Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “mRNA stability”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 775 records · Page 43Linked to original sources
PubMed2001

Molecular mechanisms of IL-2 gene regulation following costimulation through LFA-1.

The integrin LFA-1 serves as an accessory molecule in T cell activation. In addition to its well-known role as an adhesion molecule, LFA-1 can contribute to T cell activation and up-regulation of IL-2 gene expression. However, the specific mechanisms by which LFA-1 influences T cell activation have not been elucidated. Therefore, we examined the impact of LFA-1:ICAM-1 interactions on transcriptional and posttranscriptional IL-2 gene regulation, using a costimulation-negative cell line transfected with MHC class II alone, or in combination with ICAM-1 or B7-1. IL-2 transcription was assessed utilizing transgenic mice expressing an IL-2 promoter luciferase reporter construct crossed to DO11.10 TCR-transgenic mice, and IL-2 mRNA stability was evaluated by real-time RT-PCR. Comparison of naive and previously activated T cells demonstrates a dramatic increase in IL-2-luciferase transcription in activated T cells that can, in part, be attributed to downstream signaling events. Costimulation through LFA-1 enhances transcription of the transgenic reporter construct across a wide Ag dose range, but does not affect IL-2 mRNA stability. In contrast, CD28 costimulation is clearly mediated through up-regulation of IL-2 transcription and through enhancement of mRNA stability. These results indicate that the primary pathway whereby engagement of LFA-1 through its ligand ICAM-1 up-regulates IL-2 gene expression is through enhanced IL-2 transcription, in the absence of any effect on IL-2 mRNA stabilization.

CD28 Antigens↗
PubMed1992

Alteration of translation and stability of mRNA for the poly(A)-binding protein during myogenesis.

The regulation of synthesis of various factors involved in mRNA translation during differentiation of muscle cells was examined. The steady-state levels of mRNAs coding for eukaryotic initiation factor (eIF) 2 alpha, 2 beta and elongation factor (eEF)-1 alpha were measured in both proliferating rat L6 myoblast and differentiated myotubes. The steady-state levels of these mRNAs were not altered during myogenesis. Furthermore, the distribution of these mRNAs between repressed and translated populations remained unchanged. Recent studies suggest a role for poly(A)-binding protein (PABP) in translation initiation. Therefore, we also examined the expression of PABP mRNA during myogenesis. The PABP mRNA was less abundant in myotubes compared to myoblasts. However, the synthesis of PABP remained unchanged. In myoblasts, approximately 50-60% of the total mRNA was associated with polyribosomes, whereas in myotubes more than 80% of the mRNA was associated with polyribosomes. These results, therefore, suggest that the PABP mRNA was more efficiently translated in differentiated myotubes than in the proliferating myoblasts. Measurement of the stability and transcription of PABP mRNA showed that, while transcription was not affected during myogenesis, the stability of the mRNA was reduced in differentiated cells. The t1/2 of PABP mRNA in myoblasts was 13 h compared to 7.5 h in myotubes. This observation suggests that the reduced steady-state level of PABP mRNA in myotube were largely due to the change in stability of this mRNA during myogenesis.

Animals↗
PubMed1998

Regulation of the stability of heat-stable antigen mRNA by interplay between two novel cis elements in the 3' untranslated region.

The heat-stable antigen (HSA) is a costimulatory molecule for T-cell activation. Its expression is strictly regulated during lymphocyte development and differentiation. Recent studies using HSA-transgenic mice have demonstrated that this regulated expression is critical for normal development of T and B lymphocytes. However, the mechanisms that control the expression of HSA are largely unknown. HSA mRNA is comprised of a 0.23-kb open reading frame and a 1.5-kb 3' untranslated region (3'UTR). The function of the long 3'UTR has not been addressed. Here we investigate the role of the 3'UTR of HSA mRNA. We show that a 160-bp element, located in the region of nucleotides 1465 to 1625 in the 3'UTR of HSA mRNA, promotes RNA degradation and that this effect is neutralized by a 43-bp fragment approximately 1 kb upstream of the negative cis element. Both positive and negative cis elements in the HSA mRNA are distinct from other sequences that are known to modulate mRNA stability. These results provide direct evidence that the interplay between two novel cis elements in the 3'UTR of HSA mRNA determines cell surface HSA expression by modulating its RNA stability.

Animals↗
PubMed1997

c-fms mRNA is regulated posttranscriptionally by 1,25(OH)2D3 in HL-60 cells.

Macrophage colony-stimulating factor (MCSF) is required for normal osteoclast and macrophage development. The receptor for MCSF (c-fms) is expressed on the pluripotent precursor and mature osteoclasts and macrophages. We have previously shown in myelomonocytic HL-60 cells that phorbol myristate acetate (PMA) upregulates c-fms mRNA expression. This induction of c-fms is inhibited by 1,25(OH)2D3. The major regulatory control of c-fms mRNA levels by PMA has been identified as posttranscriptional. However, a role of transcript elongation in controlling levels of c-fms mRNA has also been suggested. To better understand the 1,25(OH)2D3 regulation of c-fms mRNA expression we studied nuclear run on, mRNA stability, and transcript elongation in HL-60 cells treated with 10 ng/ml phorbol myristate acetate, 10 nM 1,25(OH)2D3 alone or combined. We demonstrated by nuclear run on that c-fms was constitutively transcribed in 1,25(OH)2D3 as well as control and PMA-treated cells. Transcript elongation was evaluated by RT-PCR for exon 2 or exon 3. Both exons were minimally expressed in control and 1,25(OH)2D3-treated cells, and increased in PMA-treated cells; this increased expression was inhibited by the addition of 1,25(OH)2D3. These results fail to show differential transcript elongation. Measurement of mRNA stability demonstrated decreased mRNA half-life to 5 hours in cells treated with PMA and 1,25(OH)2D3 compared with a half-life of 8 hours in cells treated with PMA alone. Our findings demonstrate that c-fms is regulated by 1,25(OH)2D3 at the posttranscriptional level by changes in mRNA stability. This gives the cell the ability to respond to local signals with rapid changes in c-fms levels altering the ability of the cell to respond to MCSF.

Calcitriol↗
PubMed1997

VEGF mRNA is stabilized by ras and tyrosine kinase oncogenes, as well as by UV radiation--evidence for divergent stabilization pathways.

Vascular Endothelial Growth Factor (VEGF) is a pivotal endothelial cell mitogen that mediates both normal and pathological angiogenesis. Although expressed at very low levels in cells not undergoing vascularization, VEGF mRNA is transiently upregulated and stabilized by a variety of extracellular stimuli, and is persistently upregulated and stabilized in many human tumor cell lines (White et al., 1995). Here we demonstrate that oncogenic activation of tyrosine protein kinases and Ras proteins induce a 6- to 16-fold increase in the abundance of VEGF mRNA and a 3- to 5-fold increase in the stability of VEGF mRNA, suggesting that persistent activation of signaling pathways induced by these oncoproteins accounts for overexpression of VEGF in a significant fraction of human tumors. In addition to these oncoproteins, ultraviolet (UV) radiation upregulated and stabilized VEGF mRNA 15- and 5-fold, respectively. While the tyrosine kinase inhibitor, genistein, blocked VEGF upregulation by activated tyrosine protein kinases, and the Ras inhibitor, N-Acetyl-S-trans-farnesyl-L-cysteine (AFC), eliminated VEGF expression in cells transformed by v-Ras, neither agent blocked upregulation by hypoxia or UV radiation. These data argue that multiple divergent pathways upregulate and stabilize VEGF mRNA.

3T3 Cells↗
PubMed1994

Postmortem stability of mRNA for glucocorticoid and mineralocorticoid receptor in rodent brain.

The relative postmortem stability of the mRNA's for glucocorticoid (GR) and mineralocorticoid (MR) receptor in rodent brain was determined using semi-quantitative in situ hybridization (ISH). Rats were killed by CO2 asphyxiation and their brains removed immediately (0 h) or following 12 h or 24 h delays. Specific hybridization of GR and MR anti-sense [35S]RNA-probe to tissue mRNA encoding these receptors was detected using film and emulsion autoradiography. The most intense labeling for GR mRNA was in the dentate gyrus followed by the CA1 hippocampal region. Lower, but still detectable signal, was apparent over CA3-CA4 pyramidal cell regions. MR mRNA was detected throughout the CA1-4 pyramidal cell fields of the hippocampus and the granular cells of the dentate gyrus. Film images demonstrated that even in the 24 h postmortem delay group intense specific signal was present in sections hybridized with both anti-sense GR and MR probes, although there was some diminution in signal intensity in cortical areas at this later postmortem delay. These initial experiments with rat brain demonstrate that the mRNA's for both GR and MR, as detected with ISH, are stable for up to 24 h following death.

Animals↗
PubMed1983

Transcriptional and cell cycle-mediated regulation of myosin heavy chain gene expression during muscle cell differentiation.

The molecular mechanisms regulating the induction of myosin heavy chain (MHC) gene expression during muscle cell differentiation were studied using a MHC cDNA recombinant plasmid. During in vitro L6E9 cell myogenesis, cytoplasmic MHC mRNA content/cell nucleus increases a minimum of 500-fold during the first 6 days of differentiation. Two independent parameters regulating MHC mRNA accumulation were directly measured. (i) Intrinsic (chemical) MHC mRNA stability (t1/2 = 55-60 h) is the same in both myotubes and when first detected in myoblasts. This suggests that the intrinsic stability of the MHC mRNA molecule does not change during myogenesis. (ii) The rate of MHC gene transcription and MHC mRNA synthesis increases approximately 100-fold during myogenesis but is insufficient to account for the entire MHC mRNA accumulation. An additional independent parameter was found to profoundly affect MHC mRNA accumulation. Withdrawal from the cell cycle, as occurs during terminal myogenic differentiation, increases the final accumulation of stable mRNAs, such as MHC, by increasing mRNA effective stability. During L6E9 myogenesis, the transition from mitotically active myoblasts (doubling time = 16 h) to postmitotic myotubes results in a 4-5-fold increase in the effective stability of cytoplasmic MHC mRNA. This cell cycle-mediated effect, combined with the induction of MHC mRNA synthesis, completely accounts for MHC mRNA accumulation. A parallel effect occurs in the total cytoplasmic poly(A)+ mRNA population. The rates of synthesis of each of the two major stability components (t1/2 = 5 and 50 h, respectively) are equally increased 2-3-fold during myogenesis. However, the composition of the cytoplasmic mRNA population changes due to the preferential accumulation of stable mRNAs. We conclude that both transcriptional and cell cycle-mediated regulation of MHC gene expression is necessary, but either alone is not sufficient to produce the differentiated muscle cell phenotype.

Animals↗
PubMed1999

ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system.

We have developed an in vitro mRNA stability system using HeLa cell cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that reproduces regulated aspects of mRNA decay. The addition of cold poly(A) competitor RNA activated both a sequence-specific deadenylase activity in the extracts as well as a potent, ATP-dependent ribonucleolytic activity. The rates of both deadenylation and degradation were up-regulated by the presence of a variety of AU-rich elements in the body of substrate RNAs. Competition analyses demonstrated that trans-acting factors were required for RNA destabilization by AU-rich elements. The approximately 30-kD ELAV protein HuR specifically bound to RNAs containing an AU-rich element derived from the TNF-alpha mRNA in the in vitro system. Interaction of HuR with AU-rich elements, however, was not associated with RNA destabilization. Interestingly, recombinant ELAV proteins specifically stabilized deadenylated intermediates generated from the turnover of AU-rich element-containing substrate RNAs. These data suggest that mammalian ELAV proteins play a role in regulating mRNA stability by influencing the access of degradative enzymes to RNA substrates.

Adenosine Triphosphate↗
PubMed1989

Expression of a glucose transporter gene cloned from brain in cellular models of insulin resistance: dexamethasone decreases transporter mRNA in primary cultured adipocytes.

In two cellular models of insulin resistance we measured glucose transport activity, total glucose transporter number using the cytochalasin B binding assay, and expression of a transporter mRNA species specifically hybridizing with cDNA cloned from brain. In primary cultured adipocytes, chronic exposure to glucose plus insulin (24 h), but neither agent alone, markedly decreased (less than 50%) glucose transport activity; however, neither glucose nor insulin regulated the number of glucose transporters or levels of transporter mRNA whether normalized per total RNA, RNA per cell, or as a fraction of CHO-B mRNA. On the other hand, chronic treatment with 30 nM dexamethasone (24 h) decreased basal and maximal transport rates (approximately 75%), led to a 40% depletion in total cellular glucose transporters, and decreased transporter mRNA by 57-59% (t 1/2 = 10 h; ED50 = 4-5 nm). Dexamethasone's effects to decrease transport rates, transporter protein, and mRNA were inhibited by coincubation with insulin. Dexamethasone did not alter the degradation rate of transporter mRNA relative to that in control cells indicating a lack of effect on mRNA stability. Also, suppression of transporter mRNA did not appear to require ongoing protein synthesis since the effect was observed when dexamethasone was added to cycloheximide-treated cells; however, cycloheximide per se specifically increased transporter mRNA 4-fold. We conclude in adipocytes: 1) glucose and insulin (24 h) do not regulate the total number of glucose transporters or expression of mRNA encoding a transporter species cloned from brain. 2) Long-term dexamethasone treatment reduces the cellular abundance of both glucose transporters and the specific transporter mRNA; these effects may be due to inhibition of gene transcription since dexamethasone does not influence transporter mRNA stability. 3) Insulin heterologously inhibits regulation of the glucose transport system by dexamethasone. 4) Dexamethasone-mediated insulin resistance is due in part to regulation of a glucose transporter species encoded by cDNA cloned from brain. These observations may be relevant to mechanisms of insulin resistance in clinical states of hypercortisolism.

Adipose Tissue↗
PubMed2001

Autoregulation allows Escherichia coli RNase E to adjust continuously its synthesis to that of its substrates.

The Escherichia coli endonuclease RNase E plays a key role in rRNA maturation and mRNA decay. In particular, it controls the decay of its own mRNA by cleaving it within the 5'-untranslated region (UTR), thereby autoregulating its synthesis. Here, we report that, when the synthesis of an RNase E substrate is artificially induced to high levels in vivo, both the rne mRNA concentration and RNase E synthesis increase abruptly and then decrease to a steady-state level that remains higher than in the absence of induction. Using rne-lacZ fusions that retain or lack the rne 5'UTR, we show that these variations reflect a transient mRNA stabilization mediated by the rne 5'UTR. Finally, by putting RNase E synthesis under the control of an IPTG-controlled promoter, we show that a similar, rne 5'UTR-mediated mRNA stabilization can result from a shortage of RNase E. We conclude that the burst in substrate synthesis has titrated RNase E, stabilizing the rne mRNA by protecting its 5'UTR. However, this stabilization is self-correcting, because it allows the RNase E pool to expand until its mRNA is destabilized again. Thus, autoregulation allows RNase E to adjust its synthesis to that of its substrates, a behaviour that may be common among autoregulated proteins. Incidentally, this adjustment cannot occur when translation is blocked, and we argue that the global mRNA stabilization observed under these conditions originates in part from this defect.

5' Untranslated Regions↗
PubMed2004

Poly(A)-binding protein-interacting protein 2, a strong regulator of vascular endothelial growth factor mRNA.

Expression of vascular endothelial growth factor (VEGF) is tightly regulated, particularly at the level of its mRNA stability, which is essentially mediated through the 3'-untranslated region (3'-UTR) of VEGF mRNA. To identify new protein partners regulating VEGF mRNA stability, we screened a cDNA expression library with an RNA probe corresponding to the entire VEGF mRNA 3'-UTR. We identified the "poly(A)-binding protein-interacting protein 2" (PAIP2) as a new VEGF mRNA 3'-UTR interacting protein. By RNA electromobility shift assays, we showed that PAIP2 binds to two distinct regions of a domain encompassing base 1 to 1280 of the VEGF 3'-UTR. Such in vitro interaction was confirmed using cell extracts in which PAIP2 expression is induced by tetracycline (Tet-on cells). Moreover, we demonstrated by RNA affinity purification as well as by ribonucleoprotein complexes immunoprecipitation, that PAIP2 interacts with VEGF mRNA in vivo. Using an in vitro RNA degradation assay, the half-life of VEGF 3'-UTR was found to be increased by overexpressing PAIP2. PAIP2 stabilizes endogenous VEGF mRNA in Tet-on cells, leading to increased VEGF secretion. Moreover, RNAi-mediated knock-down of PAIP2 significantly reduces the steady-state levels of endogenous VEGF mRNA. We also showed, by in vitro protein-protein interactions and co-immunoprecipitation experiments, that PAIP2 interacts with HuR, an already known VEGF mRNA-binding protein, suggesting cooperation of both proteins for VEGF mRNA stabilization. Hence, PAIP2 appears to be a crucial regulator of VEGF mRNA and as a consequence, any variation in its expression could modulate angiogenesis.

3' Untranslated Regions↗
PubMed1976

Effect of arginine on the stability and size of argECBH messenger ribonucleic acid in Escherichia coli.

The chemical stability of argECBH messenger ribonucleic acid (mRNA) produced by Escherichia coli was found to be unaltered during steady-state repression by arginine. During extreme arginine deprivation, the increase in argECBH mRNA stability was related to general effects of amino acid starvation on mRNA stability. Thus a mechanism whereby argECBH gene expression is regulated by altering the decay rate of this mRNA is not consistent with our data. Sucrose gradient analysis followed by hybridization revealed that both heavy (14S) and light (8S) components of argECBH mRNA were produced by cells of E. coli K-12 grown without arginine, whereas predominantly light (8S) mRNA was produced by cells grown with arginine. A functional argR gene and the EC portion of the argECBH cluster were found essential for the arginine restriction of heavy-mRNA production. Experiments suggest that light argECBH mRNA did not arise from heavy message, and 8u% of both light and heavy mRNA was found bound to ribosomes. The data appear most consistent with the notion that a second site of control by arginine regulates the amounts of light and heavy arginine mRNA in the cell either by early termination of transcription or by endonucleolytic processing. Consideration of these data in conjunction with those of the accompanying report (Krzyzek and Rogers, 1976) permits the tentative conclusion that light argECBH mRNA is not translated into active enzymes and is thus responsible for the discrepancy between the high content of hybridizable mRNA and low rates of enzyme synthesis found during arginine repression.

Amidohydrolases↗
PubMed1997

Transferrin gene expression and secretion in rat sertoli cells.

Transferrin secretion and expression were studied in cultured Sertoli cells recovered from fats at days 10 and 17 postpartum. Transferrin biosynthesis as measured by radioimmunoassay showed a dramatic, 3.9-fold increase between days 10 and 17. The majority of the transferrin was secreted, and a kinetic study revealed that the production was four times higher at day 17 than at day 10. This difference was not the result of altered transferrin degradation as the protein was shown to be very stable at both ages. To determine if this regulation was at the transcriptional or translation level, Northern blot analysis, nuclear run-on assays, mRNA stability (half-life) measurements, and mRNA intracytoplasmic distribution analyses were carried out. The Northern blots analysis, the nuclear run-on assays, and the half-life measurements revealed that transferrin mRNA levels, gene transcription rates, and mRNA stability were indistinguishable at both ages. Interestingly, the intracytoplasmic mRNA distribution analyses showed that most of the transferrin mRNA (80%) was associated with the 40 S and 60 S protein particles at both ages and was, therefore, theoretically untranslatable. However, approximately twice as much transferrin in RNA was found to be associated with polysomes at day 17 as compared to day 10. We have shown that the increase in transferrin biosynthesis by rat Sertoli cells during testicular development is not due to an increase in the amount of transferrin mRNA or an increase in its half-life, but appears to be due to an increase in translation rate of the transferrin mRNA.

Animals↗
PubMed1995

A role for increased lutropin/choriogonadotropin receptor (LHR) gene transcription in the follitropin-stimulated induction of the LHR in granulosa cells.

Follitropin (FSH) has been shown in previous studies to stimulate the induction of the LH/CG receptor (LHR) and LHR mRNA in the granulosa cells of diethylstilbesterol-primed immature rats. The present studies were undertaken to identify the mechanisms underlying the hormone-dependent induction of the LHR in rat granulosa cells. The effect of FSH on LHR mRNA stability was determined by measuring the decay of LHR mRNA after removal of FSH under conditions where transcription was inhibited. Under these conditions, readdition of FSH had little effect on mRNA stability. However, inhibitors of transcription themselves were found to have a marked effect on stabilizing the LHR mRNA, thus potentially masking an effect of FSH. These results suggest that there is a labile destabilizing factor that constitutively degrades LHR mRNA. At present, it cannot be ascertained whether FSH has any effect on this destabilizing factor. Transcriptional activity of the LHR gene was examined using nuclear run-on assays. It was found that 1) in the absence of FSH, LHR-binding activity and mRNA levels were negligible, but the LHR gene was transcriptionally active in granulosa cells of immature rats; 2) incubations of granulosa cells with FSH or 8-bromo-cAMP significantly increased endogenous LHR gene transcription (approximately 10-fold) under conditions where increases in LHR mRNA were observed; 3) the continuous presence of FSH or 8-bromo-cAMP was required to maintain elevated levels of LHR gene transcription and LHR mRNA; and 4) exogenous estradiol alone had no effect on transcription of the LHR gene although it was able to synergistically enhance FSH-induced LHR expression. These experiments suggest that while the effects of estradiol on LHR induction do not appear to be mediated by an increase in LHR gene transcription, the effects of FSH (or cAMP) on LHR induction are clearly mediated, at least in part, by significant increases in the rate of LHR gene transcription.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Effects of polyamines, polyamine analogs, and inhibitors of protein synthesis on spermidine-spermine N1-acetyltransferase gene expression.

The key polyamine catabolizing enzyme spermidine-spermine N1-acetyltransferase (SSAT) is among the few genes known to be inducible by the natural polyamines. Certain polyamine analogs markedly exaggerate this response and thus provide useful tools for studying the underlying regulatory mechanisms. As shown here, the analog which most potently induces SSAT activity, N1, N11-diethylnorspermine (DENSPM), increases SSAT mRNA in MALME-3M human melanoma cells to a maximum of > 20-fold and immunodetectable SSAT protein to > 300-fold. By comparison, the natural polyamine spermine is far less effective, increasing SSAT mRNA by approximately 3-fold and protein by approximately 7-fold. In particular, the difference in mRNA accumulation by spermine and the analog was shown to be due to differential effects on both gene transcription and mRNA stabilization. Although the analog DENSPM has been regarded as the most potent inducer of SSAT activity and mRNA, we now report that inhibitors of protein synthesis are capable of increasing SSAT mRNA to nearly comparable levels. Inhibitor-induced accumulation in SSAT mRNA was shown to involve increased gene transcription and mRNA stabilization. This suggests that, under basal conditions, SSAT gene expression is suppressed by a labile protein (or proteins). While induction of SSAT mRNA by inhibitors of protein synthesis only occurred at concentrations which blocked protein synthesis, that by DENSPM took place at concentrations which did not. The combination of either protein inhibitor with DENSPM or spermine produced an additive increase in SSAT mRNA. Taken together, these findings suggest the involvement of two separate but possibly converging pathways in the regulation of SSAT mRNA, one mediated by polyamines and their analogs and the other mediated by a labile repressor of SSAT gene transcription and/or mRNA stabilization. In addition to its apparent regulatory importance, induction of SSAT mRNA by inhibitors of protein synthesis represents a potentially useful system for studying the posttranscriptional regulation of this interesting gene.

Acetyltransferases↗
PubMed1995

Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes.

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is upregulated in response to a hypoxic or hypoglycemic stress. Here we show that the increase in steady-state levels of VEGF mRNA is partly due to transcriptional activation but mostly due to increase in mRNA stability. Both oxygen and glucose deficiencies result in extension of the VEGF mRNA half-life in a protein synthesis-dependent manner. Viewing VEGF as a stress-induced gene, we compared its mode of regulation with that of other stress-induced genes. Results showed that under nonstressed conditions, VEGF shares with the glucose transporter GLUT-1 a relatively short half-life (0.64 and 0.52 h, respectively), which is extended fourfold and more than eightfold, respectively, when cells are deprived of either oxygen or glucose. In contrast, the mRNAs of another hypoxia-inducible and hypoglycemia-inducible gene, grp78, as well as that of HSP70, were not stabilized by these metabolic insults. To show that VEGF and GLUT-1 are coinduced in differentially stressed microenvironments, multicell spheroids representing a clonal population of glioma cells in which each cell layer is differentially stressed were analyzed by in situ hybridization. Cellular microenvironments conducive to induction of VEGF and GLUT-1 were completely coincidental. These findings show that two different consequences of tissue ischemia, namely, hypoxia and glucose deprivation, induce VEGF and GLUT-1 expression by similar mechanisms. These proteins function, in turn, to satisfy the tissue needs through expanding its vasculature and improving its glucose utilization, respectively.

Animals↗
PubMed2001

Activation of the IL-10 gene promoter following photodynamic therapy of murine keratinocytes.

Photodynamic therapy (PDT), an anticancer treatment modality, has recently been shown to be an effective treatment for several autoimmune disease models including antigen-induced arthritis. PDT was found to induce the expression of IL-10 messenger RNA (mRNA) and protein in the skin, and this expression has similar kinetics to the appearance of PDT-induced suppression of skin-mediated immune responses such as the contract hypersensitivity (CHS) response. Some aspects of the UVB-induced suppression of the immune response have been linked to the induction of IL-10. IL-10 has been shown to inhibit the development and activation of Th1 cells, which are critical for many cell-mediated immune responses, including CHS. We have examined the effect of PDT and UVB irradiation on the activity of the IL-10 gene promoter and on IL-10 mRNA stability using the murine keratinocyte line, PAM 212. In vitro PDT induces IL-10 mRNA and protein expression from PAM 212 cells, which can be correlated with an increase in AP-1 DNA binding activity and activation of the IL-10 gene promoter by PDT. Deletion of an AP-1 response element from the IL-10 gene promoter was shown to abrogate the PDT-induced promoter activity indicating that the AP-1 response element is critical to IL-10 induction by PDT. In addition, PDT results in an increase in IL-10 mRNA stability, which may also contribute to the increased IL-10 expression in PAM 212 cells following PDT. In vitro UVB irradiation also results in activation of the IL-10 promoter. However, in contrast to PDT, UVB-induced activation of the IL-10 promoter is not AP-1 dependent and did not increase IL-10 mRNA stability.

Animals↗
PubMed2001

Identification of a 43-kDa protein in human liver cytosol that binds to the 3'-untranslated region of CYP2A6 mRNA.

Hepatic expression of cytochrome P450 2A6 (CYP2A6) varies widely in humans and is induced during hepatitis; however, the mechanism regulating CYP2A6 has not been established. The murine orthologue Cyp2a5 is regulated post-transcriptionally by mRNA stabilization. A 43-kDa protein that binds to the 3'-untranslated region (3'-UTR) of Cyp2a5 mRNA has been identified, but its role in mRNA stabilization is unclear. We hypothesized that similar interactions occur between cytosolic proteins in human liver and CYP2A6 3'-UTR mRNA. We identified, by RNA electrophoretic mobility shift assay, an hepatic cytosolic protein that binds specifically to sequences in the 3'-UTR of CYP2A6. Complexes did not form with denatured proteins and were eliminated with proteinase K digestion. Complex formation was inhibited with a molar excess of unlabeled CYP2A6 RNA but not by non-specific competitor RNA. Protein-mRNA interactions were not affected by probe denaturation, suggesting that RNA secondary structure is not essential for binding. UV cross-linking of complexes revealed RNA-binding proteins in both human and mouse liver cytosols with molecular masses of approximately 43 kDa. Using truncated RNA probes corresponding to various lengths of CYP2A6 mRNA, the protein-binding site was localized to a 50-nucleotide region between bases 1478 and 1527 of the 3'-UTR. Complex formation with hepatic cytosolic protein from four human subjects correlated with levels of hepatic CYP2A6 microsomal protein, suggesting a possible regulatory role. Further characterization of the RNA-binding protein, the primary binding site, and the influence of this interaction on CYP2A6 mRNA stability will help to elucidate the relevance of these findings to the post-transcriptional control of CYP2A6.

3' Untranslated Regions↗