共查询到20条相似文献,搜索用时 15 毫秒
1.
We investigated the regulation of synthesis of low density lipoprotein (LDL) receptor in cultured luteinized human granulosa cells using a monoclonal antibody recognizing the human LDL receptor (IgG-C7). Cells cultured under serum-free conditions were treated with human chorionic gonadotropin (hCG) or 8-bromo-cAMP alone or in combination with aminoglutethimide (to block conversion of cholesterol to steroid hormones) and 5-cholesten-3 beta, 25-diol (25-hydroxycholesterol, a potent suppressor of LDL receptor expression in human fibroblasts) and pulse-labeled with [35S]methionine. A labeled protein immunoisolated with IgG-C7 was identified as the mature LDL receptor in 7.5% sodium dodecyl sulfate-polyacrylamide gels on the basis of an apparent molecular mass of 160 kDa, absence of the protein from immunoisolates prepared with a monoclonal antibody against an irrelevant antigen, and an apparent decrease in molecular weight of the mature receptor upon treatment with neuraminidase or electrophoresis under nonreducing conditions. hCG and 8-bromo-cAMP consistently increased the incorporation of radioactivity into the mature LDL receptor by 2-6-fold. The effect of hCG on LDL receptor synthesis was observed with as little as 10 mIU of hCG/ml and was apparent within 2 h of addition of the hormone. A combination of 25-hydroxycholesterol and aminoglutethimide resulted in a 60% suppression of label incorporation into mature LDL receptor compared to untreated cells. This would suggest some regulation of LDL receptor synthesis by negative feedback of sterol. However, both hCG and 8-bromo-cAMP increased label incorporation into the LDL receptor in the face of these agents. We conclude that in human granulosa cells, hCG, through the intermediacy of cAMP, rapidly increases LDL receptor synthesis by a mechanism which is, at least in part, independent of alterations in cellular cholesterol balance. 相似文献
2.
Kinetic studies were performed on two-day cultures of rat ovarian granulosa cells to follow the fate of surface-bound 125I-labeled human chorionic gonadotropin (125I-hCG). Low pH was used to release hCG from its surface receptor, allowing us to distinguish between surface-bound and internalized hormone. Because our results indicated that hormone is lost from the cell surface by dissociation as well as internalization, equations were derived to determine independent rate constants for each process. We calculate that if hormone binding were irreversible, the t 1/2 for internalization would be 8.5 hour. Morphometric studies on the uptake of horseradish peroxidase indicate that the t 1/2 for internalization of bulk membrane in granulosa cells is 55 to 77 minutes. Thus, the rate of uptake of surface-bound hCG appears to be seven to nine times slower than the rate of uptake of bulk plasma membrane, which suggests that the LH/hCG receptor may be selectively excluded from the endocytic vesicles of granulosa cells. 相似文献
3.
Treatment of human granulosa cells with human CG (hCG) or an analog of its second messenger, cAMP, promotes a rapid increase in low density lipoprotein (LDL) receptor mRNA content. After 1 h of treatment with 8-bromo-cAMP, an appreciable increase in hybridizable LDL receptor mRNA was found which increased to apparently maximal levels within 4-6 h. Treatment of the granulosa cells with 25-hydroxycholesterol, in the presence of aminoglutethimide, resulted in a reduction in LDL receptor mRNA content within 6 h of treatment. However, hCG or 8-bromo-cAMP were able to stimulate an increase in LDL receptor mRNA content in the presence of this inhibitory signal. We further investigated the mechanism by which tropic agents increased mRNA content. While inhibition of RNA synthesis with actinomycin D blocked the hCG or cAMP-induced rise in LDL receptor mRNA content, inhibition of protein synthesis with cycloheximide augmented basal or hCG- or cAMP-stimulated LDL receptor mRNA levels. We conclude that human steroidogenic cells possess a cAMP-mediated mechanism for rapid upregulation of LDL receptor mRNA which is distinct from, and supercedes, cholesterol negative feedback of LDL receptor gene expression. The actions of hCG and 8-bromo-cAMP do not require ongoing protein synthesis. Indeed, a cycloheximide-sensitive mechanism modulates receptor mRNA levels in these cells such that the effects of hCG and 8-bromo-cAMP are enhanced when cells are pretreated with this drug. 相似文献
4.
Effect of cyclic AMP on low density lipoprotein binding and internalization by cultured human fibroblasts 总被引:2,自引:0,他引:2
J C Mazière C Mazière J Gardette L Mora J Polonovski 《Biochemical and biophysical research communications》1983,112(3):795-800
Pretreatment of cultured human fibroblasts by cyclic AMP resulted in a marked decrease in the binding and internalization of the low density lipoproteins (about 55% of controls for cyclic AMP 2.10(-3) M). This effect was dose dependent and increased by theophyllin. DL propranolol, an inhibitor of adenylcyclase, had an opposite effect. Isoproterenol, which stimulates adenylcyclase, reproduced the effect of cyclic AMP. The cholesterol synthesis from [2-14C] acetate was decreased by cyclic AMP, theophyllin and isoproterenol, and increased by propranolol. The incorporation of [1-14C] oleate into cholesteryl esters was reduced by cyclic AMP, theophyllin, isoproterenol and propranolol. 相似文献
5.
Free sterol metabolism and low density lipoprotein receptor expression as differentiation markers of cultured human keratinocytes 总被引:2,自引:0,他引:2
M L Williams A M Mommaas-Kienhuis S L Rutherford S Grayson B J Vermeer P M Elias 《Journal of cellular physiology》1987,132(3):428-440
In contrast to most tissues, epidermis and its derivatives appear to lack low density lipoprotein (LDL) receptors and exhibit sterologenesis rates unaffected by circulating lipoprotein (LP) cholesterol content. Since LDL receptors have been demonstrated in both cultured squamous cell carcinoma cells and human foreskin keratinocytes, when maintained in low-calcium media, LDL receptor expression may be related to keratinocyte differentiation. We compared receptor binding and internalization of LDL-gold in normal keratinocytes at different stages of growth at physiological calcium concentrations (early, 3-5 days; preconfluent, 6-10 days; postconfluent, 12-17 days), and correlated receptor expression with sterologenesis in LP-replete vs.-depleted media. Whereas in early cultures about 60% of sterologenesis was LP dependent, this fraction declined in preconfluent and confluent cultures despite continued culture growth and little decline in total sterologenesis. Accordingly, LDL receptors were most evident in early cultures, declining in preconfluent cultures in parallel with the decrease in LP-dependent sterol synthesis. In contrast, sterologenesis in human foreskin fibroblasts was profoundly influenced by exogenous LP at all stages of confluence; total and LP-dependent sterologenesis declined in parallel with growth cessation. These studies represent the first demonstration that normal keratinocytes express functional LDL receptors at physiologic calcium concentrations. Moreover, they demonstrate that LDL receptor expression in keratinocytes, in contrast to fibroblasts, can only in part be attributed to growth requirements. Instead, loss of LDL receptor expression serves as a distinctive marker of keratinocyte differentiation and may reflect the specific functional requirements of the epidermis in vivo. 相似文献
6.
K Rajkumar H Ly P J Chedrese B D Murphy 《Canadian journal of physiology and pharmacology》1988,66(11):1450-1454
The present study examines the effect of prolactin (PRL) and N6-2(1)-O-dibutyryladenosine 3'5'-cyclic monophosphate (cAMP) on low density lipoprotein (LDL) uptake and metabolism by luteinized porcine granulosa cells in culture. Granulosa cells from preovulatory follicles were plated with 1% serum and 1 microgram/mL of insulin for the first 48 h. Following plating (day 3) the cells were cultured in serum-free media with the same dose of insulin. The next day the medium was replaced with serum- and insulin-free medium, and to some cultures 1.23 IU/mL of human chorionic gonadotrophin (hCG) was added. On day 5 the medium was again replaced and graded amounts of PRL (0, 0.03, 0.3, and 3 micrograms/mL) were added. Following 48 h of incubation with PRL, 20 micrograms/mL of 125I-labelled LDL was added to cultures. Surface-bound, internalized, and degraded LDLs were quantitated at 12 h following addition of LDL. To examine the effect of cAMP on LDL metabolism, the cells were exposed for 24 h to cAMP (3mM) on day 6 of culture. PRL had a stimulatory effect on LDL degradation by luteinized granulosa cells. Pre-exposure of cells to hCG augmented the stimulatory effect of PRL. Addition of cAMP also enhanced LDL degradation by luteinized granulosa cells. Both PRL and cAMP increased surface binding of LDL in cells pre-exposed to hCG, but there was no effect on internalization. The increase in cell surface binding of LDL with PRL and cAMP was less than their effect on LDL degradation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis. 相似文献
8.
9.
10.
It is now well recognized that hCG-induced luteolysis is associated with hCG-induced desensitization, but the physiological significance of luteal cell GnRH, PGs and beta-receptors is still undefined. Therefore, we intend in this study to observe the effects of prostaglandin F2 alpha and prostaglandin E2 and the interactions between epinephrine, a potent LHRH agonist [(D-Ser-(TBu)6, des-Gly-NH10(2) LHRH ethylamide: Buserelin] and hCG in normal and in vitro hCG-desensitized rat immature luteal cells in monolayer culture, on basal, hCG or cholera toxin stimulated intracellular and extracellular cAMP and progesterone secretion. The present report shows that incubation of immature rat luteal cells in monolayer culture with Buserelin, led to 25-50% inhibition of the epinephrine-as well as PGE2-induced cAMP and progesterone responses. The LHRH agonist can also reverse the stimulatory effects of cholera toxin in the presence of hCG and led with PGF2 alpha, to additive inhibitory effects on extracellular cAMP accumulation induced by cholera toxin. Both Buserelin and PGF2 alpha can reverse the hCG-induced cAMP and progesterone release but no effect could be observed when the incubation was carried out with either substance in the absence of hCG. Prostaglandin E2, in acute conditions of incubation, seems to share agonist properties with hCG when both were incubated with luteal cells. Buserelin reversed the stimulatory effects of PGE2, hCG, epinephrine and cholera toxin on cAMP and progesterone responses to these substances. These results suggest that Buserelin and PGF2 alpha have luteolytic-like effects and that there may be a complementary action for the two substances. Preincubation of rat luteal cells in monolayer culture with 1 nM hCG for a 24 h period led to the inhibition of cAMP and progesterone responses after a subsequent exposure to hCG and epinephrine. Luteal cells were no longer responsive to hCG while the presence of epinephrine in hCG-desensitized cells led to a 40% stimulation of cAMP and progesterone production. These observations suggest that occurred a partial alteration of the N component activity of the adenylyl cyclase system. 相似文献
11.
12.
Insulin, somatomedin-C, human chorionic gonadotropin, and forskolin enhance glucose oxidation by granulosa cells 总被引:1,自引:0,他引:1
The long exposure times required to observe stimulatory effects of insulin on steroidogenesis and protein synthesis in granulosa cells suggested that these effects might be secondary to stimulation of another metabolic process. The present studies examined the effects of insulin, the insulin-like growth factor somatomedin-C (Sm-C), human chorionic gonadotropin (hCG), and forskolin, a compound that activates adenylyl cyclase independently of a receptor, on glucose metabolism. Granulosa cells from preovulatory porcine ovarian follicles were incubated at 37 degrees C in Dulbecco's phosphate-buffered saline supplemented with bovine serum albumin, vitamins, amino acids, and glucose (0.01-20 mM). Cells were incubated with [14C]glucose for up to 23 h with or without a prior 20-h preincubation. Oxidation of glucose, assessed by quantitation of 14CO2 produced, was dependent on time and concentration of glucose. Optimal glucose concentrations for glucose oxidation were 3 mM in the absence or presence of insulin and correlated well with the measured glucose concentrations in follicular fluid (3 mM). After a 20-h preincubation in the absence or presence of insulin (1 microM), the rates of CO2 production were 10.6 and 21.6 pmol/micrograms DNA/h for control and insulin-treated cells, respectively. Insulin had an EC50 of 164 nM. Sm-C and hCG were more potent stimulators than insulin with EC50s of 768 pM and 161 pM, respectively. The greater sensitivity of granulosa cells to Sm-C than to insulin supports the concept that insulin exerts its effect via reactivity with the Sm-C receptor. The effect of hCG may have been mediated by cyclic adenosine 3',5'-monophosphate (cAMP), since forskolin also enhanced 14CO2 production.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Inactivation of the cyclic nucleotide signal in granulosa cells depends on a complex array of cyclic nucleotide phosphodiesterases (PDE). In order to examine the role of PDE in cyclic AMP (cAMP) signaling in granulosa cells, the present study examined the expression of PDE4D proteins and regulation of cAMP-PDE activities in cultured rat granulosa cells. The results of immunoblot analyses showed that two predominant PDE4D subtypes of approximately 80 and 70 kDa appeared when immature rat granulosa cells were treated with FSH. However, these two new subtypes presumed to be PDE4D proteins were not influenced by treatments of DETA/NO, cGMP and PKB inhibitor, LY294002. Immature rat granulosa cells treated with medium alone displayed low cAMP-PDE activity throughout 48 h of culture while those treated with FSH (2 ng.mL-1) showed a marked increase in cAMP-PDE activity between 6 and 12 h of culture, followed by a decline. The findings from the present study indicate that the increased cAMP-PDE activity by FSH is mainly related to the changes of PDE4D protein levels. However, the inhibitory effects of NO on cAMP accumulation in rat granulosa cells are not via the increased cAMP-PDE activity. 相似文献
14.
Macrophage-derived factors increase low density lipoprotein uptake and receptor number in cultured human liver cells. 总被引:1,自引:0,他引:1
R I Grove C Mazzucco N Allegretto P A Kiener G Spitalny S F Radka M Shoyab M Antonaccio G A Warr 《Journal of lipid research》1991,32(12):1889-1897
Recent evidence suggests the possibility that macrophages can influence lipoprotein metabolism. Therefore we investigated the ability of cultured macrophages to alter low density lipoprotein (LDL) uptake in a human liver cell line (HepG2). Conditioned media from phlogogenic-induced mouse peritoneal macrophages or from a human macrophage cell line stimulated with endotoxin increased HepG2 LDL uptake by as much as 60-70%. The increase was due, in part, to a significant macrophage-induced 40% increase in the number of LDL receptors per cell. Although macrophage conditioned media inhibited HepG2 cholesterol synthesis, the LDL receptor up-regulation did not appear to be due to the effects on cholesterol synthesis. The LDL receptor stimulatory activity was sensitive to proteolysis and heat. Its molecular mass was approximately 20 kDa based on gel filtration. Several macrophage secretory proteins were tested in HepG2 cultures for LDL uptake stimulation. Of these, oncostatin M (approximately 18 kDa by gel filtration) gave the strongest response. The rank order for LDL uptake stimulation was oncostatin M much greater than interleukin 6 = interleukin 1 = transforming growth factor-beta 1. A neutralizing antibody directed against oncostatin M inhibited the ability of conditioned media to up-regulate LDL receptors by 85%. Thus, our results indicate that macrophages can secrete several proteins that up-regulate LDL receptors in HepG2 cells and that most of the up-regulatory activity in macrophage conditioned media appears to be due to oncostatin M. 相似文献
15.
16.
The actions of follicle-stimulating hormone (FSH), 8-bromo-cyclic AMP (8-Br-cAMP), and low density lipoprotein (LDL) to stimulate the production of progesterone and the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450ssc) and adrenodoxin were investigated in bovine granulosa cells maintained in primary monolayer culture. Treatment of granulosa cells in culture with FSH resulted in an increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc in a concentration-dependent fashion with a maximal effect being obtained at an FSH concentration of 500 ng/ml. Treatment of granulosa cells with FSH also resulted in the induction of synthesis of adrenodoxin. The cyclic AMP analog, 8-Br-cAMP, induced the synthesis of both cytochrome P-450scc and adrenodoxin to a greater extent than did FSH. LDL also stimulated the synthesis of both cytochrome P-450scc and adrenodoxin, when added to cells maintained in the presence of lipoprotein-poor serum. The presence of FSH or 8-Br-cAMP together with LDL resulted in a higher rate of enzyme synthesis than that observed with each effector alone. FSH, 8-Br-cAMP, and LDL also stimulated progesterone production by cultured granulosa cells. The results of this study offer a possible mechanism whereby granulosa cells undergo cytodifferentiation in vivo into luteal cells. The concentration of LDL in follicular fluid is very low. Following ovulation, vascularization of the follicle occurs and thus the granulosa cells are exposed to high levels of LDL, allowing for provision of substrate cholesterol, as well as stimulation of the synthesis of the enzymes involved in cholesterol side chain cleavage. 相似文献
17.
Expression of low density lipoprotein receptor gene in human placenta during pregnancy 总被引:1,自引:0,他引:1
M Furuhashi H Seo S Mizutani O Narita Y Tomoda N Matsui 《Molecular endocrinology (Baltimore, Md.)》1989,3(8):1252-1256
Mammalian cells require cholesterol as a structural component of plasma membranes. It is also required for placental steroid synthesis. De novo synthesis of cholesterol is limited in human placenta and cholesterol is obtained mainly from plasma low density lipoprotein (LDL). Cholesterol delivery from LDL is mediated by receptor-mediated uptake and the receptor amount is the most important factor for cellular delivery. Thus, the regulation of receptor synthesis is important for placental development and function. Since the regulation of LDL receptor gene expression has not been studied in human placenta, LDL receptor mRNA was measured in placentae of 5-40 weeks of gestation by hybridization of RNA with 32P-labeled cDNA for human LDL receptor. Two mRNA species for LDL receptor were demonstrated by Northern blot analysis. The longer mRNA [5.3 kilobases (kb)] was much more abundant than the shorter mRNA (3.7 kb). The amount of 5.3 kb mRNA was highest early in gestation and decreased during pregnancy. However, the amount of 3.7 kb mRNA did not change appreciably during gestation. Dot blot analysis of 26 placental mRNAs obtained from various stages of gestation revealed a negative correlation between LDL receptor mRNA and gestation (r = -0.76, P less than 0.001). Considering the rapid growth of the trophoblast during gestation, especially in the first and the second trimester, increased expression of the LDL receptor gene and subsequent translation are expected for efficient cholesterol uptake to provide a sufficient substrate for cell growth. Possible mechanisms for the appearance of two mRNA species for LDL receptor are also discussed. 相似文献
18.
J R Philippot S P Verma P S Lin 《Biochemical and biophysical research communications》1986,138(2):938-944
Specific binding and degradation of native and gamma-rays irradiated (100-2000 rad; 100 rad/min; 137Cs) human low density lipoprotein by Chinese hamster V79 cells and mouse peritoneal macrophage line, J774G were studied. Low density lipoproteins were labeled with 125I for studying the specific binding and subsequent degradation. The specific binding and degradation of irradiated 125I-low density lipoproteins (mixed with irradiated native lipoprotein) by Chinese hamster V79 cells are considerably reduced. The uptake depends on the concentration of thiobarbutaric acid-reactive products generated in the irradiated lipoproteins which in turn depends on the concentration of carotenoids. In contrast the rate of uptake of oxidized low density lipoproteins is enhanced by Chinese hamster macrophages. The alteration in the surface amino groups of apo-B of low density lipoprotein either due to direct damage of peptide bonds by gamma-rays or via interaction with lipid peroxides (generated in the core upon irradiation) are invoked as possible mechanisms for the reduction in specific binding and subsequent degradation by V79 cells. 相似文献
19.
S Chatterjee N Ghosh E Castiglione P O Kwiterovich 《The Journal of biological chemistry》1988,263(26):13017-13022
We have shown previously that low density lipoproteins (LDL) suppressed the synthesis of lactosylceramide in normal human proximal tubular cells, but stimulated such synthesis in proximal tubular cells from LDL receptor negative subjects (Chatterjee, S., Clarke, K., and Kwiterovich, P.O., Jr. (1986) J. Biol. Chem. 261, 13474-13479). To understand the mechanism(s) of this effect of LDL, we have studied here the effects of LDL on the activity of UDP-GalCer:beta-galactosyltransferase (GalT-2). Maximum suppression (70-80%) of the activity of GalT-2 in normal proximal tubular cells at 37 degrees C occurred at a LDL concentration of 25 micrograms/ml medium. Such suppression was not observed either when the cells were incubated with LDL at 4 degrees C, or when the cells were preincubated with leupeptin, followed by incubation with LDL at 37 degrees C. High density lipoproteins and fetuin did not suppress the activity of GalT-2 in normal proximal tubular cells. In contrast LDL modified by reductive methylation (M-LDL, 100 micrograms/ml) stimulated the activity of GalT-2, approximately 3-fold. The effects of LDL and M-LDL were not related to their glycosphingolipid content. Much less suppression and stimulation of the activity of GalT-2 in proximal tubular cells by LDL and M-LDL, respectively, was found in normal human skin fibroblasts, Chinese hamster ovary cells, and bovine smooth muscle cells, suggesting that the LDL-mediated effect may be tissue-specific. In cells grown to very high density, the activity of the LDL receptor is decreased, and there was less suppression of GalT-2 activity by LDL. In normal proximal tubular cells, LDL stimulated the activity of UDP-Gal:LacCer, alpha-galactosyltransferase activity, UDP-Gal:LcOse3Cer, beta-galactosyltransferase, and CMP-NeuAc:LacCer,alpha-sialyltransferase activity but did not alter the activity of sulfotransferase. In conclusion, LDL that entered the normal proximal tubular cells via the LDL receptor-mediated pathway decreased GalT-2 activity, an effect that was dependent upon the binding, internalization, and degradation of receptor-bound LDL. In contrast LDL that entered normal or LDL receptor-negative proximal tubular cells via an LDL receptor-independent pathway failed to suppress GalT-2 activity, and led to a stimulation of LacCer synthesis. 相似文献
20.
Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed. 相似文献