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At least 973 records · Page 54Linked to original sources
PubMed2000

A conserved AU-rich element in the 3' untranslated region of bcl-2 mRNA is endowed with a destabilizing function that is involved in bcl-2 down-regulation during apoptosis.

The control of mRNA stability is becoming recognized as a crucial point of gene expression regulation. A common element responsible for mRNA decay modulation is the adenine- and uracil-rich element that is found in the 3' untranslated region of numerous mRNAs subjected to fast expression changes in response to various stimuli. Previously we identified a post-transcriptional regulation level for the antiapoptotic bcl-2 gene, which could be involved in t(14;18) lymphoma-associated bcl-2 overexpression. Here we demonstrate that bcl-2 mRNA is endowed with an adenine- and uracil-rich element (ARE) characterized by high evolutionary conservation not only among all chordates examined, but even between chordates and the nematode Caenorhabditis elegans (ced-9 gene). As for other well-established destabilizing AREs, the insertion of the bcl-2 ARE downstream from stable beta-globin mRNA causes an enhanced decay of the beta-globin transcript, which proves its functional role. This possibility is corroborated by the fact that the pathway leading to the modulating activity of bcl-2 ARE is influenced by PKC, since the addition of DAG and TPA markedly attenuated the bcl-2 ARE destabilizing potential. Conversely, it is noteworthy that when C(2)-ceramide is added to the culture medium as the apoptotic agent, the beta-globin transcript harboring the bcl-2 ARE undergoes a dramatic increase in decay. This observation clearly indicates that the destabilizing function of bcl-2 ARE is enhanced by apoptotic stimuli and suggests that this element could be involved in a post-transcriptional mechanism of bcl-2 down-regulation during apoptosis. The half-life of the mRNA of bcl-2 in Jurkat cells is prolonged by PKC stimulation and shortened by C(2)-ceramide addition, strongly supporting the view that bcl-2 mRNA stability plays a physiological role in modulating bcl-2 expression, particularly in its down-regulation during apoptosis. Thus, this element becomes a new candidate for mediating those bcl-2 gene expression changes-from apoptosis-associated down-regulation to tumor-associated overexpression-observed thus far that profoundly influence single cell fate and tissue homeostasis.

3' Untranslated Regions↗
PubMedDate not supplied

Evidence that an endothelial cytosolic protein binds to the 3'-untranslated region of endothelial nitric oxide synthase mRNA.

Changes in the endothelial nitric oxide synthase (eNOS) expression could be involved in the endothelium-dependent vasorelaxing dysfunction associated with cardiovascular diseases. We have recently demonstrated the existence of endothelial cytosolic proteins that bind to the 3'-untranslated region (3'-UTR) of eNOS mRNA and could be involved in eNOS mRNA stabilization. In the present work, we have characterized the cytosolic proteins that bind to 3'-UTR eNOS mRNA. An endothelial cytosolic protein (MW 60-kD) specifically bound to 3'-UTR eNOS mRNA as determined by a cross-linking assay followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The endothelial cytosolic protein recognized a cytidine (C)-rich region within 3'-UTR eNOS mRNA. Furthermore, tumor necrosis factor-alpha (TNF-alpha) increased the level of the 60-kD endothelial cytosolic protein. In addition, TNF-alpha reduced eNOS mRNA levels and this was prevented by coincubation with cycloheximide. Cycloheximide also prevented the binding activity of the endothelial cytosolic protein to 3'-UTR eNOS mRNA. In summary, these data suggest that a 60-kD endothelial cytosolic protein binds to 3'-UTR eNOS mRNA. TNF-alpha increased the 60-kD protein levels. Cycloheximide prevented the binding activity of the cytosolic protein to 3'-UTR eNOS mRNA related to TNF-alpha; this effect was associated with greater eNOS mRNA levels. Further specific studies are needed to determine the involvement of this 60-kD endothelial cytosolic protein in the regulation of eNOS mRNA stabilization and in the endothelial dysfunction associated with cardiovascular diseases.

3' Untranslated Regions↗
PubMed2004

Interspecies comparison of neuroglobin, cytoglobin and myoglobin: sequence evolution and candidate regulatory elements.

Neuroglobin and cytoglobin are two novel members of the vertebrate globin family. Their physiological role is poorly understood, although both proteins bind oxygen reversibly and may be involved in cellular oxygen homeostasis. Here we investigate the selective constraints on coding and non-coding sequences of the neuroglobin and cytoglobin genes in human, mouse, rat and fish. Neuroglobin and cytoglobin are highly conserved, displaying very low levels of non-synonymous nucleotide substitutions. An oxygen supply function predicts distinct modes of gene regulation, involving hypoxia-responsive transcription factors. To detect conserved candidate regulatory elements, we compared the neuroglobin and cytoglobin genes in mammals and fish. The myoglobin gene was included to test if it also contains hypoxia-responsive regulatory elements. Long conserved non-coding sequences, indicative of gene-regulatory elements, were found in the cytoglobin and myoglobin, but not in the neuroglobin gene. Sequence comparison and experimental data allowed us to delimit upstream regions of the neuroglobin and cytoglobin genes that contain the putative promoters, defining candidate regulatory regions for functional tests. The neuroglobin and the myoglobin genes both lack conserved hypoxia-responsive elements (HREs) for transcriptional activation, but contain conserved hypoxia-inducible mRNA stabilization signals in their 3' untranslated regions. The cytoglobin gene, in contrast, harbors both conserved HREs and mRNA stabilization sites, strongly suggestive of an oxygen-dependent regulation.

Animals↗
PubMed2005

Tumor necrosis factor-alpha induces transforming growth factor-beta1 expression in lung fibroblasts through the extracellular signal-regulated kinase pathway.

Increased expression of transforming growth factor (TGF)-beta(1) and tumor necrosis factor (TNF)-alpha are thought to play important roles in the development of pulmonary fibrosis. We recently reported that TNF-alpha upregulates TGF-beta(1) expression in primary mouse lung fibroblasts (MLFs), a key cell population in fibrogenesis. In the present study, we have investigated signal transduction pathways involved in TNF-alpha upregulation of TGF-beta(1) in both primary MLFs and the Swiss 3T3 fibroblast cell line. Treatment of fibroblasts with TNF-alpha resulted in a significant increase in TGF-beta(1) protein as measured by ELISA. The increase in protein was preceded by a 200-400% increase in TGF-beta(1) mRNA detected by quantitative, real-time, reverse transcriptase-polymerase chain reaction. Western blot analysis showed that TNF-alpha activated the extracellular signal-regulated kinase (ERK), and inhibitors of the ERK-specific mitogen-activated protein kinase pathway (PD98059 or U0126) blocked TNF-alpha induction of TGF-beta(1) mRNA and protein. mRNA stability experiments showed that TNF-alpha increased the half-life of TGF-beta(1) mRNA to more than 24 h compared with approximately 15 h in unstimulated cells. Expression of constitutively active MEK1 that selectively phosphorylates ERK was sufficient for TGF-beta(1) mRNA stabilization in Swiss 3T3 fibroblasts. These results indicate that TNF-alpha activates the ERK-specific mitogen-activated protein kinase pathway leading to increased TGF-beta(1) production in fibroblasts, primarily via a post-transcriptional mechanism that involves stabilization of the TGF-beta(1) transcript.

Animals↗
PubMed1999

A role of insulin-like growth factor I in luteinizing hormone receptor expression in granulosa cells.

The present study was undertaken to identify the mechanisms underlying the effect of insulin-like growth factor (IGF-I) on LH receptor in rat granulosa cells. Treatment with FSH, as expected, produced a substantial increase in LH receptor messenger RNA (mRNA) level, and concurrent treatment with increasing concentrations of IGF-I brought about dose-dependent increases in FSH-induced LH receptor mRNA, with a maximal response 2.5-fold greater than that induced by FSH alone. IGF-I, either alone or in combination with FSH, did not affect intracellular cAMP levels, whereas it enhanced the effect of 8-bromo-cAMP on LH receptor mRNA production. We then investigated whether the effects of IGF-I and FSH on LH receptor mRNA levels are the results of increased transcription and/or altered mRNA stability. To determine whether the LH receptor 5'-flanking region plays a role in directing LH receptor mRNA expression, the proximal area of the LH receptor 5'-flanking regions were inserted into a transient expression vector, pGL-Basic, which contains luciferase as the reporter gene, and the resulting plasmids were transiently transfected into rat granulosa cells. Our studies show that the FSH-induced luciferase activity varied dependent upon the length of the 5'-flanking region sequence in the reporter gene. In addition, FSH (30 ng/ml) significantly enhanced the activity of 1379 bp of the LH receptor 5'-flanking region, but treatment with 10 ng/ml IGF-I alone did not significantly influence the activity of the LH receptor promoter or affect the increased promoter activity induced by FSH. The rates of LH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, were not increased by the addition of IGF-I. On the other hand, the decay curves for LH receptor mRNA transcript in primary granulosa cells showed a significant increase in the half-life after the addition of IGF-I. These data suggest a possible role for changes in LH receptor mRNA stability in the IGF-I-induced regulation of LH receptor in rat granulosa cells. This interface between circulating hormones and paracrine/autocrine systems could provide an important mechanism to amplify the effects of gonadotropic hormones at the local level.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Functional mapping of the translation-dependent instability element of yeast MATalpha1 mRNA.

The determinants of mRNA stability include specific cis-acting destabilizing sequences located within mRNA coding and noncoding regions. We have developed an approach for mapping coding-region instability sequences in unstable yeast mRNAs that exploits the link between mRNA translation and turnover and the dependence of nonsense-mediated mRNA decay on the activity of the UPF1 gene product. This approach, which involves the systematic insertion of in-frame translational termination codons into the coding sequence of a gene of interest in a upf1delta strain, differs significantly from conventional methods for mapping cis-acting elements in that it causes minimal perturbations to overall mRNA structure. Using the previously characterized MATalpha1 mRNA as a model, we have accurately localized its 65-nucleotide instability element (IE) within the protein coding region. Termination of translation 5' to this element stabilized the MATalpha1 mRNA two- to threefold relative to wild-type transcripts. Translation through the element was sufficient to restore an unstable decay phenotype, while internal termination resulted in different extents of mRNA stabilization dependent on the precise location of ribosome stalling. Detailed mutagenesis of the element's rare-codon/AU-rich sequence boundary revealed that the destabilizing activity of the MATalpha1 IE is observed when the terminal codon of the element's rare-codon interval is translated. This region of stability transition corresponds precisely to a MATalpha1 IE sequence previously shown to be complementary to 18S rRNA. Deletion of three nucleotides 3' to this sequence shifted the stability boundary one codon 5' to its wild-type location. Conversely, constructs containing an additional three nucleotides at this same location shifted the transition downstream by an equivalent sequence distance. Our results suggest a model in which the triggering of MATalpha1 mRNA destabilization results from establishment of an interaction between translating ribosomes and a downstream sequence element. Furthermore, our data provide direct molecular evidence for a relationship between mRNA turnover and mRNA translation.

Alleles↗
PubMed2000

Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding.

The adaptation of mRNA stability to environmental changes is a means of cells to adjust the level of gene expression. The Escherichia coli ompA mRNA has served as one of the paradigms for regulated mRNA decay in prokaryotes. The stability of the transcript is known to be correlated inversely with the bacterial growth rate. Thus, the regulation of ompA mRNA stability meets the physiological needs to adjust the level of ompA expression to the rate of cell division. Recently, host factor I (Hfq/HF1) was shown to be involved in the regulation of ompA mRNA stability under slow growth conditions. Here, we present the first direct demonstration that 30S ribosomes bound to the ompA 5'-UTR protect the transcript from RNase E cleavage in vitro. However, the 30S protection was found to be abrogated in the presence of Hfq. Toeprinting and in vitro translation assays revealed that translation of ompA is repressed in the presence of Hfq. These in vitro studies are corroborated by in vivo expression studies demonstrating that the reduced synthesis rate of OmpA effected by Hfq results in functional inactivation of the ompA mRNA. The data are discussed in terms of a model wherein Hfq regulates the stability of ompA mRNA by competing with 30S ribosomes for binding to the ompA 5'-UTR.

5' Untranslated Regions↗
PubMed2003

Gastrin and EGF synergistically induce cyclooxygenase-2 expression in Swiss 3T3 fibroblasts that express the CCK2 receptor.

Over-expression of cyclooxygenase-2 (COX-2) has been demonstrated to be tumorigenic in transgenic mice. Chronic treatment with NSAIDs is chemoprotective for colorectal cancer. Gastrin is a growth factor for gastric mucosa and has been shown to promote proliferation of colorectal cells. Recent studies suggest that COX-2 expression levels could mediate the growth effects of gastrin. Here, we report that gastrin increased PGE2 secretion in Swiss 3T3 cells expressing the CCK2 receptor. Gastrin dose dependently induced COX-2 protein levels in a time dependent manner. COX-2 mRNA levels were rapidly induced by a dose dependent increase in gastrin. Prior treatment of the cells with the CCK2 receptor specific antagonist, L365,260, inhibited gastrin-induced COX-2 protein and mRNA expression. Pretreatment with L364,714, the CCK1 receptor specific antagonist did not block COX-2 induction by gastrin. Inhibition of de novo protein synthesis by cycloheximide did not block COX-2 mRNA induction by gastrin. Also, gastrin-dependent COX-2 expression did not require PKC activity, activation of ERK, or transactivation of EGFR. However, co-stimulation with EGF and gastrin synergistically induced COX-2 protein and mRNA expression and PGE2 secretion. Measurements of COX-2 mRNA stability and COX-2 gene transcription reveal that EGF significantly increased the half-life of COX-2 mRNA with only a slight increase in the COX-2 transcription rate. Conversely, gastrin significantly increased COX-2 gene transcription rates but did not enhance COX-2 mRNA stability.

3T3 Cells↗
PubMed1997

Regulated expression of endothelin converting enzymes in glomerular endothelial cells.

Endothelin converting enzyme (ECE) constitutes a potential regulatory site for the production of active mature endothelins. Two cDNAs (ECE-1 and -2) encoding ECE have recently been cloned, but the regulation of the expression of these ECE has not been clarified. In the study presented here, an attempt was made to determine whether or not glomerular endothelial cells (GEN) express ECE-1 and -2, and to learn how the expression of ECE-1 and -2 is regulated by kinase-mediated signaling systems. Ribonuclease protection assay revealed the expression of ECE-1 and -2 in cultured GEN, and the expression was increased approximately 2.5- and approximately 1.8-fold, respectively, by treatment with 10(-7) M 12-O-tetradecanocyl-phorbol-13-acetate (TPA) for 4 hours. These increases in ECE-1 and -2 expression with TPA were inhibited by cotreatment with calphostin C (10(-7) M). In contrast, 24-h treatment with 10(-7) M TPA significantly decreased the expression of ECE-1 and -2, indicating that the expression was tightly regulated by protein kinase C (PKC)-dependent mechanism(s). Actinomycin D (1 microgram/mL) abolished the TPA-induced increase of ECE-1 and -2 mRNA, whereas TPA treatment did not affect the mRNA stability of ECE-1 and -2, thus suggesting that TPA-induced increases of ECE-1 and -2 mRNA resulted from the transcriptional activation of ECE-1 and -2, gene, rather than from the increase of mRNA stability. In addition to the regulation by PKC, the effects of protein kinase A and G on ECE-1 and -2 expression were also examined. Treatment with chlorophenyl-thio cyclic AMP (200 microM) for 24 h decreased ECE-1 and -2 expression to approximately 50% and approximately 40% of the control value, respectively. 8-bromo-3', 5'-cyclic GMP also decreased ECE-1 and -2 expression to approximately 80% and approximately 25% of the control value, respectively. These results demonstrate that the expression of ECE-1 and -2 is regulated by kinase-mediated signaling systems, with the most prominent regulatory effect shown by protein kinase C.

Animals↗
PubMed1989

Structure and stability of mRNA synthesized by vaccinia virus-encoded bacteriophage T7 RNA polymerase in mammalian cells. Importance of the 5' untranslated leader.

We have analyzed the structure and stability of RNA synthesized by bacteriophage T7 RNA polymerase in mammalian cells. The T7 polymerase, expressed by a recombinant vaccinia virus, transcribed the Escherichia coli lacZ gene flanked by T7 promoter and terminator signals. The lacZ gene cassette was introduced into infected cells within either a transfected plasmid or a second recombinant vaccinia virus. The T7-lacZ transcripts, which had a half-life of approximately 75 minutes, represented approximately 30% of total cytoplasmic RNA after a 24 hour period. The latter estimation indicated a disparity between the levels of lacZ RNA and beta-galactosidase synthesis. Analysis of the T7 transcripts indicated that they were initiated correctly but that only 5 to 10% contained terminal cap structures, providing an explanation for the low translatability of the RNA. Since the 5' end of the T7 transcripts can form a stem-loop structure that might interfere with capping by vaccinia virus RNA guanylyltransferase, as well as ribosome binding and scanning, a similar vector lacking such sequences was constructed. In vitro experiments demonstrated that T7 RNA polymerase transcribed both templates with similar efficiency and that the RNA lacking the potential to form the stem-loop was capped more rapidly by the purified vaccinia virus enzyme. Nevertheless, when the stem-loop was removed, beta-galactosidase was not expressed in infected cells; moreover, no T7 transcripts could be detected, suggesting that the RNA was not made or more likely was degraded during or shortly after synthesis. There is previous evidence that vaccinia virus RNA guanylyltransferase is associated with the viral transcription complex, thereby allowing RNA synthesis and capping to occur concurrently. We suggest that a lack of coupling between the vaccinia viral RNA guanylyltransferase and bacteriophage T7 RNA polymerase delays capping of T7 transcripts and that, under these conditions, the 5'-terminal double-stranded stem is required to stabilize the nascent RNA against degradation. Although deletion of the 3' palindromic sequence specifying T7 transcriptional termination from the expression cassette resulted in RNA of more heterogeneous lengths, neither the apparent turnover rate nor translation of the RNAs was diminished appreciably.

Animals↗
PubMed1997

Regulation of antioxidant enzyme expression by NGF.

The rapid decreases in viability seen in H2O2-treated PC12 cells reflect enhanced susceptibility of neural cell types to oxidant injury. The dose-response relationship between NGF concentration and survival after H2O2 treatment resembles that for NGF effects on PC12 survival in serumless medium. Previously we have shown that NGF treatment enhances the activity of GSH-Px and catalase which catalyze the degradation of H2O2. Here in order to ascertain whether NGF stimulates transcription, affects mRNA stability, or acts post-transcriptionally, we measured catalase and GSH-Px mRNA half-lives. While both catalase and GSH-Px transcripts are stable with a relatively long half life and a gradual decay in mRNA levels, NGF had different effects on their stability. NGF had marked effects on catalase mRNA stability. The catalase gene has a 3' flanking region with T-rich clusters and CA repeats known to be susceptible to regulation by destabilization or ubiquination. NGF maintained catalase mRNA levels of actinomycin D (ACT-D) treated PC12 cells at twice that of cells exposed to ACT-D alone, delaying the rate of decay for catalase mRNA for 24 h. The NGF induction of GSH-Px and catalase mRNA was inhibited by cycloheximide (CHX) treatment with a slight decrease in their mRNA levels due to prolonged exposure to CHX. When the CHX treatment was delayed relative to the NGF treatment there was no effect on NGF effects on catalase and GSH-Px. The GSH-Px gene has conserved sequences in the open reading frame and 3' untranslated region which forms a stem-loop structure necessary for the incorporation of Se into this selenoprotein. While Se is important in stabilizing GSH-Px transcripts, it did not affect transcription rates or mRNA stability. These results are consistent with the hypothesis that NGF regulates catalase and GSH-Px expression via a primary effect on transcription factor pathways.

Animals↗
PubMed1988

Premature translation termination mediates triosephosphate isomerase mRNA degradation.

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.

Alleles↗
PubMed2007

Gene transcription acceleration: main cause of hepatitis B surface antigen production improvement by dimethyl sulfoxide in the culture of Chinese hamster ovary cells.

The production and specific productivity of hepatitis B surface antigen (HBsAg) in recombinant Chinese hamster ovary (CHO) cells were increased by 81% and threefold, respectively, when supplemented with 1.5% dimethyl sulfoxide (DMSO) in the culture medium. To investigate the mechanism of DMSO effect on HBsAg production improvement, HBsAg mRNA level was measured by real-time PCR. HBsAg mRNA was increased by about 1.5-fold at 1.5% DMSO. The increase could derive from the increase of HBsAg gene copy number, the improvement of HBsAg mRNA stability, or the acceleration of HBsAg gene transcription. It was found that HBsAg gene copy number was not significantly changed in the cells stimulated with DMSO. HBsAg mRNA stability of cells with DMSO treatment was also not obviously different from control, and the mRNA half-life of 5.58 h in the cells at 1.5% DMSO was comparable to that of 5.36 h in the control culture. DMSO resulted in 80% increase in HBsAg gene transcription activity assessed using a nuclear run-on transcription assay. It could be deduced that the acceleration of HBsAg gene transcription is the main cause of HBsAg production improvement.

Animals↗
PubMed2004

Trans-splicing adeno-associated viral vector-mediated gene therapy is limited by the accumulation of spliced mRNA but not by dual vector coinfection efficiency.

Therapeutic application of recombinant adeno-associated virus (AAV) has been limited by its small carrying capacity. To overcome this limitation trans-splicing vectors were developed recently. However, the transduction efficiency of trans-splicing vectors is considerably lower than that of a single intact vector in skeletal muscle. To improve trans-splicing vectors for skeletal muscle gene therapy, we examined whether coinfection efficiency is a rate-limiting factor in the mdx mouse, a model for Duchenne muscular dystrophy. Two different AAV viruses were delivered to the mdx muscle. Similar to previous reports in normal muscle, coinfection efficiency reached approximately 90% in the diseased muscle. This result suggests that coinfection is not a hurdle in dystrophic muscle. Another critical step in the trans-splicing method is the transcription and splicing across the inverted terminal repeat (ITR) junction in the reconstituted genome. To test whether this represented a significant obstacle, we systematically evaluated the transcription, pre-mRNA stability and splicing, and translation in a synthetic lacZ construct that mimicked the reconstituted genome. Although inserting an intron in the lacZ gene had no effect on its expression, inclusion of the ITR junction in the intron reduced expression by 50%. In construct containing the ITR junction, the mRNA transcript level was significantly reduced. This mRNA level reduction was associated with decreased pre-mRNA stability. These data suggest that the accumulation of mRNA is a rate-limiting factor in trans-splicing vector-mediated gene therapy.

Animals↗
PubMed1994

Agonist-induced down-regulation of the m4 muscarinic acetylcholine receptor occurs without changes in receptor mRNA steady-state levels.

The regulation of m4 muscarinic acetylcholine receptor mRNA expression by receptor activation was studied in N1E-115 neuroblastoma and AtT-20 pituitary cells that endogeneously express the m4 muscarinic receptor. Receptor concentration was measured by binding of the muscarinic receptor radioligand [3H]quinuclidinyl benzilate, and RNA-RNA solution hybridization/RNase protection assay with a m4 receptor-specific [32P]-cRNA probe was used to evaluate the levels of receptor mRNA. Treatment of both cell lines with a receptor-saturating concentration of the agonist carbachol decreased receptor number. However, there was no change in steady-state levels of m4 mAChR mRNA in both cell lines. Determination of mRNA stability in the presence of the transcription blocker actinomycin D revealed that carbachol treatment increased half-life of receptor mRNA in N1E-115 cells, but not in AtT-20 cells, suggesting that receptor activation can regulate m4 receptor mRNA stability dependently on cell type. Analysis of receptor degradation kinetics in the presence of the protein synthesis inhibitor cycloheximide showed that receptor down-regulation in N1E-115 and AtT-20 cells is sufficiently accounted for by increased receptor degradation. These results indicate than m4 muscarinic receptor down-regulation is substantially different from that of the muscarinic receptor subtypes m2 and m3 which is reported to be associated with agonist-induced reduction in receptor mRNA.

Animals↗
PubMed2005

Cyclin T1 expression is regulated by multiple signaling pathways and mechanisms during activation of human peripheral blood lymphocytes.

Stimulation of primary human T lymphocytes results in up-regulation of cyclin T1 expression, which correlates with phosphorylation of the C-terminal domain of RNA polymerase II (RNAP II). Up-regulation of cyclin T1 and concomitant stabilization of cyclin-dependent kinase 9 (CDK9) may facilitate productive replication of HIV in activated T cells. We report that treatment of PBLs with two mitogens, PHA and PMA, results in accumulation of cyclin T1 via distinct mechanisms. PHA induces accumulation of cyclin T1 mRNA and protein, which results from cyclin T1 mRNA stabilization, without significant change in cyclin T1 promoter activity. Cyclin T1 mRNA stabilization requires the activation of both calcineurin and JNK because inhibition of either precludes cyclin T1 accumulation. In contrast, PMA induces cyclin T1 protein up-regulation by stabilizing cyclin T1 protein, apparently independently of the proteasome and without accumulation of cyclin T1 mRNA. This process is dependent on Ca2+-independent protein kinase C activity but does not require ERK1/2 activation. We also found that PHA and anti-CD3 Abs induce the expression of both the cyclin/CDK complexes involved in RNAP II C-terminal domain phosphorylation and the G1-S cyclins controlling cell cycle progression. In contrast, PMA alone is a poor inducer of the expression of G1-S cyclins but often as potent as PHA in inducing RNAP II cyclin/CDK complexes. These findings suggest coordination in the expression and activation of RNAP II kinases by pathways that independently stimulate gene expression but are insufficient to induce S phase entry in primary T cells.

Calcineurin↗
PubMed2004

How strong is the case for regulation of the initiation step of translation by elements at the 3' end of eukaryotic mRNAs?

The belief that initiation of translation requires communication between the 5' and 3' ends of the mRNA guides--or misguides--the interpretation of many experiments. The closed-loop model for initiation creates the expectation that sequences at the 3' end of eukaryotic mRNAs should regulate translation. This review looks closely at the evidence in three prominent cases where such regulation is claimed. The mRNAs in question encode 15-lipoxygenase, ceruloplasmin, and histones. Vertebrate histone mRNAs lack a poly(A) tail, instead of which a 3' stem-loop structure is said to promote translation by binding a protein which purportedly binds initiation factors. The proffered evidence for this hypothesis has many flaws. Temporal control of 15-lipoxygenase production in reticulocytes is often cited as another well-documented example of translational regulation via the 3' untranslated region, but inspection of the evidence reveals significant gaps and contradictions. Solid evidence is lacking also for the idea that a ribosomal protein binds to and shuts off translation of ceruloplasmin mRNA. Some viral RNAs that lack a poly(A) tail have alternative 3' structures which are said to promote translation via circularization of the mRNA, but in no case has this been shown convincingly. Interpretation of many experiments is compromised by possible effects of the 3' structures on mRNA stability rather than translation. The functional-half-life assay, which is often employed to rule out effects on mRNA stability, might not be adequate to settle the question. Other issues, such as the possibility of artifacts caused by overexpression of RNA-binding proteins, can complicate studies of translational regulation. There is no doubt that elements at the 3' end of eukaryotic mRNAs can regulate gene expression in a variety of ways. It has not been shown unequivocally that one of these ways involves direct participation of the 3' untranslated region in the initiation step of translation.

3' Untranslated Regions↗
PubMed2005

Stimulus-dependent glucocorticoid-resistance of GM-CSF production in human cultured airway smooth muscle.

For a subpopulation of asthmatics, symptoms persist even with high doses of glucocorticoids. Glucocorticoids reduce the levels of the proinflammatory and fibrogenic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by human cultured airway smooth muscle (ASM). We have contrasted the effects of a synthetic glucocorticoid, dexamethasone, on thrombin- and IL-1alpha-stimulated GM-CSF production in human ASM cells. Although IL-1alpha stimulated three-fold higher levels of GM-CSF mRNA and protein compared to thrombin, dexamethasone concentration-dependently reduced IL-1alpha-stimulated GM-CSF more potently and to a greater extent than the response to thrombin. This pattern of glucocorticoid regulation was also observed at the GM-CSF mRNA level and was reproduced with other glucocorticoids such as fluticasone propionate. IL-1alpha and thrombin stimulated NF-kappa B-dependent luciferase expression equally. Dexamethasone treatment reduced luciferase expression stimulated by both IL-1alpha and thrombin. The GM-CSF mRNA half life was markedly prolonged by IL-1alpha compared to thrombin. This IL-1alpha-induced GM-CSF mRNA stability was prevented by either dexamethasone or the p38(MAPK) inhibitor, SB203580, neither of which influenced GM-CSF mRNA stability in thrombin-treated cells. Dexamethasone inhibited p38(MAPK) phosphorylation in IL-1alpha-stimulated ASM, whereas thrombin does not stimulate p38(MAPK) phosphorylation. These data suggest that the mechanism underlying the greater potency and efficacy of glucocorticoids in reducing GM-CSF synthesis stimulated by IL-1alpha depends on inhibition of the involvement of p38(MAPK)-induced increases in GM-CSF message stability.

Androstadienes↗