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)

In order to study the effect of phosphorylation on the function of the steroidogenic acute regulatory protein (StAR), 10 putative phosphorylation sites were mutated in the hamster StAR. In pcDNA3.1-StAR transfected COS-1 cells, decreases in basal activity were found for the mutants S55A, S185A and S194A. Substitution of S185 by D or E to mimic phosphorylation resulted in decreased activity for all mutants; we concluded that S185 was not a phosphorylation site and we hypothesized that mutations on S185 created StAR conformational changes resulting in a decrease in its binding affinity for cholesterol. In contrast, the mutation S194D resulted in an increase in StAR activity. We have calculated the relative rate of pregnenolone formation (App. V(max)) in transfected COS-1 cells with wild type (WT) and mutant StAR-pcDNA3.1 under control and (Bu)(2)-cAMP stimulation. The App. V(max) values refer to the rate of cholesterol transported and metabolized by the cytochrome P450scc enzyme present in the inner mitochondrial membrane. The App. V(max) was 1.61 +/- 0.28 for control (Ctr) WT StAR and this value was significantly increased to 4.72 +/- 0.09 for (Bu)(2)-cAMP stimulated preparations. App. V(max) of 5.53 (Ctr) and 4.82 ((Bu)(2)-cAMP) found for S194D StAR preparations were similar to that of the WT StAR stimulated preparations. At equal StAR quantity, an anti-phospho-(S/T) PKA substrate antibody revealed four times more phospho-(S/T) in (Bu)(2)-cAMP than in control preparations. The intensity of phosphorylated bands was decreased for the S55A, S56A and S194A mutants and it was completely abolished for the S55A/S56A/S194A mutant. StAR activity of control and stimulated preparations were diminished by 73 and 72% for the mutant S194A compared to 77 and 83% for the mutant S55A/S56A/S194A. The remaining activity appears to be independent of phosphorylation at PKA sites and could be due to the intrinsic activity of non-phosphorylated StAR or to an artefact due to the pharmacological quantity of StAR expressed in COS-1. In conclusion we have shown that (Bu)(2)-cAMP provokes an augmentation of both the quantity and activity of StAR, and that an enhancement in StAR phosphorylation increases its activity. The increased quantity of StAR upon (Bu)(2)-cAMP stimulation could be due to an augmentation of its mRNA or protein synthesis stability, or both; this is yet to be determined.
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PMID:Phosphorylation and function of the hamster adrenal steroidogenic acute regulatory protein (StAR). 1533 3

We have determined the effects of LH on the expression of transforming growth factor-alpha (TGFalpha) and epidermal growth factor receptor (EGFR) system in rat Leydig cells and investigated its role in steroidogenesis. LH and TGFalpha/epidermal growth factor (EGF) significantly increased the levels of TGFalpha mRNA and protein, and the levels of EGFR protein in immature rat Leydig cells (ILC). Treatment with TGFalpha or EGF for 24h resulted in significant increase in androgen production in ILC. The increase in androgen production in response to TGFalpha was associated with increased mRNA levels of SR-BI, steroidogenic acute regulatory (StAR) and P450scc but not of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and P450c17. TGFalpha also caused a marked increase in the levels StAR protein in ILC. EGFR inhibitor (AG1478) blocked the effects of TGFalpha while MEK-inhibitor (PD98059) potentiated TGFalpha or LH effects on steroidogenesis. A PKA inhibitor (H89) blocked both TGFalpha and LH effects on steroidogenesis. We conclude that TGFalpha plays an autocrine role in LH dependent development and function of Leydig cells.
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PMID:Autocrine regulation of steroidogenic function of Leydig cells by transforming growth factor-alpha. 1535 78

The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.
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PMID:Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro. 1712 51

Transforming growth factor (TGF) beta1 facilitates FSH-induced differentiation of rat ovarian granulosa cells. The signaling crosstalk between follicle stimulating hormone (FSH) and TGFbeta receptors remains unclear. This study was to investigate the interplay of cAMP/protein kinase A (PKA) and phosphatidylinositol-3-kinase (PI3K) signaling including mammalian target of rapamycin (mTOR)C1 dependence in FSH- and TGFbeta1-stimulated steroidogenesis in rat granulosa cells. To achieve this aim, inhibitors of PKA (PKAI), PI3K (wortmannin), and mTORC1 (rapamycin) were employed. PKAI and wortmannin suppressions of the FSH-increased progesterone production were partly attributed to decreased level of 3beta-HSD, and their suppression of the FSH plus TGFbeta1 effect was attributed to the reduction of all the three key players, steroidogenic acute regulatory (StAR) protein, P450scc, and 3beta-HSD. Further, FSH activated the PI3K pathway including increased integrin-linked kinase (ILK) activity and phosphorylation of Akt(S473), mTOR(S2481), S6K(T389), and transcription factors particularly FoxO1(S256) and FoxO3a(S253), which were reduced by wortmannin treatment but not by PKAI. Interestingly, PKAI suppression of FSH-induced phosphorylation of cAMP regulatory element-binding protein (CREB(S133)) disappeared in the presence of wortmannin, suggesting that wortmannin may affect intracellular compartmentalization of signaling molecule(s). In addition, TGFbeta1 had no effect on FSH-activated CREB and PI3K signaling mediators. We further found that rapamycin reduced the TGFbeta1-enhancing effect of FSH-stimulated steroidogenesis, yet it exhibited no effect on FSH action. Surprisingly, rapamycin displayed a suppressive effect at concentrations that had no effect on mTORC1 activity. Together, this study demonstrates a delicate interplay between cAMP/PKA and PI3K signaling in FSH and TGFbeta1 regulation of steroidogenesis in rat granulosa cells. Furthermore, we demonstrate for the first time that TGFbeta1 acts in a rapamycin-hypersensitive and mTORC1-independent manner in augmenting FSH-stimulated steroidogenesis in rat granulosa cells.
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PMID:Interplay of PI3K and cAMP/PKA signaling, and rapamycin-hypersensitivity in TGFbeta1 enhancement of FSH-stimulated steroidogenesis in rat ovarian granulosa cells. 1728 41

Trichlorfon, a widely used organophosphorus pesticide, has been reported to disrupt reproductive function in humans. However, the mechanisms have not been clearly elucidated. In the present study, mouse Leydig tumor cells (MLTC-1) was used to evaluate the effect of trichlorfon on progesterone synthesis. After the various concentrations of trichlorfon treatment (0, 0.04, 0.2, 1, 5 or 25 micromol/l) for 24h, the progesterone production presented a dose-dependent decrease in the presence of some stimulators such as human chorionic gonadotrophin (hCG), cholera toxin (CT) or forskolin. However, the levels of intracellular cAMP remained unaltered, which suggested that trichlorfon suppressed steroidogenesis occurred after PKA activation along cAMP/PKA pathway. Moreover, trichlorfon suppressed the expression of the steroidogenic acute regulatory protein (StAR) mRNA and protein, and also profoundly inhibited the activity of P450 side chain cleavage enzyme (P450scc), rather than 3beta-hydorxysteroid dehydrogenase (3beta-HSD). The suppressive expression of P450scc mRNA and protein further accounted for the inhibitory action of trichlorfon on steroidogenesis. These results indicated that trichlorfon suppressed progesterone synthesis in MLTC-1 cells, at least in part, via inhibiting StAR expression and P450scc activity.
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PMID:Study on the mechanism of trichlorfon-induced inhibition of progesterone synthesis in mouse leydig tumor cells (MLTC-1). 1735 Jul 42

We have demonstrated that dehydroepiandrosterone (DHEA) acts directly on rat zona fasciculata-reticularis (ZFR) cells to diminish corticosterone secretion by an inhibition of post-cAMP pathway, and decreases functions of steroidogenic enzymes after P450(scc) as well as steroidogenic acute regulatory (StAR) protein expression. However, the mechanisms by which DHEA engages with environmental messenger signals which translate into interfering StAR protein expression are still unclear. This study explored the effects of DHEA on the phosphorylation/activation of extracellular signal-regulated kinases (ERKs). ERK activation resulted in enhancing phosphorylation of steroidogenic factor-1 (SF-1) and increased StAR protein expression. ZFR cells were incubated in the presence or absence of adrenocorticotropin (ACTH), forskolin (FSK), 25-OH-cholesterol, U0126, and H89 at 37 degrees C. The concentration of corticosterone released into the media was measured by radioimmunoassay (RIA). The cells were used to extract protein for Western blot analysis of ERKs or StAR protein expression or immunoprecipitation of SF-1 analysis. The results suggested that (1) ERK pathway of rat ZFR cells might be PKA dependent, (2) ERK activity was required for SF-1 phosphorylation to upregulate steroidogenesis in rat ZFR cells, and (3) DHEA did not affect ERK phosphorylation, however, it attenuated forskolin-stimulated SF-1 phosphorylation to affect StAR protein expression.
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PMID:Mechanisms of inhibition of dehydroepiandrosterone upon corticosterone release from rat zona fasciculata-reticularis cells. 1800 94

Previous studies demonstrated significantly lower plasma cortisol level in homozygous halothane-positive (Hal nn) pigs, as compared with homozygous halothane-negative (Hal NN) pigs. To determine whether such difference is attributed to the fundamental alterations in adrenocortical function, F1 offsprings from Pietrain (Hal nn)xErhualian (Hal NN) were intercrossed to produce F2 sibling pigs with segregated genotypes. Adrenocortical cells were isolated from the Hal nn and Hal NN F2 pigs, respectively, and cultured with or without ACTH challenge. Cortisol levels in culture medium, as well as the content of MC2R, cAMP, CREB, phosphorylated CREB (pCREB), StAR and P450scc in adrenocortical cell lysates, were determined. Cortisol, cAMP, StAR and P450scc levels were significantly lower in Hal nn adrenocortical cells under basal condition without ACTH challenge. ACTH significantly increased cortisol level in the medium and the protein content of MC2R, StAR, P450scc in adrenocortical cell lysates, regardless of genotypes. Total CREB protein content was not different between genotypes and treatments, whereas pCREB content exhibited significant effects of genotype and treatment, being higher in Hal NN than in Hal nn under basal condition and in response to ACTH challenge. These results indicate that the compromised cAMP/PKA/pCREB signaling pathway of ACTH and diminished expression of limiting factors in adrenocortical steroidogenesis (StAR and P450scc) may contribute to the significantly lower plasma cortisol levels in Hal nn pigs.
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PMID:Diminished expression of ACTH signaling proteins and steroidogenic limiting factors in adrenocortical cells isolated from halothane nn pigs. 1830 76

Our previous study demonstrated significant difference in the basal plasma cortisol levels between Erhualian (EHL) and Pietrain (PIE) pigs, implicating fundamental breed difference in adrenocortical function. The objectives of the present study were therefore to characterize the expression pattern of proteins involved in adrenal ACTH signaling and, including melanocortin type 2 receptor (MC2R), cAMP response element binding protein (CREB) and phosphorylated CREB (pCREB), steroidogenic acute regulatory protein (StAR), as well as that of the key enzymes involved in steroidogenesis in EHL and PIE pigs, in association with the plasma corticotrophin (ACTH) and cortisol levels. The plasma concentrations of the substrates for adrenal steroidogenesis, cholesterol and low-density lipoprotein (LDL) cholesterol, did not differ between breeds. Plasma concentration of ACTH and the adrenal contents of MC2R mRNA and protein were similar in two breeds of pigs, whereas the basal plasma concentrations of cortisol in EHL pigs were 1.5 folds higher than that in PIE pigs. The higher basal plasma cortisol levels in EHL pigs were found to be accompanied with the higher expression of ACTH post-receptor signaling components, cAMP, pCREB and StAR, as well as the higher expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17alpha-hydroxylase cytochrome P450 (P450(17alpha)), 21-hydroxylase cytochrome P450 (P450c21) and 11beta-hydroxylase cytochrome P450 (P450(11beta)). These results indicated that the enhanced cAMP/PKA/pCREB-signaling system and augmented expression of StAR and steroidogenic enzymes are major attributes to the higher basal plasma cortisol concentrations in pigs.
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PMID:Characterization of adrenal ACTH signaling pathway and steroidogenic enzymes in Erhualian and Pietrain pigs with different plasma cortisol levels. 1843 13

Activation of the protein kinase A (PKA) signaling system is necessary for FSH-induced granulosa cell differentiation, but it is not known whether activation of PKA is sufficient to account for the complex pattern of gene expression that occurs during this process. We addressed this question by infecting granulosa cells with a lentiviral vector that directs the expression of a constitutively active mutant of PKA (PKA-CQR) and compared the cellular responses to PKA-CQR with cells stimulated by FSH. Expression of PKA-CQR in undifferentiated granulosa cells resulted in the induction of both estrogen and progesterone production in the absence of cAMP. The stimulatory effects of both PKA-CQR and FSH on estrogen and progesterone production were suppressed by the PKA inhibitor H-89 and were mimicked by PKA-selective cAMP agonists. mRNA levels for P450scc and 3beta-HSD were induced to a similar extent by FSH and PKA-CQR, whereas mRNA levels for P450arom and the LHr were induced to a greater extent by FSH. Microarray analysis of gene expression profiles revealed that the majority of genes appeared to be comparably regulated by FSH and PKA-CQR but that some genes appear to be induced to a greater extent by FSH than by PKA-CQR. These results indicate that the PKA signaling pathway is sufficient to account for the induction of most genes (as identified by microarray analysis), including those of the progesterone biosynthetic pathway during granulosa cell differentiation. However, optimal induction of aromatase, the LHr, and other genes by FSH appears to require activation of additional signaling pathways.
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PMID:Constitutively active protein kinase A qualitatively mimics the effects of follicle-stimulating hormone on granulosa cell differentiation. 1853 49

The peripheral-type benzodiazepine receptor (PBR), a putative receptor in Leydig cells, modulates steroidogenesis. Since benzodiazepines are commonly used in regional anesthesia, their peripheral effects need to be defined. Therefore, this study set out to investigate in vitro effects of the benzodiazepine midazolam (MDZ) on Leydig cell steroidogenesis, and the possible underlying mechanisms. The effects of MDZ on steroidogenesis in primary mouse Leydig cells and MA-10 Leydig tumor cells were determined by radioimmunoassay. PBR, P450scc, 3beta-HSD and StAR protein expression induced by MDZ was determined by Western blotting. Inhibitors of the signal transduction pathway and a MDZ antagonist were used to investigate the intracellular cascades activated by MDZ. In both cell types, MDZ-stimulated steroidogenesis in dose- and time-dependent manners, and induced the expression of PBR and StAR proteins, but had no effect on P450scc and 3beta-HSD expressions. Moreover, H89 (PKA inhibitor) and GF109203X (PKC inhibitor) attenuated MDZ-stimulated steroid production. Interestingly, the MDZ antagonist (flumazenil) did not decrease MDZ-induced steroid production in both cell types. These results highly indicated that MDZ-induced steroidogenesis in mouse Leydig cells via PKA and PKC pathways, along with the expression of PBR and StAR proteins. In addition, MDZ at high dosages induced rounding-up, membrane blebbing, and then death in MA-10 cells. In conclusion, midazolam could induce Leydig tumor cell steroidogenesis, and high dose of midazolam could induce apoptosis in Leydig tumor cells.
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PMID:The effect of midazolam on mouse Leydig cell steroidogenesis and apoptosis. 1985 60


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