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

---

Query: EC:2.7.11.1 (protein kinase)
81,284 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.
...
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

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.
...
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

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.
...
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

The role of protein kinase-C-dependent mechanisms in steroidogenic enzyme gene expression was studied in primary cultures of human fetal and adult adrenals. Cells were first cultured for 7-10 days and then stimulated with ACTH or 12-O-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase-C activator, for 1-2 days. Cytoplasmic RNA was extracted and analyzed by Northern and dot blotting with 32P-labeled cDNA probes for P450scc (cholesterol side-chain cleavage enzyme/20,22-desmolase), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase); for P450c11 (11 beta-hydroxylase/18-hydroxylase/18-methyl oxidase), a 30-mer oligonucleotide was used as a probe. ACTH (200 ng/ml) increased the accumulation of all of the studied steroidogenic enzyme mRNAs in both fetal and adult cultures by several-fold. TPA inhibited this accumulation in a dose-dependent manner (0.01-100 ng/ml), whereas the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate was without effect. On the other hand, in the absence of ACTH, TPA slightly increased all steroidogenic P450 mRNAs in adult cultures. In fetal cultures TPA slightly increased P450scc, P450c11, and P450c21 mRNA levels, whereas it decreased P450c17 mRNA. (Bu)2cAMP and cholera toxin increased steroidogenic enzyme mRNAs such as ACTH. TPA down-regulated (Bu)2cAMP- and cholera toxin-induced P450mRNAs in the same way as ACTH-induced mRNAs. The secretion of ACTH-stimulated cortisol, dehydroepiandrosterone sulfate, and aldosterone was decreased by TPA in both fetal and adult cultures. The basal steroid production was slightly increased by TPA in both culture types. The changes in steroid production correlated well with the alterations in the steroidogenic enzyme gene expression. Our results show that the inhibitory effect of TPA on ACTH-stimulated adrenal steroidogenesis is mediated at the mRNA level of steroidogenic enzymes. Thus, it seems likely that both protein kinase-C- and cAMP-dependent mechanisms are involved in the long term maintenance of steroidogenic enzymes and hormone production in adrenocortical cells.
...
PMID:Interaction of phorbol ester and adrenocorticotropin in the regulation of steroidogenic P450 genes in human fetal and adult adrenal cell cultures. 184 59

FSH is the primary hormonal inducer of ovarian follicle maturation and a critically important regulator of steroidogenesis in granulosa cells. We examined possible molecular mechanisms subserving FSH action by assessing concentrations of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA in porcine granulosa cells maintained in serum-free culture. Cellular concentrations of specific P450scc mRNA were measured by Northern blot hybridization using a 32P-labeled 1-kilobase porcine cDNA clone. Specificity was tested by estimating the granulosa cell mRNA content of the constitutively expressed enzyme, glyceraldehyde-3-phosphate dehydrogenase. Steroidogenesis was evaluated by measuring concomitant progesterone accumulation in the culture medium. Treatment with ovine FSH (100 ng/ml) increased P450scc mRNA concentrations in a time-dependent fashion, with significant effects on both P450scc mRNA concentrations and progesterone accumulation by 4 h and a maximal increase (8- to 10-fold) at 48 h. FSH dose-response studies at 48 h revealed a significant stimulatory effect of 30 ng/ml FSH on P450scc mRNA accumulation and progesterone production, with a maximal effect at 100 ng/ml FSH. To examine the role of cAMP in mediating granulosa cell P450scc mRNA accumulation, granulosa cells were treated with forskolin, cholera toxin, 8-bromo-cAMP, 8-bromo-cGMP, 5'AMP, or cAMP analogs that differentially stimulate the two isoenzymes of protein kinase-A. Increased specific P450scc mRNA accumulation and progesterone production occurred in response to each agent except 5'AMP and 8-bromo-cGMP. No effects of these agents were observed on glyceraldehyde-3-phosphate dehydrogenase mRNA. To assess possible feedback effects of steroid or sterol on FSH-stimulated P450scc mRNA concentrations, granulosa cells were treated with aminoglutethimide to block or with low density lipoprotein to stimulate steroid production. Inhibition of sterol utilization by the cholesterol side-chain cleavage enzyme had no effect on basal or FSH-stimulated concentrations of P450scc mRNA, but markedly suppressed progesterone production. Low density lipoprotein, which increases intracellular sterol, also did not alter basal or FSH-stimulated P450scc mRNA accumulation, suggesting that neither the utilization nor the availability of sterol regulates specific P450scc mRNA levels. Estradiol alone did not increase P450scc mRNA accumulation, but did augment progesterone production. Treatment of granulosa cells with estradiol and FSH produced a synergistic increase in progesterone concentrations, but did not affect FSH-stimulated P450scc mRNA accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Follicle-stimulating hormone increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells. 184 8

The following studies were conducted with the goal of understanding some of the molecular mechanisms by which cAMP alters granulosa cell function. In this regard, we characterized the expression of messages for the cholesterol side-chain cleavage cytochrome P450 (P450scc) enzyme and a type II cAMP-dependent protein kinase subunit (RII beta) in granulosa cells isolated from small antral follicles and cultured in 1% fetal bovine serum-containing medium. Forskolin (FSK) stimulated cAMP production followed by accumulation of RII beta mRNAs, induction of P450scc mRNA, and, finally, progesterone biosynthesis. The regulation of each mRNA displayed a different sensitivity to actinomycin-D treatment. To determine if the modulation of RII beta or the induction of P450scc could be mediated by the enhancer activity of a cAMP-responsive DNA element (CRE), plasmid DNA containing the CRE and TATA box of the human glycoprotein alpha-subunit (alpha G) gene fused to the chloramphenicol acetyl transferase (CAT) gene was introduced into cultured granulosa or interstitial cells, and the ability of FSK to stimulate the expression of the CAT enzyme was measured. When granulosa cells were isolated from preantral/small antral follicles and maintained for at least 5 days in culture, 5 or 10 microM FSK reversibly stimulated expression of the CAT enzyme within 18 h posttransfection. Under similar conditions, interstitial ovarian cells (prepared from the residual ovarian tissue remaining after granulosa cell isolation) were unable to express the template unless the cells were pretreated for 24 h with FSK before transfection. Thereafter, CAT gene expression by interstitial cells was maintained in a FSK-insensitive manner. To determine if cAMP-dependent transcription of the reporter gene required the same sequences that had been characterized for placenta-derived cells, a truncated plasmid lacking the CRE of the alpha G gene was transfected. Under no condition was expression of the CAT gene observed from a CRE-deficient template. The acute transcriptional activation of a CRE/TATA box-containing gene by cAMP in granulosa cells indicated that transcription-activating proteins interacting with the CRE were either present in these cells or were rapidly synthesized after stimulation. To distinguish between these two possibilities, protein synthesis was transiently inhibited after transfection, before the addition of FSK, by incubating the cells for 2 h with 25 micrograms/ml cycloheximide. Cycloheximide treatment alone did not stimulate transcription from the CRE-containing molecule. Incubation with cycloheximide, followed by treatment with FSK, increased CAT activity 2-fold compared to t
...
PMID:An adenosine 3'5'-monophosphate-responsive deoxyribonucleic acid element confers forskolin sensitivity on gene expression by primary rat granulosa cells. 247 38

LH has been shown to be the principal hormone regulating ovarian thecal-interstitial cell (TIC) differentiation. It has been well documented that LH stimulates cAMP production and that cAMP analogs mimick the stimulatory actions of LH, but the mechanisms by which LH and cAMP stimulate TIC differentiation are unknown. The purpose of these studies was to characterize LH-stimulated differentiation of isolated TIC in serum-free medium and examine the role of cAMP-dependent protein kinase (PKA) isoenzymes in TIC differentiation. Highly purified (greater than 90%) TIC which were free from granulosa cell contamination were isolated from collagenase-dispersed ovaries of hypophysectomized immature rats by Percoll gradient centrifugation. When the purified TIC (20,000 viable cells/well) were cultured (2 days) in serum-free medium (0.2 ml in 96-well plates), low levels of steroids were produced. LH stimulated a dose-related (ED50 = 2.6 +/- 0.4 ng/ml) increase (50-fold) in androsterone, the principal androgen produced. LH stimulated an immediate dose-related increase in cAMP production, but there was a 20-h lag before LH stimulated an increase in androsterone production, which reached maximum levels at 30 h. LH-stimulated progesterone production increased immediately to a maximum at 10 h, then progesterone levels decreased as androsterone production increased. To determine the role of PKA in stimulating androsterone and progesterone production, TIC were cultured (2 days) with 8-aminohexylamino-cAMP (100 microM) plus N6-benzoyl-cAMP (30 microM) or 8-thiomethyl-cAMP (30 microM) plus N6-benzoyl-cAMP (30 microM) to directly and selectively activate type I or type II PKA, respectively. Selective activation of either isoenzyme increased androsterone and progesterone production by TIC. Immunoblots revealed that either type I or type II PKA increased the contents of P450scc and P45017 alpha in TIC. This is the first demonstration that direct activation of either type I or type II PKA stimulates TIC differentiation. These results indicate that LH stimulates TIC differentiation by a mechanism mediated by activation of one or both PKA isoenzymes.
...
PMID:Evidence that luteinizing hormone-stimulated differentiation of purified ovarian thecal-interstitial cells is mediated by both type I and type II adenosine 3',5'-monophosphate-dependent protein kinases. 254 87

The conversion of cholesterol to pregnenolone by adrenocortical mitochondria is the rate-limiting step in steroidogenesis. This process is stimulated dramatically by the action of ACTH through the sequential reactions, in which adenyl cyclase, cAMP-dependent protein kinase, cholesterol esterase and ribosomal protein synthesis are all involved. The de novo synthesized protein, the so-called labile protein with a half-life of approx 10 min, is believed to stimulate the cholesterol side chain cleavage reaction by an unknown mechanism. Available evidence indicates that the electron on transfer reaction from NADPH to P-450scc is mediated rapidly by adrenodoxin reductase and p-450 scc. In addition, these redox components are inactivated slowly with a half-life of 3.5 days after hypophysectomy. It is known that the corticoid output from adrenocortical cells starts within 5 min and reaches the maximum after 10-15 min of ACTH administration to animals. One can assume that under normal physiological conditions, both O2 and NADPH are not limiting. Additionally, mitochondrial inner membranes are poor in cholesterol. In this context, the availability of substrate cholesterol to P450scc is the most likely candidate for the regulatory mechanism.
...
PMID:Transduction of ACTH signal from plasma membrane to mitochondria in adrenocortical steroidogenesis. Effects of peptide, phospholipid, and calcium. 302 55

The rate-limiting step in adrenal steroidogenesis is associated with the mitochondrial-cytochrome-P450scc-dependent production of pregnenolone from cholesterol. This sterol side-chain cleavage reaction is influenced by the supply of cholesterol to the mitochondria. Cholesterol is stored as cholesterol esters while the cytosol contains a hormone-sensitive cholesterol ester hydrolase. This enzyme is activated by phosphorylation involving a cyclic AMP-dependent protein kinase and ATP; this enzyme preferentially attacks cholesterol oleate or cholesterol linoleate. The lipid composition of the adrenal cortex is influenced by diet so that animals on a low-fat diet tend to store cholesterol oleate and as the linoleate content of the diet is increased, the cholesterol linoleate content of the adrenal cortex increases. Animals maintained on a high erucate diet tend to store large amounts of cholesterol erucate in the adrenal cortex; such animals have an impaired adrenal cortical function. Animals maintained on a low-fat diet (marginally deficient in essential fatty acids), a linoleate-replete diet or a moderate erucate diet, all exhibited normal responses to ACTH and normal corticosterone production rates.
...
PMID:Dietary effects on certain adrenal cortical functions in the rat. 625 93

Adrenal cortical mitochondria contain a mixed function oxidase capable of converting cholesterol to pregnenolone; this enzyme requires NADPH, oxygen and cholesterol. This cholesterol side chain cleavage enzyme system contains a Flavoprotein, an iron sulphur protein and a specific cytochrome P450 termed cytochrome P450scc. ACTH stimulates the adrenal cortex by activating adenyl cyclase producing an elevated intracellular concentration of cAMP. This in turn increases the activity of a cytosolic cAMP dependent protein kinase. Adrenal cortical cytosol contains a cholesterol ester hydrolase which is activated by ATP and a protein kinase. This enzyme may be deactivated by a phosphoprotein phosphatase. The adrenal cortex contains lipid droplets that are rich in esterified cholesterol. Cholesterol ester hydrolase can release free cholesterol from the lipid droplets. The free cholesterol released may be used to supplement the mitochondrial cholesterol as a pregnenolone precursor. Steroid hormone production by the adrenal cortex exhibits a diurnal rhythm and correlates with the activity of the cytosolic cholesterol ester hydrolase. The acute steroidogenic response to ACTH may be in part attributed to the availability of free cholesterol to the mitochondrial cholesterol side chain cleavage enzyme complex. The intracellular movement of free cholesterol from lipid droplets to mitochondrial inner membranes may be impeded by protein synthesis inhibitors such as cycloheximide. The precise mechanism of this block in steroidogenesis remains to be elucidated. Various drugs and oestrogenic hormones suppress the plasma and adrenal cholesterol concentrations. If adrenal cells are deficient in cholesterol, these cells exhibit a diminished response to ACTH. The response to this hormone can be corrected by supplying cholesterol via exogenous plasma lipoproteins. The route that free cholesterol follows within the adrenal cortical cell and the physiological factors influencing free cholesterol movement in such cells are important issues to be explored in future.
...
PMID:Cholesterol metabolism in the adrenal cortex. 631 Feb 52


1 2 3 4 5 6 7 Next >>

---