Monophosphate in Tumor Leydig Cells ′ : 5 ′ for the Estrogen Receptor by Cyclic Adenosine 3 Biphasic Regulation of the Messenger Ribonucleic Acid Coding Updated
In this report we show that the mRNA level for the estrogen receptor (ER) is regulated by 8-bromo... more In this report we show that the mRNA level for the estrogen receptor (ER) is regulated by 8-bromo cyclic AMP (8-Br-cAMP) and human chorionic gonadotropin in a mouse tumor Leydig cell line (MA-IO cells). When the MA-10 cells were cultured in the presence of the cAMP analogue for varying time periods, a transient increase in the level of ER mRNA was observed. Short time incubation (0-2 h) with 8-Br-cAMP enhanced the expression of ER mRNA (2-fold), whereas longer times of incubation (6 h) had the opposite effect (the level of ER mRNA was reduced by 60-70%). The inhibitory effect of 8-Br-cAMP on ER mRNA was not counteracted by aminoglutethimide, an inhibitor of steroidogenic enzymes, indicating that this effect is not mediated via steroids (proges terone). Treatment of 8-Br-cAMP for 6 h caused a concentration-de pendent inhibition of ER mRNA with a half-maximal effect of approxi mately 150 JIM. Increasing concentrations of human chorionic gonadotropin for 6 h was also associated with a ...
Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been sho... more Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmpgt/gtmice (formerly known as Ncu-g1gt/gtmice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency ofGlmpgt/gtmice were comparable to wild type siblings, only at the age of 9 months the Glmpgt/gt siblings had significantly re-duced body weight. Reduced size of epididymal fat pads was accompanied by hepatosple-nomegaly inGlmpgt/gtmice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids inGlmpgt/gtmice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected inGlmpgt/gt primary hepatocytes, as well as elevated triacylglycerol levels inGlmpgt/gt liver homoge-nates, compared to hepatocytes ...
ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines-0
<b>Copyright information:</b>Taken from "ABCA1, ABCG1 and SR-BI: hormonal regula... more <b>Copyright information:</b>Taken from "ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines"BMC Molecular Biology 2007;8():5-5.Published online 22 Jan 2007PMCID:PMC1790708.ex), insulin (Ins), or mifepristone (Mif) in lipid-deficient medium for 24 hours and examined by real time RT-PCR and Western blotting. . The relative mRNA expression of SR-BI (black bars), ABCA1 (white bars) and ABCG1 (gray bars). . Representative Western blots of ABCA1, ABCG1, SR-BI and β-actin. Proteins (50 μg) were separated on a 7.5% polyacrylamide gel as described in materials and methods. . The relative mRNA expression of CYP7A1 (black bars) and apoA-I (white bars). The results were normalized to GAPDH mRNA expression and expressed relative to the non-treated controls (medium B only) (± STDEV). The experiments were performed at least three times. * (p &lt; 0.05) indicate significantly difference from control.
of transfection affects the cAMP-mediated induction of the RIIb subunit of protein kinase A in Se... more of transfection affects the cAMP-mediated induction of the RIIb subunit of protein kinase A in Sertoli cells: inhibition of response by increase in intracellullar calcium
Background and Aims. Vascular endothelial growth factor (VEGF) receptors (VEGFR1 and VEGFR2) bind... more Background and Aims. Vascular endothelial growth factor (VEGF) receptors (VEGFR1 and VEGFR2) bind VEGF-A with high affinity. This study sought to determine the relative contributions of these two receptors to receptor-mediated endocytosis of VEGF-A and to clarify their endocytic itineraries in rat liver sinusoidal endothelial cells (LSECs).Methods. Isolated LSECs and radiolabeled VEGF-A were used to examine surface binding and receptor-mediated endocytosis. Quantitative real time RT-PCR (Q-RT-PCR) and Western blotting were applied to demonstrate receptor expression.Results. Q-RT-PCR analysis showed that VEGFR1 and VEGFR2 mRNA were expressed in LSECs. Ligand saturation analysis at 4°C indicated two different classes of [125I]-VEGFA binding sites on LSECs with apparent dissociation constants of 8 and 210 pM. At 37°C, LSECs efficiently took up and degraded [125I]-VEGF-A for at least 2 hours. Uptake of [125I]-VEGF-A by LSECs was blocked by dynasore that inhibits dynamin-dependent intern...
Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of c... more Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels. Unstimulated Sertoli cells contain high levels of R (~9.2 ± 0.8 pmol/mg protein) and C (~7.3 ± 0.7 pmol/mg protein). Stimulation with (Bt) 2 cAMP (0.1 mMJ for 24 and 48 h revealed a time-dependent increase in [ 3 H]cAMP-binding activity. During the same time period the catalytic activity remained relatively constant, resulting in an increase in the R/C ratio from approximately 1.3 to 3.0. Using diethylaminoethyl cellulose chromatography, 8-N 3-[ 32 P]cAMP photoaffinity labeling, autophosphorylation by y-[ 32 P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RI a , 25% RII a , and very low levels of RII 3. Stimulation of Sertoli cells with (Bt) 2 cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RI O (6.6-14 pmol/mg) and a 10-to 20-fold increase in RII,, (<0.1-1.1 pmol/mg), with little or no change in RII a (1.9-2.3 pmol/ mg). Treatment with cAMP was associated with a slight increase in RI/RII ratio (3.3-4.1). mRNA levels for RII,, increased 30-to 50-fold after (Bt) 2 cAMP stimulation, whereas only minor changes in mRNA levels for RI a , RII«, and C o were observed (1.5-to 2.0-fold). mRNA levels for RI ft C^, and C y were not detected in either unstimulated or in cAMP-stimulated Sertoli cells. It is concluded that chronic treatment with cAMP changes the relative proportion of R subunits of PKA in a manner reflecting the changing levels in respective mRNAs. Furthermore, such treatment; is associated with the appearance of a new PKA R subunit (Rllf), which is absent in untreated Sertoli cells.
Down-regulation of messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) by phorbol ester requires ongoing RNA synthesis but not protein synthesis. Is hormonal control of ER mRNA degradation mediated by an RNA molecule?
Endocrinology, 1992
Treatment of MCF-7 cells, a human mammary carcinoma cell line, with phorbol ester [12-O-tetradeca... more Treatment of MCF-7 cells, a human mammary carcinoma cell line, with phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] or calcium ionophore (A23187) was associated with striking effects on levels of estrogen receptor (ER) mRNA, specific binding of 17 beta-[3H]estradiol [( 3H]E2), and immunoreactive ER. TPA (10(-7) M) caused a time-dependent reduction of ER mRNA which was below the level of detection after 9 h. Similar effects of TPA appeared at levels of specific binding of [3H]E2 as well as immunoreactive ER. In contrast, TPA induced an increase in mRNA for beta-actin. Incubation of MCF-7 cells with increasing concentrations of TPA (10(-11)-10(-7) M) was associated with biphasic effects on ER mRNA and proteins. Levels of immunoreactive progesterone receptors (PR) were induced by E2 (10(-9) M) in a time-dependent manner. In the presence of TPA (10(-7) M), where ER levels were suppressed, no induction of PR was observed. Removal of TPA (10(-7) M) after 10 h (ER mRNA) or 22 h (ER proteins) of treatment was associated with a continued suppression of both mRNA and protein levels during the entire incubation period (48 h). Treatment with A23187 (2 x 10(-7) M) also caused a time-dependent down-regulation of levels of ER mRNA and proteins. These effects occurred somewhat more slowly than those of TPA. Levels of beta-actin mRNA were not changed by this treatment. These results indicate that changes in estrogen sensitivity are mediated by calcium-dependent protein kinases in human mammary carcinoma MCF-7 cells.
Gonadotropin activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases ... more Gonadotropin activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases plays an important role in the regulation of testicular function. This study was undertaken to establish the expression of various subunits of cAMP-dependent protein kinases in different testicular cell types as well as during sexual maturation. RNA was extracted from cultured Sertoli cells, cultured peritubular cells, gern cells (pachytene spermatocytes, round spermatids), tumor Leydig cells, as well as whole testis from rats of various ages. Messenger R NA levels were studied by Northern analysis using available cDNA probes.
Autologous Down-Regulation of Messenger Ribonucleic Acid and Protein Levels for Estrogen Receptors in MCF-7 Cells: An Inverse Correlation to Progesterone Receptor Levels*
Endocrinology, 1989
In the present study we have examined the effects of estradiol on mRNA levels for estrogen (ER) a... more In the present study we have examined the effects of estradiol on mRNA levels for estrogen (ER) and progesterone receptors (PR) in the estrogen-dependent mammary carcinoma MCF-7 cell line. The changes in ER immunoactivity and specific binding of [3H]R5020 were also assessed. Estradiol (10(-7) M) caused a transient and time-dependent reduction of the level of mRNA for ER, with a maximal effect (30-40% of control; n = 3) after 72 h. This was associated with a similar decrease in ER immunoactivity. Further treatment (96 and 120 h) revealed a return of ER mRNA to control values, whereas the ER immunoactivity remained depressed. The effect on the mRNA level for PR gave almost the inverse curve. Initially (24-72 h), we observed a pronounced increase in this mRNA, with a maximal effect (6-7 times the control value; n = 3) after 72 h. Treatment beyond 72 h was associated with a gradual return of mRNA for PR toward the control level. The variation in specific binding of [3H]R5020 revealed similar changes, except that changes in specific receptor binding were delayed 24 h compared to the levels of mRNA. Incubation with low concentrations (10(-11) and 10(-10) M) of estradiol for 72 h was associated with slightly elevated levels of mRNA for ER, whereas higher concentrations gave a dose-dependent decrease. The mRNA for PR was biphasically stimulated, with a maximal effect at 10(-10)-10(-8) M, where a 10- to 13-fold stimulation was observed. The highest concentration (10(-7) M) gave a lower response. Assessment of concentration-induced variations in protein receptor levels of ER and PR reflected the effects of estradiol on their mRNAs. Low concentrations of estradiol slightly enhanced the ER level, whereas high concentrations clearly reduced ER immunoactivity. The PR level was stimulated by all concentrations used, and 10(-8) M estradiol raised the PR level more than 11-fold. Our results indicate autologous regulation of estrogen receptor gene transcripts and proteins and a clear induction of PR mRNA and receptor proteins by estradiol.
Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator-0
<b>Copyright information:</b>Taken from "Human NCU-G1 can function as a transcri... more <b>Copyright information:</b>Taken from "Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator"http://www.biomedcentral.com/1471-2199/8/106BMC Molecular Biology 2007;8():106-106.Published online 16 Nov 2007PMCID:PMC2233640.nserved amino acids are shown against a black background. Highly conserved amino acids are shown against a grey background. The positions of conserved prolines are marked with black bullets. Panel B: Potential functional motifs in human NCU-G1.
Multifactorial metabolic diseases, such as non-alcoholic fatty liver disease, are a major burden ... more Multifactorial metabolic diseases, such as non-alcoholic fatty liver disease, are a major burden to modern societies, and frequently present with no clearly defined molecular biomarkers. Herein we used system medicine approaches to decipher signatures of liver fibrosis in mouse models with malfunction in genes from unrelated biological pathways: cholesterol synthesis—Cyp51, notch signaling—Rbpj, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling—Ikbkg, and unknown lysosomal pathway—Glmp. Enrichment analyses of Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome and TRANScription FACtor (TRANSFAC) databases complemented with genome-scale metabolic modeling revealed fibrotic signatures highly similar to liver pathologies in humans. The diverse genetic models of liver fibrosis exposed a common transcriptional program with activated estrogen receptor alpha (ERα) signaling, and a network of interactions between regulators of lipid metabolism and transcr...
Background: Novel, uncharacterised proteins represent a challenge in biochemistry and molecular b... more Background: Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1. Results: NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa. It is localized on chromosome 1 and consists of 6 exons. Analysis of the amino acid sequence revealed no known transcription activation domains or DNA binding regions, however, four nuclear receptor boxes (LXXLL), and four SH3-interaction motives in addition to numerous potential phosphorylation sites were found. Two nuclear export signals were identified, but no nuclear localization signal. In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney. Electrophoretic mobility shift analysis showed specific binding of NCU-G1 to an oligonucleotide representing the footprin...
Lysosomes are major sites for intracellular, acidic hydrolase-mediated proteolysis and cellular d... more Lysosomes are major sites for intracellular, acidic hydrolase-mediated proteolysis and cellular degradation. The export of low-molecular-weight catabolic end-products is facilitated by polytopic transmembrane proteins mediating secondary active or passive transport. A number of these lysosomal transporters, however, remain enigmatic. We present a detailed analysis of MFSD1, a hitherto uncharacterized lysosomal family member of the major facilitator superfamily. MFSD1 is not N-glycosylated. It contains a dileucine-based sorting motif needed for its transport to lysosomes. Mfsd1 knockout mice develop splenomegaly and severe liver disease. Proteomics of isolated lysosomes from Mfsd1 knockout mice revealed GLMP as a critical accessory subunit for MFSD1. MFSD1 and GLMP physically interact. GLMP is essential for the maintenance of normal levels of MFSD1 in lysosomes and vice versa. Glmp knockout mice mimic the phenotype of Mfsd1 knockout mice. Our data reveal a tightly linked MFSD1/GLMP l...
In the present study we have examined the cellular localization and developmental changes of mRNA... more In the present study we have examined the cellular localization and developmental changes of mRNAs for retinoid-binding proteins in rat testis. We demonstrate that mRNA (0.7 kb) for cellular retinol-binding protein (CRBP) is expressed only in Sertoli cells and peritubular cells. The mRNA for CRBP could not be detected in other testicular cells. In contrast, mRNA for cellular retinoic acid-binding protein (CRABP) was detected primarily in germ cells and to a small extent in tumor Leydig cells. The mR.NA for CRARP in germ cells revealed distinct size heterogeneity and three distinct mRNA species were observed (1.0, 1.8, and 1.9 kb), in contrast to previous data for somatic cells where only the 1.0-kb mRNA has been reported. Messenger RNAs for retinoic acid receptor-a (RARa) were detected in both somatic and haploid germ cells. The highest level of RARa was seen in Sertoli cells, round spermatids, and tumor Leydig cells. Lower, but distinct, levels were observed in peritubular cells. Furthermore, we observed germ cell-specific species of RARa mRNA (4 kb and approximately 7 kb). The smallest mRNA for RARa (2.7 kb) in somatic cells was absent in germ cells. The levels of mRNAs for the various retinoid-binding proteins in whole testis obtained from rats of various ages confirmed this cellular localization. The mRNAs for CRBP, the small molecular size (2.7 kb) mRNA for RARu (localized to somatic cells), and the 1-kb mRNA for CRABP showed an age-dependent decrease. In contrast, the 1.8-kb and 1.9-kb mRNA5 for CRABP (localized to germ cells) revealed a dramatic increase as a function of age. The 3.4-kb mRNA and RARa (present in both germ cells and somatic cells) did not change with age. Thus, the mRNAs for retinoid-binding proteins revealed distinct cell-specific expression. Furthermore germ cell-specific mRNA species for retinoid-binding proteins were demonstrated.
The liver X receptors a and b (LXRa and LXRb) are members of the nuclear receptor superfamily of ... more The liver X receptors a and b (LXRa and LXRb) are members of the nuclear receptor superfamily of proteins which are highly expressed in metabolically active tissues. They regulate gene expression of critical genes involved in cholesterol catabolism and transport, lipid and triglyceride biosynthesis, and carbohydrate metabolism in response to distinct oxysterol intermediates in the cholesterol metabolic pathway. Several LXR target genes have been identified, but there is limited information on how expression of the LXRs themselves is controlled. In this study we have characterized the upstream flanking region of the mouse LXRa gene. Transient transfections show that the LXRa promoter is able to drive transcription of a luciferase reporter gene, however, the transcriptional potential of the promoter in the cell lines used was low. The)2143 to)1513 region of the promoter mediates repression of reporter gene activity in all cells analyzed and multiple DNA-protein interactions were detected in this region by DNase I footprinting. The Zta, Ets, and Hes1 transcription factors were all shown to mediate alterations in reporter gene activity driven by LXRa promoter deletion constructs. These factors have been linked to cell cycle and differentiation processes suggesting that expression of LXRa might be under control of signalling mechanisms regulating cell proliferation. Several putative binding sites of the glucocorticoid receptor (GR) were identified in the LXRa promoter and transient cotransfections of the GR and LXRa promoter deletion constructs induced reporter gene activity. Addition of dexamethasone, a GR agonist, abolished this effect suggesting cross talk between GR and LXR signalling.
Background: Mice lacking glycosylated lysosomal membrane protein (Glmp gt/gt mice) have liver fib... more Background: Mice lacking glycosylated lysosomal membrane protein (Glmp gt/gt mice) have liver fibrosis as the predominant phenotype due to chronic liver injury. The Glmp gt/gt mice grow and reproduce at the same rate as their wild-type siblings. Life expectancy is around 18 months. Methods: Wild-type and Glmp gt/gt mice were studied between 1 week and 18 months of age. Livers were analyzed using histological, immunohistochemical, biochemical, and qPCR analyses. Results: It was shown that Glmp gt/gt mice were not born with liver injury; however, it appeared shortly after birth as indicated by excess collagen expression, deposition of fibrous collagen in the periportal areas, and increased levels of hydroxyproline in Glmp gt/gt liver. Liver functional tests indicated a chronic, mild liver injury. Markers of inflammation, fibrosis, apoptosis, and modulation of extracellular matrix increased from an early age, peaking around 4 months of age and followed by attenuation of these signals. To compensate for loss of hepatocytes, the oval cell compartment was activated, with the highest activity of the oval cells detected at 3 months of age, suggesting insufficient hepatocyte proliferation in Glmp gt/gt mice around this age. Although constant proliferation of hepatocytes and oval cells maintained adequate hepatic function in Glmp gt/gt mice, it also resulted in a higher frequency of liver tumors in older animals. Conclusions: The Glmp gt/gt mouse is proposed as a model for slowly progressing liver fibrosis and possibly as a model for a yet undescribed human lysosomal disorder.
Biochimica Et Biophysica Acta Molecular Cell Research, Feb 22, 1988
Endocytosis of formaldehyde-treated serum albumin (f-albumin) in isolated liver sinusoidal endoth... more Endocytosis of formaldehyde-treated serum albumin (f-albumin) in isolated liver sinusoidal endothelial cells was studied. Uptake occurs via the scavenger receptor and was found to be very sensitive to the ionophore monensin. Binding at 4 °C of f-albumin was reduced to 50% of control values by preincuhation for 2 min with 2 ?tM monensin. Both uptake and degradation of f-albumin were more sensitive to monensin. No lag-phase in the inhibitory effect on uptake and degradation was detected. A concentration of 0.1 #M monensin reduced uptake of f-albumin by 50%. Degradation of internalized f-albumin was reduced by 50% in the presence of 0.2 ~M monensin. Since uptake and degradation of f-albumin were very sensitive to monensin, the effect of introducing the drug during endocytosis of the ligand was tested. All processing of f-albumin stopped instantly upon addition of monensin; hence, there seems to be no step in the endocytie process beyond which monensin is ineffective. The data suggest that the scavenger receptor of liver endothelial cells is internalized and recycled very rapidly. Abbreviations: LDL, low-density lipoprotein; f-albumin, formaldehyde-treated albumin.
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