TWEAK and its receptor Fn14 in the synovium of patients with rheumatoid arthritis compared to psoriatic arthritis and its response to tumour necrosis factor blockade

Objective: To investigate the expression of tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor inducible 14 (Fn14) in the inflamed synovium of patients with arthritis, as TWEAK blockade has been observed to have a beneficial effect in an animal model of rheumatoid arthritis (RA). Methods: Synovial tissue (ST) biopsies were obtained from 6 early, methotrexate-naive patients with RA as well as 13 patients with RA and 16 patients with psoriatic arthritis (PsA) who were matched for treatment and disease duration. Serial ST samples were obtained from a separate cohort of 13 patients with RA before and after infliximab treatment. TWEAK and Fn14 expression was evaluated by immunohistochemistry and digital image analysis. Results: TWEAK and Fn14 were clearly expressed in ST of patients with RA and PsA. TWEAK expression was significantly higher in RA (sub) lining samples compared to PsA (p = 0.005 and p = 0.014, respectively), but Fn14 expression was comparable. Double immunofluorescence showed TWEAK and Fn14 expression on fibroblast-like synoviocytes and macrophages, but not T cells. Of interest, persistent TWEAK and Fn14 expression was found after anti-TNF therapy. Conclusions: TWEAK and Fn14 are abundantly expressed in the inflamed synovium of patients with RA and PsA. This raises the possibility that blocking TWEAK/Fn14 signalling could be of therapeutic benefit in inflammatory arthriti

Promotion of macrophage activation by Tie2 in the context of the inflamed synovia of rheumatoid arthritis and psoriatic arthritis patients

OBJECTIVE: To examine the role of Tie2 signalling in macrophage activation within the context of the inflammatory synovial microenvironment present in patients with RA and PsA. METHODS: Clinical responses and macrophage function were examined in wild-type and Tie2-overexpressing (Tie2-TG) mice in the K/BxN serum transfer model of arthritis. Macrophages derived from peripheral blood monocytes from healthy donors, RA and PsA patients, and RA and PsA synovial tissue explants were stimulated with TNF (10 ng/ml), angiopoietin (Ang)-1 or Ang-2 (200 ng/ml), or incubated with an anti-Ang2 neutralizing antibody. mRNA and protein expression of inflammatory mediators was analysed by quantitative PCR, ELISA and Luminex. RESULTS: Tie2-TG mice displayed more clinically severe arthritis than wild-type mice, accompanied by enhanced joint expression of IL6, IL12B, NOS2, CCL2 and CXCL10, and activation of bone marrow-derived macrophages in response to Ang-2 stimulation. Ang-1 and Ang-2 significantly enhanced TNF-induced expression of pro-inflammatory cytokines and chemokines in macrophages from healthy donors differentiated with RA and PsA SF and peripheral blood-derived macrophages from RA and PsA patients. Both Ang-1 and Ang-2 induced the production of IL-6, IL-12p40, IL-8 and CCL-3 in synovial tissue explants of RA and PsA patients, and Ang-2 neutralization suppressed the production of IL-6 and IL-8 in the synovial tissue of RA patients. CONCLUSION: Tie2 signalling enhances TNF-dependent activation of macrophages within the context of ongoing synovial inflammation in RA and PsA, and neutralization of Tie2 ligands might be a promising therapeutic target in the treatment of these diseases

Selective involvement of ERK and JNK mitogen-activated protein kinases in early rheumatoid arthritis (1987 ACR criteria compared to 2010 ACR/EULAR criteria): a prospective study aimed at identification of diagnostic and prognostic biomarkers as well as therapeutic targets

Objectives To investigate the expression and activation of mitogen-activated protein kinases in patients with early arthritis who are disease-modifying antirheumatic drug (DMARD) naive. Methods A total of 50 patients with early arthritis who were DMARD naive (disease duration <1 year) were prospectively followed and diagnosed at baseline and after 2 years for undifferentiated arthritis (UA), rheumatoid arthritis (RA) (1987 American College of Rheumatology (ACR) and 2010 ACR/European League Against Rheumatism (EULAR) criteria), or spondyloarthritis (SpA). Synovial biopsies obtained at baseline were examined for expression and phosphorylation of p38, extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by immunohistochemistry and digital analysis. Synovial tissue mRNA expression was measured by quantitative PCR (qPCR). Results ERK and JNK activation was enhanced at inclusion in patients meeting RA criteria compared to other diagnoses. JNK activation was enhanced in patients diagnosed as having UA at baseline who eventually fulfilled 1987 ACR RA criteria compared to those who remained UA, and in patients with RA fulfilling 2010 ACR/EULAR criteria at baseline. ERK and JNK activation was enhanced in patients with RA developing progressive joint destruction. JNK activation in UA predicted 1987 ACR RA classification criteria fulfilment (R-2=0.59, p=0.02) after follow-up, and disease progression in early arthritis (R-2=0.16, p <0.05). Enhanced JNK activation in patients with persistent disease was associated with altered synovial expression of extracellular matrix components and CD44. Conclusions JNK activation is elevated in RA before 1987 ACR RA classification criteria are met and predicts development of erosive disease in early arthritis, suggesting JNK may represent an attractive target in treating RA early in the disease proces

Binding Properties and Stability of the Ras-Association Domain of Rap1-GTP Interacting Adapter Molecule (RIAM)

The Rap1-GTP interacting adapter protein (RIAM) is an important protein in Rap1-mediated integrin activation. By binding to both Rap1 GTPase and talin, RIAM recruits talin to the cell membrane, thus facilitating talin-dependent integrin activation. In this article, we studied the role of the RIAM Ras-association (RA) and pleckstrin-homology (PH) domains in the interaction with Rap1. We found that the RA domain was sufficient for GTP-dependent interaction with Rap1B, and the addition of the PH domain did not change the binding affinity. We also detected GTP-independent interaction of Rap1B with the N-terminus of RIAM. In addition, we found that the PH domain stabilized the RA domain both in vitro and in cells

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting.

Background and objective S ystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes. Methods C hromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFNresponsive gene expression. Results 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFN\u3b1 induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression. Conclusion SS c monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc

Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells

While the adaptor SKAP-55 mediates LFA-1 adhesion on T-cells, it is not known whether the adaptor regulates other aspects of signaling. SKAP-55 could potentially act as a node to coordinate the modulation of adhesion with downstream signaling. In this regard, the GTPase p21ras and the extracellular signal-regulated kinase (ERK) pathway play central roles in T-cell function. In this study, we report that SKAP-55 has opposing effects on adhesion and the activation of the p21ras -ERK pathway in T-cells. SKAP-55 deficient primary T-cells showed a defect in LFA-1 adhesion concurrent with the hyper-activation of the ERK pathway relative to wild-type cells. RNAi knock down (KD) of SKAP-55 in T-cell lines also showed an increase in p21ras activation, while over-expression of SKAP-55 inhibited activation of ERK and its transcriptional target ELK. Three observations implicated the p21ras activating exchange factor RasGRP1 in the process. Firstly, SKAP-55 bound to RasGRP1 via its C-terminus, while secondly, the loss of binding abrogated SKAP-55 inhibition of ERK and ELK activation. Thirdly, SKAP-55−/− primary T-cells showed an increased presence of RasGRP1 in the trans-Golgi network (TGN) following TCR activation, the site where p21ras becomes activated. Our findings indicate that SKAP-55 has a dual role in regulating p21ras-ERK pathway via RasGRP1, as a possible mechanism to restrict activation during T-cell adhesion

A Novel Role for PECAM-1 (CD31) in Regulating Haematopoietic Progenitor Cell Compartmentalization between the Peripheral Blood and Bone Marrow

Although the expression of PECAM-1 (CD31) on vascular and haematopoietic cells within the bone marrow microenvironment has been recognized for some time, its physiological role within this niche remains unexplored. In this study we show that PECAM-1 influences steady state hematopoietic stem cell (HSC) progenitor numbers in the peripheral blood but not the bone marrow compartment. PECAM-1−/− mice have higher levels of HSC progenitors in the blood compared to their littermate controls. We show that PECAM-1 is required on both progenitors and bone marrow vascular cells in order for efficient transition between the blood and bone marrow to occur. We have identified key roles for PECAM-1 in both the regulation of HSC migration to the chemokine CXCL12, as well as maintaining levels of the matrix degrading enzyme MMP-9 in the bone marrow vascular niche. Using intravital microscopy and adoptive transfer of either wild type (WT) or PECAM-1−/− bone marrow precursors, we demonstrate that the increase in HSC progenitors in the blood is due in part to a reduced ability to migrate from blood to the bone marrow vascular niche. These findings suggest a novel role for PECAM-1 as a regulator of resting homeostatic progenitor cell numbers in the bloo

SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes

Myosin IIA Modulates T Cell Receptor Transport and CasL Phosphorylation during Early Immunological Synapse Formation

Activation of T cell receptor (TCR) by antigens occurs in concert with an elaborate multi-scale spatial reorganization of proteins at the immunological synapse, the junction between a T cell and an antigen-presenting cell (APC). The directed movement of molecules, which intrinsically requires physical forces, is known to modulate biochemical signaling. It remains unclear, however, if mechanical forces exert any direct influence on the signaling cascades. We use T cells from AND transgenic mice expressing TCRs specific to the moth cytochrome c 88–103 peptide, and replace the APC with a synthetic supported lipid membrane. Through a series of high spatiotemporal molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA, transiently drives TCR transport during the first one to two minutes of immunological synapse formation. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated protein kinase 70) with TCR, revealing an influence on signaling activity. More tellingly, its inhibition also significantly reduces phosphorylation of the mechanosensing protein CasL (Crk-associated substrate the lymphocyte type), raising the possibility of a direct mechanical mechanism of signal modulation involving CasL