Troponin T isoform expression is modulated during Atlantic Halibut metamorphosis

<p>Abstract</p> <p>Background</p> <p>Flatfish metamorphosis is a thyroid hormone (TH) driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in <it>Troponin T </it>(<it>TnT</it>), a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied.</p> <p>Results</p> <p>In the present study five cDNAs for halibut <it>TnT </it>genes were cloned; three were splice variants arising from a single <it>fast TnT </it>(<it>fTnT</it>) gene; a fourth encoded a novel teleost specific fTnT-like cDNA (<it>AfTnT</it>) expressed exclusively in slow muscle and the fifth encoded the teleost specific <it>sTnT2</it>. THs modified the expression of halibut <it>fTnT </it>isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, <it>AfTnT </it>and <it>sTnT2</it>, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of <it>TnT </it>genes and muscle fibre organization occurred in halibut musculature.</p> <p>Conclusion</p> <p>Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in <it>TnT </it>gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.</p

Exacerbated inflammatory arthritis in response to hyperactive gp130 signalling is independent of IL-17A

Objective Interleukin (IL)-17A producing CD4 T-cells (TH-17 cells) are implicated in rheumatoid arthritis (RA). IL-6/STAT3 signalling drives TH-17 cell differentiation, and hyperactive gp130/STAT3 signalling in the gp130F/F mouse promotes exacerbated pathology. Conversely, STAT1-activating cytokines (eg, IL-27, IFN-γ) inhibit TH-17 commitment. Here, we evaluate the impact of STAT1 ablation on TH-17 cells during experimental arthritis and relate this to IL-17A-associated pathology. Methods Antigen-induced arthritis (AIA) was established in wild type (WT), gp130F/F mice displaying hyperactive gp130-mediated STAT signalling and the compound mutants gp130F/F:Stat1−/− and gp130F/F: Il17a−/− mice. Joint pathology and associated peripheral TH-17 responses were compared. Results Augmented gp130/STAT3 signalling enhanced TH-17 commitment in vitro and exacerbated joint pathology. Ablation of STAT1 in gp130F/F mice (gp130F/F: Stat1−/− ) promoted the hyperexpansion of TH-17 cells in vitro and in vivo during AIA. Despite this heightened peripheral TH-17 cell response, disease severity and the number of joint-infiltrating T-cells were comparable with that of WT mice. Thus, gp130-mediated STAT1 activity within the inflamed synovium controls T-cell trafficking and retention. To determine the contribution of IL-17A, we generated gp130F/F:IL-17a−/− mice. Here, loss of IL-17A had no impact on arthritis severity. Conclusions Exacerbated gp130/STAT-driven disease in AIA is associated with an increase in joint infiltrating T-cells but synovial pathology is IL-17A independent

Deletion of P58^IPK, the cellular inhibitor of the protein kinases PKR and PERK, causes bone changes and joint degeneration in mice

Objective: Protein kinase-like endoplasmic reticulum kinase (PERK) and protein kinase R (PKR) are implicated in endoplasmic reticulum stress-induced arthritis and pro-inflammatory cytokine-mediated cartilage degradation in vitro, respectively. We determined whether knockout of the cellular inhibitor of PERK and PKR, P58IPK causes joint degeneration in vivo and whether these molecules are activated in human osteoarthritis (OA). Materials and Methods: Sections of knee joints from P58IPK-null and wild-type mice aged 12–13 and 23–25 months were stained with toluidine blue and scored for degeneration using the osteoarthritis research society international (OARSI) system. Bone changes were assessed by radiology and high-resolution micro-computed tomography of hind limbs. Sections from the medial tibial plateaus of two human knees, removed in total knee replacement surgery for OA, were immunolabelled for phosphorylated PERK and PKR and P58IPK. Results: Knockout mice exhibited narrower tibiae (p = 0.0031) and smaller epiphyses in tibiae (p = 0.0004) and femora (p = 0.0214). Older knockout mice had reduced total volume inside the femoral periosteal envelope (p = 0.023), reduced tibial (p = 0.03), and femoral (p = 0.0012) bone volumes (BV) and reduced femoral BV fraction (p = 0.025). Compared with wild-types, younger P58IPK-null mice had increased OARSI scores in medial femoral condyles (p = 0.035). Thirty four percent of null mice displayed severe joint degeneration with complete articular cartilage loss from the medial compartment and heterotopic chondro-osseous tissue in the medial joint capsule. Phosphorylated PERK and PKR were localized throughout human osteoarthritic tibial plateaus but, in particular, in areas exhibiting the most degeneration. There was limited expression of P58IPK. Conclusion: This study is the first to reveal a critical role for P58IPK in maintaining joint integrity in vivo, implicating the PKR and PERK stress signaling pathways in bony changes underlying the pathogenesis of joint degeneration

The interleukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145 confers increased proteolytic conversion rates by ADAM proteases

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Intra-articular AMPA/kainate glutamate receptor antagonists alleviate inflammation, pain and pathology in rat antigen induced arthritis [Abstract]

Introduction: Concentrations of the neurotransmitter glutamate are greatly increased in synovial fluids of RA and OA patients, where they correlate with cytokine concentrations. Previously, we demonstrated that human synoviocytes express functional glutamate receptors (GluRs) and that activation of NMDA GluRs decreases proMMP2 expression, whilst activation of kainate GluRs increases IL-6 release (Flood et al. 2007). High glutamate concentrations in the joint cause arthritic pain which is alleviated by intra-articular injection of GluR antagonists. However, the effects of such inhibitors on arthritis progression via GluRs on other joint tissues have not been considered. We are investigating the hypothesis that specific GluR subunits in the arthritic synovium mediate proinflammatory, degradative and proliferative responses, and may be therapeutically targeted to reduce disease rogression and pain. Materials and Methods: Using the mono-articular antigen induced arthritis (AIA) rat model, we used intra-articular injection of NBQX to inhibit AMPA/kainate receptors at the time of arthritis induction, prior to peak IL-6 levels. Over a 21 day period, we measured knee swelling and gait patterns from AIA, AIA + NBQX and normal rats (n = 6 per group). A combination of motion analysis, using Qualisys Pro-Reflex MCU-1000 cameras, and footprint characteristics revealed limping and abnormal movements as an indirect measure of pain. On day 21, joint tissues were taken for reverse transcription PCR, x-ray, immunohistochemistry and histology to examine GluR expression and joint destruction. Results: Less knee swelling (ANOVA with Fisher’s post hoc test, P < 0.0006) was found in NBQX treated rats compared to AIA rats. Using footprints to indicate abnormal loading and movements, NBQX treated rats displayed less pain related behaviour during the initial flare of arthritis compared to AIA rats (Mann–Whitney test, P = 0.0002). Radiological pathology appeared reduced in NBQX rats compared to AIA rats (8.33 and 12, mean x-ray erosion/osteophyte score respectively). Ionotropic and metabotropic GluRs mRNA was differentially expressed in cartilage, synovium, meniscus,fat pad, patella, femoral head and shaft of rat knees. Discussion: We have shown that intra-articular NBQX treatment alleviates inflammation, pain and pathology in arthritis in vivo and that GluRs are differentially expressed in knee joint tissues. This supports our hypothesis that kainate GluRs may be specifically targeted to ease pain, inflammation and pathology in arthritis

Soluble IL-6 Receptor Governs IL-6 Activity in Experimental Arthritis: Blockade of Arthritis Severity by Soluble Glycoprotein 130

Studies in IL-6-deficient (IL-6-/-) mice highlight that IL-6 contributes to arthritis progression. However, the molecular mechanism controlling its activity in vivo remains unclear. Using an experimental arthritis model in IL-6-/- mice, we have established a critical role for the soluble IL-6R in joint inflammation. Although intra-articular administration of IL-6 itself was insufficient to reconstitute arthritis within these mice, a soluble IL-6R-IL-6 fusion protein (HYPER-IL-6) restored disease activity. Histopathological assessment of joint sections demonstrated that HYPER-IL-6 increased arthritis severity and controlled intrasynovial mononuclear leukocyte recruitment through the CC-chemokine CCL2. Activation of synovial fibroblasts by soluble IL-6R and IL-6 emphasized that these cells may represent the source of CCL2 in vivo. Specific blockade of soluble IL-6R signaling in wild-type mice using soluble gp130 ameliorated disease. Consequently, soluble IL-6R-mediated signaling represents a promising therapeutic target for the treatment of rheumatoid arthritis

Interleukin-6 signalling in juvenile idiopathic arthritis is limited by proteolytically cleaved soluble interleukin-6 receptor

OBJECTIVES: Interleukin-6 (IL-6) exerts multiple effects on chondrocytes and fibroblasts within the joint and is associated with disease activity in juvenile idiopathic arthritis (JIA). Although these cells express the ubiquitous signalling receptor for all IL-6-related cytokines, gp130, they do not express a cognate IL-6 receptor. Consequently, IL-6 responses within these cells occur via IL-6 trans-signalling relying on the presence of a soluble receptor (sIL-6R). Levels of sIL-6R in vivo are governed by either proteolytic cleavage (PC) of cognate receptor or by differential sIL-6R mRNA splicing (DS). The aim of this study was to evaluate the contribution of both isoforms to clinical parameters associated with IL-6 signalling in JIA. METHODS: IL-6, sIL-6R and DS-sIL-6R were measured by ELISA in serum and synovial fluid (SF) samples from 86 JIA patients. These data were related to indicators of inflammation-erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) and compared between patients stratified by subtype, age and disease duration. RESULTS: SF IL-6 significantly correlated with general indicators of activity (ESR and CRP) and SF PC-sIL-6R to a lesser degree with CRP. When the IL-6:sIL-6R ratio was calculated as an indicator of the potential for IL-6 signalling within the joint, 33% of SF samples showed a ratio >1 indicating saturation of sIL-6R by IL-6. Mean DS-sIL-6R levels were 0.71 ng/ml, whereas PC-sIL-6R levels constituted the majority of sIL-6R at 20.89 ng/ml. CONCLUSIONS: IL-6 trans-signalling within the joints of JIA patients is predominantly governed by the presence of PC-sIL-6R, and the data provided suggest that synovial levels of IL-6 and sIL-6R would be sufficient to drive IL-6 responses in chondrocytes and synovial fibroblasts