Biomedical subjects

J Saklatvala

Publications and source records attributed to J Saklatvala.

At least 19 recordsLinked to original sources

PubMed2002

The mechanical joint score: a new clinical index of joint damage in rheumatoid arthritis.

OBJECTIVES: To evaluate the mechanical joint score (MJS) in terms of its reliability between observers and over time, its ease of use and its relationship with conventional measures of rheumatoid arthritis (RA) disease activity, severity and functional outcome. METHODS: The MJS was evaluated in 103 patients with reference to the following joints: total proximal interphalangeal (PIP) joints, total metacarpophalangeal (MCP) joints, wrists, elbows, shoulders, hips, knees, ankles and total metatarsophalangeal (MTP) joints. The score was based on the appearance of the joints on a scale of 0-3, 0 representing no abnormality and 3 severe abnormality or previous surgery. The MJS was evaluated in terms of its intra- and inter-observer variability and its content, construct and criterion validities. A subset of 29 patients were re-evaluated after 5 yr to examine change in MJS over time. RESULTS: The MJS performed well in terms of inter-observer and intra-observer reliability. The MJS showed strong correlation with the Larsen X-ray score of hands and feet (Spearman correlation coefficient 0.74) and with the modified Health Assessment Questionnaire (Spearman correlation coefficient 0.56) and only weak correlation with indices of disease activity, such as the Ritchie index and erythrocyte sedimentation rate. The MJS showed highly significant positive change over time. CONCLUSION: The MJS is a reliable clinical index of joint damage and may be a useful new outcome measure in RA.

Adult↗

PubMed2001

Analysis of mitogen-activated protein kinase pathways used by interleukin 1 in tissues in vivo: activation of hepatic c-Jun N-terminal kinases 1 and 2, and mitogen-activated protein kinase kinases 4 and 7.

The effects of interleukin 1 (IL-1) are mediated by the activation of protein kinase signalling pathways, which have been well characterized in cultured cells. We have investigated the activation of these pathways in rabbit liver and other tissues after the systemic administration of IL-1alpha. In liver there was 30-40-fold activation of c-Jun N-terminal kinase (JNK) and 5-fold activation of both JNK kinases, mitogen-activated protein kinase (MAPK) kinase (MKK)4 and MKK7. IL-1alpha also caused 2-3-fold activation of p38 MAPK and degradation of the inhibitor of nuclear factor kappaB ('IkappaB'), although no activation of extracellular signal-regulated protein kinase (ERK) (p42/44 MAPK) was observed. The use of antibodies against specific JNK isoforms showed that, in liver, short (p46) JNK1 and long (p54) JNK2 are the predominant forms activated, with smaller amounts of long JNK1 and short JNK2. No active JNK3 was detected. A similar pattern of JNK activation was seen in lung, spleen, skeletal muscle and kidney. Significant JNK3 activity was detectable only in the brain, although little activation of the JNK pathway in response to IL-1alpha was observed in this tissue. This distribution of active JNK isoforms probably results from a different expression of JNKs within the tissues, rather than from a selective activation of isoforms. We conclude that IL-1alpha might activate a more restricted set of signalling pathways in tissues in vivo than it does in cultured cells, where ERK and JNK3 activation are often observed. Cultured cells might represent a 'repair' phenotype that undergoes a broader set of responses to the cytokine.

Animals↗

PubMed2001

The 3' untranslated region of tumor necrosis factor alpha mRNA is a target of the mRNA-stabilizing factor HuR.

Posttranscriptional regulation is important for tumor necrosis factor alpha (TNF-alpha) expression in monocytes and macrophages, and an AU-rich element (ARE) in the 3' untranslated region (UTR) of TNF-alpha mRNA is implicated in control of its translation and mRNA stability. Regulation of mRNA turnover is thought to be mediated by trans-acting proteins, which bind the ARE and stabilize or destabilize the transcript. However, with the exception of the destabilizing factor tristetraprolin, the identity and function of the proteins binding the TNF-alpha mRNA ARE have not been established. To identify other proteins involved in the posttranscriptional control of TNF-alpha, the subcellular location of TNF-alpha mRNA was determined in the macrophage-like cell line RAW 264.7. TNF-alpha mRNA was located in the pellet following centrifugation of cytoplasm at 100,000 x g (P100 fraction). This fraction also contained proteins which formed two distinct ARE-specific complexes with the TNF-alpha mRNA 3' UTR in electrophoretic mobility shift assays (EMSAs). A protein present in these two complexes was purified and identified by peptide mass mapping and tandem mass spectrometry as HuR. In EMSAs both complexes were supershifted by an anti-HuR antibody, while Western blotting also demonstrated the presence of HuR in the P100 extract. A HeLa cell tetracycline-regulated reporter system was used to determine the effect of HuR on mRNA stability. In this system, overexpression of HuR resulted in stabilization of an otherwise unstable reporter-mRNA containing the TNF-alpha ARE. These results demonstrate that the TNF-alpha ARE is a target of the mRNA-stabilizing factor HuR.

3' Untranslated Regions↗

PubMed2001

Dexamethasone destabilizes cyclooxygenase 2 mRNA by inhibiting mitogen-activated protein kinase p38.

The stability of cyclooxygenase 2 (Cox-2) mRNA is regulated positively by proinflammatory stimuli acting through mitogen-activated protein kinase (MAPK) p38 and negatively by anti-inflammatory glucocorticoids such as dexamethasone. A tetracycline-regulated reporter system was used to investigate mechanisms of regulation of Cox-2 mRNA stability. Dexamethasone was found to destabilize beta-globin-Cox-2 reporter mRNAs by inhibiting p38. This inhibition occurred at the level of p38 itself: stabilization of reporter mRNA by a kinase upstream of p38 was blocked by dexamethasone, while stabilization by a kinase downstream of p38 was insensitive to dexamethasone. Inhibition of p38 activity by dexamethasone was observed in a variety of cell types treated with different activating stimuli. Furthermore, inhibition of p38 was antagonized by the anti-glucocorticoid RU486 and was delayed and actinomycin D sensitive, suggesting that ongoing glucocorticoid receptor-dependent transcription is required.

3' Untranslated Regions↗

PubMed2001

Mitogen-activated protein kinase p38 controls the expression and posttranslational modification of tristetraprolin, a regulator of tumor necrosis factor alpha mRNA stability.

Signal transduction pathways regulate gene expression in part by modulating the stability of specific mRNAs. For example, the mitogen-activated protein kinase (MAPK) p38 pathway mediates stabilization of tumor necrosis factor alpha (TNF-alpha) mRNA in myeloid cells stimulated with bacterial lipopolysaccharide (LPS). The zinc finger protein tristetraprolin (TTP) is expressed in response to LPS and regulates the stability of TNF-alpha mRNA. We show that stimulation of RAW264.7 mouse macrophages with LPS induces the binding of TTP to the TNF-alpha 3' untranslated region. The p38 pathway is required for the induction of TNF-alpha RNA-binding activity and for the expression of TTP protein and mRNA. Following stimulation with LPS, TTP is expressed in multiple, differentially phosphorylated forms. We present evidence that phosphorylation of TTP is mediated by the p38-regulated kinase MAPKAPK2 (MAPK-activated protein kinase 2). Our findings demonstrate a direct link between a specific signal transduction pathway and a specific RNA-binding protein, both of which are known to regulate TNF-alpha gene expression at a posttranscriptional level.

3' Untranslated Regions↗

PubMed2000

Regulation of tumour necrosis factor alpha mRNA stability by the mitogen-activated protein kinase p38 signalling cascade.

The translation of tumour necrosis factor alpha (TNFalpha) mRNA is regulated by the stress-activated protein kinase p38, which also controls the stability of several pro-inflammatory mRNAs. The regulation of TNFalpha gene expression in a mouse macrophage cell line RAW264.7 was re-examined using an inhibitor of stress-activated protein kinases. Stimulation of these cells with bacterial lipopolysaccharide resulted in stabilisation of TNFalpha mRNA, which was reversed by specific inhibition of p38. An adenosine/uridine-rich element from the TNFalpha 3' untranslated region conferred p38-sensitive decay in a tetracycline-regulated mRNA stability assay. Therefore the p38 pathway also controls TNFalpha mRNA turnover.

3' Untranslated Regions↗

PubMed2000

D-2-hydroxyglutaric aciduria in association with spondyloenchondromatosis.

D-2-hydroxyglutaric aciduria is a rare metabolic disorder, first reported in 1980, and does not yet have a clinically specific presentation pattern nor any specific treatment regime. We report a girl born with this uncommon metabolic disorder, who, at the age of 12 months, was also found to have a severe crippling form of skeletal dysplasia, spondyloenchondromatosis.

Bone Diseases, Developmental↗

PubMed2000

Do patients with rheumatoid arthritis established on methotrexate and folic acid 5 mg daily need to continue folic acid supplements long term?

BACKGROUND: It is postulated that some aspects of methotrexate toxicity may be related to its action as an anti-folate. Folic acid (FA) is often given as an adjunct to methotrexate therapy, but there is no conclusive proof that it decreases the toxicity of methotrexate and there is a theoretical risk that it may decrease the efficacy of methotrexate. OBJECTIVES: To look at the effect of stopping FA supplementation in UK rheumatoid arthritis (RA) patients established on methotrexate <20 mg weekly and FA 5 mg daily, to report all toxicity (including absolute changes in haematological and liver enzyme indices) and to report changes in the efficacy of methotrexate. METHODS: In a prospective, randomized, double-blind, placebo-controlled study, 75 patients who were established on methotrexate <20 mg weekly and FA 5 mg daily were asked to stop their FA and were randomized to one of two groups: placebo or FA 5 mg daily. Patients were evaluated for treatment toxicity and efficacy before entry and then at intervals of 3 months for 1 yr. RESULTS: Overall, 25 (33%) patients concluded the study early, eight (21%) in the group remaining on FA and 17 (46%) in the placebo group (P = 0.02). Two patients in the placebo group discontinued because of neutropenia. At 9 months there was an increased incidence of nausea in the placebo group (45 vs. 7%, P = 0.001). The placebo group had significantly lower disease activity on a few of the variables measured, but these were probably not of clinical significance. CONCLUSIONS: It is important to continue FA supplementation over the long term in patients on methotrexate and FA in order to prevent them discontinuing treatment because of mouth ulcers or nausea and vomiting. Our data suggest that FA supplementation is also helpful in preventing neutropenia, with very little loss of efficacy of methotrexate.

Aged↗

PubMed2000

Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade.

A tetracycline-regulated reporter system was used to investigate the regulation of cyclooxygenase 2 (Cox-2) mRNA stability by the mitogen-activated protein kinase (MAPK) p38 signaling cascade. The stable beta-globin mRNA was rendered unstable by insertion of the 2, 500-nucleotide Cox-2 3' untranslated region (3' UTR). The chimeric transcript was stabilized by a constitutively active form of MAPK kinase 6, an activator of p38. This stabilization was blocked by SB203580, an inhibitor of p38, and by two different dominant negative forms of MAPK-activated protein kinase 2 (MAPKAPK-2), a kinase lying downstream of p38. Constitutively active MAPKAPK-2 was also able to stabilize chimeric beta-globin-Cox-2 transcripts. The MAPKAPK-2 substrate hsp27 may be involved in stabilization, as beta-globin-Cox-2 transcripts were partially stabilized by phosphomimetic mutant forms of hsp27. A short (123-nucleotide) fragment of the Cox-2 3' UTR was necessary and sufficient for the regulation of mRNA stability by the p38 cascade and interacted with a HeLa protein immunologically related to AU-rich element/poly(U) binding factor 1.

Anti-Bacterial Agents↗

PubMed1999

Synergistic activation of JNK/SAPK by interleukin-1 and platelet-derived growth factor is independent of Rac and Cdc42.

The c-Jun N-terminal kinases (JNKs) are activated strongly by inflammatory cytokines and environmental stresses, but only weakly by growth factors. Here we show that platelet-derived growth factor (PDGF) strongly potentiates activation of JNK by interleukin 1 (IL-1) in human fibroblasts and a pig aortic endothelial (PAE) cell line. This synergistic activation of JNK by IL-1 and PDGF was unaffected by bacterial toxins that inactivate Rho proteins and Ras. Since Rho proteins have been implicated in JNK activation, their possible involvement was investigated further using stably expressed, inducible N17 or V12 mutants in PAE cell lines. N17 Rac non-selectively reduced JNK activity by 30% in resting or stimulated cells (IL-1 alone, or with PDGF). N17 Cdc42 had no effect. V12 Rac weakly activated JNK and synergized with IL-1, but not with PDGF. V12 Cdc42 weakly activated JNK, but synergized with PDGF and not IL-1. Our results imply that Rho GTPases are not directly involved in mediating IL-1-induced JNK activation, or in the potentiation of this activation by PDGF.

Bacterial Toxins↗

PubMed1999

p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes.

p38 mitogen-activated protein kinase (MAPK) is activated by inflammatory stimuli such as bacterial lipopolysaccharide (LPS), interleukin-1, and tumor necrosis factor. We have previously shown that the pyridinyl imidazole SB 203580, which inhibits it, blocks the interleukin-1 induction of cyclooxygenase-2 (COX-2) and matrix metalloproteinase 1 and 3 mRNAs in fibroblasts. Here we explore the role of p38 MAPK in the response of human monocytes to LPS. 0.1 microM SB 203580 significantly inhibited the LPS induction of COX-2 and tumor necrosis factor protein and mRNAs. The activity of MAPK-activated protein kinase-2 (a substrate of p38 MAPK) in the cells was commensurately reduced. Some isoforms of c-jun N-terminal kinase (which is also activated by LPS) are sensitive to SB 203580; the inhibitor had little effect on monocyte c-jun N-terminal kinases up to 2 microM. We investigated the mechanism of inhibition of COX-2 induction. Transcription (measured by a nuclear run-on assay) was 60% inhibited by SB 203580 (2 microM). Importantly, we found that p38 MAPK was essential for stabilizing COX-2 mRNA: when cells stimulated for 4 h with LPS were treated with actinomycin D, COX-2 mRNA decayed slowly. Treatment of stimulated cells with 2 microM SB 203580 caused a rapid disappearance of COX-2 mRNA, even with actinomycin D present. We conclude p38 MAPK plays a role in the transcription and stabilization of COX-2 mRNA.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

The proinflammatory and chondral activities of leukemia inhibitory factor in goat joints are partially a function of interleukin-1.

We wished to determine if the effects of injected recombinant human leukemia inhibitory factor (LIF) are a function of endogenous goat interleukin-1 (IL-1) production and, conversely, if the effects of injected recombinant human IL-1 are a function of endogenous LIF production in goat radiocarpal joints (RCJ). In preliminary experiments, murine LIF binding protein (MuLBP) and recombinant HuIL-1RA were found to independently attenuate the cartilage proteoglycan resorbing activity of goat synovial membrane-conditioned medium (GSMCM), implying activity against goat LIF and goat IL-1, respectively. The present study shows that the proinflammatory and chondral actions of rHuLIF in goat RCJ are partially attenuated by rHuIL-1RA. This implies that a small but important component of the in vivo activity of rHuLIF is a result of IL-1 production in the synovial joint. With the exception of proteoglycan synthesis, the absence of significant effects by MuLBP on the actions of rHuIL-1alpha in goat RCJ suggests that the proinflammatory and chondral effects of IL-1alpha in vivo are probably not mediated by LIF.

Animals↗

PubMed1999

High-density lipoproteins differentially modulate cytokine-induced expression of E-selectin and cyclooxygenase-2.

Atherogenesis is a multifactorial chronic inflammatory disease in which low plasma levels of HDLs are a strong predictor of the condition. Although the mechanism of protection by HDLs is not precisely known, HDLs have been shown to influence many of the events involved in the development of atherosclerosis. Previously we have shown that HDLs inhibited the cytokine-induced expression of adhesion molecules (E-selectin, VCAM-1, and ICAM-1) by endothelial cells (ECs). As the complete transcriptional regulation of all 3 genes requires the NF-kappaB family of transcription factors, we examined the effect of HDLs on activation of NF-kappaB. We also investigated the effect of HDLs on 2 other cytokine-induced genes, granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclooxygenase (Cox-2; prostaglandin H2 synthase, EC 0.1.14.99.1). E-selectin expression in response to tumor necrosis factor-alpha (TNFalpha) was, as expected, inhibited in ECs that had been preincubated with HDLs. However, the level of secretion of GM-CSF in the same cultures was no different from control. In a similar manner, although HDLs had no effect on steady-state mRNA levels of GM-CSF, the levels of E-selectin were significantly inhibited by HDLs. In transient cotransfection experiments we found that HDLs inhibited the cytokine-induced expression of a reporter gene driven by the E-selectin proximal promoter (-383 to 80) but had no effect on the expression of a reporter gene driven under the control of the proximal promoter of GM-CSF (-627 to 28). As would be predicted from this differential response, HDLs did not influence the nuclear translocation or DNA binding of NF-kappaB, or alter the kinetics of degradation and resynthesis of the inhibitory protein IkappaBalpha. We found that HDLs synergized with cytokine to enhance the expression of Cox-2 and induce the synthesis of its main EC product, prostacyclin (PGI2), a potent inhibitor of platelet and leukocyte functions. In conclusion, HDL induces an antiinflammatory phenotype in cytokine-induced ECs, synergizing with cytokine to induce elevation of Cox-2 in addition to inhibiting adhesion molecule expression. Our studies show that these differential effects are mediated in a manner that is likely to be independent of NF-kappaB per se.

Adult↗

PubMed1999

Protein kinase cascades in intracellular signalling by interleukin-I and tumour necrosis factor.

Interleukin 1 (IL-1) and tumour necrosis factor (TNF) are major mediators of inflammation, with similar actions. Their receptor mechanisms and downstream pathways are reviewed. They activate several protein kinases in fibroblasts, including the three types of mitogen-activated protein kinase (MAPK), the kinase of the inhibitor of nuclear factor-kappa B (I kappa BK), and the TNF-/IL-1-activated beta-casein kinase. Cultured cells show a broader spectrum of kinase activation by IL-1 than tissues in vivo, suggesting that the receptors connect to more pathways in proliferating cells than in resting differentiated cells. The c-Jun N-terminal kinase (JNK) is strongly activated by IL-1 in tissues. In rabbit liver this is mediated by MAPK kinase 7; the upstream kinase is unidentified. Little is known of downstream MAPK targets in inflammation. Inhibitor experiments suggest that p38MAPK mediates induction of cyclo-oxygenase-2 and metalloproteinases by IL-1, and of TNF, IL-1 and cyclo-oxygenase-2 by endotoxin (in monocytes). p38MAPK is needed for induction of the mRNAs (except IL-1 mRNA).

Animals↗

PubMed1998

A p38 MAP kinase inhibitor regulates stability of interleukin-1-induced cyclooxygenase-2 mRNA.

The mechanism by which p38 mitogen-activated protein kinase (MAPK) regulates the induction of cyclooxygenase (COX)-2 by interleukin-1 (IL-1) has been investigated in HeLa cells. SB 203580, an inhibitor of p38 MAPK, in the range 0.1-1 microM inhibited IL-1-stimulated PGE2 (but not arachidonic acid) release and this was associated with inhibition of induction of COX-2 protein and mRNA. IL-1 stimulated COX-2 transcription in HeLa cells about 2-fold as judged by both reporter gene and nuclear run-on assays. The inhibitor had no significant effect on this. However, in cells previously stimulated with IL-1 it caused rapid destabilisation of COX-2 mRNA independently of on-going transcription. The results suggest a novel function for p38 MAPK in the regulation of mRNA stability.

Arachidonic Acids↗

PubMed1998

p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes.

We have investigated the activation of the p38 MAPK pathway in response to CD40 engagement in multiple B cell lines and in human tonsillar B cells to define the role of p38 MAPK in proliferation, NF-kappaB activation and gene expression. Cross-linking CD40 rapidly stimulates both p38 MAPK and its downstream effector, MAPKAPK-2. Inhibition of p38 MAPK activity in vivo with the specific cell-permeable inhibitor, SB203580, under conditions that completely prevented MAPKAPK-2 activation, strongly perturbed CD40-induced tonsillar B cell proliferation while potentiating the B cell receptor (BCR)-driven proliferative response. SB203580 also significantly reduced expression of a reporter gene driven by a minimal promoter containing four NF-kappaB elements, indicating a requirement for the p38 MAPK pathway in CD40-induced NF-kappaB activation. However, CD40-mediated NF-kappaB binding was not affected by SB203580, suggesting that NF-kappaB may not be a direct target for the CD40-induced p38 MAPK pathway. In addition, SB203580 selectively reduced CD40-induced CD54/ICAM-1 expression, whereas CD40-dependent expression of CD40 and CD95/Fas and four newly defined CD40-responsive genes cIAP2, TRAF1, TRAF4/CART and DR3 were unaffected. Our observations show that the p38 MAPK pathway is required for CD40-induced proliferation and that CD40 induces gene expression via both p38 MAPK-dependent and -independent pathways.

Animals↗

PubMed1998

Stress-activated protein kinase/Jun N-terminal kinase is required for interleukin (IL)-1-induced IL-6 and IL-8 gene expression in the human epidermal carcinoma cell line KB.

The cytokine interleukin-1 (IL-1) is a major inflammatory hormone which activates a broad range of genes during inflammation. The signaling mechanisms triggered by IL-1 include activation of several distinct protein kinase systems. The stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), is activated particularly strongly by the cytokine. In an attempt to delineate its role in activation of gene expression by IL-1, we inhibited the IL-1-induced SAPK/JNK activity by stable overexpression of either a catalytically inactive mutant of SAPKbeta (SAPKbeta(K-R)) or antisense RNA to SAPKbeta in human epidermal carcinoma cells. A detailed analysis of signal transduction in those cells showed that activation of neither NFkappaB nor p38 mitogen-activated protein kinase was affected, suggesting that we achieved specific blockade of the SAPK/JNK. In untransfected and vector-transfected KB cells, IL-1 induced a strong increase in expression of IL-6 and IL-8 mRNA, along with the synthesis of high amounts of the proteins. In two KB cell clones stably overexpressing the mutant SAPKbeta(K-R), and three clones stably overexpressing antisense RNA to SAPKbeta, expression of IL-6 and IL-8 in response to IL-1 was strongly reduced at both the mRNA and protein level. These data indicate that the SAPK/JNK pathway provides an indispensable signal for IL-1-induced expression of IL-6 and IL-8.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

Interleukin-1.

Interleukin 1 (IL1) is a primary regulator of inflammatory and immune responses. Via its type I receptor it activates specific protein kinases, including the NF kappa B inducing kinase (NIK) and three distinct mitogen-activated protein (MAP) kinase cascades. These modulate a number of transcription factors including NF kappa B, AP1 and CREB each of which regulate a plethora of immediate early genes central to the inflammatory response. Phase I clinical trials of the soluble type I receptor and IRAP indicate that these have potential anti-inflammatory effects.

Adaptor Protein Complex 1↗