Decrease In Gene Transcription Research Articles

Molecular analysis of the regulation of muscarinic receptor expression and function.

Several systems are being used to determine the molecular and cellular basis for the regulation of expression and function of the muscarinic receptors. Treatment of chick heart cells in culture results in decreased levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription which requires concomitant mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. Site-directed mutagenesis was used to demonstrate that the single tyrosine residue in the carboxyl-terminal cytoplasmic tail of the m2 receptor is involved in agonist-induced down-regulation but not sequestration. Activation of heterologous receptors in chick heart cells can also regulate mAChR mRNA levels. A cAMP-regulated luciferase reporter gene, has been used to demonstrate that the m4 receptor preferentially couples to Gi alpha-2 or Go alpha over Gi alpha-1 or Gi alpha-3 to mediate inhibition of adenylyl cyclase activity. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we are beginning to use the method of targeted gene disruption by homologous recombination to generate mice deficient in specific receptor subtypes.

Protein Restriction Specifically Decreases the Abundance of Serum Albumin and Transthyretin Nuclear Transcripts in Rat Liver

The expression of the genes for serum albumin and several other plasma proteins is decreased in animals consuming inadequate amounts of dietary protein. To define the specificity of this phenomenon, we examined the effect of dietary protein restriction on the abundance of the mRNA for nine genes in rat liver. The results of this and previous studies indicate that genes in liver can be divided into two classes based on their response to protein restriction. Group I genes (albumin, transthyretin, carbamyl phosphate synthetase-I, class I alcohol dehydrogenase, insulin-like growth factor-I) exhibit decreased expression in response to protein restriction. In contrast, the expression of group II genes (hypoxanthine-guanine phosphoribosyl transferase, ubiquitin, H-ferritin, insulin-like growth factor binding proteins-1, -2 and -4) is either unchanged or increased in response to protein restriction. To investigate the molecular mechanism(s) leading to the decreased level of albumin and transthyretin mRNA in protein-restricted animals, the effect of protein restriction on the abundance of albumin and transthyretin nuclear transcripts was examined. The results demonstrated that protein restriction specifically decreased the abundance of albumin and transthyretin nuclear transcripts, indicating that the reduction in mRNA levels is caused at least partly by a decrease in gene transcription.

Inhibition of protein synthesis in baby-hamster kidney cells blocks oxysterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA at a post-transcriptional level.

The effects of the protein-synthesis inhibitor cycloheximide on 25-hydroxycholesterol-mediated suppression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA levels were evaluated in the baby-hamster kidney cell line C100. Cells cultured in medium supplemented with delipidized fetal bovine serum and 25 microM lovastatin for 12-24 h had a 5-fold higher level of HMG-CoA reductase mRNA than cells grown in medium supplemented with non-delipidized fetal bovine serum (FBS). The higher level was due to increased transcription, as determined by run-on assays with isolated nuclei. Addition of 25-hydroxycholesterol to lovastatin-treated cells lowered HMG-CoA reductase mRNA levels within 4 h of treatment to those of cells grown in FBS-supplemented medium. This decrease was due in part to a decrease in gene transcription. Cycloheximide added in conjunction with 25-hydroxycholesterol to lovastatin-treated cells blocked the suppression of mRNA levels, but did not block oxysterol-mediated suppression of transcription. In addition, cycloheximide added to cells grown in FBS-supplemented medium rapidly increased mRNA levels by 10-fold relative to untreated cells, with no comparable increase in transcription. No comparable increase in either the mRNA level or rate of transcription for beta-actin was observed under such conditions. These results indicate that cycloheximide specifically stabilizes HMG-CoA reductase mRNA in the presence of oxysterols and suggests that continuous synthesis of a short lived protein regulator is required for oxysterol-mediated suppression of HMG-CoA reductase mRNA at a post-transcriptional level.

Abnormal regulation of low density lipoprotein-sensitive events in a cholesterol transport mutant

We have isolated and characterized Chinese hamster ovary cell mutants defective in the intracellular transport of low density lipoprotein (LDL)-derived cholesterol (Dahl, N. K., Reed, K. L., Daunais, M. A., Faust, J. R., and Liscum, L. (1992) J. Biol. Chem. 267, 4889-4896). Mutant 2-2, which exhibits a cholesterol transport defect indistinguishable from the Niemann-Pick C phenotype, shows impaired but not absent LDL-mediated suppression of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. In parental cells, LDL suppression of HMG-CoA reductase is modulated by two mechanisms, decreased gene transcription and accelerated protein turnover. Using the chimeric protein HMGal as a reporter protein for LDL-mediated turnover and Northern blot analysis to monitor HMG-CoA reductase mRNA levels, we have dissected the contributions of these two regulatory responses to LDL-mediated suppression of HMG-CoA reductase activity. Kinetic modeling using the kinlsq program showed the following. Mutant 2-2 exhibits normal LDL-mediated acceleration of HMGal degradation, coupled with relatively abnormal regulation of mRNA. This suggests that the LDL-cholesterol signaling pathway to the nucleus is defective relative to the signal that results in HMG-CoA reductase turnover. In addition, LDL-mediated acceleration of HMGal turnover occurs well before LDL stimulation of cholesterol esterification in mutant 2-2, whereas these events occur synchronously in the parental cell line. This suggests that more than one pathway or mechanism exists for LDL-cholesterol signaling to the endoplasmic reticulum.

Biphasic glucocorticoid regulation of pulmonary SP-A: characterization of inhibitory process.

Pulmonary surfactant, which is necessary for normal lung function, is under both developmental and hormonal regulation. Glucocorticoids induce all components of surfactant and have a unique biphasic effect on surfactant protein A (SP-A), either stimulating or inhibiting accumulation in cultured fetal lung depending on dose and time of exposure. In this study we further characterized glucocorticoid inhibition of SP-A in cultured explants of human fetal lung. Decreased content of SP-A mRNA was the dominant response to dexamethasone added either early or later during culture. Inhibition occurred at < or = 1 nM dexamethasone on prolonged exposure, was blocked by RU 486, and was observed with other glucocorticoids but not sex steroids. When cortisol was removed from the culture medium, inhibition was rapidly reversed. The immediate inhibitory effect of 100 nM dexamethasone on SP-A mRNA content was completely blocked in the presence of cycloheximide. SP-A gene transcription, measured by nuclear elongation assay, was decreased by 60% after 4- to 8-h exposure to 100 nM dexamethasone. Stability of SP-A mRNA, determined both by addition of actinomycin D and by label-chase experiments, was transiently decreased immediately after adding dexamethasone (t1/2 approximately 3 h). In tissue treated with dexamethasone for > or = 8 h the stability of SP-A mRNA in control and treated explants was not different (t1/2 approximately 8 h). Our findings indicate that inhibition of SP-A is the dominant response to glucocorticoid. This effect is receptor mediated and apparently involves induction of a labile protein(s) that decreases gene transcription and transiently reduces mRNA stability.

Regulation of expression and function of muscarinic receptors

The regulation of expression and function of the muscarinic acetylcholine receptor has been studied using several different systems. The role of glycosylation of the m2 receptor was examined by removal of glycosylation sites using site-directed mutagenesis followed by expression in stably transfected cells. The results demonstrated that glycosylation was not required for the synthesis and appearance of the receptors on the cell surface or for the coupling of the receptors to inhibition of adenylyl cyclase activity. Site-directed mutagenesis also was used to demonstrate that the single cysteine in the carboxy terminal domain of the m2 receptor was not required for receptor function, thus rendering unlikely a model suggesting a requirement for palmitoylation of this cysteine in receptor function. The muscarinic receptors expressed in embryonic chick heart were identified by molecular cloning. Two genes were initially identified which are expressed in chick heart and correspond to the chick m2 and m4 receptors. Experiments using the polymerase chain reaction to identify low abundance mRNAs indicate that at least one addition receptor gene is expressed in chick heart. In cell culture, activation of the muscarinic receptors decreases the levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription due to both mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. The elucidation of the factors which regulate the expression and function of muscarinic acetylcholine receptors (mAChR) is of obvious importance in understanding the mechanisms underlying cholinergic transmission. In this chapter, we will describe studies on the expression and function of wild type and mutant muscarinic receptors, the molecular characterization of mAChR expressed in chick heart, and the regulation of mAChR gene expression in response to muscarinic receptor activation.

Apolipoprotein B mRNA abundance is decreased by eicosapentaenoic acid in CaCo-2 cells. Effect on the synthesis and secretion of apolipoprotein B.

The regulation of apolipoprotein B (apo B) metabolism by eicosapentaenoic acid was investigated in CaCo-2 cells. Cells cultured on semipermeable membranes that separated an upper from a lower well were incubated for 48 hours with albumin alone or 1 mM eicosapentaenoic acid or oleic acid attached to albumin (4:1, mol/mol). Compared with cells incubated with oleic acid, cells incubated with eicosapentaenoic acid synthesized and secreted less [3H]glycerol-labeled triglycerides. Although both fatty acids increased cellular triglyceride mass compared with control cells, less triglycerides accumulated in cells incubated with the n-3 fatty acid. The secretion of triglyceride and apo B mass by cells incubated with eicosapentaenoic acid was less than that observed by cells incubated with oleate. The amount of apo B mass within cells, however, was not altered by either of the fatty acids and was similar to amounts found in control cells. Apo B mRNA abundance was decreased fourfold in cells exposed for 48 hours to eicosapentaenoic acid. In contrast, in cells incubated with oleic acid, apo B mRNA levels were not significantly altered. Pulse-chase experiments were performed to investigate the regulation of apo B synthesis and degradation by the fatty acids. In cells incubated with eicosapentaenoic acid, the synthesis and basolateral secretion of newly synthesized apo B-100 and apo B-48 were significantly less compared with control cells or cells incubated with oleic acid. In contrast, the synthesis and secretion of newly synthesized apo B in cells exposed to oleic acid were similar to control cells. Rates of apo A-I synthesis were similar in cells incubated with either of the fatty acids. Compared with control cells and cells incubated with eicosapentaenoic acid, the residence time of labeled apo B in cells incubated with oleic acid was prolonged. The percentage of newly synthesized apo B that was degraded was less in cells incubated with oleic acid. In contrast, residence times and the percentages of apo A-I and apo B-48 degraded were similar in control cells and cells incubated with the fatty acids. Thus, in CaCo-2 cells, compared with the effects of oleic acid, eicosapentaenoic acid impairs triglyceride transport in part by inhibiting apo B synthesis and secretion. The inhibition of apo B synthesis by eicosapentaenoic acid may be related to a decrease in gene transcription or a decrease in mRNA stability, as apo B mRNA levels were significantly decreased in cells incubated with this fatty acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of lipopolysaccharide-induced interleukin-1 beta mRNA expression in mouse macrophages by oxidized low density lipoprotein.

Recent studies have demonstrated the expression of messenger RNA (mRNA) for several cytokines within atherosclerotic arteries. Since cytokines have been shown to modulate functions of cultured arterial wall cells in a manner that could influence atherogenesis, this suggests that factors that modulate cytokine production would influence the atherosclerotic process. To examine whether lipoproteins can modulate cytokine production, the effect of lipoproteins on mouse macrophage interleukin-1 beta (IL-1 beta) mRNA expression was examined by dot blot and Northern blot analyses. Low density lipoprotein (LDL), acetylated-LDL, or malondialdehyde-LDL did not induce IL-1 beta mRNA expression or affect the expression in response to lipopolysaccharide (LPS). Similarly, copper ion-oxidized LDL did not stimulate the production of IL-1 beta mRNA. However, oxidized LDL inhibited the LPS-induced expression in a concentration- and time-dependent manner with a maximum inhibition (greater than 90%) observed after a 2.5 h preincubation with 25 micrograms protein/ml. These conditions did not affect protein synthesis or phagocytosis and the inhibition was partially reversible after 24 h, which together suggest that the inhibition was not due to cell death. An inhibition of IL-1 alpha and IL-6 mRNA expression was also observed while there was no change in gamma-actin mRNA levels. The level of inhibition of IL-1 beta mRNA was dependent upon the extent of LDL oxidation, but did not correlate with recognition by the scavenger receptor. A non-receptor pathway was supported by two lines of evidence: 1) the inhibition could be reproduced with a lipid extract, and 2) oxidized LDL also inhibited scavenger receptor negative THP-1 cell IL-1 beta mRNA expression. Finally, oxidized LDL had no effect on the turnover of IL-1 beta mRNA, suggesting that the decreased accumulation of IL-1 beta mRNA is due to a decrease in gene transcription. Together these studies suggest that as macrophages become foam cells their immune responsiveness is attenuated.

Modulation of complement gene expression by glucocorticoids

The addition of dexamethasone, prednisolone or cortisol (in order of efficacy) to human monocytes in culture produced dose-related increases in the synthesis rates of the complement components C1 inhibitor (C1-inh), factor B (B) and C2. In contrast, concentrations of C3 and lysozyme in the culture supernatants were decreased. Indomethacin stimulated synthesis of C1-inh, C2 and B, but had little effect on synthesis of C3 or lysozyme. The simultaneous addition of cycloheximide (2.5 micrograms/ml) abrogated the effects of dexamethasone on synthesis of C2, B and C1-inh, but the effect of indomethacin on the synthesis of these components was unchanged. These data suggest that protein synthesis is required for the effects of glucocorticoids on the synthesis of C2, B and C1-inh to occur. Dexamethasone and indomethacin increased the abundances of C1-inh mRNA, B mRNA and C2 mRNA in parallel with changes in the synthesis rates of these proteins. The changes in mRNA abundance were not transcriptional, but were shown to be due to increased mRNA stability. In contrast, dexamethasone decreased the expression of C3 and lysozyme by decreasing the rate of transcription of these genes. Indomethacin had no effect on transcription of the C3 and lysozyme genes. The half-lives of C3 mRNA, lysozyme mRNA and actin mRNA were not altered by dexamethasone or indomethacin. It is concluded that the effects of glucocorticoids on monocyte synthesis of C2, B and C1-inh are due to increased mRNA stability and may be related to inhibition of prostaglandin synthesis, as these effects are similar to those produced by indomethacin. The effects of dexamethasone on the synthesis of C3 and lysozyme differ from those on C2, B and C1-inh as they depend upon a decrease in gene transcription, which is not affected by indomethacin.

Dietary cholesterol, membrane cholesterol and cholesterol synthesis

After describing the main steps of cholesterol biosynthesis the author recalls that the cholesterogenesis rate is feedback-induced by dietary cholesterol and examines the various processes of modulation. Hydroxy-3-methylglutaryl (HMG) CoA reductase. the key rate-limiting enzyme, is a 97 kDa endoplasmic reticulum glycoprotein, anchored 7-fold in this membrane. The N-terminal membrane-bound domain plays a fundamental role in the modulation of reductase activity. This modulation is essentially mediated by decreased gene transcription and enhanced degradation of the protein. The possible modulation by a bicyclic cascade system involving phosphorylation (inactivation) and dephosphorylation (activation) of reductase does not seem to play an essential role in vivo. Finally, recent data show that the lipid composition (C/P molar ratio) of some reticular membranes (fibroblasts, for example) can strongly modulate the activity of this ubiquitous enzyme.

Regulation of ornithine decarboxylase in murine erythroleukemic cells by N,N'-diacetyl-1,6-hexanediamine and N-acetyl-1,6-hexanediamine.

The levels at which the ornithine decarboxylase gene is regulated in murine erythroleukemic cells treated with N,N'-hexamethylene bisacetamide (N,N' diacetyl-1,6-hexanediamine) and with N-acetyl-1,6-hexanediamine, the monoacetylated catabolite of this inducer of erythrodifferentiation, have been investigated. Although these two molecules are structurally related and both cause a decrease in ornithine decarboxylase activity, they did not similarly affect ornithine decarboxylase mRNA. In the presence of 4 mM N,N'-hexamethylenebisacetamide, at the concentration generally used to induce differentiation in these cells, a decreased steady-state level of ornithine decarboxylase mRNA, due to decreased gene transcription, was observed. In the presence of N-acetyl-1,6-hexanediamine the decrease in enzyme activity was shown to be associated with a decrease in the half-life of the enzyme in the absence of a change in gene transcription. These results show that in proliferating cultured cells changes in ornithine-decarboxylase-gene transcription can be uncoupled from changes in cell growth and that N-acetyl-1,6-hexanediamine only regulates ornithine decarboxylase expression post-transcriptionally.

Triiodothyronine Rapidly Decreases Transcription of the Thyrotropin Subunit Genes in Thyrotropic Tumor Explants*

We have investigated the direct effect of the thyroid hormone T3 on the TSH subunit genes in tissue explants. Minces of TtT 97 thyrotropic tumor were treated with 5 nM T3 for varying periods of time. Nuclei were then isolated from the tumor cells and allowed to continue RNA synthesis in the presence of [alpha-32P]UTP. Newly synthesized RNA sequences were quantified by hybridization to immobilized cloned cDNAs containing sequences specific for either TSH beta or alpha-subunit mRNA. Basal TSH beta and alpha-subunit mRNA synthesis rates were both approximately 300-400 parts/million, the same as in vivo values. After 15 min of T3 treatment, TSH beta mRNA synthesis was significantly decreased by 42% and was maximally decreased by 95% after 1 or more hours of T3. Synthesis of alpha-subunit mRNA was decreased by 38% after 30 min of T3 treatment and by 78% after 1 h or more of T3. The suppressive effects of T3 on transcription correlated with the time course of T3 binding to its nuclear receptor. These changes are quantitatively similar to those observed after in vivo T3 treatment. Decreases in mRNA synthesis preceded significant decreases in tissue steady state mRNA levels or subunit protein levels. The presence of the protein synthesis inhibitor cycloheximide (25 micrograms/ml) during a 4-h incubation with T3 did not change the T3-mediated decreases in TSH beta or alpha-subunit mRNA synthesis or the decreases in cellular mRNA levels. Therefore, T3 can act directly on the thyrotrope to suppress TSH beta and alpha-subunit mRNA synthesis, and protein synthesis is not necessary for the T3-mediated decreases in gene transcription. The data suggest that T3 may act directly at the level of the TSH subunit genes to modulate their expression.

Transcription and replication of influenza virus RNA

Methods are described for measuring the amounts of virus-specific polyadenylated (A+) cRNA, nonpolyadenylated (A−) cRNA, and virus genome RNA (vRNA) in nucleus and cytoplasm of cells infected with influenza virus. The amounts of A(+) cRNA to individual virus genes accumulated during infection at different rates, not related to gene molecular weight, indicating transcriptional control. The pattern of accumulation of each gene transcript was the same in the nucleus as in the cytoplasm. Addition of cycloheximide during infection abolished the transcriptional control and resulted in linear accumulation of each gene transcript. In cells treated with 0.1 μg/ml actinomycin D, which blocked late but not early virus-specific protein synthesis, there was a significant accumulation of all gene transcripts in the nucleus and a relative decrease in transcripts of genes coding for late proteins (M and HA) in the cytoplasm. Analysis of A(−) cRNA and vRNA accumulations in infected cells showed that the A(−) cRNA synthesis precedes vRNA synthesis and net accumulation of A(−) cRNA ceased by 3 hr postinfection (pi). Significant amounts of vRNA in excess of input vRNA were detected by about 2 hr pi and continued to accumulate in both nucleus and cytoplasm at least up to 6 hr pi. Protein synthesis was required for A(−) cRNA production, since in the presence of cycloheximide very little A(−) cRNA, and no vRNA, could be detected. Actinomycin D (0.1 μg/ml) reduced the amount of A(−) cRNA, and completely inhibited the increase in vRNA which occurred from 2 hr pi in normal infection.