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At least 1,027 records · Page 57Linked to original sources
PubMed2007

GADD153 mediates celecoxib-induced apoptosis in cervical cancer cells.

Celecoxib, a selective cyclooxygenase 2 inhibitor, is known to have anti-inflammatory activity and to induce apoptosis in various types of cancer cells. Here, we examined the molecular mechanism of celecoxib-induced apoptosis in cervical cancer cell lines (HeLa, CaSki and C33A). Screening of a microarray cDNA-chip containing 225 different genes revealed that growth arrest and DNA damage inducible gene (GADD153), a transcription factor involved in apoptosis, showed the strongest differential expression following celecoxib treatment in all three cervical cancer cell lines. Notably, siRNA-induced silencing of GADD153 suppressed celecoxib-induced apoptosis in all the three cell lines, and exogenous expression of GADD153 triggered apoptosis in cervical cancer cells in the absence of other apoptotic stimuli. A luciferase reporter gene assay and mRNA stability tests revealed that expression of GADD153 was regulated at both the transcriptional and post-transcriptional levels following celecoxib treatment. The region between -649 and -249, containing an intact C/EBP-ATF binding site, was required for the basal activity and celecoxib-induced stimulation of GADD153 promoter activity. Also, mRNA stability test showed that celecoxib prolonged the half-life of GADD153 mRNA. In terms of signaling pathway, addition of the NF-kappaB inhibitor, N-tosyl-L phenylalanyl-chloromethyl ketone (TPCK), had no effect on GADD153 expression levels. Celecoxib treatment induced Bak expression, whereas cell treated with siGADD153 or TPCK showed lower levels of celecoxib-induced Bak up-regulation. These novel findings collectively suggest that GADD153 may play a key role in celecoxib-induced apoptosis in cervical cancer cells by regulating the expression of proapoptotic proteins such as Bak.

Apoptosis↗
PubMed2006

Regulation of urokinase receptor expression by protein tyrosine phosphatases.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play a major role in several physiological processes such as cell migration, proliferation, morphogenesis, and regulation of gene expression. Many of the biological activities of uPA depend on its association with uPAR. uPAR expression and its induction by uPA are regulated at the posttranscriptional level. Inhibition of protein tyrosine phosphatase-mediated dephosphorylation by sodium orthovanadate induces uPAR expression and, with uPA, additively induces cell surface uPAR expression. Sodium orthovanadate induces uPAR by increasing uPAR mRNA in a time- and concentration-dependent manner. Both sodium orthovanadate and uPA induce uPAR mRNA stability, indicating that dephosphorylation could contribute to uPA-induced posttranscriptional regulation of uPAR expression. Induction of the tyrosine phosphatase SHP2 in Beas2B and H157 cells inhibits basal cell surface uPAR expression and uPA-induced uPAR expression. Sodium orthovanadate also increases uPAR expression by decreasing the interaction of a uPAR mRNA coding region sequence with phosphoglycerate kinase (PGK) as well as by enhancing the interaction between a uPAR mRNA 3' untranslated sequence with heterogeneous nuclear ribonucleoprotein C (hnRNPC). On the contrary, overexpression of SHP2 in Beas2B cells increased interaction of PGK with the uPAR mRNA coding region and inhibited hnRNPC binding to the 3' untranslated sequence. These findings confirm a novel mechanism by which uPAR expression of lung airway epithelial cells is regulated at the level of mRNA stability by inhibition of protein tyrosine phosphatase-mediated dephosphorylation of uPAR mRNA binding proteins and demonstrate that the process involves SHP2.

Cells, Cultured↗
PubMed2000

Regulation of interleukin-1beta -induced platelet-derived growth factor receptor-alpha expression in rat pulmonary myofibroblasts by p38 mitogen-activated protein kinase.

The potential role of p38 mitogen-activated protein (MAP) kinase in platelet-derived growth factor receptor-alpha (PDGF-Ralpha) gene expression was investigated using cultured rat pulmonary myofibroblasts. p38 MAP kinase was constitutively expressed in myofibroblasts and activated by interleukin (IL)-1beta. A pyridinylimidazole compound, SB203580, completely inhibited the ability of p38 MAP kinase activity to phosphorylate PHAS-1 substrate. SB203580 inhibited IL-1beta-induced up-regulation of PDGF-Ralpha mRNA and protein in a concentration-dependent manner. Other kinase inhibitors, including the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor PD98059, did not block up-regulation of PDGF-Ralpha. The IL-1beta-induced increase in the number of (125)I-PDGF-AA-binding sites at the cell surface was reduced >70% by pretreatment with SB203580. Accordingly, an enhancement of PDGF-AA-stimulated DNA synthesis following IL-1beta pretreatment was blocked >70% by SB203580. SB203580 did not affect IL-1beta-induced ERK activation, yet enhanced IL-1beta-induced JNK activation approximately 2-fold. Treatment of cells with SB203580 after inhibition of transcription by actinomycin D decreased the half-life of IL-1beta-induced PDGF-Ralpha mRNA from >4 to approximately 1.5 h. Moreover, pretreatment of cells with cycloheximide blocked induction of PDGF-Ralpha mRNA by IL-1beta, suggesting that de novo protein synthesis was required for PDGF-Ralpha mRNA stabilization. These data indicate that p38 MAP kinase regulates PDGF-Ralpha expression at the translational level by signaling the synthesis of an mRNA-stabilizing protein.

Animals↗
PubMed2004

Identification and characterization of cis-acting elements in the human and bovine PTH mRNA 3'-untranslated region.

UNLABELLED: The human PTH mRNA 3'-UTR has a cis element homologous to the rat cis-acting instability element and a more proximal element identical to the single binding element identified in bovine PTH mRNA 3'-UTR. The function of the elements was shown in vitro. INTRODUCTION: In the rat, Ca(2+) and phosphate regulate PTH mRNA stability by the interaction of trans-acting proteins with a defined cis-acting instability element in the distal region of the PTH mRNA 3'-untranslated region (UTR). This element has been characterized in the rat and is conserved in human, canine, feline, and murine 3'-UTRs but not in bovine and porcine 3'-UTRs. MATERIALS AND METHODS: Parathyroid protein-binding assays to the PTH mRNA transcripts were performed. Functionality was studied in reporter genes that were transiently transfected into HEK293 cells. RESULTS: Protein-RNA binding experiments identified an element in bovine PTH mRNA at the proximal end of the 3'-UTR that is different from the rat protein-binding element. The human 3'-UTR contains both elements, but only the distal element binds proteins. Functional studies with HEK293 cells transiently transfected with reporter genes containing the different elements and flanking nucleotides (nt) showed that the human distal element destabilized a reporter mRNA similar to the effect of this element in the rat. A reporter mRNA containing the single bovine PTH mRNA protein-binding element was also destabilized, and this was prevented by coexpression of AU-rich element binding factor 1 (AUF1). CONCLUSION: Our results identify a new protein-binding element in the PTH mRNA 3'-UTR. In bovine PTH mRNA, it is the only element, and it is functional in destabilizing a reporter gene. It is also present in other species, including human PTH mRNA, where it is not functional, possibly because of differences in flanking sequences. The human PTH mRNA 3'-UTR distal element is highly homologous to the rat cis-acting instability element and destabilized a reporter gene, indicating its functionality. Therefore, different species have alternative cis-acting protein-binding elements that may determine the regulation of PTH mRNA stability in response to changes in serum calcium and phosphate.

3' Untranslated Regions↗
PubMed2002

Post-transcriptional regulation of ura4+ gene expression by glucose in Schizosaccharomyces pombe.

Glucose-inducible gene expression is a fundamental cellular response for optimal cell growth, but identities of glucose-inducible genes and its regulatory mechanism remain largely elusive in Schizosaccharomyces pombe. Here we report that ura4+, encoding orotidine monophosphate decarboxylase (OMPdecase), shows glucose-inducible expression regulated at post-transcriptional level. The ura4+ mRNA level was rapidly decreased by approximately 50% within 20 min after glucose depletion and it was readily recovered upon glucose-readdition within 1 h. Glucose at above 2% similarly raised the transcript level of ura4+, while low concentration (0.1%) was not effective. Interestingly, control of mRNA turnover would be the main regulatory step of the glucose-dependent expression of ura4+. Moreover, stress-activated MAPK (SAPK) pathway was partially responsible for the glucose-regulated expression of ura4+ and rrg1+, another example of glucose-dependent mRNA stability control in S. pombe. These results suggest that the SAPK pathway might participate in the glucose-dependent regulation of ura4+ and rrg1+ mRNA stabilities.

Blotting, Northern↗
PubMed1994

Regulation of apolipoprotein A-I gene expression by phenobarbital in the human hepatocarcinoma cell line, Hep3B.

Apolipoprotein (apo) A-I is the major protein constituent of plasma high density lipoprotein (HDL), which has been suggested to play a protective role against the development of atherosclerosis. The effect of phenobarbital on apo A-I mRNA and protein levels was studied in the human hepatoma cell line, Hep3B. Exposure of Hep3B cells to the drug (200 micrograms/ml) for 16 h resulted in a 4-fold and 8-fold increase in apo A-I mRNA and secreted protein levels, respectively. The induction of apo A-I mRNA level caused by phenobarbital could be due to increased rates of transcription and/or alteration in mRNA stability. To test these possibilities, nuclear run-off transcription assays and pulse-chase deinduction experiments were performed. We have demonstrated that phenobarbital treatment is associated with a 2-fold induction in apo A-I transcriptional activity. The estimated half-lives for apo A-I mRNA are 2 h and 3.6 h in the absence or presence of phenobarbital, respectively. The combination of increase in apo A-I transcription rate and mRNA stabilization could explain the 4-fold induction in apo A-I mRNA levels caused by phenobarbital treatment. However, these events could not be solely responsible for the 8-fold increase in secreted apo A-I protein level observed. The results suggest that the mechanism(s) by which phenobarbital induces apo A-I production operate at both pre- and either co- or post-translational mechanisms. The induction of apo A-I is specific since no significant alteration in apo E mRNA and proteins was observed in drug-treated cells.

Animals↗
PubMed2004

Butyrate suppresses Cox-2 activation in colon cancer cells through HDAC inhibition.

Cox-2 plays an important role in colon carcinogenesis and inflammation. Studying the HT-29 colon cancer cell line as a model, we found that Cox-2 expression and activity is increased approximately 25-fold by TNF-alpha. As previously reported for other Cox-2 inducers, this activation appears to result from a p38-mediated mRNA stabilization rather than an increase in promoter activity. The HDAC inhibitors butyrate and TSA blocked the TNF-alpha activation of Cox-2 protein and mRNA synthesis, and dramatically suppressed Cox-2 activity in HT-29 cells. The suppression of Cox-2 synthesis did not involve promoter inactivation and could be achieved even when applied after the TNF-alpha stimulus. The effect of the HDAC inhibitors was observed prior to the activation of p21 expression and did not require new protein synthesis. Finally, butyrate did not prevent p38 phosphorylation, so the block is likely to occur at a later step in the activation pathway. We propose that a component of the cytokine-induced Cox-2 mRNA stabilization pathway is sensitive to acetylation.

Butyrates↗
PubMed2004

The androgen receptor mRNA.

Androgens (testosterone), acting via the androgen receptor (AR) a nuclear transcription factor, regulate male sexual development and body composition. In addition, AR expression plays an important role in the proliferation of human prostate cancer and confers a better prognosis in breast cancer. AR mRNA stability is central to the regulation of AR expression in prostate and breast cancer cells, and recent studies have demonstrated binding by members of the ELAV/Hu and poly(C) RNA-binding protein families to a highly conserved UC-rich element in the 3'-untranslated region of AR mRNA, with functional impact on AR protein expression. Remarkably, a CAG trinucleotide repeat in exon 1 of the AR, the length of which has been linked to prostate cancer survival, is also a target for multiple RNA-binding proteins from a variety of human and murine tissues. In this review, we will detail the current knowledge of the mechanisms involved in regulating AR mRNA stability, the nature, potential role and structural biology of several novel AR mRNA-protein interactions, and the implications for novel therapeutics in human prostate cancer.

Amino Acid Motifs↗
PubMed1990

Stabilization of mRNA in the cell-free translation system from Escherichia coli.

In the RNA directed cell-free protein synthesizing system from E. coli, there is a problem of contaminating 3'-exonucleases which attack the mRNAs. Thus, we tried the following two methods to stabilize mRNAs against nucleases: (A) To use mRNAs having hairpin structures at their termini and (B) To hybridize mRNAs with small DNA fragments to the 3'-termini of mRNAs. It was found that degradation of a mRNA was inhibited by the method B rather than the method A in the translation system.

Amino Acid Sequence↗
PubMed2001

Healthy mice with an altered c-myc gene: role of the 3' untranslated region revisited.

c-Myc is a protooncogene involved in the control of cellular proliferation, differentiation and apoptosis. Like many other early response genes, regulation of c-myc expression is mainly controlled at the level of mRNA stability. Multiple cis-acting destabilizing elements have been described that are located both in the protein-coding region and in the 3' untranslated region (3' UTR). However, it is not known when they function during development and whether they act as partly redundant or independent elements to regulate c-myc mRNA level of expression. To begin to address these questions, we created a series of c-myc alleles modified in the 3' UTR, using homologous recombination and the Cre/loxP system, and analysed the consequences of these modifications in ES cells and transgenic animals. We found that deletion of the complete 3' UTR, including runs of Us and AU-rich elements proposed, on the basis of cell-culture assays, to be involved in the control of c-myc mRNA stability, did not alter the steady-state level of c-myc mRNA in any of the various situations analysed in vivo. Moreover, mice homozygous for the 3' UTR-deleted gene were perfectly healthy and fertile. Our results therefore strongly suggest that the 3' UTR of c-myc mRNA does not play a major role in the developmental control of c-myc expression.

3' Untranslated Regions↗
PubMed1997

Transcriptional activation of the Glut1 gene in response to oxidative stress in L6 myotubes.

Exposure of L6 myotubes to prolonged low grade oxidative stress results in increased Glut1 expression at both the protein and mRNA levels, leading to elevated glucose transport activity. To further understand the cellular mechanisms responsible for this adaptive response, the Glut1 transcription rate and mRNA stability were assessed. Nuclear run-on assays revealed 2.0- and 2.4-fold increases in Glut1 transcription rates in glucose oxidase- and xanthine/xanthine oxidase-pretreated cells, respectively. Glut1 mRNA stability was increased with both treatments compared with the control (t1/2 = 7.8 +/- 1.3, 6.0 +/- 2.0, and 2.4 +/- 0.5 h, respectively). The serum-responsive element and AP-1 (but not the cAMP-responsive element) showed increased binding capacity following oxidative stress. Both activation of AP-1 binding and elevation of Glut1 mRNA were prevented by cycloheximide. The involvement of enhancer 1 of the Glut1 gene was demonstrated using transfected 293 cells. Induction of Glut1 mRNA in response to oxidative stress differed from its activation by chronic insulin exposure as demonstrated by the ability of rapamycin to inhibit the latter without an effect on the former. In conclusion, oxidative stress increases the Glut1 transcription rate by mechanisms that may involve activation of AP-1 binding to enhancer 1 of the Glut1 gene.

Animals↗
PubMed2005

The peptidyl-prolyl isomerase Pin1 regulates the stability of granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils.

The infiltration, accumulation and degranulation of eosinophils in the lung represents a hallmark of active asthma. In vivo or in vitro eosinophil activation triggers the secretion of the antiapoptotic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). We now identify Pin1, a cis-trans isomerase, as an essential component of the ribonucleoprotein complex responsible for GM-CSF mRNA stabilization, cytokine secretion and the survival of activated eosinophils. Pin1 regulated the association of the AU-rich element-binding proteins AUF1 and hnRNP C with GM-CSF mRNA, accelerating or slowing decay, respectively. These data indicate Pin1 is a key mediator of GM-CSF production.

Allergens↗
PubMed1988

Sequence changes preceding a Shine-Dalgarno region influence trpE mRNA translation and decay.

In studies with a trpE promoter-strength measuring system we observed that constructs containing the Escherichia coli trp promoter and its adjacent transcribed region yielded lower levels of trpE protein than were expected. To analyze this observation we introduced mutational changes in the nucleotide sequence preceding the trpE Shine-Dalgarno region and examined their effects on trpE mRNA synthesis, translation and decay. We found that certain deletion, insertion and substitution mutations in the pre-Shine-Dalgarno region caused a two- to fivefold increase in trpE enzyme activity. These increases were accompanied by increases in steady-state levels of trpE mRNA. Pulse-chase analyses of trpE mRNA degradation revealed that the observed steady-state trpE mRNA levels correlated with changes in trpE mRNA stability. These findings are interpreted in terms of alternative models in which the primary effect of mutational changes that elevate trpE expression is to increase trpE mRNA translation, versus increasing trpE mRNA stability.

Anthranilate Synthase↗
PubMed2006

Trichostatin A down-regulate DNA methyltransferase 1 in Jurkat T cells.

Histone deacetylase inhibitor Trichostatin A (TSA), alone, is able to activate the transcription of DNA methylation-mediated silenced genes in human cancer cells. Increase in expression and half-life of the DNA methyltransferase DNMT1 has been found in carcinomas of the colon, lung, liver, prostate, and breast cancer. This overexpression of DNMT1 is responsible for hypermethylation of regulatory sequences of many genes involved in tumorigenesis. Using quantitative real-time PCR and Western blot analysis, we found that TSA down-regulate DNMT1 mRNA and protein expression in Jurkat T leukemia cells clone E6-1. We also observed that TSA decreased DNMT1 mRNA stability and reduced this transcript half-life from approximately 7 to 2h. We also found that protein biosynthesis is needed for posttranscriptional regulation of DNMT1 mRNA, which suggests the involvement of an RNase and/or mRNA stabilization protein entity in DNMT1 transcript stabilization. Our findings suggest that TSA not only alters histone acetylation, but also may affect DNA methylation.

Analysis of Variance↗
PubMed2000

Cell type specificity and mechanism of control of a gene may be reverted in different strains of Dictyostelium discoideum.

Twelve genes which are expressed exclusively in pre-spore cells of Dictyostelium strain AX3 are expressed exclusively in pre-stalk cells of strain AX2. One gene has the opposite behavior: it is expressed in pre-stalk cells in AX3 and in pre-spore cells in AX2. The change in cell type specificity involves a change in the mechanism of control of gene expression. When they are expressed in pre-stalk cells, genes are controlled at the level of transcription, whilst in pre-spore cells, they are controlled at the level of mRNA stability. Genes expressed in pre-stalk cells in strain AX2, fused with an AX2 pre-spore specific promoter, become regulated at the level of mRNA stability. These findings indicate that at least a group of pre-stalk mRNAs possess the cis-destabilizing element typical of pre-spore mRNAs, though they are not destabilized in disaggregated cells. This is due to the fact that ribosomal protein S6, phosphorylation of which is responsible for controlling the stability of pre-spore mRNAs, is not dephosphorylated in disaggregated pre-stalk cells. These cells lack an S6 phosphatase activity which has been purified from disaggregated pre-spore cells.

Animals↗
PubMed1999

Tumor necrosis factor-alpha stimulates lactate dehydrogenase A expression in porcine cultured sertoli cells: mechanisms of action.

In the present study, we investigated the regulatory action of tumor necrosis factor-alpha (TNFalpha) on lactate dehydrogenase A (LDH A), a key enzyme involved in lactate production. To this end, use was made of a primary culture system of porcine testicular Sertoli cells. TNFalpha stimulated LDH A messenger RNA (mRNA) expression in a dose (ED50 = 2.5 ng/ml; 0.1 nM TNFalpha)-dependent manner. This stimulatory effect was time dependent, with an effect detected after 6 h of TNFalpha treatment and maximal after 48 h of exposition (5-fold; P<0.001). The direct effect of TNFalpha on LDH A mRNA could not be accounted for by an increase in mRNA stability (half-life = 9 h), but was probably due to an increase in LDH A gene transcription. Inhibitors of protein synthesis (cycloheximide), gene transcription (actinomycin D and dichlorobenzimidazole riboside), tyrosine kinase (genistein), and protein kinase C (bisindolylmaleimide) abrogated completely (actinomycin D, dichlorobenzimidazole riboside, cycloheximide, and genistein) or partially (bisindolylmaleimide) TNFalpha-induced LDH A mRNA expression. These observations suggest that the stimulatory effect of TNFalpha on LDH A mRNA expression requires protein synthesis and may involve a protein tyrosine kinase and protein kinase C. In addition, we report that LDH A mRNA levels were increased in Sertoli cells treated with FSH. However, although the cytokine enhances LDH A mRNA levels through increased gene transcription, the hormone exerts its stimulatory action through an increase in LDH A mRNA stability. The regulatory actions of the cytokine and the hormone on LDH A mRNA levels and therefore on lactate production may operate in the context of the metabolic cooperation between Sertoli and postmeiotic germ cells in the seminiferous tubules.

Animals↗
PubMed1997

Transcriptional and post-transcriptional regulation of prolactin during the turkey reproductive cycle.

The present study examined turkey prolactin (PRL) transcription and PRL mRNA stability during different reproductive stages. Nuclear run-on transcription assays were performed using isolated nuclei from pituitaries of turkeys at different reproductive stages. Meanwhile, cytoplasmic PRL mRNA and plasma PRL were measured by slot blot and RIA respectively. The PRL transcription, pituitary cytoplasmic PRL mRNA abundance and plasma PRL levels increased after photostimulation and peaked at the incubating stage (P < 0.05). A decrease in PRL transcription, pituitary cytoplasmic PRL mRNA and plasma PRL (P < 0.05) was observed during the transition from incubation to photorefractoriness. Nest-deprivation reduced circulating PRL (P < 0.05), whereas pituitary cytoplasmic PRL mRNA and PRL transcription were not significantly altered from those in incubating birds (P > 0.05). The half-life of PRL mRNA was determined in pituitaries of non-photostimulated, laying, incubating and photorefractory hens. Primary pituitary cell cultures were treated with the transcription inhibitor actinomycin-D and the decay of the pre-existing PRL mRNA was quantified using Northern blot analysis. The PRL mRNA half-life was 1.5- and 1.4-fold greater in incubating and laying birds respectively than in non-photostimulated turkeys (P < 0.05). The half-life of PRL mRNA in photorefractory and incubating hens was similar in spite of great differences in pituitary PRL mRNA steady-state levels and PRL transcription. Our data suggest that photoinduced changes in pituitary PRL mRNA and plasma PRL are due to changes in both PRL transcription and PRL mRNA stability. Nest-deprivation inhibits the PRL releasing mechanism(s) independently of PRL transcription in turkeys.

Animals↗
PubMed1997

Nitric oxide decreases stability of mRNAs encoding soluble guanylate cyclase subunits in rat pulmonary artery smooth muscle cells.

Nitric oxide stimulates soluble guanylate cyclase (sGC) to convert GTP to the intracellular second messenger cGMP. In rat pulmonary artery smooth muscle cells, sGC is an obligate heterodimer composed of alpha1 and beta1 subunits. We investigated the effect of NO donor compounds on sGC subunit gene expression in rat pulmonary artery smooth muscle cells. Sodium nitroprusside and S-nitroso-glutathione decreased sGC subunit mRNA and protein levels, as well as sGC enzyme activity. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor, blocked the effect of sodium nitroprusside on sGC subunit gene expression, whereas 8-bromo cGMP decreased subunit mRNA levels, demonstrating that NO-mediated decrease in sGC subunit mRNA levels is cGMP-dependent. sGC subunit mRNA levels decreased more rapidly in rat pulmonary artery smooth muscle cells exposed to NO than in cells exposed to actinomycin D, suggesting that NO decreases sGC subunit mRNA stability. Actinomycin D and cycloheximide blocked the ability of NO to decrease sGC subunit mRNA levels. These results demonstrate that NO decreases sGC subunit mRNA stability via a transcription- and translation-dependent mechanism.

Animals↗