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)

Production and secretion of steroid hormones throughout the ovarian cycle occurs in a highly episodic and coordinated fashion that requires precise and finely tuned regulatory mechanisms. The regulation of ovarian steroidogenesis by the gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as by other factors occurs, at least in part, at the level of expression of the genes encoding steroidogenic enzymes. The present study is aimed at the elucidation of regulatory mechanisms by which cyclic adenosine monophosphate (cAMP) and protein kinase C regulate cytochrome P450scc (CYP11A) gene expression in bovine granulosa cells in primary culture. As a first step we characterized the bovine granulosa cell cultures with regard to regulation of P450scc activity and mRNA levels upon treatment with forskolin and/or the phorbol ester TPA. Forskolin, a potent stimulator of cAMP generation, increased both progesterone secretion and P450scc mRNA levels. In contrast, treatment with TPA alone decreased both basal progesterone production and P450scc mRNA accumulation. Co-treatment with forskolin and TPA decreased progesterone and P450scc mRNA levels as compared to forskolin treatment alone. The possibility that TPA interfered with the forskolin-stimulated cAMP production could be excluded because simultaneous treatment of granulosa cells with TPA and forskolin potentiated the formation of cAMP. In order to identify regulatory sequences within the 5' flanking region of the bovine CYP11A gene, chimeric DNA constructs comprizing regions of the CYP11A gene fused to a beta-globin-derived reporter gene were transfected into granulosa cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of CYP11A gene expression in bovine ovarian granulosa cells in primary culture by cAMP and phorbol esters is conferred by a common cis-acting element. 822 26

In rat thecal-interstitial cells (TIC), treatment with the synthetic androgen mibolerone has led to the documentation of an autoregulatory process for androgen production. In the present study, accumulated evidence has provided insight into the mechanisms of mibolerone action that control this process. Investigations using the nonsteroidal antiandrogen hydroxyflutamide were conducted to characterize mibolerone's mode of action. Hydroxyflutamide had differential effects on hCG action, the 1-microM dose stimulating hCG-induced androsterone synthesis by 27% and the 10-microM concentration decreasing the androgen levels by 84%. In addition, treatment with 1 microM hydroxyflutamide was effective in partially reversing the inhibitory action of mibolerone on hCG-stimulated androsterone production. Thus, the data indicated that mibolerone's mode of action may be mediated, at least in part, via the androgen receptor. The possibility that mibolerone had multiple sites of action prompted studies on the effectiveness of this androgen to alter various signaling pathways. Treatment with increasing concentrations (0.01-100 nM) of the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C, resulted in a 75% decrease in hCG-stimulated androgen production at a dose of 100 nM TPA. Treatment with mibolerone (100 nM) was unable to alter the action of TPA on androgen synthesis when doses of 1 and 10 nM TPA were employed. It was also found that Ca2+ can serve as a mediator of mibolerone action. Treatment with a 0.01-microM dose of A23187, a Ca2+ ionophore known to increase intracellular Ca2+, was ineffective in altering hCG-stimulated androsterone synthesis. The concurrent treatment of mibolerone (100 nM) and A23187 (0.01 microM) resulted in the potentiation of mibolerone's inhibitory effects on hCG-stimulated androgen production, thereby suggesting that mibolerone can stimulate Ca2+ influx. Additional studies revealed that the administration of a 1-microM dose of the L-type Ca2+ channel blocker verapamil to TIC cultures was able to partially block the inhibitory effect of mibolerone on androgen synthesis. Evidence for an additional site of mibolerone action was revealed through an analysis of the mRNA levels of P450scc and P450(17) alpha enzymes. Although hCG and insulin-like growth factor I treatment resulted in 20- and 32-fold increases in the amount of P450scc and P450(17) alpha mRNA, respectively, the addition of mibolerone (100 nM) reduced only P450(17) alpha mRNA levels by 91%. Overall, the evidence indicates that mibolerone has multiple sites of action in exerting its regulatory effect on androgen synthesis.
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PMID:Mechanisms of action for an androgen-mediated autoregulatory process in rat thecal-interstitial cells. 828 1

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

Steroid hormones are synthesized from cholesterol in the adrenals, gonads, and placenta by a complex series of reactions. The human genes encoding each of these biosynthetic enzymes have been cloned, permitting study of their regulation. Tropic hormones, such as corticotropin and the gonadotropins, exert their chronic effects on steroidogenesis by increasing the amounts of steroidogenic enzymes; this in turn occurs primarily through increased gene transcription. Our studies have emphasized the cholesterol side-chain cleavage enzyme P450scc, which catalyzes the first and rate-limiting step in steroidogenesis, and P450c17, which determines what class of steroids is synthesized. By fusing the promoters of the genes for these enzymes to readily assayed reporter genes and transiently transfecting cultured cells with these constructions, we have identified the regions of each promoter that confer basal expression, induction by cAMP, and repression by activators of protein kinase C. Different segments of the P450scc promoter are used for each of these purposes in different cell types, indicating that the regulation of this gene is very complex. Transcription is not the only level at which steroidogenesis is regulated. The abundance of mRNA for adrenodoxin reductase, a flavoprotein needed for P450scc activity, is post-transcriptionally regulated by cAMP.
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PMID:Transcriptional regulation of human genes for steroidogenic enzymes. 843 24

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

Earlier studies in immature porcine granulosa cells cultured in serum-free medium showed dual actions of the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In cells incubated for 24 h, TPA inhibited follicle-stimulating hormone (FSH)-stimulated cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA accumulation. In contrast, at 4 h, TPA increased P450scc mRNA concentration in the absence and presence of FSH or 8-bromo-cAMP; in addition, TPA augmented FSH-stimulated cAMP accumulation. The actions of TPA were then examined in the presence of the phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). With IBMX present, TPA caused a smaller relative augmentation of cAMP accumulation during a 4-h incubation period, suggesting that TPA may both increase cAMP synthesis and inhibit its degradation. The stimulatory effect of FSH or 8-bromo-cAMP on P450scc mRNA concentration was not modified by IBMX. However, TPA no longer augmented the FSH- or 8-bromo-cAMP-stimulated P450scc mRNA accumulation when IBMX was present. In cells treated with FSH for 24 h, IBMX augmented progesterone production, but paradoxically accentuated the inhibitory effect of TPA on steroidogenesis. These results indicate that IBMX converts TPA from a stimulatory into an inhibitory agent by an action unrelated to cAMP, and points to the need for caution in interpreting experiments with this drug.
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PMID:Paradoxical effect of 3-isobutyl-1-methylxanthine on cytochrome P450 cholesterol side-chain cleavage mRNA accumulation in porcine granulosa cells. 873 81

Previous studies of human adrenocortical cells have given inconsistent findings concerning the effects of angiotensin II (AII) alone or in combination with activators of the protein kinase A-signaling pathway on expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450c17), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), as well as the corresponding effects on adrenocortical cell steroid secretory products. We have used the human adrenocortical carcinoma H295R cell to evaluate further this question and determine the role of protein kinase C in each of these responses to AII. Treatment with AII alone (10 nmol/L, 48 h) resulted in a significant increase in cortisol production (1.8-fold), as well as a much greater effect on aldosterone production. This increased formation of 17 alpha-hydroxysteroids was accompanied by increased expression of P450c17 as determined at the level of messenger RNA (mRNA) and enzyme activity. Similar increases in expression of P450scc were observed at the level of mRNA. Increases in 3 beta-HSD expression were also seen at the level of mRNA and, to a lesser extent, at the level of enzyme activity. Because of the comparatively low basal 17 alpha-hydroxylase and high basal 3 beta-HSD activity of H295R cells, however, the overall effect of AII treatment was actually a rise in the 17 alpha-hydroxylase/3 beta-HSD activity ratio, resulting in increased formation of 17 alpha-hydroxysteroids such as cortisol. Whereas treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) reproduced the effect of AII on 3 beta-HSD expression, TPA failed to reproduce the effects of AII on P450c17 and P450scc and even resulted in a marked decrease in expression of P450c17. Thus, the stimulatory effect of AII alone on P450c17 expression was not mediated via protein kinase C but, like the action of K+, was probably mediated via the Ca(2+)-signaling pathway. Treatment with forskolin (10 mumol/L, 48 h) resulted in a dramatic increase in both cortisol and dehydroepiandrosterone production together with increases in expression of P450c17, P450scc, and 3 beta-HSD as measured at the level of mRNA and activity. Consistent with the increase in 17 alpha-hydroxysteroid formation, the effect on 17 alpha-hydroxylase expression was greater than that on 3 beta-HSD at the level of enzyme activity, so a larger 17 alpha-hydroxylase/3 beta-HSD activity ratio was achieved. Cotreatment with forskolin and AII, however, resulted in a dose-dependent reduction in cortisol and DHEA production concomitant with a marked attenuation of P450scc and P450c17 expression. Although forskolin-induced expression of 3 beta-HSD was not further increased at the level of mRNA by cotreatment with AII, additivity was observed at the level of changes in enzyme activity. Thus, AII cotreatment resulted in a marked reduction of the forskolin-induced increase in 17 alpha-hydroxylase/3 beta-HSD activity ratio, and so, 17 alpha-hydroxysteroid synthesis was attenuated. These effects of AII cotreatment on expression of P450c17 and P450scc were reproduced by cotreatment with TPA (10 nmol/L), suggesting the involvement of protein kinase C in these attenuative responses. Furthermore, the effect of AII cotreatment on changes in forskolin-induced 17 alpha-hydroxylase and 3 beta-HSD activities were blocked by the AII Type 1 (AT1) receptor antagonist DuP753 (Losartan), confirming the involvement of an AT1 receptor-linked phospholipase C in activating protein kinase C.
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PMID:Differential control of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase expression in human adrenocortical H295R cells. 896 47

Ligand- and second messenger-regulated expression of the gene for steroidogenic acute regulatory protein (StAR) was evaluated in luteinized porcine granulosa cells. For comparison, cytochrome P450 side-chain cleavage (P450scc) was examined. Northern hybridization with homologous cDNA probes demonstrated three StAR mRNA species, of 2.7, 1.6, and 0.8 kilobases (kb), with the smallest variably present, and a single P450scc band at 1.9 kb. FSH elevated both STAR and P450scc messages in a dose-dependent manner over 6 h and continually stimulated both over 24 h (p < 0.001). STAR message induction depended on transcription, as did that of P450scc. Over 6 h, actinomycin D eliminated constitutive StAR message and reduced that of P450scc by two thirds, indicating briefer persistence of StAR. Pretreatment with cycloheximide prevented FSH induction of StAR and P450scc mRNA, implicating intermediate protein synthesis in expression of both genes. Dibutyryl cAMP caused time-dependent increases in StAR and P450 mRNAs over 24 h (p < 0.001), indicating the importance of the protein kinase A (PKA) pathway in their gene expression. Activation of the protein kinase C (PKC) pathway by a phorbol ester eliminated FSH induction of STAR mRNA increases (p < 0.01) while only reducing P450scc induction (p < 0.05). Thus, StAR gene expression, as reflected in mRNA abundance, is regulated by FSH via the PKA pathway and is dependent on transcription and translation. Conversely, the PKC pathway inhibits induction of these important steroid synthetic genes in luteinized granulosa cells.
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PMID:Follicle-stimulating hormone and intracellular second messengers regulate steroidogenic acute regulatory protein messenger ribonucleic acid in luteinized porcine granulosa cells. 928 5

Trophoblast giant cells are the steroidogenic cells of the rat placenta. In this study, the role of protein kinase C signalling pathways in the control of DNA synthesis and differentiation-dependent progesterone biosynthesis by trophoblast cells were investigated. Rcho-1 trophoblast cells, derived from a rat choriocarcinoma, can be experimentally manipulated to proliferate or differentiate and provide a useful model for studying trophoblast giant cell endocrine differentiation. The role of protein kinase C signal transduction was examined through the treatment of Rcho-1 trophoblast cells with isoquinolinesulfonamide derivatives (H7, a protein kinase C inhibitor; HA1004, a control compound), chelytherine (a protein kinase C inhibitor), and phorbol esters (protein kinase C activators). Treatment with H7 significantly attenuated DNA synthesis in proliferating and differentiating trophoblast cells and accelerated the acquisition of progesterone biosynthetic capabilities by trophoblast cells. Treatment with HA1004, the related but functionally distinct isoquinolone, did not significantly affect trophoblast DNA synthesis or proliferation and only weakly increased progesterone accumulation. Chelytherine significantly inhibited trophoblast cell proliferation but failed to influence trophoblast progesterone production significantly. The phorbol ester, 12-O-tetradecanoylphorbol acetate, did not significantly influence progesterone accumulation. H7 did not significantly influence the concentration of either P450scc or the mRNA encoding it in Rcho-1 trophoblast cells, or the transcriptional activity of the P450scc gene. The results indicate that signalling pathways sensitive to protein kinase C are involved in the control of trophoblast cell proliferation. Differentiation-dependent production of progesterone is sensitive to H7 but appears to be independent of protein kinase C and occurs at a stage other than P450scc expression.
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PMID:Protein kinase C dependent and independent mechanisms controlling rat trophoblast cell DNA synthesis and differentiation. 937 Sep 62

Steroidogenic acute regulatory (StAR) protein plays a crucial role in the regulation of cholesterol transport from the outer mitochondrial membrane to the inner membrane, where P450scc participates in a rate-limiting step of adrenal steroidogenesis. We have already reported that both of cAMP- and protein kinase C-dependent processes may play a crucial role in the regulation of expression of StAR protein when bovine fasciculata cells are stimulated with ACTH. In the present study, ACTH increased cytosolic calcium movement and activated expression of StAR protein, resulting in enhancing cortisol production by bovine adrenal fasciculata cells. The role of the calcium/calmodulin-dependent protein kinase process in the regulation of expression of the StAR protein by ACTH was studied. The activating effects of ACTH on the StAR protein and cortisol production were inhibited by pretreatment with KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II. These findings suggest that ACTH can enhance expression of the StAR protein as well as cortisol synthesis in bovine adrenal fasciculata cells, in part via a calcium/calmodulin-dependent protein kinase process.
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PMID:Possible involvement of calcium/calmodulin-dependent protein kinase in ACTH-induced expression of the steroidogenic acute regulatory (StAR) protein in bovine adrenal fasciculata cells. 962 8


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