PubMed Health⌕ Search

Biomedical subjects

Jeremy Saklatvala

Publications and source records attributed to Jeremy Saklatvala.

At least 19 recordsLinked to original sources
PubMed2007

Heat shock protein 27 functions in inflammatory gene expression and transforming growth factor-beta-activated kinase-1 (TAK1)-mediated signaling.

Heat shock protein (HSP) 27 has long been known to be a component of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. p38 MAPK has important functions in the inflammatory response, but the role of HSP27 in inflammation has remained unknown. We have used small interfering RNAs to suppress HSP27 expression in HeLa cells and fibroblasts and found that it is required for pro-inflammatory cell signaling and the expression of pro-inflammatory genes. HSP27 is needed for the activation by interleukin (IL)-1 of TAK1 and downstream signaling by p38 MAPK, JNK, and their activators (MKK-3, -4, -6, -7) and IKKbeta. IL-1-induced ERK activation appears to be independent of HSP27. HSP27 is required for both IL-1 and TNF-induced signaling pathways for which the most upstream common signaling protein is TAK1. HSP27 is also required for IL-1-induced expression of the pro-inflammatory mediators, cyclooxygenase-2, IL-6, and IL-8. HSP27 functions to drive cyclooxygenase-2 and IL-6 expression by augmenting the activation of the kinase downstream of p38 MAPK, MK2, resulting in stabilization of cyclooxygenase-2 and IL-6 mRNAs. The mechanism may not occur in cells of myeloid lineage because HSP27 protein was undetectable in human monocytes and murine macrophages.

Cells, Cultured↗
PubMed2006

Antiinflammatory effects of dexamethasone are partly dependent on induction of dual specificity phosphatase 1.

Glucocorticoids (GCs), which are used in the treatment of immune-mediated inflammatory diseases, inhibit the expression of many inflammatory mediators. They can also induce the expression of dual specificity phosphatase 1 (DUSP1; otherwise known as mitogen-activated protein kinase [MAPK] phosphatase 1), which dephosphorylates and inactivates MAPKs. We investigated the role of DUSP1 in the antiinflammatory action of the GC dexamethasone (Dex). Dex-mediated inhibition of c-Jun N-terminal kinase and p38 MAPK was abrogated in DUSP1-/- mouse macrophages. Dex-mediated suppression of several proinflammatory genes (including tumor necrosis factor, cyclooxygenase 2, and interleukin 1alpha and 1beta) was impaired in DUSP1-/- mouse macrophages, whereas other proinflammatory genes were inhibited by Dex in a DUSP1-independent manner. In vivo antiinflammatory effects of Dex on zymosan-induced inflammation were impaired in DUSP1-/- mice. Therefore, the expression of DUSP1 is required for the inhibition of proinflammatory signaling pathways by Dex in mouse macrophages. Furthermore, DUSP1 contributes to the antiinflammatory effects of Dex in vitro and in vivo.

Animals↗
PubMed2006

Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway, acting through the downstream kinase MK2, regulates the stability of many proinflammatory mRNAs that contain adenosine/uridine-rich elements (AREs). It is thought to do this by modulating the expression or activity of ARE-binding proteins that regulate mRNA turnover. MK2 phosphorylates the ARE-binding and mRNA-destabilizing protein tristetraprolin (TTP) at serines 52 and 178. Here we show that the p38 MAPK pathway regulates the subcellular localization and stability of TTP protein. A p38 MAPK inhibitor causes rapid dephosphorylation of TTP, relocalization from the cytoplasm to the nucleus, and degradation by the 20S/26S proteasome. Hence, continuous activity of the p38 MAPK pathway is required to maintain the phosphorylation status, cytoplasmic localization, and stability of TTP protein. The regulation of both subcellular localization and protein stability is dependent on MK2 and on the integrity of serines 52 and 178. Furthermore, the extracellular signal-regulated kinase (ERK) pathway synergizes with the p38 MAPK pathway to regulate both stability and localization of TTP. This effect is independent of kinases that are known to be synergistically activated by ERK and p38 MAPK. We present a model for the actions of TTP and the p38 MAPK pathway during distinct phases of the inflammatory response.

Animals↗
PubMed2005

Two-stage affinity purification for inducibly phosphorylated membrane proteins.

Characterisation of tyrosine phosphorylations induced in immune cells in response to inflammatory stimuli may help elucidate the molecular bases of the diversity of immune responses. We have used anti-phosphotyrosine antibodies in combination with cell surface biotinylation in a two-step affinity purification procedure to recover pervanadate-induced tyrosine phosphorylated proteins from sub-cellular compartments, including the cell surface, of murine T cells and macrophages prior to separation by solution-phase isoelectric focussing and one-dimensional gel electrophoresis and identification by tandem mass spectrometry.

Animals↗
PubMed2004

Proteomic analysis of articular cartilage shows increased type II collagen synthesis in osteoarthritis and expression of inhibin betaA (activin A), a regulatory molecule for chondrocytes.

We show that proteomic analysis can be applied to study cartilage pathophysiology. Proteins secreted by articular cartilage were analyzed by two-dimensional SDS-PAGE and mass spectrometry. Cartilage explants were cultured in medium containing [35S]methionine/cysteine to radiolabel newly synthesized proteins. To resolve the cartilage proteins by two-dimensional electrophoresis, it was necessary to remove the proteoglycan aggrecan by precipitation with cetylpyridinium chloride. 50-100 radiolabeled protein spots were detected on two-dimensional gels of human cartilage cultures. Of 170 silver-stained proteins identified, 19 were radiolabeled, representing newly synthesized gene products. Most of these were known cartilage constituents. Several nonradiolabeled cartilage proteins were also detected. The secreted protein pattern of explants from 12 osteoarthritic joints (knee, hip, and shoulder) and 14 nonosteoarthritic adult joints were compared. The synthesis of type II collagen was strongly up-regulated in osteoarthritic cartilage. Normal adult cartilage synthesized little or no type II collagen in contrast to infant and juvenile cartilage. Potential regulatory molecules novel to cartilage were identified; pro-inhibin betaA and processed inhibin betaA (which dimerizes to activin A) were produced by all the osteoarthritic samples and half of the normals. Connective tissue growth factor and cytokine-like protein C17 (previously only identified as an mRNA) were also found. Activin induced the tissue inhibitor for metalloproteinases-1 in human chondrocytes. Its expression was induced in isolated chondrocytes by growth factors or interleukin-1. We conclude that type II collagen synthesis in articular cartilage is down-regulated at skeletal maturity and reactivated in osteoarthritis in attempted repair and that activin A may be an anabolic factor in cartilage.

Activins↗
PubMed2004

The stability of tristetraprolin mRNA is regulated by mitogen-activated protein kinase p38 and by tristetraprolin itself.

Tristetraprolin (TTP) is an mRNA-destabilizing protein that negatively regulates the expression of proinflammatory mediators such as tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor, and cyclooxygenase 2. Here we investigate the regulation of TTP expression in the mouse macrophage cell line RAW264.7. We show that TTP mRNA is expressed in a biphasic manner following stimulation of cells with lipopolysaccharide and that the second phase of expression, like the first, is dependent on mitogen-activated protein kinase (MAPK) p38. MAPK p38 acts through a downstream kinase to stabilize TTP mRNA, and this stabilization is mediated by an adenosine/uridine-rich region at the 3'-end of the TTP 3'-untranslated region. Hence TTP is post-transcriptionally regulated in a similar manner to several proinflammatory genes. We also demonstrate that TTP is able to bind to its own 3'-untranslated region and negatively regulate its own expression, forming a feedback loop to limit expression levels.

3' Untranslated Regions↗
PubMed2004

Structural and functional dissection of a conserved destabilizing element of cyclo-oxygenase-2 mRNA: evidence against the involvement of AUF-1 [AU-rich element/poly(U)-binding/degradation factor-1], AUF-2, tristetraprolin, HuR (Hu antigen R) or FBP1 (far-upstream-sequence-element-binding protein 1).

COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a beta-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant sequences were identified that were incapable of destabilizing a reporter mRNA, yet showed unimpaired binding of FBP1 and AUF-1 and/or -2. TTP was absent from the HeLa cell line used in this analysis. Finally, RNA interference experiments argued against a prominent role for HuR in the CR1-mediated regulation of mRNA stability. We conclude that at least one critical regulator of COX-2 mRNA stability is likely to remain unidentified at present.

3' Untranslated Regions↗
PubMed2004

Basic fibroblast growth factor mediates transduction of mechanical signals when articular cartilage is loaded.

OBJECTIVE: To determine whether the basic fibroblast growth factor (bFGF) mediates signal transduction in articular cartilage in response to mechanical loading. METHODS: Articular cartilage from porcine metacarpophalangeal or knee joints was cyclically loaded (62.5-250N) for 2 minutes in the absence or presence of a bFGF receptor inhibitor, SB 402451 (250 nM). Activation of the extracellularly regulated kinase MAP kinase ERK was measured by Western blot analysis. Changes in protein synthesis were assessed by measuring the incorporation of (35)S-Met/Cys into proteins secreted by cartilage explants or by isolated chondrocytes. RESULTS: Rapid activation of the ERK MAP kinase occurred when articular cartilage was loaded. This was dependent upon release of the bFGF because it was restricted by the FGF receptor inhibitor. Loaded explants were shown to release bFGF. Loading or bFGF stimulation of explants induced synthesis and secretion of tissue inhibitor of metalloproteinases 1 (TIMP-1), which was inhibited by SB 402451. CONCLUSION: Cyclical loading of articular cartilage causes bFGF-dependent activation of ERK and synthesis of TIMP-1.

Animals↗
PubMed2004

Induction of interleukin-1 in articular cartilage by explantation and cutting.

OBJECTIVE: To investigate the effect of explantation and fine cutting of articular cartilage upon intracellular inflammatory signaling pathways and expression of interleukin-1 (IL-1). METHODS: Cartilage from porcine metacarpophalangeal joints was cultured in serum-free medium. Tissue extracts were examined for ERK activation by phosphorylated-Western blotting, for JNK and p38 MAPK activity by kinase assay, and for IkappaBalpha. IL-1alpha and IL-1beta messenger RNA (mRNA) was measured by reverse transcriptase-polymerase chain reaction. IL-1 activity was measured by the induction of serum amyloid A protein in cultured chondrocytes. RESULTS: All 3 MAPKs (p38, JNK, and ERK) were rapidly activated upon dissection and explantation of the cartilage. IL-1alpha and IL-1beta mRNA was also induced: the speed and magnitude of induction were increased if the explants had been finely cut. IL-1 activity that could be inhibited by IL-1 receptor antagonist or antibodies to IL-1alpha was found in extracts of explants cultured for 20 hours or lysates of cells isolated from them. This activity was likely due to intracellular proIL-1alpha that was not secreted. ProIL-1beta would not be detected because it is biologically inactive. The mechanism of inflammatory signaling pathway activation underlying the induction of IL-1 is unknown. CONCLUSION: Explantation and cutting of articular cartilage activates intracellular inflammatory signaling pathways and induces expression of mRNA for IL-1alpha and IL-1beta. Biologically active IL-1alpha protein was detectable in cartilage lysates and was probably intracellular proIL-1alpha. We were unable to show that IL-1 was secreted by chondrocytes.

Animals↗
PubMed2004

The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation.

The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in the post-transcriptional regulation of inflammatory genes. p38 has been found to regulate both the translation and the stability of inflammatory mRNAs. The mRNAs regulated by p38 share common AU-rich elements (ARE) present in their 3'-untranslated regions. AREs act as mRNA instability determinants but also confer stabilisation of the mRNA by the p38 pathway. In recent years, AREs have shown to be binding sites for numerous proteins including HuR, TTP, AUF1, AUF2, FBP1, FBP2 (KSRP), TIA-1, and TIAR. However, it is unclear which protein is responsible for mRNA stabilisation by p38. This review gives an overview of the major ARE-binding proteins and discusses reasons for and against their involvement in p38-mediated mRNA stabilisation.

Animals↗
PubMed2004

The p38 MAP kinase pathway as a therapeutic target in inflammatory disease.

The p38 MAPK signalling pathway plays an important role in inflammation and other physiological processes. Specific inhibitors of p38 alpha and beta MAPK block production of the major inflammatory cytokines (i.e. tumour necrosis factor-alpha and interleukin-1) and other proteins (e.g. cyclooxygenase-2), and are anti-inflammatory in animal models of disease. A major function of the pathway is post-transcriptional control of inflammatory gene expression. Many of the mRNAs are unstable (or untranslatable) because of AU-rich elements in the 3'untranslated region. Signalling in the p38 pathway counteracts these and stabilizes the mRNAs by preventing their otherwise rapid de-adenylation.

Animals↗
PubMed2003

Expression profiling of lymphocyte plasma membrane proteins.

The physicochemical properties of plasma membrane proteins of mammalian cells render them refractory to systematic analysis by two-dimensional electrophoresis. We have therefore used in vivo cell surface labeling with a water-soluble biotinylation reagent, followed by cell lysis and membrane purification, prior to affinity capture of biotinylated proteins. Purified membrane proteins were then separated by solution-phase isoelectric focusing and SDS-PAGE and identified by high-pressure liquid chromatography electrospray/tandem mass spectrometry. Using this approach, we identified 42 plasma membrane proteins from a murine T cell hybridoma and 46 from unfractionated primary murine splenocytes. These included three unexpected proteins; nicastrin, osteoclast inhibitory lectin, and a transmembrane domain-containing hypothetical protein of 11.4 kDa. Following stimulation of murine splenocytes with phorbol ester and calcium ionophore, we observed differences in expression of CD69, major histocompatibility complex class II molecules, the glucocorticoid-induced TNF receptor family-related gene product, and surface immunoglobulin M and D that were subsequently confirmed by Western blot or flow cytometric analysis. This approach offers a generic and powerful strategy for investigating differential expression of surface proteins in many cell types under varying environmental and pathophysiological conditions.

Animals↗
PubMed2003

p38 Mitogen-activated protein kinase stabilizes mRNAs that contain cyclooxygenase-2 and tumor necrosis factor AU-rich elements by inhibiting deadenylation.

AU-rich elements (AREs) in 3'-untranslated regions of mRNAs confer instability. They target mRNAs for rapid deadenylation and degradation and may enhance decapping. The p38 MAPK pathway stabilizes many otherwise unstable ARE-containing mRNAs encoding proteins involved in inflammation; however, the mRNA decay step(s) regulated by the signaling pathway are unknown. To investigate whether it regulates deadenylation or the decay of the mRNA body, we used a tetracycline-regulated beta-globin mRNA reporter system to transcribe pulses of mRNA of uniform length. We measured on Northern gels the migration of reporter mRNAs isolated from cells transfected only with reporter plasmid or co-transfected with an active mutant of MAPK kinase-6, and treated either with or without the p38 MAPK inhibitor SB 203580. Differences in migration were shown by RNase H mapping with oligo(dT) to be due to poly(A) shortening. Insertion of an ARE into the beta-globin reporter mRNA promoted rapid deadenylation and decay of hypo-adenylated reporter mRNA. p38 MAPK activation inhibited the deadenylation of reporter mRNAs containing either the cyclooxygenase-2 or tumor necrosis factor AREs. The regulation of deadenylation by p38 MAPK was found to be specific because deadenylation of the beta-globin reporter mRNA either lacking an ARE or containing the c-Myc 3'-untranslated region (which is not p38 MAPK-responsive) was unaffected by p38 MAPK. It was concluded that the p38 MAPK pathway predominantly regulates deadenylation, rather than decay of the mRNA body, and this provides an explanation for why p38 MAPK regulates mRNA stability in some situations and translation in others.

Base Sequence↗
PubMed2003

Post-transcriptional regulation of gene expression by mitogen-activated protein kinase p38.

The mitogen-activated protein kinase p38 pathway was originally identified as a signalling cascade activated by pro-inflammatory stimuli and cellular stresses, and playing a critical role in the translational regulation of pro-inflammatory cytokine synthesis. In almost a decade since this discovery, a great deal has been learned about the role of the p38 pathway in the post-transcriptional regulation of pro-inflammatory gene expression. However, important questions remain to be answered concerning the specificity and mechanism or mechanisms of action of p38. This review describes recent progress and remaining puzzles in the field of post-transcriptional regulation by p38.

Animals↗
PubMed2003

Control of the expression of inflammatory response genes.

The expression of genes involved in the inflammatory response is controlled both transcriptionally and post-transcriptionally. Primary inflammatory stimuli, such as microbial products and the cytokines interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF alpha), act through receptors of either the Toll and IL-1 receptor (TIR) family or the TNF receptor family. These cause changes in gene expression by activating four major intracellular signalling pathways that are cascades of protein kinases: namely the three mitogen-activated protein kinase (MAPK) pathways, and the pathway leading to activation of the transcription factor nuclear factor kappa B (NF kappa B). The pathways directly activate and induce the expression of a limited set of transcription factors which promote the transcription of inflammatory response genes. Many of the mRNAs are unstable, and are stabilized by the p38 MAPK pathway. Instability is mediated by clusters of the AUUUA motif in the 3' untranslated regions of the mRNAs. Control of mRNA stability provides a means of increasing the amplitude of a response and allows rapid adjustment of mRNA levels. Not all mRNAs stabilized by p38 contain AUUUA clusters; for example, matrix metalloproteinase-1 and -3 mRNAs lack these clusters, but are stabilized. Inflammatory gene expression is inhibited by glucocorticoids. These suppress MAPK signalling by inducing a MAPK phosphatase. This may be a significant mechanism additional to that by which the glucocorticoid receptor interferes with transcription factors.

Animals↗