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At least 127 records · Page 7Linked to original sources
PubMed1991

Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells.

Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region.

Animals↗
PubMed1994

Role of differential mRNA stability in the regulated expression of IgM and IgD.

The expression of IgM and IgD heavy chain mRNA in resting vs activated B cells offers a unique tool for the assessment of the effect of translation on mRNA stability because mu and delta mRNA have identical VDJ sequences but differ substantially in the rest of the molecule. We have shown that despite the 5' UTR identity that allows equal access to the translation machinery, mu mRNA has a significantly higher turnover rate than delta mRNA. However, the short t1/2 of mu mRNA increases significantly after B cell activation. Furthermore, the induction of microS mRNA after B cell activation provides yet another related molecule for comparison. Thus, despite the fact that microS and microM mRNA differ at their 3' ends, they have identical turnover rates in activated B cells. In addition, because the turnover rates of delta mRNA and beta 2 and GAPDH mRNA remain unchanged, these experiments suggest that B cell activation results in the induction of regulatory factor(s) that target specific sequences within mRNA-mu to confer greater stability. They also argue against a more passive regulation of mRNA stability that is a consequence of alterations in the secretory machinery.

Animals↗
PubMed1995

Interleukin-3 mRNA stabilization by a trans-acting mechanism in autocrine tumors lacking interleukin-3 gene rearrangements.

Tumors obtained from v-Ha-ras-transformed PB-3c cells are characterized by autocrine interleukin-3 (IL3) expression, which occurs either without (class I tumors) or with enhanced transcription (class II tumors). To address possible post-transcriptional mechanisms of IL3 expression, IL3 mRNA stability was examined in both tumor classes. Increased stability of IL3 mRNA was detected in class I tumor lines (t1/2 > 3 h), whereas rapid decay of IL3 transcripts (t1/2 < 0.5 h) was found in class II tumor lines. In both tumor classes, the c-myc and interleukin-6 transcripts were short-lived. Transcripts of a constitutively expressed IL3 reporter gene were stable in class I tumor cells but unstable in class II tumor cells, suggesting that IL3 mRNA stabilization involved a trans-acting mechanism. Rapid decay of IL3 reporter transcripts was observed in untransformed PB-3c as well as in v-Ha-ras expressing precursor cells linking transcript stabilization to the tumor stage. Reporter transcript stabilization in class I tumor cells correlated with increased IL3 production. Deletion of the AU-rich element from the IL3 reporter gene further augmented IL3 mRNA levels as well as IL3 production, suggesting that the stabilizing mechanism in class I tumor cells is not equivalent to AU-rich element deletion.

Animals↗
PubMed2006

PDGF-BB enhances monocyte chemoattractant protein-1 mRNA stability in smooth muscle cells by downregulating ribonuclease activity.

Platelet-derived growth factor (PDGF) has protean manifestations, including the regulation of growth and migration, in many cell types. We have previously reported that PDGF-BB induces the accumulation of monocyte chemoattractant protein (MCP)-1 mRNA in smooth muscle cells (SMC), in large part due to an increase in mRNA stability. To elucidate the mechanism by which PDGF-BB stabilizes MCP-1 mRNA, we have employed in vitro RNA gel mobility shift and decay assays. Cytoplasmic extracts from PDGF-BB-treated SMC increased the half-life of in vitro transcribed MCP-1 mRNA from approximately 45 min to >2 h. PDGF-BB-inhibitable degradation was not dependent on specific regions of the MCP-1 mRNA and was equally effective on a variety of in vitro transcribed mRNAs. Angiotensin II had a similar effect on MCP-1 mRNA stability, whereas tumor necrosis factor-alpha and basic fibroblast growth factor did not. The PDGF-BB-inhibitable RNAse activity was active at pH 6.6 and heat stable, but was sensitive to proteinase K. Extracts from PDGF-BB- or angiotensin II-treated cells inhibited the RNAse activity of control extracts, suggesting that the effect of PDGF-BB and angiotensin II are due to activation of a soluble inhibitor of the RNAse. The effect of PDGF-BB was blocked by inhibitors of tyrosine phosphorylation, but not by inhibitors of phosphatidylinositol 3-kinase or mitogen-activated protein kinases. These studies provide new insights into the mechanisms by which PDGF-BB enhances mRNA accumulation.

Angiotensin II↗
PubMed2000

Gene-specific trans-regulatory functions of magnesium for chloroplast mRNA stability in higher plants.

In higher plant chloroplasts the accumulation of plastid-encoded mRNAs during leaf maturation is regulated via gene-specific mRNA stabilization. The half-lives of chloroplast RNAs are specifically affected by magnesium ions. psbA mRNA (D1 protein of photosystem II), rbcL mRNA (large subunit of ribulose-1,5-bisphosphate carboxylase), 16 S rRNA, and tRNA(His) gain stability at specific magnesium concentrations in an in vitro degradation system from spinach chloroplasts. Each RNA exhibits a typical magnesium concentration-dependent stabilization profile. It shows a cooperative response of the stability-regulated psbA mRNA and a saturation curve for the other RNAs. The concentration of free Mg(2+) rises during chloroplast development within a range sufficient to mediate gene-specific mRNA stabilization in vivo as observed in vitro. We suggest that magnesium ions are a trans-acting factor mediating differential mRNA stability.

5' Untranslated Regions↗
PubMed2002

Interleukin-1beta elevates cyclooxygenase-2 protein level and enzyme activity via increasing its mRNA stability in human endometrial stromal cells: an effect mediated by extracellularly regulated kinases 1 and 2.

We investigated the regulation of PG production in human endometrial stromal cells (ESC) by IL-1beta. We found that cyclooxygenase-2 (COX-2) mRNA and protein levels and PGE(2) production in ESC were significantly increased by IL-1beta. COX-2 mRNA, protein, and PGE(2) levels in IL-1beta-treated ESC were decreased by a PKA inhibitor, a nuclear factor (NF-kappaB) inhibitor, and an ERK1/2 inhibitor, but not by a p38 MAPK inhibitor or a PKC inhibitor, suggesting the possible involvement of PKA, NF-kappaB, and/or the ERK1/2 signaling pathway(s) in IL-1beta-mediated COX-2 gene induction in ESC. We then transiently transfected deletion mutants of the COX-2 promoter fused to the luciferase reporter gene and variants of -360/+56 bp promoter construct carrying different site-directed mutations of selected cis-acting elements. We determined that a NF-kappaB site (-222/-213 bp), a nuclear factor for IL-6 expression site (NF-IL6, -132/-124 bp), and a cAMP response element (-59/-52 bp) were essential for the baseline COX-2 gene promoter regulation. The addition of IL-1beta, however, did not affect the activity of these COX-2 promoter constructs. To investigate the potential effects of IL-1beta on COX-2 mRNA stability, ESC were treated with actinomycin D, a general transcription inhibitor, in the absence or presence of IL-1beta. We found that 1) IL-1beta significantly increased COX-2 mRNA stability; 2) continuous transcription was not required to sustain the IL-1beta-induced COX-2 mRNA levels; and 3) COX-2 mRNA was highly unstable in the absence of IL-1beta. Additionally, we found that the ERK1/2 signaling pathway was essential for stabilizing COX-2 mRNA. We conclude that levels of COX-2 mRNA, protein, and enzyme activity in ESC are controlled by various signaling pathways, including PKA, ERK1/2, and NF-kappaB. Moreover, posttranscriptional mRNA stability is an important mechanism for IL-1beta-induced elevation of COX-2 expression in ESC.

Butadienes↗
PubMed2001

Cold accumulation of SCOF-1 transcripts is associated with transcriptional activation and mRNA stability.

Cold acclimation enhances the transcription of several cold regulated (COR) genes. However, little is known about whether the elevation of the transcriptional level of the COR genes is due to transcriptional activation, or mRNA stability by a low temperature. Recently, we cloned a novel cold-inducible zinc finger protein gene from soybean, SCOF-1, which may function as a positive regulator of the COR gene expression . Here we report that the elevation of the SCOF-1 transcript level by cold stress is associated with both transcriptional activation and post-transcriptional mRNA stability under a low temperature. A nuclear run-on assay reveals that cold acclimation elevates the SCOF-1 transcript about three-fold compared to that of non-acclimated soybean nuclei. Furthermore, SCOF-1 transcripts increased substantially by a low temperature in transgenic tobacco plants that constitutively expressed SCOF-1 under the control of a constitutive cauliflower mosaic virus (CaMV) 35S promoter. When a transcription inhibitor, cordycepin, was treated with the deacclimating soybean cell, the decay level of the SCOF-1 transcripts was delayed significantly. This suggests that it may affect de novo protein synthesis, which degrades the SCOF-1 mRNA at room temperature. In addition, a secondary structure may be involved in the mRNA stability of SCOF-1 under a low temperature.

Acclimatization↗
PubMed2003

Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization.

Mycobacterial infections of macrophages have been shown to inhibit the ability of the macrophage to respond to IFN-gamma. We previously reported that Mycobacterium avium infection of mouse macrophages decreases IFN-gamma-induced STAT1 tyrosine phosphorylation and STAT1 DNA binding. Because macrophages respond to M. avium through Toll-like receptor 2 (TLR2), we determined whether TLR2 stimulation inhibits the response to IFN-gamma. Treatment of mouse RAW264.7 macrophages with TLR2 agonists inhibited the induction of IFN-gamma-inducible genes by IFN-gamma. In contrast to M. avium infection, TLR2 agonists did not inhibit the IFN-gamma induction of DNA-binding activity of STAT1 and the tyrosine phosphorylation of STAT1alpha. Instead, IFN-gamma induction of RAW264.7 cells treated with TLR2 agonists resulted in an increase in the tyrosine phosphorylation of the dominant-negative STAT1beta. TLR2 stimulation of RAW264.7 cells increased both STAT1beta protein and mRNA expression, suggesting that the increased STAT1beta phosphorylation results from increased STAT1beta expression. Because STAT1alpha and STAT1beta mRNA have different 3' untranslated regions, and 3' untranslated regions can regulate mRNA stability, we examined the effects of TLR2 stimulation on mRNA stability. TLR2 stimulation of RAW264.7 cells increased the stability of STAT1beta mRNA, while not affecting the stability of STAT1alpha mRNA. The ability of STAT1beta to function as a dominant negative was confirmed by overexpression of STAT1beta in RAW264.7 macrophages by transient transfection, which inhibited IFN-gamma-induced gene expression. These findings suggest that M. avium infection of mouse macrophages inhibits IFN-gamma signaling through a TLR2-dependent increase in STAT1beta expression by mRNA stablization and a TLR2-independent inhibition of STAT1 tyrosine phosphorylation.

Animals↗
PubMed2003

3'UTRs of glutathione peroxidases differentially affect selenium-dependent mRNA stability and selenocysteine incorporation efficiency.

Selenoprotein mRNAs are particular in several aspects. They contain a specific secondary structure in their 3'UTR, called Secis (selenocysteine inserting sequence), which is indispensable for selenocysteine incorporation, and they are degraded under selenium-limiting conditions according to their ranking in the hierarchy of selenoproteins. In the familiy of selenium-dependent glutathione peroxidases (GPx) the ranking is GI-GPx > or = PHGPx > cGPx = pGPx. This phenomenon was studied by mutually combining the coding regions of GI-GPx, PHGPx and cGPx with their 3'UTRs. HepG2 cells were stably transfected with the resulting constructs. Expression of glutathione peroxidases was estimated by activity measurement and Western blotting, the selenium-dependent mRNA stability by real-time PCR. Whereas 3'UTRs from stable PHGPx and GI-GPx could be exchanged without loss of stability, they were not able to stabilize cGPx mRNA. cGPx 3'UTR rendered GI-GPx and PHGPx mRNA unstable. Thus, cGPx mRNA contains selenium-responsive instability elements in both the translated and the untranslated region, which cannot be compensated by one of the stable homologs. Stabilizing efficiency of an individual GPx 3'UTR did not correlate with the efficiency of selenocysteine incorporation. PHGPx 3'UTR was equally effective as cGPx 3'UTR in enhancing GPx activity in all constructs, while GI-GPx 3'UTR showed a markedly lower efficacy. We conclude that different mRNA sequences and/or RNA-binding proteins might regulate mRNA stability and translation of selenoprotein mRNA.

3' Untranslated Regions↗
PubMed2004

Activation of adenosine receptors inhibits tumor necrosis factor-alpha release by decreasing TNF-alpha mRNA stability and p38 activity.

Adenosine receptor agonists have anti-inflammatory properties and modulate immune responses partly by inhibiting pro-inflammatory cytokine production by monocytes. We investigated signal transduction mechanisms by which adenosine receptor activation inhibits tumor necrosis factor-alpha (TNF-alpha) production. Phorbol-12-myristate-13-acetate (PMA) and phytohemagglutinin treatment of human pro-monocytic U937 cells increased TNF-alpha protein release. Activation of adenosine receptors up to 1 hr following stimulation with PMA/phytohemagglutinin significantly inhibited TNF-alpha protein release indicating that inhibition of TNF-alpha occurred post-transcriptionally. The adenosine receptor agonist 2-p-(carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) decreased stability and half-life of PMA/phytohemagglutinin-induced TNF-alpha mRNA from 80 to 37 min. p38 signaling pathways control TNF-alpha mRNA stability in macrophages and we confirmed in our cells that p38 was involved in controlling TNF-alpha release post-transcriptionally. Activation of adenosine receptors with CGS 21680 decreased phospho-p38 protein levels. These data suggest that adenosine receptor activation regulates TNF-alpha release post-transcriptionally by decreasing mRNA stability through a mechanism involving inhibition of p38 activity.

Extracellular Signal-Regulated MAP Kinases↗
PubMed2000

Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation.

Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.

Amino Acid Sequence↗
PubMed1994

Gonadotropin-releasing hormone stimulates glycoprotein hormone alpha-subunit messenger ribonucleic acid (mRNA) levels in alpha T3 cells by increasing transcription and mRNA stability.

Pulsatile GnRH stimulates gonadotropin secretion, whereas continuous exposure to GnRH causes pituitary desensitization and suppressed levels of LH and FSH. At the level of gene expression, continuous GnRH also causes partial or complete suppression of the LH beta and FSH beta genes, but expression of the alpha-subunit gene is stimulated without evidence of desensitization. In this report, we examined the transcriptional and posttranscriptional mechanisms by which GnRH controls alpha-gene expression using the gonadotrope-derived alpha T3 cell line. Continuous GnRH caused a 4- to 5-fold accumulation of alpha mRNA over 72 h, without evidence of a decline. In contrast, measurements of alpha-gene transcription, either by nuclear run-on assays or using a stably integrated alpha LUC reporter gene, revealed that GnRH caused a transient increase in alpha-promoter activity, followed by a decline after 4-6 h. The prolonged accumulation of alpha mRNA at a time when transcriptional activity had abated was accounted for by independent effects of GnRH on alpha mRNA stability. After prior treatment with GnRH, its removal either by washout or using a GnRH receptor antagonist caused an abrupt decline in steady state alpha mRNA levels (t1/2, < 2 h). Readdition of GnRH prevented the decay in alpha mRNA, and experiments using the transcriptional inhibitor actinomycin-D confirmed that this effect of GnRH did not require transcription. Consistent with these results, pulse-chase analyses of mRNA stability demonstrated that GnRH increased the alpha mRNA half-life 6.7-fold, from 1.2 h in the absence of GnRH to 8.0 h in the presence of GnRH. We conclude that GnRH induces a transient burst of alpha-gene transcription that is accompanied by marked induction of mRNA stability.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2006

LFA-1-dependent HuR nuclear export and cytokine mRNA stabilization in T cell activation.

Lymphokine gene expression is a precisely regulated process in T cell-mediated immune responses. In this study we demonstrate that engagement of the beta(2) integrin LFA-1 in human peripheral T cells markedly extends the half-life of TNF-alpha, GM-CSF, and IL-3 mRNA, as well as a chimeric beta-globin mRNA reporter construct containing a strongly destabilizing class II AU-rich element from the GM-CSF mRNA 3'-untranslated region. This integrin-enhanced mRNA stability leads to augmented protein production, as determined by TNF-alpha ELISPOT assays. Furthermore, T cell stimulation by LFA-1 promotes rapid nuclear-to-cytoplasmic translocation of the mRNA-stabilizing protein HuR, which in turn is capable of binding an AU-rich element sequence in vitro. Abrogation of HuR function by use of inhibitory peptides, or marked reduction of HuR levels by RNA interference, prevents LFA-1 engagement-mediated stabilization of T cell TNF-alpha or IFN-gamma transcripts, respectively. Thus, HuR-mediated mRNA stabilization, stimulated by integrin engagement and controlled at the level of HuR nuclear export, is critically involved in T cell activation.

Active Transport, Cell Nucleus↗
PubMed2003

Heterogeneity in control of mRNA stability by AU-rich elements.

AU-rich elements (AREs), located in the 3'-untranslated region of unstable cytokine and chemokine mRNAs, promote rapid decay of otherwise stable mRNAs and may mediate selective mRNA stabilization in response to stimulation with interleukin-1 (IL-1). AREs vary considerably, however, in both size and sequence context. To assess the heterogeneity involved in control of mRNA stability by ARE motifs, human mRNA sequences from IL-1alpha-stimulated HEK293 cells and T98G cells were screened for either instability or stability using both cDNA (950 ARE containing sequences) and Affymetrix oligonucleotide (U95Av2 GeneChip) array analysis. Although ARE-containing mRNAs exhibited a broad range of stability, IL-1alpha promoted stability in a subset of mRNAs that were unstable when transcriptionally induced by tumor necrosis factor alpha. Stabilization of granulocyte/macrophage-colony stimulating factor and IL-8 mRNAs by IL-1alpha was achieved only after 2 h of stimulation, required ongoing protein synthesis, and depended on the activation of p38 MAPK. In contrast, stabilization of Gro3 mRNA in response to IL-1alpha was achieved immediately and was insensitive to inhibitors of protein synthesis and p38 MAPK activation. In concert, these findings demonstrate that ARE sequences are functionally heterogeneous; only a subset of unstable mRNAs is sensitive to stabilization by IL-1alpha. Moreover, IL-1alpha promotes stabilization of unstable mRNAs through distinct mechanistic pathways that distinguish between specific mRNA sequences.

3' Untranslated Regions↗
PubMed1991

Changes in Chloroplast mRNA Stability during Leaf Development.

During spinach leaf development, chloroplast-encoded mRNAs accumulate to different steady-state levels. Their relative transcription rates alone, however, cannot account for the changes in mRNA amount. In this study, we examined the importance of mRNA stability for the regulation of plastid mRNA accumulation using an in vivo system to measure mRNA decay in intact leaves by inhibiting transcription with actinomycin D. Decay of psbA and rbcL mRNAs was assayed in young and mature leaves. The psbA mRNA half-life was increased more than twofold in mature leaves compared with young leaves, whereas rbcL mRNA decayed with a similar relative half-life at both leaf developmental stages. The direct in vivo measurements demonstrated that differential mRNA stability in higher plant plastids can account for differences in mRNA accumulation during leaf development. The role of polysome association in mRNA decay was also investigated. Using organelle-specific translation inhibitors that force mRNAs into a polysome-bound state or deplete mRNAs of ribosomes, we measured mRNA decay in vivo in either state. The results showed that rbcL and psbA mRNAs are less stable when bound to polysomes relative to the polysome-depleted mRNAs and that their stabilities are differentially affected by binding to polysomes. The results suggested that ribosome binding and/or translation of the psbA and rbcL mRNAs may function to modulate the rate of their decay in chloroplasts.

Journal Article↗
PubMed1987

Inhibition of protein synthesis in LLC-PK1 cells increases calcitonin-induced plasminogen-activator gene transcription and mRNA stability.

The peptide hormone calcitonin induces the accumulation of urokinase-type plasminogen activator (uPA) mRNA in pig kidney LLC-PK1 cells. By itself, inhibition of protein synthesis had a negligible effect on uPA mRNA accumulation. Inhibition of protein synthesis led to two superinductive effects: an increase in calcitonin-induced uPA mRNA accumulation over time, and a shift in the dose-response curve so that lower calcitonin doses became more potent. To explain these two superinductive effects of protein-synthesis inhibition on calcitonin treatment, we demonstrated that the inhibition of protein synthesis increased both calcitonin-induced uPA-gene transcription and uPA-mRNA stability. Different protein-synthesis inhibitors had similar actions, arguing against the possibility that the results were attributable to an anomalous action of a particular inhibitor. The superinductive effects of protein-synthesis inhibition could not be mimicked when a tumour promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), was used instead of calcitonin as an inducer. Calcitonin and TPA exert their effects through different pathways, suggesting a clue to the mechanism of superinduction. Although inhibition of protein synthesis has been reported to increase transcription and mRNA stability in a number of other systems, the one described here appeared unique in combining both effects in the context of hormonal regulation.

Animals↗
PubMed2003

Regulation of chemokine mRNA stability by lipopolysaccharide and IL-10.

IL-10 has been reported to inhibit the expression of LPS-induced proinflammatory cytokines and chemokines by altering the rate of specific mRNA decay although the molecular target(s) for its action remain unknown. In the present study, using primary peritoneal exudate macrophages and a cell culture model in which a tetracycline-responsive promoter controls transcription of CXC ligand 1 (KC) mRNA, we demonstrate that LPS promotes a time-dependent increase in KC mRNA stability. Although IL-10 had no direct effect on mRNA decay, this treatment antagonized the stabilizing action of LPS. The mechanisms involved were further explored using a cell-free mRNA degradation system. A 5'-capped, polyadenylated in vitro transcript derived from the 3'-untranslated region of KC mRNA exhibited time-dependent decay in the presence of protein extracts prepared from untreated RAW264.7 macrophages. Extracts prepared from LPS-treated RAW264.7 cells had reduced decay activity and this change was antagonized if the cells were costimulated with IL-10. A substrate in which the AU-rich element motifs were mutated exhibited minimal decay that did not vary using extracts prepared from cells treated with LPS or LPS and IL-10. A nonadenylated RNA substrate was also degraded and that activity was diminished by LPS. In concert, these findings demonstrate that KC mRNA stability is regulated by LPS-induced alterations in activities that govern both deadenylation and degradation of the mRNA body. The effects of IL-10 on KC mRNA stability reflect antagonism of the response to LPS.

Animals↗
PubMed1993

Physiologic concentrations of glucose regulate fatty acid synthase activity in HepG2 cells by mediating fatty acid synthase mRNA stability.

Carbohydrate feeding of animals results in striking increases in hepatic fatty acid synthesis but much of this induction is presumed to be hormone mediated. To clarify the mechanisms responsible for the specific effect of carbohydrate on fatty acid synthesis, the effects of D-glucose on the expression of human fatty acid synthase (FAS) in HepG2 cells cultured in serum-free medium were studied. Northern blots of total RNA from these cells showed a single FAS mRNA band of 9.3 kilobases that was increased 2.7-5.4-fold in the presence of D-glucose. Lactate and citrate but not L-glucose mimicked this effect. Glucose induction of FAS mRNA was time- and concentration-dependent and most of the increase in FAS message was detected within the range of physiologic glucose concentrations. Glucose induction of FAS mRNA, protein synthetic rate, and enzyme activity were similar suggesting that glucose regulates FAS expression at a pretranslational level in this system. Transcription run-off experiments showed that glucose feeding was associated with no change in the FAS transcription initiation rate despite a 5-fold increase in FAS mRNA. FAS mRNA stability as determined by actinomycin D chase was 7-fold greater in the presence of glucose. Differences between FAS mRNA levels in cells grown in the presence versus the absence of glucose were inhibited by cycloheximide but not puromycin, suggesting that glucose regulation of FAS mRNA stability is not dependent on translation. Glucose, at physiologic concentrations and in the absence of hormones, appears to regulate FAS gene expression in HepG2 cells predominantly by mediating FAS mRNA stability.

Base Sequence↗