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Results for “RNA Stability”

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PubMed1992

Superinduction of c-fos gene expression by estrogen in cultured guinea-pig endometrial cells requires priming by a cycloheximide-dependent mechanism.

The c-fos gene expression is rapidly induced by various mitogenic agents and protein synthesis inhibitors in many cell types. Estradiol-17 beta can induce c-fos gene expression in breast cancer cell lines and in the uterus in vivo, but not in cultured guinea-pig endometrial cells. Using this model, we investigated whether a protein synthesis inhibitor, cycloheximide, could induce the c-fos gene and permit a superinduction by estrogens. In the presence of cycloheximide (10 micrograms/ml), protein synthesis was inhibited at 95% within the first hour. From 190 min after the addition of estradiol-17 beta or diethylstilbestrol (10(-8) M) and cycloheximide (10 micrograms/ml), there was a significant increase (ranging from 3- to 5-fold) of the c-fos messenger RNA level (2.2 kilobase in size), compared with the level in cells treated with cycloheximide alone. Nonestrogenic steroid hormones and estradiol-17 alpha were unable to induce c-fos gene expression in the presence of cycloheximide. The effect of estradiol-17 beta observed in the presence of cycloheximide was completely abolished by 4-hydroxy-tamoxifen or by Ly 156758 or by ICI 164384 (10(-6) M). The c-fos mRNAs were rather stable in cells treated with cycloheximide for 2 h (half-life = 51 +/- 6 min) and there was no further increase in the c-fos messenger RNA stability after the addition of cycloheximide plus estradiol-17 beta (half-life = 40 +/- 3 min). The overall results suggest a response at the transcriptional level. In conclusion, cycloheximide transmits activating signals to the c-fos gene which act as priming elements to allow the estrogen action in cultured guinea-pig endometrial cells.

Androstenedione↗

PubMed1995

mRNA decay mediated by two distinct AU-rich elements from c-fos and granulocyte-macrophage colony-stimulating factor transcripts: different deadenylation kinetics and uncoupling from translation.

Poly(A) tail removal is a critical first step in the decay pathway for many yeast and mammalian mRNAs. Poly(A) shortening rates can be regulated by cis-acting sequences within the transcribed portion of mRNA, which in turn control mRNA turnover rates. The AU-rich element (ARE), found in the 3' untranslated regions of many highly labile mammalian mRNAs, is a well-established example of this type of control. It represents the most widespread RNA stability determinant among those characterized in mammalian cells. Here, we report that two structurally different AREs, the c-fos ARE and the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE, both direct rapid deadenylation as the first step in mRNA degradation, but by different kinetics. For c-fos-ARE-mediated decay, the mRNA population undergoes synchronous poly(A) shortening and is deadenylated at the same rate, implying the action of distributive or nonprocessive ribonucleolytic digestion of poly(A) tails. In contrast, the population of granulocyte-macrophage colony-stimulating factor ARE-containing mRNAs is deadenylated asynchronously, with the formation of fully deadenylated intermediates, consistent with the action of processive ribonucleolytic digestion of poly(A) tails. An important general implication of this finding is that different RNA-destabilizing elements direct deadenylation either by modulating the processivity at which a single RNase functions or by recruiting kinetically distinct RNases. We have also employed targeted inhibition of translation initiation to demonstrate that the RNA-destabilizing function of both AREs can be uncoupled from translation by ribosomes. In addition, a blockade of ongoing transcription has been used to further probe the functional similarities and distinctions of these two AREs. Our data suggest that the two AREs are targets of two distinct mRNA decay pathways. A general model for ARE-mediated mRNA degradation involving a potential role for certain heterogeneous nuclear ribonucleoproteins and ARE-binding proteins is proposed.

3T3 Cells↗

PubMed1987

Organization and expression of the Plasmodium knowlesi circumsporozoite antigen gene.

To investigate the mechanisms regulating the stage specific expression of the Plasmodium knowlesi circumsporozoite (CS) antigen gene, the sporozoite genomic DNA copy of the CS gene has been isolated and compared with the blood stage genomic DNA and the sporozoite cDNA copies. The genomic DNA sequences of the two developmental stages are identical across 4 kilobase pairs of the chromosome containing the entire CS gene transcriptional unit. From restriction enzyme mapping no DNA rearrangements over 15 kilobase pairs of the chromosome containing the CS gene appear to be involved in its stage-specific expression and its regulation appears to be at the level of transcription or RNA stability. S1 nuclease and primer extension transcript mapping studies suggest that the CS mRNA has multiple start sites, that the leader sequence is devoid of introns, and is approximately 270 bases long. Consensus eukaryotic TATA and CAAT box sequences and potential regulatory elements, including sequences highly homologous to the reiterated and core enhancer sequences of SV40 precede the gene.

Animals↗

PubMed1996

Structure and characterization of the human tissue inhibitor of metalloproteinases-2 gene.

We report here the characterization of the human tissue inhibitor of metalloproteinases-2 (TIMP-2) gene. The gene is 83 kilobase pairs (kb) long with exon-intron splicing sites located in preserved positions among the three members of the TIMP family. A 2.6-kb genomic DNA fragment flanking the 5'-end of the gene contains several regulatory elements including five Sp1, two AP-2, one AP-1, and three PEA-3 binding sites. Despite the presence of a complete AP-1 consensus at position -281, the promoter did not respond to 12-O-tetradecanoylphorbol-13-acetate treatment. However, 12-O-tetradecanoylphorbol-13-acetate response was generated by insertion of a similar AP-1 consensus at position -71, indicating the importance of the positioning of this motif. The promoter contains a typical CpG island; however, methylation of this island did not seem to influence gene expression. Analysis of the 3'-end of the gene revealed that the two mRNAs for TIMP-2 (1.2 and 3.8 kb) differ by the selection of their polyadenylation signal sites, but selection of these sites does not affect RNA stability. In summary, the TIMP-2 gene has several features observed in housekeeping genes, and differs significantly from TIMP-1 and TIMP-3 genes. These differences are likely to explain the specific roles that these inhibitors play in the regulation of matrix metalloproteinases.

Amino Acid Sequence↗

PubMed2007

Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases.

PURPOSE: Long QT Syndrome, Marfan Syndrome, hypertrophic and dilated cardiomyopathy are caused by mutations in large, multi-exon genes that are principally expressed in cardiovascular tissues. Genetic testing for these disorders is labor-intensive and expensive. We sought to develop a more rapid, comprehensive, and cost-effective approach. METHODS: Paired whole blood samples were collected into tubes with or without an RNA-preserving solution, and harvested for whole blood RNA or leukocyte DNA, respectively. Large overlapping cDNA fragments from KCNQ1 and KCNH2 (Long QT Syndrome), MYBPC3 (hypertrophic and dilated cardiomyopathy), or FBN1 (Marfan Syndrome) were amplified from RNA and directly sequenced. Variants were confirmed in leukocyte DNA. RESULTS: All 4 transcripts were amplified and sequenced from whole blood mRNA. Six known and 2 novel mutations were first identified from RNA of 10 probands, and later confirmed in genomic DNA, at considerable savings in time and cost. In one patient with MFS, RNA sequencing directly identified a splicing mutation. Results from RNA and DNA were concordant for single nucleotide polymorphisms at the same loci. CONCLUSION: Taking advantage of new whole blood RNA stabilization methods, we have designed a cost-effective, comprehensive method for mutation detection that should significantly facilitate clinical genetic testing in four lethal cardiovascular disorders.

Adolescent↗

PubMed2004

RNA integrity as a quality indicator during the first steps of RNP purifications : a comparison of yeast lysis methods.

BACKGROUND: The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor. RESULTS: We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins. CONCLUSION: We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells.

Cell Fractionation↗

PubMed2005

Regulation of finP transcription by DNA adenine methylation in the virulence plasmid of Salmonella enterica.

DNA adenine methylase (Dam(-)) mutants of Salmonella enterica serovar Typhimurium contain reduced levels of FinP RNA encoded on the virulence plasmid. Dam methylation appears to regulate finP transcription, rather than FinP RNA stability or turnover. The finP promoter includes canonical -10 and -35 modules and depends on the sigma(70) factor. Regulation of finP transcription by Dam methylation does not require DNA sequences upstream from the -35 module, indicating that Dam acts at the promoter itself or downstream. Unexpectedly, a GATC site overlapping with the -10 module is likewise dispensable for Dam-mediated regulation. These observations indicate that Dam methylation regulates finP transcription indirectly and suggest the involvement of a host factor(s) responsive to the Dam methylation state of the cell. We provide evidence that one such factor is the nucleoid protein H-NS, which acts as a repressor of finP transcription in a Dam(-) background. H-NS also restrains transcription of the overlapping traJ gene, albeit in a Dam-independent fashion. Hence, the decreased FinP RNA content found in Dam(-) hosts of S. enterica appears to result from H-NS-mediated repression of finP transcription.

Adenine↗

PubMed2006

Evaluation of FTA paper and phenol for storage, extraction and molecular characterization of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is an important poultry pathogen and is distributed world wide that can cause immune suppression and lesions of the bursa of Fabricius. The main component of the virus, VP2, is not only responsible for the bird's immune response, but is important for the molecular identification of this virus as well. The nucleic acid of the virus must be adequately preserved to be analyzed by reverse-transcriptase PCR (RT-PCR) and sequenced for the molecular characterization of the field strain. Phenol inactivation has been the standard for IBDV tissue collection and international shipment; however, there have been some reports of interference with molecular detection capabilities when using phenol. Phenol is also a hazardous chemical and must be handled and shipped carefully. The ability to use the Flinders Technology Associates filter paper (FTA card) for inactivation of several avian pathogens has been proven previously, however no work has been published on its use in IBDV nucleic acid detection. Bursas from experimentally infected birds was imprinted on FTA cards, and then placed in phenol. Samples were evaluated and compared based on molecular detection capabilities between the two inactivation methods. The nucleic acid of the virus was detected in 85% of the FTA card inactivated samples compared to 71% in the phenol inactivated samples. Sequence analysis was performed on samples inactivated by both methods and no differences were found. When comparing the RNA stability at different temperatures, euthanized IBDV infected birds were held at two different temperatures before sampling. No differences were detected for FTA sampling; however, for tissues in phenol the nucleic acid was only detectable up to 2 h post-mortem in the tissues held at 4 degrees C prior to sampling. These findings indicate that the FTA card is an efficient and reliable alternative collection method for molecular detection and characterization of IBDV.

Animals↗

PubMed1993

Repression of the herpes simplex virus 1 alpha 4 gene by its gene product occurs within the context of the viral genome and is associated with all three identified cognate sites.

The infected cell protein 4 (ICP-4), the major regulatory protein encoded by the a4 gene of the herpes simplex virus 1, binds two sites (alpha 4-1 proximal, alpha 4-1 distal) at the 5'-untranscribed domain and at the transcription initiation site (alpha 4-2) of the alpha 4 gene. Chimeric genes consisting of the 5'-untranscribed and transcribed noncoding domains of the alpha 4 gene fused to the coding sequences of the thymidine kinase gene were mutagenized to abolish binding of ICP-4 by substitution of bases, including the guanines whose methylation interferes with binding of the protein, and recombined into the viral genome. The cytoplasmic RNAs extracted from infected cells treated with cycloheximide, from untreated infected cells maintained for 4 or 8 hr, and from cells infected first with a virus deleted in the alpha 22 gene and 3 hr later with the test viruses were tested in RNase protection assay for amounts of the chimeric gene RNA relative to amounts of alpha 22 gene RNA. We report the following: (i) Mutation of the alpha 4-2 binding site resulted in a 5-to 6-fold higher accumulation of chimeric gene RNA at 4 hr and as much as 15-fold higher accumulation by 8 hr after infection. (ii) Mutations of alpha 4-1 sites by themselves had no effect on RNA accumulation. However, mutagenesis of all three sites significantly increased mRNA amounts above the levels seen in cells infected with alpha 4-2 site mutants. (iii) The mutations have no effect on accumulation of alpha 4 mRNA in the absence of ICP-4 synthesis and, therefore, the mutations had no effect on RNA stability or transcription rate. (iv) Accumulation of alpha 4 mRNA relative to that of alpha 22 mRNA is highest in the presence of cycloheximide and decreases with time after infection. We conclude that ICP-4 autoregulates the transcription of its own gene in infected cells and that binding of ICP-4 to three sites in its promoter is additive in its effects on this process.

Animals↗

PubMed1994

Ontogeny and regulation of platelet-derived growth factor gene expression in distal fetal rat lung epithelial cells.

Using flow cytometry, thymidine uptake into DNA, and expression of two growth-related genes, histone 3 and c-myc, we found an increase in the proportion of distal lung epithelial cells in the G0/G1 phase of the cell cycle with advancing gestation. Since our previous studies had demonstrated that platelet-derived growth factor (PDGF) is essential for the progression of these cells from the G0/G1 to the S phase of cell cycle, we investigated the gene and protein expression of PDGF-related genes (PDGF-A, PDGF-B, alpha-receptor, and beta-receptor) in distal fetal lung epithelial cells. The cells transcribed all the PDGF-related genes and translated the PDGF-A and PDGF-B mRNAs into protein, as demonstrated by immunocytochemistry and immunoprecipitation. To explore an autocrine role for PDGF in distal fetal lung epithelial cells, intervention studies using PDGF-A and -B chain-specific antisense oligodeoxynucleotides (ODN) were carried out. Antisense PDGF-B ODN, but not antisense PDGF-A ODN, significantly reduced the DNA synthesis of these cells. The inhibitory effect of antisense PDGF-B ODN on DNA synthesis was reversed by the addition of exogenous PDGF-BB, which supports an autocrine role in the DNA synthesis of these cells. We also examined the expression of PDGF genes in distal fetal lung epithelial cells during late gestation. PDGF-A chain and beta-receptor gene expressions declined with advancing gestation, whereas expression of message for PDGF-B chain and alpha-receptor increased. The increases in message for PDGF-B chain and alpha-receptor with advancing gestation were due to a greater rate of transcription, whereas the developmental decrease of PDGF-A chain and beta-receptor mRNAs was caused by a decrease in RNA stability. Taken together with the ODN data, these results suggest that the G0/G1 cell cycle arrest of distal lung epithelial cells during late fetal gestation is due to a decrease in PDGF beta-receptor expression by the cells.

Animals↗

PubMed1995

Reverse transcriptase: mediator of genomic plasticity.

Reverse transcription has been an important mediator of genomic change. This influence dates back more than three billion years, when the RNA genome was converted into the DNA genome. While the current cellular role(s) of reverse transcriptase are not yet completely understood, it has become clear over the last few years that this enzyme is still responsible for generating significant genomic change and that its activities are one of the driving forces of evolution. Reverse transcriptase generates, for example, extra gene copies (retrogenes), using as a template mature messenger RNAs. Such retrogenes do not always end up as nonfunctional pseudogenes but form, after reinsertion into the genome, new unions with resident promoter elements that may alter the gene's temporal and/or spatial expression levels. More frequently, reverse transcriptase produces copies of nonmessenger RNAs, such as small nuclear or cytoplasmic RNAs. Extremely high copy numbers can be generated by this process. The resulting reinserted DNA copies are therefore referred to as short interspersed repetitive elements (SINEs). SINEs have long been considered selfish DNA, littering the genome via exponential propagation but not contributing to the host's fitness. Many SINEs, however, can give rise to novel genes encoding small RNAs, and are the migrant carriers of numerous control elements and sequence motifs that can equip resident genes with novel regulatory elements [Brosius J. and Gould S.J., Proc Natl Acad Sci USA 89, 10706-10710, 1992]. Retrosequences, such as SINEs and portions of retroelements (e.g., long terminal repeats, LTRs), are capable of donating sequence motifs for nucleosome positioning, DNA methylation, transcriptional enhancers and silencers, poly(A) addition sequences, determinants of RNA stability or transport, splice sites, and even amino acid codons for incorporation into open reading frames as novel protein domains. Retroposition can therefore be considered as a major pacemaker for evolution (including speciation). Retroposons, with their unique properties and actions, form the molecular basis of important evolutionary concepts, such as exaptation [Gould S.J. and Vrba E., Paleobiology 8, 4-15, 1982] and punctuated equilibrium [Elredge N. and Gould S.J. in Schopf T.J.M. (ed). Models in Paleobiology. Freeman, Cooper, San Francisco, 1972, pp. 82-115].

Animals↗

PubMed1992

Processing of a polycistronic mRNA requires a 5' cis element and active translation.

We have characterized a major processed species of mRNA in the his operon of Salmonella typhimurium. In vivo and in vitro analyses of the his transcripts from wild-type and mutant strains using S1 nuclease protection assays, measurements of RNA stability, deletion mapping, gel retardation, and in vitro translation assays demonstrate that the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis-acting element and translation of the cistron immediately downstream of the 5' end of the processed species. The cistrons contained in this segment are also independently transcribed from an internal promoter which is maximally active in the absence of readthrough transcription from the primary promoter.

Base Sequence↗

PubMed1999

The internal equilibrium of the hairpin ribozyme: temperature, ion and pH effects.

The hairpin ribozyme reversibly cleaves phosphodiesters of RNA substrates to generate products with 5' hydroxyl and 2',3'-cyclic phosphate termini. We previously found that the rate constant for ligation is tenfold faster than the rate constant for cleavage under standard conditions. The hammerhead ribozyme catalyzes the same reactions but is reported to favor cleavage relative to ligation by more than 100-fold under the same conditions. To explore the basis for this difference, we examined the influence of temperature, ions and pH on the hairpin ribozyme internal equilibrium. Under the same conditions, the loss of entropy associated with ligation is less for the hairpin than for the hammerhead ribozyme, consistent with the notion that a more rigid hairpin structure undergoes a smaller decrease in dynamics upon ligation than the more flexible hammerhead structure. Increased salt and reduced temperature shift the equilibrium toward ligation while pH has little effect, suggesting that conditions that stabilize RNA structure tend to promote ligation. The hairpin ribozyme appears to take up at least one tri- or divalent cation or two monovalent cations upon ligation. The efficiency with which different cations promote ligation depends strongly on valence and, less strongly, on ionic radius or electronegativity. This pattern of cation selectivity suggests that cations promote ligation through delocalized electrostatic shielding, perhaps interacting with a region of especially high charge density in the ligated ribozyme. Changes in ionic conditions produce large but compensating changes in enthalpy and entropy for cleavage and ligation. Thus, in addition to any increase in ribozyme dynamics associated with cleavage, re-organization of associated cations contributes significantly to hairpin ribozyme thermodynamics.

Base Sequence↗

PubMed1993

Differential expression of the partially duplicated chloroplast S10 ribosomal protein operon.

The chloroplast S10 ribosomal protein operon is partially duplicated in many plants because it initiates within the inverted repeat of the circular chloroplast genome. In spinach, the complete S10 operon (S10B) spans the junction between inverted repeat B (IRB) and the large single-copy (LSC) region. The S10 operon is partially duplicated in the inverted repeat A (IRA), but the sequence of S10A completely diverges from S10B at the junction of S10A and the LSC region. The DNA sequence shared by S10A and S10B includes trnI1, the rpl23 pseudogene (rpl23 psi), the intron-containing rpl2 and rps19, which is truncated in S10A at the S10A/LSC junction (rps19'). Transcription of rps19' from the promoter region of S10A could result in the synthesis of a mutant S19 protein. Analysis of RNA accumulation and run-on transcription from S10A and S10B using unique probes from the S10A/LSC and S10B/LSC junctions reveals that expression of S10A is reduced. The difference in S10A and S10B expression appears to be the result of reduced transcription from S10A, rather than differences in RNA stability. Transcription of S10B can initiate at three distinct promoter regions, P1, P2 and P3, which map closely to transcripts detected by S1 nuclease analysis. P1 is located upstream of trnI1 and has the highest transcription initiation frequency in vitro of the three promoter regions. The DNA sequence of P1 is most similar to the chloroplast promoter consensus DNA sequence. Interference by the highly and convergently transcribed psbA-trnH1 operon is considered as a mechanism to explain the reduced activity of the S10A promoters.

Chloroplasts↗

PubMed1997

A nodule-specific gene family from Alnus glutinosa encodes glycine- and histidine-rich proteins expressed in the early stages of actinorhizal nodule development.

Two cDNAs representing different members (agNt84 and ag164) of a gene family encoding glycine- and histidine-rich proteins have been isolated from cDNA libraries from Alnus glutinosa root nodules. Expression of the corresponding genes could only be detected in nodules. With in situ hybridization, the expression in nodules was found to occur in young, infected cells of the prefixation zone (zone 2). The encoded proteins contain putative signal peptides for targeting to the endomembrane system, sharing sequence similarity with signal peptides from plant glycine-rich proteins, among them nodulin 24, a nodule-specific protein from soybean. This similarity suggests that, analogous to nodulin-24, proteins encoded by agNt84/ag164 may be located at the interface between the host plant membrane and the matrix surrounding the endosymbiont. The 3' untranslated regions of the cDNAs contain unusual poly(AT)n stretches that may play a role in the regulation of RNA stability. The protein encoded by agNt84 cDNA was expressed in Escherichia coli as a fusion with maltose-binding protein, and was shown to have the ability to bind to a nickel-chelating resin, indicating that it may function as a metal-binding protein.

Actinomycetales↗

PubMed2002

The macrophage response to endotoxin requires platelet activating factor.

Platelet activating factor (PAF) is a key proinflammatory mediator of septic shock and is metabolized by PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of autodestructive excessive inflammatory responses such as post-injury multiple organ failure, and recombinant PAF-AH is being studied for the prevention of acute respiratory distress syndrome (ARDS). However, the potential role of PAF as an autocrine mediator of macrophage activation is unclear. We wanted to examine the role of PAF in the endotoxin- (LPS) induced macrophage response using PAF-AH. Rabbit alveolar macrophages were stimulated with LPS (10 ng/mL) with or without PAF-AH (0.1-10 microg/mL). Supernatants were collected to measure the production of tumor necrosis factor (TNF), interleukin 8 (Il-8), and prostaglandin E2 (PGE2). Cell monolayers were assessed for procoagulant activity (PCA). TNF mRNA production was determined by Northern blot and RNA stability was assessed. Evaluation of intracellular signaling pathways for LPS included western blots for phosphorylated p38 and ERK kinases and gel shift for nuclear factor-kappaB. There was a dose-response inhibition of TNF, PCA, Il-8, and PGE2 production following pretreatment with PAF-AH. Time course studies revealed effective inhibition of TNF production with administration of PAF-AH up to 2 h after LPS challenge. TNF mRNA production was inhibited, while mRNA stability was not affected. There was no effect on the phosphorylation of p38 or ERK 1 kinases; however, the nuclear translocation of NF-kappaB was inhibited. Macrophage cytokine production in response to endotoxin is PAF dependent. This effect involves the inhibition of TNF gene transcription and concomitant inhibition of NF-kappaB.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1998

Aminoglutethimide suppresses adrenocorticotropin receptor expression in the NCI-h295 adrenocortical tumor cell line.

The adrenostatic compound aminoglutethimide (AG), a potent inhibitor of the P450 side chain cleavage enzyme, is used in the treatment of ACTH-dependent or adrenal Cushing's syndrome. Recently, AG has been shown to inhibit ACTH receptor (ACTH-R) mRNA expression in ovine adrenocortical cells in a time-dependent fashion. To investigate whether ACTH-R down-regulation will also be induced in tumor cells, we studied the effect of AG on ACTH-R expression in the human NCI-h295 adrenocortical carcinoma cell line, which expresses functional ACTH receptors and produces steroids of the glucocorticoid, mineralocorticoid and androgen pathway. The cells were incubated in triplicate with increasing doses of AG (3, 30, 300 microM) which suppressed steroid secretion dose-dependently. After 48 h, cells were harvested, and total RNA was extracted, electrophoresed, blotted and hybridized with a human ACTH-R cDNA probe. In parallel experiments, after preincubation with AG the cells were stimulated with ACTH (10 nM) for 10 min and the intracellular cAMP accumulation was determined by RIA. AG significantly suppressed the baseline ACTH-R mRNA expression in a dose-dependent fashion (300 microM AG, 5+/-1%; 30 microM AG, 64+/-1%; 3 microM AG, 108+/-19% compared with control cells, 100+/-11%). The reduced ACTH-R mRNA expression was paralleled by low ACTH-induced cAMP accumulation indicating reduced expression of the ACTH-R protein. The adrenostatic compound metyrapone, an inhibitor of 11beta-hydroxylase activity, also suppressed ACTH-R mRNA expression in a similar fashion. Stimulation of the protein kinase A pathway by simultaneous incubation of ACTH (10 nM) or forskolin (10 microM) together with AG was not able to overcome the steroid biosynthesis blockade, but reversed the inhibitory effects of AG on the ACTH-R mRNA expression. Also, cortisol (12 microM) reversed the AG-induced ACTH-R mRNA expression. We conclude that AG induces profound ACTH-R down-regulation in the NCI-h295 cell line either by affecting the gene expression or by decreasing transcript accumulation via an effect on RNA stability. This novel action of AG can be reversed by stimulation of the cAMP pathway and of the glucocorticoid-mediated signal transduction cascade. As the down-regulation occurs in vitro at concentrations which are reached during treatment with AG in humans it may contribute to its therapeutic activity in adrenal disease.

Adrenal Cortex Neoplasms↗

PubMed1997

Tumour necrosis factor-alpha up-regulates decay-accelerating factor gene expression in human intestinal epithelial cells.

The increased expression of decay-accelerating factor (DAF) has been detected in intestinal epithelial cells at the inflamed mucosa. In this study, we examined the effects of tumour necrosis factor (TNF)-alpha on DAF expression in three intestinal epithelial cell lines. DAF mRNA expression was evaluated by Northern blot analysis, and DAF protein expression was analysed by biotin labelling and immunoprecipitation. TNF-alpha induced a marked increase in DAF mRNA and protein expression in HT-29, T84 and Caco-2 cells. In HT-29 cells, the effects of TNF-a on DAF mRNA accumulation were observed in a dose-dependent manner; DAF mRNA accumulation reached a maximum at 3-6 hr, and then gradually decreased. These effects of TNF-alpha required de novo protein synthesis. Messenger RNA stability studies suggested that TNF-alpha partially regulated DAF gene expression by a posttranscriptional mechanism. Moreover, the combination of TNF-alpha and interleukin (IL)-4 induced an additive increase in DAF mRNA accumulation in HT-29 and T84 cells. In human intestinal epithelial cells, TNF-alpha acts as a potent inducer of DAF mRNA expression, indicating an important role for TNF-alpha in the regulation of DAF expression at the inflamed mucosa.

Blotting, Northern↗