Untranslated Region Reporter Constructs Research Articles

MiR-125b downregulates macrophage scavenger receptor type B1 and reverse cholesterol transport

ObjectiveTo determine whether miR-125b regulates cholesterol efflux in vivo and in vitro through the regulation of scavenger receptor type B1 (SR-B1). Approach and resultsWe demonstrated that miR-125b is up-regulated in the human aortas of patients with CAD and is located in macrophages and vascular smooth muscle cells (VSMCs). We identified SCARB1 as a direct target of miR-125b by repressing the activity of the SCARB1 3′-untranslated region reporter construct. Moreover, the overexpression of miR-125b in both human and mouse macrophages as well as VSMCs was found to downregulated the expression of the SCARB1 and the SR-B1 protein levels, thereby impairing α-HDL-mediated macrophage cholesterol efflux in vitro. The in vivo reverse cholesterol transport (RCT) rate from non-cholesterol-loaded macrophages transfected with miR-125b to feces was also found to be decreased when compared with that of control mimic-transfected macrophages. ConclusionsTogether, these results provide evidence that miR-125b downregulates SCARB1 and SR-B1 in both human and mouse macrophages as well as VSMCs, thereby impairing macrophage cholesterol efflux in vitro and the whole macrophage-specific RCT pathway in vivo.

MicroRNA-150 enhances radiosensitivity by inhibiting the AKT pathway in NK/T cell lymphoma

BackgroundRadioresistance is a major challenge during the treatment of NK/T cell lymphoma. This study aimed to investigate the potential role of MicroRNA-150 (miR-150) in increase the sensitivities of NK/T cell lymphoma to ionizing radiation.ResultsIn this study, we found that miR-150 was significantly decreased in NK/T cell lymphoma tissues and cell lines. Low expression of miR-150 was positively associated with therapeutic resistance in 36 NK/T cell lymphoma cases. Our further in vitro and in vivo studies illustrated that overexpression of miR-150 substantially enhanced the sensitivity of NK/T cell lymphoma cells to ionizing radiation treatment. Furthermore, luciferase reporter assays in NK/T cell lymphoma cells transfected with the AKT2 or AKT3 three prime untranslated region reporter constructs established AKT2 and AKT3 as direct targets of miR-150. The phosphatidylinositol 3-kinase inhibitor LY294002 was used to inhibit Akt to verify miR-150 increase NK/T cell lymphoma cell radiorsensitivity through suppress the PI3K/AKT/mTOR pathway.ConclusionsTaken together, this study demonstrates that miR-150 might serve as a potential therapeutic sensitizer through inhibition of the AKT pathway in NK/T cell lymphoma treatment.

MiR-181b-5p Modulates Cell Migratory Proteins, Tissue Inhibitor of Metalloproteinase 3, and Annexin A2 During In Vitro Decidualization in a Human Endometrial Stromal Cell Line.

Decidualization is essential for successful embryo implantation and is regulated by concerted actions of growth factors and hormones. More recently, microRNAs, small RNA molecules that regulate posttranscriptional gene expression, have been implicated to play a role in the decidualization process. Of these microRNAs, miR-181b-5p has been associated with decidualization but its precise role and targets are not well established. To address this gap in our knowledge, we assessed the expression of miR-181b-5p, and its target tissue inhibitor of metalloproteinase 3 (TIMP-3), during in vitro decidualization using the well-characterized human endometrial stromal cell line, t-HESC. miR-181b-5p expression was highest prior to decidualization and significantly decreased in response to decidualization stimulus. In contrast, TIMP-3 expression was absent prior to in vitro decidualization and increased during decidualization. Regulation of TIMP-3 expression by miR-181b-5p was confirmed in vitro by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot analysis, and 3' untranslated region reporter constructs. To identify unforeseen targets of miR-181b-5p during in vitro decidualization, t-HESC cells were transfected with pre- miR-181b-5p, and protein profiles were determined by 2-dimensional differential in-gel electrophoresis followed by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI TOF/TOF) tandem mass spectrometry. Of these proteins, several downregulated proteins associated with cell migration were identified including annexin A2, which we subsequently confirmed by qRT-PCR and Western blot analysis to be regulated by miR-181b-5p. In conclusion, miR-181b-5p is downregulated during the process of in vitro decidualization and may regulate the expression of proteins associated with cell migration including TIMP-3 and annexin A2.

MicroRNA-20a/b regulates cholesterol efflux through post-transcriptional repression of ATP-binding cassette transporter A1

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in reverse cholesterol transport and exhibits anti-atherosclerosis effects. Some microRNAs (miRs) regulate ABCA1 expression, and recent studies have shown that miR-20a/b might play a critical role in atherosclerotic diseases. Here, we attempted to clarify the potential contribution of miR-20a/b in post-transcriptional regulation of ABCA1, cholesterol efflux, and atherosclerosis. We performed bioinformatics analysis and found that miR-20a/b was highly conserved and directly bound to ABCA1 mRNA with low binding free energy. Luciferase-reporter assay also confirmed that miR-20a/b significantly reduced luciferase activity associated with the ABCA1 3′ untranslated region reporter construct. Additionally, miR-20a/b decreased ABCA1 expression, which, in turn, decreased cholesterol efflux and increased cholesterol content in THP-1 and RAW 264.7 macrophage-derived foam cells. In contrast, miR-20a/b inhibitors increased ABCA1 expression and cholesterol efflux, decreased cholesterol content, and inhibited foam-cell formation. Consistent with our in vitro results, miR-20a/b-treated ApoE−/− mice showed decreased ABCA1expression in the liver and reductions of reverse cholesterol transport in vivo. Furthermore, miR-20a/b regulated the formation of nascent high-density lipoprotein and promoted atherosclerotic development, whereas miR-20a/b knockdown attenuated atherosclerotic formation. miR-20 is a new miRNA capable of targeting ABCA1 and regulating ABCA1 expression. Therefore, miR-20 inhibition constitutes a new strategy for ABCA1-based treatment of atherosclerosis.

A single nucleotide polymorphism in osteonectin 3' untranslated region regulates bone volume and is targeted by miR-433.

Osteonectin/SPARC is one of the most abundant noncollagenous extracellular matrix proteins in bone, regulating collagen fiber assembly and promoting osteoblast differentiation. Osteonectin-null and haploinsufficient mice have low-turnover osteopenia, indicating that osteonectin contributes to normal bone formation. In male idiopathic osteoporosis patients, osteonectin 3' untranslated region (UTR) single-nucleotide polymorphism (SNP) haplotypes that differed only at SNP1599 (rs1054204) were previously associated with bone mass. Haplotype A (containing SNP1599G) was more frequent in severely affected patients, whereas haplotype B (containing SNP1599C) was more frequent in less affected patients and healthy controls. We hypothesized that SNP1599 contributes to variability in bone mass by modulating osteonectin levels. Osteonectin 3' UTR reporter constructs demonstrated that haplotype A has a repressive effect on gene expression compared with B. We found that SNP1599G contributed to an miR-433 binding site, and miR-433 inhibitor relieved repression of the haplotype A, but not B, 3' UTR reporter construct. We tested our hypothesis in vivo, using a knock-in approach to replace the mouse osteonectin 3' UTR with human haplotype A or B 3' UTR. Compared with haplotype A mice, bone osteonectin levels were higher in haplotype B mice. B mice displayed higher bone formation rate and gained more trabecular bone with age. When parathyroid hormone was administered intermittently, haplotype B mice gained more cortical bone area than A mice. Cultured marrow stromal cells from B mice deposited more mineralized matrix and had higher osteocalcin mRNA compared with A mice, demonstrating a cell-autonomous effect on differentiation. Altogether, SNP1599 differentially regulates osteonectin expression and contributes to variability in bone mass, by a mechanism that may involve differential targeting by miR-433. This work validates the findings of the previous candidate gene study, and it assigns a physiological function to a common osteonectin allele, providing support for its role in the complex trait of skeletal phenotype. © 2014 American Society for Bone and Mineral Research.

Key Role of MicroRNA in the Regulation of Granulocyte Macrophage Colony-stimulating Factor Expression in Murine Alveolar Epithelial Cells during Oxidative Stress

GM-CSF is an endogenous pulmonary cytokine produced by normal alveolar epithelial cells (AEC) that is a key defender of the alveolar space. AEC GM-CSF expression is suppressed by oxidative stress through alternations in mRNA turnover, an effect that is reversed by treatment with recombinant GM-CSF. We hypothesized that specific microRNA (miRNA) would play a key role in AEC GM-CSF regulation. A genome-wide miRNA microarray identified 19 candidate miRNA altered in primary AEC during oxidative stress with reversal by treatment with GM-CSF. Three of these miRNA (miR 133a, miR 133a*, and miR 133b) are also predicted to bind the GM-CSF 3'-untranslated region (UTR). PCR for the mature miRNA confirmed induction during oxidative stress that was reversed by treatment with GM-CSF. Experiments using a GM-CSF 3'-UTR reporter construct demonstrated that miR133a and miR133b effects on GM-CSF expression are through interactions with the GM-CSF 3'-UTR. Using lentiviral transduction of specific mimics and inhibitors in primary murine AEC, we determined that miR133a and miR133b suppress GM-CSF expression and that their inhibition both reverses oxidant-induced suppression of GM-CSF expression and increases basal expression of GM-CSF in cells in normoxia. In contrast, these miRNAs are not active in regulation of GM-CSF expression in murine EL4 T cells. Thus, members of the miR133 family play key roles in regulation of GM-CSF expression through effects on mRNA turnover in AEC during oxidative stress. Increased understanding of GM-CSF gene regulation may provide novel miRNA-based interventions to augment pulmonary innate immune defense in lung injury.

A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

Neurodegeneration associated with amyloid β (Aβ) peptide accumulation, synaptic loss, and memory impairment are pathophysiological features of Alzheimer's disease (AD). Numerous microRNAs regulate amyloid precursor protein (APP) expression and metabolism. We previously reported that miR-101 is a negative regulator of APP expression in cultured hippocampal neurons. In this study, a search for predicted APP metabolism-associated miR-101 targets led to the identification of a conserved miR-101 binding site within the 3′ untranslated region (UTR) of the mRNA encoding Ran-binding protein 9 (RanBP9). RanBP9 increases APP processing by β-amyloid converting enzyme 1 (BACE1), secretion of soluble APPβ (sAPPβ), and generation of Aβ. MiR-101 significantly reduced reporter gene expression when co-transfected with a RanBP9 3′-UTR reporter construct, while site-directed mutagenesis of the predicted miR-101 target site eliminated the reporter response. To investigate the effect of stable inhibition of miR-101 both in vitro and in vivo, a microRNA sponge was developed to bind miR-101 and derepress its targets. Four tandem bulged miR-101 responsive elements (REs), located downstream of the enhanced green fluorescence protein (EGFP) open reading frame and driven by the synapsin promoter, were placed in a lentiviral vector to create the pLSyn-miR-101 sponge. Delivery of the sponge to primary hippocampal neurons significantly increased both APP and RanBP9 expression, as well as sAPPβ levels in the conditioned medium. Importantly, silencing of endogenous RanBP9 reduced sAPPβ levels in miR-101 sponge-containing hippocampal cultures, indicating that miR-101 inhibition may increase amyloidogenic processing of APP by RanBP9. Lastly, the impact of miR-101 on its targets was demonstrated in vivo by intrahippocampal injection of the pLSyn-miR-101 sponge into C57BL6 mice. This study thus provides the basis for studying the consequences of long-term miR-101 inhibition on the pathology of AD.

MicroRNA-150 inhibits expression of adiponectin receptor 2 and is a potential therapeutic target in patients with chronic heart failure

Adiponectin is an anti-inflammatory adipocytokine believed to be involved in the pathogenesis of chronic heart failure (CHF). We aimed to characterize the expression of adiponectin and its receptors in CHF and to assess the impact of microRNAs on the cardiac adiponectin system. Expression of adiponectin and adiponectin receptors (ADIPOR1 and ADIPOR2) was studied by qPCR and immunohistochemistry in myocardial tissues of patients with end-stage CHF and control subjects. MicroRNA binding was evaluated by cloning of an ADIPOR2 3´-untranslated-region reporter construct and subsequent transfection experiments. Effects of miRNA transfection were analyzed in cardiomyocyte cell cultures by qPCR and Western blotting. Gene silencing of ADIPOR2 was performed by siRNA transfection, and the effects of hypoxia/serum starvation were analyzed by flow cytometry. Although CHF patients displayed elevated plasma adiponectin levels, myocardial adiponectin expression generally was very low. In CHF, cardiac ADIPOR1 expression increased by >4-fold, whereas the increase in ADIPOR2 was less than 2-fold. Reporter gene assays on constructs containing the ADIPOR2-3'-untranslated region suggest that microRNA-150 specifically repressed ADIPOR2 expression. Transfection of cardiomyocytes with premiR-150 precursor molecules resulted in 60% down-regulation of ADIPOR2 mRNA and a significant reduction of ADIPOR2 protein expression. MicroRNA-150 was substantially expressed in both normal and CHF myocardium, with a 1.7-fold higher expression in CHF. Finally, knock-down experiments elucidated a stress-protective role of ADIPOR2 in cardiomyocytes. MicroRNA-150 counteracts ADIPOR2 up-regulation in CHF and thus may contribute to adiponectin resistance. Targeting microRNA-150 may be a future strategy to restore cardioprotective adiponectin effects.

IGF-I 3' untranslated region: strain-specific polymorphisms and motifs regulating IGF-I in osteoblasts.

Reduced IGF-I is associated with low bone mass in humans and mice. C3H/He/J (C3H) mice have higher skeletal IGF-I and greater bone mass than C57BL/6J (B6). We hypothesized that strain-related genotypic differences in Igf1 affected skeletal function. The Igf1 coding region is nonpolymorphic, but its 3' untranslated region (UTR) is polymorphic between C3H and B6. Luciferase-Igf1 3' UTR reporter constructs showed that these polymorphic regions did not affect UTR function. IGF-I splice variants give rise to a common mature IGF-I peptide, but different E peptides. We identified two splice products, exon 4+6 (Ea) and exon 4+5+6 (Eb, mechano-growth factor) and found that their abundance was unchanged during osteoblastic differentiation. The Igf1 3' UTR encoded by exon 6 contains alternative polyadenylation sites. Proximal site use produces a short 3' UTR of approximately 195 bases, whereas distal site usage results in an approximately 6300-base UTR. Although Igf1 mRNA levels did not change during osteoblastic differentiation, distal polyadenylation site usage was increased in B6 cells but not in C3H. The resulting long Igf1 RNA isoform is less stable and has decreased translation efficiency, which may be one mechanism contributing to decreased IGF-I in B6 vs. C3H mice. Although the long UTR contains a conserved [GU](18) repeat, which is a positive regulator of UTR activity, it is also targeted by negative regulators, miR-29 and miR-365. These microRNAs are increased in B6 and C3H cells during osteoblastic differentiation. Differential expression of the long Igf1 3' UTR isoform may be a possible mechanism for enhanced IGF-I regulation in B6 vs. C3H mice.

Corticosteroid resistance in sepsis is influenced by microRNA-124–induced downregulation of glucocorticoid receptor-α*

Acquired glucocorticoid resistance frequently complicates the therapy of sepsis. It leads to an exaggerated proinflammatory response and has been related to altered expression profiles of glucocorticoid receptor isoforms glucocorticoid receptor-α (mediating anti-inflammatory effects) and glucocorticoid receptor-β (acting as a dominant negative inhibitor). We investigated the impact of glucocorticoid receptor isoforms on glucocorticoid effects in human T-cells. We hypothesized that 1) changes of the ratio of glucocorticoid receptor isoforms impact glucocorticoid resistance and 2) glucocorticoid receptor-α expression is controlled by microRNA-mediated gene silencing. Laboratory-based study. University research laboratory. Healthy volunteers, sepsis patients. First, T-cells from healthy volunteers (native and CD3/CD28-stimulated cells with or without addition of hydrocortisone) were analyzed for the expression of glucocorticoid receptor-isoforms by quantitative polymerase chain reaction. Additionally, effects of gene silencing of glucocorticoid receptor-β by siRNA transfection were determined. Secondly, microRNA-mediated silencing was evaluated by cloning of a glucocorticoid receptor-α-specific 3'-untranslated-region reporter construct and subsequent transfection experiments in cell cultures. Effects of miRNA transfection on glucocorticoid receptor-α expression were analyzed in Jurkat T-cells and in T-cells from healthy volunteers (quantitative polymerase chain reaction and Western blotting). Finally, expression of glucocorticoid receptor-α, glucocorticoid receptor-β, and miR-124 was tested in T-cells of sepsis patients (n=24). Stimulation of T-cells induced a significant upregulation of glucocorticoid receptor-α (not glucocorticoid receptor-β) thereby possibly rendering T-cells more sensitive to glucocorticoids; this T-cell response was hindered by hydrocortisone. Silencing of glucocorticoid receptor-β doubled the inhibitory effects of glucocorticoids on interleukin-2 production. MicroRNA-124 was proved to specifically downregulate glucocorticoid receptor-α. Furthermore, a glucocorticoid-induced three-fold upregulation of microRNA-124 was found. T-cells of sepsis patients exhibited slightly decreased glucocorticoid receptor-α and slightly increased miR-124 expression levels, whereas glucocorticoid receptor-β expression was two-fold upregulated (p<.01) and exhibited a remarkable interindividual variability. Glucocorticoid treatment induces expression of miR-124, which downregulates glucocorticoid receptor-α thereby limiting anti-inflammatory effects of glucocorticoids. Steroid treatment might aggravate glucocorticoid resistance in patients with high glucocorticoid receptor-β levels.

MicroRNA-29c Is a Signature MicroRNA under High Glucose Conditions That Targets Sprouty Homolog 1, and Its in Vivo Knockdown Prevents Progression of Diabetic Nephropathy

Although several recent publications have suggested that microRNAs contribute to the pathogenesis of diabetic nephropathy, the role of miRNAs in vivo still remains poorly understood. Using an integrated in vitro and in vivo comparative miRNA expression array, we identified miR-29c as a signature miRNA in the diabetic environment. We validated our profiling array data by examining miR-29c expression in the kidney glomeruli obtained from db/db mice in vivo and in kidney microvascular endothelial cells and podocytes treated with high glucose in vitro. Functionally, we found that miR-29c induces cell apoptosis and increases extracellular matrix protein accumulation. Indeed, forced expression of miR-29c strongly induced podocyte apoptosis. Conversely, knockdown of miR-29c prevented high glucose-induced cell apoptosis. We also identified Sprouty homolog 1 (Spry1) as a direct target of miR-29c with a nearly perfect complementarity between miR-29c and the 3'-untranslated region (UTR) of mouse Spry1. Expression of miR-29c decreased the luciferase activity of Spry1 when co-transfected with the mouse Spry1 3'-UTR reporter construct. Overexpression of miR-29c decreased the levels of Spry1 protein and promoted activation of Rho kinase. Importantly, knockdown of miR-29c by a specific antisense oligonucleotide significantly reduced albuminuria and kidney mesangial matrix accumulation in the db/db mice model in vivo. These findings identify miR-29c as a novel target in diabetic nephropathy and provide new insights into the role of miR-29c in a previously unrecognized signaling cascade involving Spry1 and Rho kinase activation.

MicroRNA regulation in Ames dwarf mouse liver may contribute to delayed aging

The Ames dwarf mouse is well known for its remarkable propensity to delay the onset of aging. Although significant advances have been made demonstrating that this aging phenotype results primarily from an endocrine imbalance, the post-transcriptional regulation of gene expression and its impact on longevity remains to be explored. Towards this end, we present the first comprehensive study by microRNA (miRNA) microarray screening to identify dwarf-specific lead miRNAs, and investigate their roles as pivotal molecular regulators directing the long-lived phenotype. Mapping the signature miRNAs to the inversely expressed putative target genes, followed by in situ immunohistochemical staining and in vitro correlation assays, reveals that dwarf mice post-transcriptionally regulate key proteins of intermediate metabolism, most importantly the biosynthetic pathway involving ornithine decarboxylase and spermidine synthase. Functional assays using 3'-untranslated region reporter constructs in co-transfection experiments confirm that miRNA-27a indeed suppresses the expression of both of these proteins, marking them as probable targets of this miRNA in vivo. Moreover, the putative repressed action of this miRNA on ornithine decarboxylase is identified in dwarf mouse liver as early as 2 months of age. Taken together, our results show that among the altered aspects of intermediate metabolism detected in the dwarf mouse liver--glutathione metabolism, the urea cycle and polyamine biosynthesis--miRNA-27a is a key post-transcriptional control. Furthermore, compared to its normal siblings, the dwarf mouse exhibits a head start in regulating these pathways to control their normality, which may ultimately contribute to its extended health-span and longevity.

CD28 Regulates the Translation of Bcl-xL via the Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Pathway

In concert with the TCR, CD28 promotes T cell survival by regulating the expression of the antiapoptotic protein Bcl-x(L). The mechanism by which CD28 mediates the induction of Bcl-x(L) remains unknown. We show that although signaling through the TCR is sufficient to stimulate transcription of Bcl-x(L) mRNA, CD28, by activating PI3K and mammalian target of rapamycin, provides a critical signal that regulates the translation of Bcl-x(L) transcripts. We observe that CD28 induced 4E-binding protein-1 phosphorylation, an inhibitor of the translational machinery, and that CD28 costimulation directly augmented the translation of a Bcl-x(L) 5'-untranslated region reporter construct. Lastly, costimulation by CD28 shifted the distribution of Bcl-x(L) mRNA transcripts from the pretranslation complex to the translationally active polyribosomes. These results demonstrate that CD28 relieves the translational inhibition of Bcl-x(L) in a PI3K/mammalian target of rapamycin-dependent manner.

T-cell-derived Interleukin-17 Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of the p38 Mitogen-activated Protein Kinase Cascade: ROLE OF DISTAL SEQUENCES IN THE 3′-UNTRANSLATED REGION OF COX-2 mRNA

Although interleukin-17 (IL-17) is the pre-eminent T-cell-derived pro-inflammatory cytokine, its cellular mechanism of action remains poorly understood. We explored novel signaling pathways mediating IL-17 induction of the cyclooxygenase-2 (COX-2) gene in human chondrocytes, synovial fibroblasts, and macrophages. In preliminary work, recombinant human (rh) IL-17 stimulated a rapid (5-15 min), substantial (>8-fold), and sustained (>24 h) increase in COX-2 mRNA, protein, and prostaglandin E2 release. Screening experiments with cell-permeable kinase inhibitors (e.g. SB202190 and p38 inhibitor), Western analysis using specific anti-phospho-antibodies to a variety of mitogen-activated protein kinase cascade intermediates, co-transfection studies using chimeric cytomegalovirus-driven constructs of GAL4 DNA-binding domains fused to the transactivation domains of transcription factors together with Gal-4 binding element-luciferase reporters, ectopic overexpression of activated protein kinase expression plasmids (e.g. MKK3/6), or transfection experiments with wild-type and mutant COX-2 promoter constructs revealed that rhIL-17 induction of the COX-2 gene was mediated exclusively by the stress-activated protein kinase 2/p38 cascade. A rhIL-17-dependent transcriptional pulse (1.76 +/- 0.11-fold induction) was initiated by ATF-2/CREB-1 transactivation through the ATF/CRE enhancer site in the proximal promoter. However, steady-state levels of rhIL-17-induced COX-2 mRNA declined rapidly (<2 h) to control levels under wash-out conditions. Adding rhIL-17 to transcriptionally arrested cells stabilized COX-2 mRNA for up to 6 h, a process compromised by SB202190. Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3'-untranslated region reporter constructs revealed that rhIL-17 increased reporter gene mRNA stability and protein synthesis via distal regions (-545 to -1414 bases) of the 3'-untranslated region. This response was mediated entirely by the stress-activated protein kinase 2/p38 cascade. As such, IL-17 can exert direct transcriptional and post-transcriptional control over target proinflammatory cytokines and oncogenes.

Prostaglandin E2 Regulates the Level and Stability of Cyclooxygenase-2 mRNA through Activation of p38 Mitogen-activated Protein Kinase in Interleukin-1β-treated Human Synovial Fibroblasts

The p38 MAPK mediates transcriptional and post-transcriptional control of cyclooxygenase-2 (COX-2) mRNA following interleukin-1(IL-1)/lipopolysaccharide cellular activation. We explored a positive feedback, prostaglandin E(2) (PGE(2))-dependent stabilization of COX-2 mRNA mediated by the p38 MAPK cascade in IL-1 beta-stimulated human synovial fibroblasts. We observed a rapid (5 min), massive (>30-fold), and sustained (>48 h) increase in COX-2 mRNA, protein, and PGE(2) release following a recombinant human (rh) IL-1 beta signal that was inhibited by NS-398, a COX-2 inhibitor, and SB202190, a selective, cell-permeable p38 MAPK inhibitor. PGE(2) completely reversed NS-398-mediated inhibition but not SB202190-dependent inhibition. The eicosanoid didn't potentiate IL-1 beta-induced COX-2 expression nor did it activate COX-2 gene expression in quiescent cells. Transfection experiments with a human COX-2 promoter construct revealed a minor element of p38 MAPK-dependent transcriptional control after IL-1 beta stimulation. p38 MAPK synergized with the cAMP/cAMP-dependent protein kinase cascade to transactivate the COX-2 promoter. When human synovial fibroblasts were activated with rhIL-1 beta for 3-4 h (steady state) followed by washout, the elevated levels of COX-2 mRNA declined rapidly (<2 h) to control levels. If PGE(2), unlike EP2/3 agonists butaprost and sulprostone, was added to fresh medium, COX-2 mRNA levels remained elevated for up to 16 h. SB202190 or anti-PGE(2) monoclonal antibody compromised the stabilization of COX-2 mRNA by PGE(2). Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3'-untranslated region reporter constructs revealed that IL-1 beta increased reporter gene mRNA stability and translation via AU-containing distal regions of the untranslated region. This response was mediated entirely by a PGE(2)/p38 MAPK-dependent process. We conclude that the magnitude and duration of the induction of COX-2 mRNA, protein, and PGE(2) release by rhIL-1 beta is primarily the result of PGE(2)-dependent stabilization of COX-2 mRNA and stimulation of translation, a process involving a positive feedback loop mediated by the EP4 receptor and the downstream kinases p38 MAPK and, perhaps, cAMP-dependent protein kinase.