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At least 937 records · Page 52Linked to original sources
PubMed2006

Cellular retinoic acid-binding protein II is a direct transcriptional target of MycN in neuroblastoma.

Neuroblastoma is a heterogeneous disease in which 22% of tumors show MycN oncogene amplification and are associated with poor clinical outcome. MycN is a transcription factor that regulates the expression of a number of proteins that affect the clinical behavior of neuroblastoma. We report here that cellular retinoic acid-binding protein II (CRABP-II) is a novel MycN target, expressed at significantly higher levels in primary neuroblastoma tumors with mycN oncogene amplification as compared with non-MycN-amplified tumors. Moreover, regulated induction and repression of MycN in a neuroblastoma-derived cell line resulted in temporal and proportionate expression of CRABP-II. CRABP-II is expressed in several cancers, but its role in tumorigenesis has not been elucidated. We show that MycN binds to the promoter of CRABP-II and induces CRABP-II transcription directly. In addition, CRABP-II-transfected neuroblastoma cell lines show an increase in MycN protein levels resulting in increased cell motility. Gene expression profiling of CRABP-II-expressing cell lines uncovered increased expression of the HuB (Hel N1) gene. Hu proteins have been implicated in regulating the stability of MycN mRNA and other mRNAs by binding to their 3' untranslated regions. We did not, however, observe any change in MycN mRNA stability or protein half-life in response to CRABP-II expression. In contrast, de novo MycN protein synthesis was increased in CRABP-II-expressing neuroblastoma cells, thereby suggesting an autoregulatory loop that might exacerbate the effects of MycN gene amplification and affect the clinical outcome. Our findings also suggest that CRABP-II may be a potential therapeutic target for neuroblastoma.

Binding Sites↗
PubMed1999

Modulation of extracellular superoxide dismutase expression by angiotensin II and hypertension.

Angiotensin II and hypertension increase vascular oxidant stress. We examined how these might affect expression of the extracellular superoxide dismutase (ecSOD), a major form of vascular SOD. In mice, angiotensin II infusion (1.1 mg/kg for 7 days) increased systolic blood pressure from 107+/-3 to 152+/-9 mm Hg and caused a 3-fold increase in ecSOD, but there was no change in the cytosolic Cu/Zn SOD protein, as determined by Western blot analysis. This was associated with a similar increase in ecSOD mRNA as assessed by RNase protection assay and was prevented by losartan. Induction of ecSOD by angiotensin II was not due to hypertension alone, because hypertension caused by norepinephrine (5.6 mg. kg-1. d-1) had no effect on ecSOD. Similarly, exposure of mouse aortas to angiotensin II (100 nmol/L) in organoid culture increased ecSOD by approximately 2-fold. In the organoid culture, angiotensin II-induced upregulation of ecSOD was prevented by losartan (10 micromol/L) and PD985059 (30 micromol/L), a specific inhibitor of p42/44 MAP kinase kinase. Angiotensin II activates the NADH/NADPH oxidase; however, diphenyleneiodonium chloride (10 micromol/L), an inhibitor of this oxidase, did not prevent p42/44 MAP kinase phosphorylation or ecSOD induction by angiotensin II. Finally, in human aortic smooth muscle cells, angiotensin II moderately increased transcriptional rate (as assessed by nuclear run-on analysis) but markedly increased ecSOD mRNA stability. Thus, angiotensin II increases ecSOD expression independent of hypertension, and this increase involves both an increase in ecSOD transcription and stabilization of ecSOD mRNA. This effect of angiotensin II on ecSOD expression may modulate the oxidative state of the vessel wall in pathological processes in which the renin-angiotensin system is activated.

Angiotensin II↗
PubMed2005

An old story with a new twist: do NMDAR1 mRNA binding proteins regulate expression of the NMDAR1 receptor in the presence of alcohol?

NMDA receptors not only play a pivotal role in normal physiological processes in the central nervous system (CNS), but have been identified as an important target of ethanol. Chronic exposure to ethanol induces a number of adaptive processes in the CNS, including an upregulation of NMDA receptor number and function. The increase in NMDA receptor number in response to chronic ethanol exposure both in vivo and in vitro is accompanied by an increase in NMDAR1 and NMDAR2B polypeptide levels. It is widely believed that these adaptive changes play an important role in the development of alcohol dependence and withdrawal syndrome. At the molecular level, chronic ethanol exposure of fetal cortical neurons selectively increases expression of NMDAR1 splice variants lacking exon 5 and exon 22. Chronic ethanol exposure of fetal cortical neurons also increases NMDAR1 mRNA half-life in these neurons. However, when new protein synthesis is inhibited, the half-life of NR1 mRNA in these neurons returns to control values, strongly suggesting that ethanol induces the synthesis of protein(s) that may regulate the decay of NR1 mRNA. In recent years, it has become apparent that regulation of mRNA stability is an important aspect of regulation of gene expression. Changes in mRNA stability can be accomplished by interaction between cis-acting sequences in the 3' untranslated region (3'UTR) of mRNAs and trans-acting proteins expressed in cells. Such interactions may protect RNAs from degradation by ribonucleases, thereby increasing the half-life of mRNAs.

Animals↗
PubMed1989

Modulation of cytokine production by transforming growth factor-beta.

Transforming growth factor-beta 1 (TGF-beta 1) is one of a family of polypeptides involved in the regulation of cell growth and differentiation. The effects of human rTGF-beta 1 on the production of IL-1 and TNF by activated PBMC were studied. The addition of TGF-beta 1 alone caused an increase in the levels of mRNA for IL-1 alpha, IL-1 beta, and TGF-alpha. This was due to increased transcription rather than enhanced mRNA stability. The induced mRNA were of the appropriate size as assessed by Northern blotting. However, the mRNA did not appear to be translated into protein, inasmuch as the translation products of IL-1 beta and TNF-alpha were not detected by RIA or ELISA. Furthermore, in experiments utilizing a neutralizing antibody to TGF-beta 1, we were unable to unmask IL-1 biologic activities and unable to detect TNF biologic activity in the WEHI 164 cytotoxicity assay. TGF-beta inhibited in a dose-dependent manner the induction of IL-1 beta by LPS or TNF but not by PHA and PMA. Similarly, LPS induction of TNF-alpha was blocked by TGF-beta, whereas induction of PMA and PHA was completely resistant. TGF-beta 1 did not increase PGE2 secretion or cause elevated intracellular cAMP; thus, the inhibitory effects of TGF-beta 1 seem not to be mediated by PGE2 or cAMP, which have both been implicated in post-transcriptional control of cytokine gene expression. These findings suggest a dual role for TGF-beta 1 in the regulation of cytokine production at both transcriptional and translational levels.

Adjuvants, Immunologic↗
PubMed1993

Agonist-induced down-regulation and antagonist-induced up-regulation of m2- and m3-muscarinic acetylcholine receptor mRNA and protein in cultured cerebellar granule cells.

Cerebellar granule cells express m2- and m3-muscarinic acetylcholine receptors (mAChRs) and their corresponding mRNA with m3-mAChR being the predominant receptor subtype. After stimulation with the mAChR agonist, carbachol, m2- and m3-mAChR mRNA levels were decreased in a time- and concentration-dependent manner with the maximal down-regulation at 2 and 8 hr, respectively. Immunoprecipitation studies revealed that amounts of m2- and m3-mAChR protein also decreased at 8 and 24 hr, respectively. The carbachol-induced down-regulation of m3-mAChR mRNA was associated with a decrease in the transcription rate, but a substantial enhancement of the mRNA stability. Upon removal of carbachol after treatment for 8 hr, the levels of m3-mAChR mRNA and mAChR binding sites returned to their original values with a t1/2 of approximately 80 min and 6 hr, respectively. The carbachol-elicited loss of m2- and m3-mAChR mRNA was blocked by their corresponding receptor subtype-specific antagonists, AF-DX 116 (m2-selective) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (m3-selective), and was concurrent with an increase in c-fos mRNA levels. Exposure of granule cells to the nonselective mAChR antagonist, atropine, caused a time- and concentration-dependent increase in the level of both m2- and m3-mAChR mRNA and mAChR binding sites. At 24 hr, immunoprecipitable m3-mAChR protein was predominantly increased. The atropine-induced up-regulation of m3-mAChR mRNA was concurrent with a marked enhancement of the mRNA stability and its transcription rate. The elevated levels of m3-mAChR mRNA and binding sites declined to their untreated values after the removal of atropine. Treatment with AF-DX 116 and 4-DAMP also produced an increase in the level of m2- and m3-mAChR mRNA and their corresponding immunoprecipitable receptor protein. These results demonstrate that the mAChR agonist and antagonist induce a down- and up-regulation of mAChR expression, respectively, through receptor-mediated mechanisms in cerebellar granule cells. Moreover, at least for m3-mAChR mRNA, the agonist- and antagonist-induced effects are reversible and associated with corresponding changes in the transcription rate of this receptor mRNA species.

Animals↗
PubMed1999

Effect of TPA on aquaporin 4 mRNA expression in cultured rat astrocytes.

Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, localized in the astrocyte plasma membrane. The regulation of AQP4 is believed to be important for the homeostasis of water in the brain, but the AQP4 regulatory mechanisms are not yet known. In this study, we investigated the effect of a protein kinase C (PKC) activator on the expression of AQP4 mRNA in cultured rat astrocytes. Cultured rat astrocytes constitutively expressed AQP4 mRNA. Treatment of the cells with 0.1 microM of phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, caused a rapid decrease in AQP4 mRNA. This effect was time- and dose-dependent. The TPA-induced decrease in AQP4 mRNA was inhibited by a relatively specific PKC inhibitor, 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H7) in a dose-dependent manner. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decrease in AQP4 mRNA by TPA. These results strongly suggest that the TPA-induced decrease in AQP4 mRNA is mediated by PKC activation. To test whether the effect of TPA requires protein synthesis, astrocytes were pretreated with cycloheximide, an inhibitor of protein synthesis. Pretreatment of the cells with cycloheximide did not inhibit the decrease in AQP4 mRNA induced by TPA. To test whether the TPA-induced decrease in AQP4 was due to a decrease in the mRNA stability, we examined the effect of actinomycin D, an inhibitor of transcription, on TPA-treated cells. The stability of AQP4 mRNA was not decreased by the pretreatment of the cells with actinomycin D. The results suggest that AQP4 mRNA is inhibited by TPA via PKC activation without de novo protein synthesis, and that the inhibition of AQP4 mRNA could be at the transcriptional level.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Posttranscriptional regulation of p21WAF1/CIP1 expression in human breast carcinoma cells.

p21WAF1/CIP1 plays a major role in the induction of G1 arrest following DNA damage. Although p21WAF1/CIP1 expression is regulated by the tumor suppressor p53, induction of p21WAF1/CIP1 expression through p53-independent pathways has been described in numerous cell types. In this report, we describe the mechanism by which the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induces p21WAF1/CIP1 in breast carcinoma cells possessing either a wild-type (MCF-7 cells) or mutated (MDA-MB-468 cells) p53. Exposure of MDA-MB-468 cells to this retinoid results in an approximately 10-fold increase in p21WAF1/CIP1 mRNA levels, whereas less than a 2-fold increase in p21WAF1/CIP1 gene transcription was observed as indicated by transient transfection experiments utilizing a p21WAF1/CIP1 promoter firefly luciferase reporter gene construct and nuclear run-off studies. We found similar results in the MCF-7 cells (Z-M. Shao et al., Oncogene, 11: 493-504, 1995). We have now found that while enhancing p21WAF1/CIP1 gene transcription minimally, this retinoid increases p21WAF1/CIP1 mRNA stability by 3-fold in both cell types. We also demonstrate that approximately 1.5 kb of the 3' untranslated region causes enhanced instability of p21WAF1/CIP1 mRNA. The retinoid-dependent increase in p21WAF1/CIP1 mRNA stability is accompanied by an increase in p21WAF1/CIP1 protein expression, as indicated by Western blot experiments utilizing anti-p21WAF1/CIP1 monoclonal antibody. This increase in p21WAF1/CIP1 is subsequently followed by the onset of programmed cell death in both cell types. Thus, CD437 is a novel retinoid which enhances p21WAF1/CIP1 mRNA levels through stabilization of the message regardless of the p53 status of the cell.

Apoptosis↗
PubMed2006

IL-17A acts via p38 MAPK to increase stability of TNF-alpha-induced IL-8 mRNA in human ASM.

Human airway smooth muscle (ASM) plays an immunomodulatory role in asthma. Recently, IL-17A has become of increasing interest in asthma, being found at elevated levels in asthmatic airways and emerging as playing an important role in airway neutrophilia. IL-17A predominantly exerts its neutrophil orchestrating role indirectly via the induction of cytokines by resident airway structural cells. Here, we perform an in vitro study to show that although IL-17A did not induce secretion of the CXC chemokine IL-8 from ASM cells, IL-17A significantly potentiates TNF-alpha-induced IL-8 protein secretion and gene expression in a concentration- and time-dependent manner (P < 0.05). Levels of IL-8 protein produced after 24 h of incubation with TNF-alpha were enhanced 2.7-fold in the presence of IL-17A, and conditioned media significantly enhanced neutrophil chemotaxis in vitro. As IL-17A had no effect on the activity of NF-kappaB, a key transcriptional regulator of IL-8 gene expression, we then examined whether IL-17A acts at the posttranscriptional level. We found that IL-17A significantly augmented TNF-alpha-induced IL-8 mRNA stability. Interestingly, this enhanced stability occurred via a p38 MAPK-dependent pathway. The decay of IL-8 mRNA transcripts proceeded at a significantly faster rate when cells were pretreated with the p38 MAPK inhibitor SB-203580 (-0.05763 +/- 0.01964, t(1/2) = 12.0 h), compared with vehicle (-0.01030 +/- 0.007963, t(1/2) = 67.3 h) [results are expressed as decay constant (means +/- SE) and half-life (t(1/2) in h): P < 0.05]. Collectively, these results demonstrate that IL-17A amplifies the synthetic function of ASM cells, acting via a p38 MAPK-dependent posttranscriptional pathway to augment TNF-alpha-induced secretion of the potent neutrophil chemoattractant IL-8 from ASM cells.

Bronchi↗
PubMed1998

Agonist-mediated destabilization of human beta1-adrenergic receptor mRNA: role of the 3' untranslated translated region.

For proto-oncogenes and cytokines, regulation of gene expression at the level of mRNA stability is well established. In contrast, there is comparatively limited knowledge regarding this mechanism of regulation for G-protein-coupled receptors. To explore this process further, the human beta1-adrenergic receptor (AR) was stably expressed in tsAF8 cells. Treatment with beta-agonist decreased the half-life of beta1-AR mRNA by approximately 50%. Removal of the 3'UTR from the beta1-AR (coding region only) dramatically stabilized mRNA. Additionally, in a chimeric mRNA, the beta1-AR 3'UTR was able to target the normally highly stable beta-globin mRNA for accelerated decay. However, the chimera did not undergo agonist-mediated destabilization indicating that the 3'UTR may be "necessary but not sufficient" for agonist-mediated mRNA destabilization. Inhibition of translation significantly stabilized beta1-AR mRNA (approximately 2-fold); however, pretreatment of cells with beta-agonist prior to translational arrest produced the same degree of mRNA destabilization indicating that agonist-mediated destabilization may be independent of the translation process. Conversely, translational inhibition simultaneous with beta-agonist exposure abrogated agonist-mediated destabilization indicating a dependence on de novo protein synthesis.

3' Untranslated Regions↗
PubMed1993

Transcriptional down-regulation of tumor necrosis factor-alpha gene expression by a synthetic peptide homologous to retroviral envelope protein.

We have previously shown that a synthetic peptide (CKS-17) homologous to retroviral envelope protein suppresses the accumulation of superantigen staphylococcal enterotoxin-induced TNF-alpha mRNA in human PBMC and in highly purified human monocytes. The present study was designed to examine the underlying mechanism(s) by which CKS-17 down-regulates the TNF-alpha mRNA expression using a human acute monocytic leukemia cell line THP-1 stimulated with the superantigen staphylococcal enterotoxin E. A cyclooxygenase inhibitor indomethacin does not reverse the inhibition of TNF-alpha mRNA expression by CKS-17, suggesting that prostaglandins are not responsible for the suppressive action of CKS-17. The inhibitory effect of CKS-17 is, however, significantly blocked by a protein synthesis inhibitor cycloheximide, indicating that CKS-17 requires de novo protein synthesis to induce the suppressive activity. The mRNA stability assays using actinomycin D show that CKS-17 does not decrease the TNF-alpha mRNA stability. Nuclear run-on transcription assays further reveal that CKS-17 suppresses the TNF-alpha mRNA transcription rate. Taken together, these results suggest that the synthetic retroviral peptide CKS-17 down-regulates TNF-alpha mRNA expression through inhibition of transcriptional activation of the TNF-alpha gene, which requires de novo synthesis of a transcriptional repressor protein(s).

Amino Acid Sequence↗
PubMed1994

Metabolic stability of mRNA in yeast--a potential target for modulating productivity?

Attempts to improve the production of (heterologous) proteins in yeast cells have, to date, focused almost exclusively on increasing the transcriptional yield of the heterologous gene by raising the number of gene copies per cell, and/or putting the gene under the control of a strong homologous promoter. However, the cellular level of translatable mRNA is a function of the rate at which it is produced and the rate at which it is removed--or at least inactivated--by nucleolytic degradation. Recently, considerable progress has been made in unravelling the mechanism of mRNA decay in yeast cells and in identifying both the cis-acting stability determinants and the trans-acting factors involved in this process. This knowledge can be used as the basis for rational engineering of a given transcript to modulate its metabolic stability, and thus its cellular level.

Biotechnology↗
PubMed1998

Agonist-induced down-regulation of the beta2-adrenoceptor and its mRNA in human mononuclear leukocytes.

Agonist-mediated regulation of beta2-adrenoceptors in mononuclear leukocytes has been examined at the protein but not at the mRNA level. In the present study, incubation of mononuclear leukocytes with the beta-agonist (-)-isoproterenol (10(-6) M) for up to 42 hr led to a maximum decrease in both beta2-adrenoceptor mRNA concentration and total receptor number of ca. 56 and 70%, respectively. The decrease in the mRNA level, however, was slower than for the protein level. After 4 hr of incubation with the beta-agonist, the protein level decreased to a minimum of 65% of the initial amount, while an incubation of 8 hr was necessary to reach a similar decrease in the level of mRNA (69% of the initial level). Measurements of mRNA stability revealed a reduction in the half-life of beta2-adrenoceptor mRNA from 2.7 to 1.1 hr following 4 hr of incubation with (-)-isoproterenol. Our data clearly demonstrate that treatment of human mononuclear leukocytes with (-)-isoproterenol induces a beta2-adrenoceptor down-regulation together with a slower time course of mRNA down-regulation which is partly due to a reduction of mRNA stability.

Adrenergic beta-2 Receptor Agonists↗
PubMed1992

Measles virus infection enhances IL-1 beta but reduces tumor necrosis factor-alpha expression in human monocytes.

Monocytes may play a role in the immunologic abnormalities caused by measles. The effect of measles virus (MV) infection on peripheral blood monocyte functions is poorly known. We report that MV-infected PBM have an altered pattern of IL-1 beta and TNF-alpha production in response to stimulation with LPS and PMA in vitro. MV-infected peripheral blood monocytes produced higher amounts of IL-1 beta, whereas the production of TNF-alpha was reduced. The same effect was observed in the human monocytic cell line THP-1, which was used for RNA analysis. An increased steady-state level of IL-1 beta mRNA was observed in MV-infected cells, and the level of TNF-alpha mRNA was reduced. However, both IL-1 beta and TNF-alpha had about 50% increased transcription rate. Analysis of the mRNA stability after transcriptional block by actinomycin D showed that the TNF-alpha mRNA had a reduced half-life in MV-infected cells (about 30 vs 80 min in uninfected cells), whereas IL-1 beta mRNA stability was similar in uninfected and MV-infected cells. These results indicate that MV infection disturbs the immunoregulatory network by interfering with the monocyte functions.

Base Sequence↗
PubMed2004

CA repeats in the 3'-untranslated region of bcl-2 mRNA mediate constitutive decay of bcl-2 mRNA.

An AU-rich element (ARE) in the 3'-untranslated region (UTR) of bcl-2 mRNA has previously been shown to be responsible for destabilizing bcl-2 mRNA during apoptosis through increasing AUF1 binding. In the present study, we investigated the effect of the region upstream of the ARE on bcl-2 mRNA stability using serial deletion constructs of the 3'-UTR of bcl-2. Deletion of 30 nucleotides mostly consisting of the CA repeats, located upstream of the ARE, resulted in the stabilization of bcl-2 mRNA abundance, in the absence or presence of the ARE. The specificity of the CA repeats in terms of destabilizing bcl-2 mRNA was proven by the substituting the CA repeats with other alternative repeats of purine/pyrimidine, but this had no effect on the stability of bcl-2 mRNA. CA repeats alone, however, failed to confer instability to bcl-2 or gfp reporter mRNAs, indicating a requirement for additional sequences in the upstream region of the 3'-UTR. Serial deletion and replacement of a part of the region upstream of the CA repeats revealed that the entire 131-nucleotide upstream region is an essential prerequisite for the CA repeat-dependent destabilization of bcl-2 mRNA. Unlike the ARE, CA repeat-mediated degradation of bcl-2 mRNA was not accelerated upon apoptotic stimulus. Moreover, the upstream sequences and CA repeats are conserved among mammals. Collectively, CA repeats contribute to the constitutive decay of bcl-2 mRNA in the steady states, thereby maintaining appropriate bcl-2 levels in mammalian cells.

3' Untranslated Regions↗
PubMed1988

Stabilization of tubulin mRNA by inhibition of protein synthesis in sea urchin embryos.

An increased level of unpolymerized tubulin caused by depolymerization of microtubules in sea urchin larvae resulted in a rapid loss of tubulin mRNA, which was prevented by nearly complete inhibition of protein synthesis. Results of an RNA run-on assay indicated that inhibition of protein synthesis does not alter tubulin gene transcription. Analysis of the decay of tubulin mRNA in embryos in which RNA synthesis was inhibited by actinomycin D indicated that inhibition of protein synthesis prevents the destabilization of tubulin mRNA. The effect was similar whether mRNA was maintained on polysomes in the presence of emetine or anisomycin or displaced from the polysomes in the presence of puromycin or pactamycin; thus, the stabilization of tubulin mRNA is not dependent on the state of the polysomes after inhibition of protein synthesis. Even after tubulin mRNA declined to a low level after depolymerization of microtubules, it could be rescued by treatment of embryos with inhibitors of protein synthesis. Tubulin mRNA could be induced to accumulate prematurely in gastrulae but not in plutei if protein synthesis was inhibited, an observation that is indicative of the importance of the autogenous regulation of tubulin mRNA stability during embryogenesis. Possible explanations for the role of protein synthesis in the control of mRNA stability are discussed.

Animals↗
PubMed1999

Down-regulation of beta1,4-galactosyltransferase gene expression by cell-cycle suppressor gene p16.

Beta1,4-Galactosyltranferase (beta1,4GT, EC 2.4.1.38) is one of the key enzymes controlling the biosynthesis of complex-type oligosaccharides, and is also one of the best-studied glycosyltransferases. To study the molecular mechanisms involved in the regulation of beta1,4GT gene expression, we transfected cell-cycle suppressor gene p16 into A549 cell line (in which p16 is deleted), measured beta1,4GT gene expression by Northern blot hybridization, and evaluated its activity. It was found that p16 could down-regulate beta1,4GT gene expression and its activity. However, p16 decreased cell surface beta1,4GT activity more than total activity. beta1,4GT mRNA stability was also assayed. It was found that p16 could not influence beta1,4GT mRNA stability.

Cell Cycle↗
PubMed2002

Regulation of Toll-like receptor 4 expression in the lung following hemorrhagic shock and lipopolysaccharide.

The Toll-like receptor 4 (TLR4) has recently been shown to function as the major upstream sensor for LPS. In this study, a rodent model of lung injury following resuscitated hemorrhagic shock was used to examine the regulation of TLR4 gene and protein expression in vivo and in vitro. Intratracheal LPS alone induced a rapid reduction in whole lung TLR4 mRNA, an effect which is also observed in recovered alveolar macrophages. This effect appeared to be due to a lowering of TLR4 mRNA stability by approximately 69%. By contrast, while shock/resuscitation alone had no effect on TLR4 mRNA levels, it markedly altered the response to LPS. Specifically, antecedent shock prevented the LPS-induced reduction in TLR4 mRNA levels. This reversal was explained by the ability of prior resuscitated shock both to prevent the destabilization of TLR4 mRNA by LPS and also to augment LPS-stimulated TLR4 gene transcription compared with LPS alone. Oxidant stress related to shock/resuscitation appeared to contribute to the regulation of TLR4 mRNA, because supplementation of the resuscitation fluid with the antioxidant N-acetylcysteine reversed the ability of shock/resuscitation to preserve TLR4 mRNA levels following LPS. TLR4 protein levels in whole lung mirrored the changes seen for TLR4 mRNA. Considered in aggregate, these data suggest that levels of tlr4 expression are controlled both transcriptionally as well as posttranscriptionally through altered mRNA stability and that antecedent shock/resuscitation, a form of global ischemia/reperfusion, might influence regulation of this gene.

Animals↗
PubMed1999

Expression of trans-sialidase and 85-kDa glycoprotein genes in Trypanosoma cruzi is differentially regulated at the post-transcriptional level by labile protein factors.

To adapt to different environments, Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, expresses a different set of proteins during development. To begin to understand the mechanism that controls this differential gene expression, we have analyzed the levels of amastin and trans-sialidase mRNAs and the mRNAs encoding members of the 85-kDa glycoprotein gene family, which are differentially expressed in the T. cruzi stages found in the mammalian host. Amastin mRNA is expressed predominantly in intracellular and proliferative amastigotes. trans-Sialidase mRNAs are found mostly in forms undergoing transformation from amastigotes to trypomastigotes inside infected cells, whereas mRNAs encoding the 85-kDa glycoproteins appear only in the infective trypomastigotes released from the cells. The genes coding for these mRNA species are constitutively transcribed in all stages of T. cruzi cells, suggesting that expression is controlled post-transcriptionally during differentiation. Inhibition of transcription by actinomycin D revealed that each mRNA species has a relatively long half-life in stages where it accumulates. In the case of the trans-sialidase and 85-kDa glycoprotein genes, mRNA accumulation was induced by treatment with the protein synthesis inhibitor cycloheximide at the stages that preceded the normal accumulation. Therefore, mRNA stabilization may account for mRNA accumulation. mRNA degradation could be promoted by proteins with high turnover, or stabilization could be promoted by forming a complex with the translational machinery at defined times in development. Identification of the factors that induce mRNA degradation or stabilization is essential to the understanding of control of gene expression in these organisms.

Animals↗