL in adrenal energy and/or lipid metabolism and possibly in steroidogenesis.
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PMID:Expression and regulation of the lipoprotein lipase gene in human adrenal cortex. 866 37

Previous studies have shown that inhibitors of protein tyrosine kinases, tyrphostins, can markedly attenuate the steady-state levels of mRNAs of hormone-induced genes expressed in ovarian cells. To further elucidate the mechanism of tyrphostin action, rat granulosa cells were electroporated with chimeric expression vectors containing the promoters of two key steroidogenic genes, cholesterol side chain cleavage cytochrome P450 (CYP11A; P450scc) and aromatase cytochrome P450 (CYP19; P450arom), ligated to the CAT reporter gene. The electroporation method of transfection documents that the respective promoter-reporter constructs, -379sccCAT and -534aromCAT, can confer greater than 10-fold FSH/cAMP responsiveness to the reporter genes expressed in naive granulosa cells. Furthermore, the electroporation approach allows transfection of DNA into small numbers of cells and facilitates the assay of expression in cells isolated from follicles at advanced stages of differentiation. In naive granulosa cells, the functional activities of -379sccCAT, -534aromCAT, and -169 alpha CGCAT were abolished by the A-kinase specific inhibitor, H89, supporting the notion that activation of protein kinase A is obligatory for transcriptional activation of the promoter regions within these genes. Similar inhibitory effects were also observed for tyrphostin AG18, thus implicating a tyrosine kinase in the regulation of the steroidogenic genes. As a result of eCG/hCG treatments, a gradual loss of transfection efficiency accompanied by decreasing forskolin induction of CAT expression was observed in the differentiating granulosa-lutein cells. Although the reason(s) for the apparent loss in the ability of hormones to regulate chimeric gene expression remains to be determined, cell and promoter refractoriness to hormone treatment appears to reflect a fundamental change in the mechanism of promoter activation in the differentiated cells compared to the naive granulosa cells.
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PMID:Effects of hormones and protein kinase inhibitors on expression of steroidogenic enzyme promoters in electroporated primary rat granulosa cells. 883 18


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