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PubMed1994

Amyloid precursor protein mRNA stability is controlled by a 29-base element in the 3'-untranslated region.

In the accompanying paper (Zaidi, S. H. E., Denman, R., and Malter, J. S. (1994) J. Biol. Chem. 269, 24000-24006) we demonstrate that in tumor and normal cells, multiple cytosolic proteins interact with a 29-base sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. These data suggested that APP gene expression may be modulated by regulated APP mRNA decay. We have investigated this prediction by measuring the decay rates of APP mRNA in resting and mitogen-treated peripheral blood mononuclear cells and H4 and K562 tumor cell lines. In resting peripheral blood mononuclear cells, APP mRNA decayed with a half-life of 4 h. Under these conditions, the activity of APP mRNA-binding proteins was not detectable. After activation, binding protein activities were induced, and APP mRNA decay was blocked with a half-life of > 12 h. In log phase neuronal or lymphoid tumor cell lines, binding activity was constitutively present and APP mRNA displayed a half-life of > 12 h. Protein synthesis inhibition by cycloheximide had no effect on APP mRNA decay in normal or tumor cells. Transfected wild type or mutant APP mRNAs that lacked the 29-base region were stable (t1/2 > 10 h) in K562 tumor cells. Therefore, we conclude that the 29-base region functions in cis to destabilize APP mRNA in resting, normal cells. Upon activation APP mRNA-binding proteins are induced, interact with the 29-base region, and likely participate in stabilization of the mRNA.

Amyloid beta-Protein Precursor↗
PubMed2002

Inhibition of cytokine-induced matrix metalloproteinase 9 expression by peroxisome proliferator-activated receptor alpha agonists is indirect and due to a NO-mediated reduction of mRNA stability.

Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.

Animals↗
PubMed1988

A novel effect of EGF on mRNA stability.

Expression of the epidermal growth factor (EGF) receptor gene is stimulated by EGF and the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). PMA elevates EGF receptor mRNA levels in human KB epidermoid carcinoma cells, but does not significantly affect the half-life of this mRNA when its decay is examined after the addition of actinomycin D. In contrast, EGF greatly prolongs the half-life of EGF receptor mRNA suggesting a possible mechanism for the stimulatory effect of EGF on EGF receptor mRNA levels. EGF also stabilizes beta-tubulin and beta-actin mRNAs but has very little effect on the degradation of total mRNA.

Actins↗
PubMed2006

Global analysis of mRNA stability in the archaeon Sulfolobus.

BACKGROUND: Transcript half-lives differ between organisms, and between groups of genes within the same organism. The mechanisms underlying these differences are not clear, nor are the biochemical properties that determine the stability of a transcript. To address these issues, genome-wide mRNA decay studies have been conducted in eukaryotes and bacteria. In contrast, relatively little is known about RNA stability in the third domain of life, Archaea. Here, we present a microarray-based analysis of mRNA half-lives in the hyperthermophilic crenarchaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, constituting the first genome-wide study of RNA decay in archaea. RESULTS: The two transcriptomes displayed similar half-life distributions, with medians of about five minutes. Growth-related genes, such as those involved in transcription, translation and energy production, were over-represented among unstable transcripts, whereas uncharacterized genes were over-represented among the most stable. Half-life was negatively correlated with transcript abundance and, unlike the situation in other organisms, also negatively correlated with transcript length. CONCLUSION: The mRNA half-life distribution of Sulfolobus species is similar to those of much faster growing bacteria, contrasting with the earlier observation that median mRNA half-life is proportional to the minimal length of the cell cycle. Instead, short half-lives may be a general feature of prokaryotic transcriptomes, possibly related to the absence of a nucleus and/or more limited post-transcriptional regulatory mechanisms. The pattern of growth-related transcripts being among the least stable in Sulfolobus may also indicate that the short half-lives reflect a necessity to rapidly reprogram gene expression upon sudden changes in environmental conditions.

DNA, Archaeal↗
PubMed1996

Regulation of stearoyl-CoA desaturase 1 mRNA stability by polyunsaturated fatty acids in 3T3-L1 adipocytes.

The effects of arachidonic acid (20:4, n-6) and other fatty acids on the expression of stearoyl-CoA desaturase gene 1 were investigated in fully differentiated 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with arachidonic acid resulted in a decrease in stearoyl-CoA desaturase (Scd) enzyme activity and scd1 mRNA. Arachidonic acid did not alter the transcription of the scd1 gene, whereas the half-life of the scd1 mRNA was reduced from 25.1 to 8.5 h. Blocking the conversion of arachidonic acid to eicosanoids by pretreatment of the cells with cyclooxygenase, lipoxygenase, or cytochrome P-450 epoxygenase inhibitors did not reverse the inhibition caused by arachidonic acid, indicating that eicosanoid synthesis is not necessary for the repression of scd1 mRNA expression. Treatment of adipocytes with linoleic (18:2, n-6) and linolenic (18:3, n-3) acids also resulted in inhibition of scd1 mRNA accumulation. By contrast, oleic acid (18:1, n-9) and stearic acid (18:0) had no effect on scd1 mRNA levels. Taken together, these results suggest that polyunsaturated fatty acids repress the expression of the scd1 gene in mature adipocytes by reducing the stability of scd1 mRNA.

3T3 Cells↗
PubMed1989

Resistance to hydrogen peroxide associated with altered catalase mRNA stability in MCF7 breast cancer cells.

We have established a variant of the human breast cancer cell line MCF7, designated MCF7/H2O2, which is 5-fold resistant to H2O2 by clonogenic assay. The specific activity of the H2O2 disposal enzyme catalase was elevated 3-fold in MCF7/H2O2; activities of other antioxidant enzymes, including glutathione peroxidase and superoxide dismutase, were not increased. The steady-state level of catalase mRNA was only slightly elevated (approx. 1.6-fold) in MCF7/H2O2 cells; however, degradation of catalase mRNA was markedly retarded in MCF-7/H2O2 compared to MCF-7 (82% of catalase mRNA remained 24 h after inhibition of RNA synthesis by actinomycin D in MCF-7/H2O2 vs. 32% in MCF7). The degradation rates of superoxide dismutase mRNA and 28 S ribosomal RNA were not reduced in MCF-7/H2O2; however, the rate of degradation of another mRNA species, beta-actin, was also significantly decreased. These data suggest that resistance to H2O2 in MCF7/H2O2 cells is mediated by elevated catalase activity which can be explained by stabilization of certain mRNA species, including catalase mRNA.

Actins↗
PubMed1996

Posttranscriptional regulation of gene expression in liver regeneration: role of mRNA stability.

The rentry of hepatocytes and nonparenchymal cells from the normal quiescent G0 phase into the cell cycle during liver regeneration after 70% partial hepatectomy results in the discrete modulation of mRNA transcripts for many different genes. The modulation of steady-state levels of transcripts for genes involved in hepatocyte growth and replication during liver regeneration indicates that gene expression is regulated not only transcriptionally but also posttranscriptionally. In fact, posttranscriptional control appears to be the primary mechanism of regulating gene expression after the first 3 h after partial hepatectomy. Alteration in transcript stability is a key posttranscriptional regulatory mechanism used by the regenerating liver to modulate the steady-state transcript levels of multiple genes. Even genes that are transcriptionally activated during liver regeneration exhibit posttranscriptional control at the level of transcript stability. Moreover, the abundance of mRNA binding proteins, as well as translational activity and rate of poly(A) tail removal, are modulated and appear to influence transcript stability during liver regeneration. However, alteration of transcript stability is not the sole posttranscriptional mechanism regulating steady-state levels. Posttranscriptional control also occurs at the level of alternative splicing, stabilization of heterogeneous nuclear (hn) RNA, and hnRNA processing. Moreover, the role of nucleocytoplasmic transport of mature mRNA during liver regeneration is still undefined. Thus, during liver regeneration gene expression is regulated at multiple levels after the initial synthesis of hnRNA. By understanding the role of posttranscriptional mechanisms in regulating steady-state transcript levels in an in vivo model of normal growth, we will begin to appreciate its role in the genesis of abnormal growth.

Animals↗
PubMed2005

Tumor necrosis factor-alpha up-regulates the expression of beta1,4-galactosyltransferase I in primary human endothelial cells by mRNA stabilization.

During the course of an inflammatory response, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) triggers endothelial cells to increase the expression levels of adhesion molecules that are pivotal for the rolling, adhesion, and transmigration of leukocytes over the endothelial cell wall. Here we show that TNFalpha, in addition, has a regulatory function in the biosynthesis of proper carbohydrate molecules on endothelial cells that constitute ligands for adhesion molecules on leukocytes. Our data show that TNFalpha induced an increase in the expression of beta1,4-galactosyltransferase-1 (beta4GalT-1) in primary human umbilical vein endothelial cells in a time- and concentration-dependent manner. The beta4GalT-1 mRNA up-regulation correlated with an increase in the Golgi expression and catalytic activity of the enzyme. Furthermore, an enhanced incorporation of galactose was observed in newly synthesized glycoproteins. Analysis of the molecular mechanism behind the up-regulation of beta4GalT-1 showed that the increase in mRNA levels is due to an enhanced stability of the transcripts. These data strongly demonstrate that TNFalpha modulates the glycosylation of endothelial cells by a mechanism that directly enhances the stability of beta4GalT-1 mRNA transcripts.

3' Untranslated Regions↗
PubMed1995

Defining a novel cis element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA: role in transforming growth factor-beta 1 induced mRNA stabilization.

Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 1 induced message stability. Using end-labeled RNA fragments in gel shift assays and UV cross-linking analyses, we detected in the 3'-UTR a novel 9 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis element effectively competed out the formation of the R2 3'-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, we were able to predict the consensus sequences, 5'-GAGUUUNNN-3' and 5'-NNNUUUGAG-3' for optimal protein binding. These results support a model in which the 9 nt region functions in cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is involved in TGF-beta 1-mediated effects.

3T3 Cells↗
PubMedDate not supplied

CRD-BP: a c-Myc mRNA stabilizing protein with an oncofetal pattern of expression.

The Coding Region Determinant-Binding Protein (CRD-BP) is an RRM and KH-domain-containing protein that recognizes specifically at least three RNAs. It binds to one of the two c-myc mRNA instability elements, to the 5'Un Translated Region (UTR) of the leader 3 IGF-II mRNA and to the oncofetal H19 RNA. CRD-BP has been assigned a role in stabilizing c-myc mRNA by preventing its endonucleolytic cleavage and in repressing the translation of the leader 3 IGF-II mRNA, the major embryonic species of this message. CRD-BP is normally expressed only in fetal tissues. However, its expression is detected in primary tumors and transformed cell lines of different origins. The vast majority of colon (80%) and breast (60%) tumors and sarcomas (73%) express CRD-BP whereas in other tumor types, for example prostate carcinomas, its expression is rare. CRD-BP expression has also been detected in benign tumors such as breast fibroadenomas, meningiomas and other benign mesenchymal tumors, implying a role for this gene in abnormal cell proliferation. In breast carcinomas, CRD-BP expression and or gene copy number gains in the region encompassing the c-myc locus were detected in approximately 75% of tumors, implying that the deregulated expression of c-myc may be more widespread than previously believed. Infiltrated lymph nodes, corresponding to CRD-BP-positive primary tumors, were also found positive indicating that monitoring for CRD-BP could prove useful for the detection and monitoring of disseminated disease.

Animals↗
PubMed1988

A stem-loop in the 3' untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm.

Expression of the human transferrin receptor (hTR) and its mRNA is strongly induced by iron deprivation. By measuring transcription elongation rates, levels of hTR-specific nuclear RNA, and mRNA half-lives, we found this regulation to occur posttranscriptionally in the cytoplasm. Analysis of hTR cDNA mutants with deletions in the 3' untranslated region revealed the existence of two distinct domains, both of which are essential for regulation in mouse L cells. The regulated phenotype correlates with the presence of a stem-loop structure predicted by a computer algorithm. Expression of point and deletion mutants affecting the stem-loop confirmed the requirement for this secondary structure in regulation. The 3' untranslated region of hTR cDNA was sufficient to confer iron-dependent regulation on another gene.

Base Sequence↗
PubMed2000

An AU-rich sequence element (UUUN[A/U]U) downstream of the edited C in apolipoprotein B mRNA is a high-affinity binding site for Apobec-1: binding of Apobec-1 to this motif in the 3' untranslated region of c-myc increases mRNA stability.

Apobec-1, the catalytic subunit of the mammalian apolipoprotein B (apoB) mRNA-editing enzyme, is a cytidine deaminase with RNA binding activity for AU-rich sequences. This RNA binding activity is required for Apobec-1 to mediate C-to-U RNA editing. Filter binding assays, using immobilized Apobec-1, demonstrate saturable binding to a 105-nt apoB RNA with a K(d) of approximately 435 nM. A series of AU-rich templates was used to identify a high-affinity ( approximately 50 nM) binding site of consensus sequence UUUN[A/U]U, with multiple copies of this sequence constituting the high-affinity binding site. In order to determine whether this consensus site could be functionally demonstrated from within an apoB RNA, circular-permutation analysis was performed, revealing one major (UUUGAU) and one minor (UU) site located 3 and 16 nucleotides, respectively, downstream of the edited base. Secondary-structure predictions reveal a stem-loop flanking the edited base with Apobec-1 binding to the consensus site(s) at an open loop. A similar consensus (AUUUA) is present in the 3' untranslated regions of several mRNAs, including that of c-myc, that are known to undergo rapid degradation. In this context, it is presumed that the consensus motif acts as a destabilizing element. As an independent test of the ability of Apobec-1 to bind to this sequence, F442A cells were transfected with Apobec-1 and the half-life of c-myc mRNA was determined following actinomycin D treatment. These studies demonstrated an increase in the half-life of c-myc mRNA from 90 to 240 min in control versus Apobec-1-expressing cells. Apobec-1 expression mutants, in which RNA binding activity is eliminated, failed to alter c-myc mRNA turnover. Taken together, the data establish a consensus binding site for Apobec-1 embedded in proximity to the edited base in apoB RNA. Binding to this site in other target RNAs raises the possibility that Apobec-1 may be involved in other aspects of RNA metabolism, independent of its role as an apoB RNA-specific cytidine deaminase.

3' Untranslated Regions↗
PubMed2005

Interferons upregulate thymidine phosphorylase expression via JAK-STAT-dependent transcriptional activation and mRNA stabilization in human glioblastoma cells.

Overexpression of the angiogenic enzyme thymidine phosphorylase (TP) in tumor cells and/or infiltrating macrophages correlates with increased microvessel density and poor prognosis in various tumor types including glioma. The present study examined how the TP gene expression is regulated by different types of interferons (IFNs) in human T98G and A172 glioblastoma cells. Both type I (alpha, beta) and type II (gamma) IFNs upregulated TP mRNA and protein expression while inhibiting cell proliferation. IFN-induced TP mRNA accumulation was not inhibited by the protein synthesis inhibitor cycloheximide, but was strongly blocked by the transcription inhibitor actinomycin D, as well as by transcription factor decoy oligodeoxynucleotides containing the putative IFN response element or the gamma-activated sequence in the TP promoter. The Janus kinase (JAK) inhibitor AG-490 blocked both IFN-induced STAT1 (signal transducers and activators of transcription 1) phosphorylation and TP expression. All IFNs increased the stability of TP mRNA as well. In addition, IFN-evoked TP enzyme activity enhanced the cytotoxicity of 5-fluorouracil (5-FU). These findings indicate that TP expression may be upregulated by IFNs via the JAK-STAT signaling pathway and both transcriptional and posttranscriptional mechanisms. Combined treatment with IFN and 5-fluorouracil may be a useful therapeutic strategy for malignant gliomas.

Antimetabolites, Antineoplastic↗
PubMed1995

Regulation of ICAM-1 mRNA stability by cycloheximide: role of serine/threonine phosphorylation and protein synthesis.

Cycloheximide is a protein synthesis inhibitor that superinduces the expression of many genes by preventing the degradation of otherwise labile mRNAs. In some genes this depends on the presence of the AUUUA destabilizing multimers in the 3'UTR. We examined the effect of cycloheximide on the murine intercellular adhesion molecule-1 (ICAM-1; CD54) gene expression in several cell lines including A20 (B cell lymphoma), T28 (T cell hybridoma), P388D1 (monocytic cell), SVEC4-10 (lymphoid endothelial cell), and ICAM-1-transfected murine fibroblast L cells. Cycloheximide was indeed able to dramatically increase the accumulation of ICAM-1 mRNA in all the cell lines examined except T28, and this seemed to be due to the stabilization of the ICAM-1 mRNA as indicated by the half-life analysis. To determine whether this effect is dependent on the 3'UTR containing the AUUUA sequences, L cells were transfected with either the full-length ICAM-1 cDNA or a truncated form lacking the AUUUA sequences in the 3'UTR (ICAM-1Delta3). There was no discernible difference in the effect of cycloheximide on ICAM-1 mRNA accumulation or half-life between the two types of transfected cells. The effect of cycloheximide on ICAM-1 mRNA was markedly suppressed by serine/threonine (ser/thr) kinase inhibitors, H-7 and staurosporine, whereas the ser/thr phosphatase inhibitor, okadaic acid, augmented the cycloheximide effect. Inhibitors of protein tyrosine kinases and phosphatases had no effect. Unexpectedly, the level of cell surface ICAM-1 as well as de novo synthesis of ICAM-1 in SVEC4-10 and the ICAM-1-transfected L cells were also upregulated by cycloheximide, whereas the overall protein synthesis in these cells was profoundly inhibited, suggesting that ICAM-1 protein synthesis in these cells escapes the translational inhibition by cycloheximide. These results suggest that the stabilization of ICAM-1 mRNA by cycloheximide is independent of its translational inhibition and that ser/thr phosphorylation of unidentified protein(s) seems to play a crucial role in this effect.

Animals↗
PubMed2000

Differential role of tyrosine phosphorylation in adhesion-induced transcription, mRNA stability, and cytoskeletal organization in human monocytes.

Monocyte adhesion resulted in rapid tyrosine phosphorylation and subsequent cytokine mRNA induction. The objective of this study was to determine the role of specific tyrosine phosphorylation events, particularly those involving members of the MAP kinase family, in regulating adhesion-induced cytokine expression. Using nuclear run-on analyses, we demonstrated that on adhesion, monocytes rapidly transcriptionally activated numerous cytokine mRNAs, coincident with the activation of the transcription factors NF-KB and AP-1. Both an inhibitor of tyrosine phosphorylation, genistein, and the cytoplasmic tyrosine phosphatase PTP1B, were unable to prevent adhesion-mediated transcriptional activation. However, both blocked adhesion-induced ERK and JNK but not p38 kinase activation and at the same time decreased the stability of interleukin-1beta (IL-1beta) and IL-8 transcripts. In addition, whereas adhesive events occurred in the presence of genistein and PTP1B, monocyte spreading was markedly inhibited. Our results suggest that the majority of protein phosphorylation events are associated with adhesion-induced cytokine expression through transcript stabilization and cytoskeletal organization. A minority of protein phosphorylation events, not sensitive to genistein or PTP1B exposure, may be instrumental in regulating transcription. Thus the spectrum of protein tyrosine kinases required for transcription appear distinct from those involved in maintaining the stability of some cytokine mRNAs and the integrity of the cytoskeleton to which mRNA destined for translation must be associated.

Adult↗
PubMed1991

Cell adhesion regulates pro-alpha 1(I) collagen mRNA stability and transcription in mouse fibroblasts.

Adhesive interactions are important modulators of cellular phenotype. Previously, we demonstrated that quiescent, suspension-arrested cells are not equivalent to density-arrested cells in their patterns of gene expression (Dhawan, J., and Farmer, S.R. (1990) J. Biol. Chem. 265, 9015-9021). In particular, pro-alpha 1(I) collagen expression depended strongly on the extent of cell adhesion. In this paper, we demonstrate that the adhesion-induced rise in collagen gene expression is due to regulation at multiple levels. Steady state levels of pro-alpha 1(I) collagen mRNA increased up to 10-fold by 6 h after replating suspended cells, and this rise is blocked by inhibition of protein synthesis. Transcription of the pro-alpha 1(I) collagen gene was measured by run-on assay as well as by activation of a rat alpha 1(I) promoter-chloramphenicol acetyltransferase reporter gene construct. Both assays reveal a 5-fold depression of pro-alpha 1(I) collagen gene transcription in suspended cells. Reattachment of suspended cells resulted in the activation of alpha 1(I) gene transcription by 2-h postreplating, reaching a 3-5-fold level of induction by 18 h. The pro-alpha 1(I) collagen mRNA was substantially more labile in suspended cells than in adherent cells (t1/2 values of approximately 2 h in nonadherent cells and greater than 8 h in exponentially growing or density-arrested cells). Furthermore, reattachment of suspended cells for 18 h resulted in a stabilization of collagen mRNA. We conclude that cell adhesion regulates pro-alpha 1(I) collagen gene expression selectively and at transcriptional and posttranscriptional sites.

Animals↗
PubMed2002

Cytokine mRNA profile in lipoid nephrosis: evidence for increased IL-8 mRNA stability.

BACKGROUND/AIMS: Proteinuria in idiopathic minimal lesion nephrotic syndrome (IMLNS) is presumed to be due to the effect of circulating factors on glomerular permeability to plasma proteins. This study examines the expression of messenger ribonucleic acid (mRNA) for cytokines thought to mediate glomerular inflammation during different stages of the nephrotic syndrome. METHODS: Messenger RNA expression and stability from peripheral blood mononuclear cells of IMLNS patients in relapse and in remission, and age matched normal controls were measured using a ribonuclease protection assay (RPA). The spontaneous and Interleukin 2 (IL-2) stimulated mRNA expression were studied. RESULTS: Spontaneous mRNA expression for Interleukin 8 (IL-8) from IMLNS patients in relapse was significantly increased when compared to IMLNS patients in remission and normal controls (p < 0.05). After 14 h of IL-2 stimulation, mRNA IL-8 levels expressed by IMLNS PBMC patients in remission were not different from those observed in normal controls. However, after 5 days of PBMC incubation, a significant increase in mRNA for IL-8 in IMLNS patients compared to controls was found (p < 0.01). Stability assay demonstrated that IL-8 mRNA transcript from the nephrotic patients remained higher than those from controls and showed a significantly prolonged life t(1/2) (p = 0.02). CONCLUSIONS: IL-8 mRNA expression is increased in IMLNS patients in relapse. Moreover, stability studies show that IL-8 mRNA life t(1/2) is prolonged due to altered post-transcriptional regulation. This finding may explain the elevated serum IL-8 levels observed in these patients during relapse and may have pathogenic significance since IL-8 has been shown to induce proteinuria in the experimental animal.

Adolescent↗
PubMed1992

Calcium ionophore A23187 induces expression of the growth arrest and DNA damage inducible CCAAT/enhancer-binding protein (C/EBP)-related gene, gadd153. Ca2+ increases transcriptional activity and mRNA stability.

gadd153 is a CCAAT/enhancer-binding protein (C/EBP)-related gene whose expression is induced in response to growth arrest and DNA damage. This investigation explored the possibility that Ca2+ might play a role in regulating expression of gadd 153. We have demonstrated that treatment of HeLa cells with the calcium ionophores A23187 and ionomycin leads to the induction of gadd153 mRNA. The induction was rapid; increases in mRNA were detected by 90 min of treatment, and near maximum levels were achieved within 5-h exposure to A23187. Elevated mRNA levels resulted from both an increase in the rate of gadd153 transcription and an increase in the stability of the gadd153 mRNA. The response was not dependent on protein kinase C nor was it coupled to c-fos expression. Buffering intracellular and extracellular Ca2+ by combined treatment with BAPTA-AM (acetoxymethyl ester form of bis(aminophenoxy)ethane N,N'-tetraacetic acid) and EGTA prevented the induction of gadd153 mRNA by A23187. In addition, these treatments prevented the induction of gadd153 mRNA in response to the DNA damaging agent methyl methanesulfonate. We conclude that intracellular Ca2+ plays a role in regulating gadd153 expression. More specifically, Ca2+ likely plays a role in the induction of gadd153 mRNA following DNA damage.

Base Sequence↗