Synovial explant inflammatory mediator production corresponds to rheumatoid arthritis imaging hallmarks: a cross-sectional study

Introduction

Despite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study was to compare site-specific release of inflammatory mediators and evaluate the corresponding anatomical sites by examining colour Doppler ultrasound (CDUS) and MRI scans.

Methods

RA patients were evaluated on the basis of CDUS and 3-T MRI scans and subsequently underwent synovectomy using a needle arthroscopic procedure of the hand joints. The synovial tissue specimens were incubated for 72 hours, and spontaneous release of monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), macrophage inflammatory protein 1β (MIP-1β) and IL-8 was measured by performing multiplex immunoassays. Bone marrow oedema (BME), synovitis and erosion scores were estimated on the basis of the rheumatoid arthritis magnetic resonance imaging score (RAMRIS). Mixed models were used for the statistical analyses. Parsimony was achieved by omitting covariates with P > 0.1 from the statistical model.

Results

Tissue samples from 58 synovial sites were obtained from 25 patients. MCP-1 was associated with CDUS activity (P = 0.009, approximate Spearman’s ρ = 0.41), RAMRIS BME score (P = 0.01, approximate Spearman’s ρ = 0.42) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.31). IL-6 was associated with RAMRIS synovitis score (P = 0.04, approximate Spearman’s ρ = 0.50), BME score (P = 0.04, approximate Spearman’s ρ = 0.31) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.35). MIP-1β was associated with CDUS activity (P = 0.02, approximate Spearman’s ρ = 0.38) and RAMRIS synovitis scores (P = 0.02, approximate Spearman’s ρ = 0.63). IL-8 associations with imaging outcome measures did not reach statistical significance.

Conclusions

The association between imaging activity and synovial inflammatory mediators underscores the high sensitivity of CDUS and MRI in the evaluation of RA disease activity. The associations found in our present study have different implications for synovial mediator releases and corresponding imaging signs. For example, MCP-1 and IL-6 were associated with both general inflammation and bone destruction, in contrast to MIP-1β, which was involved solely in general synovitis. The lack of association of IL-8 with synovitis was likely underestimated because of a large proportion of samples above assay detection limits among the patients with the highest synovitis scores.     

A Mediator of Rho-dependent Invasion Moonlights as a Methionine Salvage Enzyme

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named “mediator of RhoA-dependent invasion (MRDI),” that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.Cutaneous malignant melanoma has doubled in incidence over the past 30 years. Stages involved in progression of melanocytes to melanoma based on clinical and histopathological features include nontumorigenic nevi, dysplastic or atypical nevi, primary radial growth phase and vertical growth phase melanoma, and metastatic melanoma (1). Metastatic melanomas are often resistant to treatment; therefore therapeutic strategies require a more complete understanding of molecular determinants of this disease, particularly those involved in the invasive phenotype (2).Rho GTPases control a wide range of cellular responses including cell movement, morphogenesis, and coordinated migration (3). These pathways are implicated in malignant cell transformation and metastasis based on in vitro evidence showing tumorigenic and invasive responses to enhanced signaling in cell lines. Studies have demonstrated that overexpression of RhoC enhances invasion and metastasis in mouse xenografts of human melanoma and lung cancer cell lines (4, 5). In addition, some human tumors show elevated expression of Rho GTPases and exchange factors and/or reduced expression of GTPase-activating factors (6–8). Signaling through RhoA promotes actin polymerization and stress fiber formation, providing cells with contractile force needed for cell movement. Rho-GTP interacts with various effectors, including Rho-activated kinase, which promotes actin-myosin assembly via phosphorylation of myosin light chain phosphatase (9), or diaphanous-related formins, which nucleate actin filaments and stabilize microtubules (10, 11). Studies of cultured melanoma cells have revealed an “amoeboid” invasion mechanism involving RhoA-dependent Rho-activated kinase activation and inactivation of Rac (12, 13).RhoA also controls the formation and turnover of focal adhesion contacts, which mediate interactions between extracellular matrix and the actin cytoskeleton (14, 15). Signaling involves activation of Src and focal adhesion kinase (FAK)¹ and subsequent tyrosine phosphorylation of proteins recruited to integrin receptor complexes (16). Embryonic cells from FAK^−/− mice lose motility and cannot be rescued with FAK harboring a Y397F autophosphorylation site mutation not because they fail to form focal adhesions but because they are unable to disassemble focal adhesions (17). Thus, Rho controls cell movement by modulating the turnover of focal adhesion complexes via FAK. However, the mechanisms by which Rho GTPases control FAK are incompletely understood.In this study, we report that RhoA was constitutively activated in melanoma cells in a stage-specific pattern with elevated activity in cells from metastatic tumors. We present a functional proteomics screen for molecular targets of RhoA from which we identified a previously uncharacterized human protein induced in response to constitutive RhoA activation. This protein promoted Rho-dependent cell invasion and cell motility and provided a novel link for regulation of FAK tyrosine phosphorylation by RhoA. Thus, we refer to it as “mediator of Rho-dependent invasion (MRDI).” Although human MRDI has not been studied previously, it shows close sequence similarity to a methylthioribose-1-phosphate isomerase, which functions in methionine salvage pathways characterized in bacteria and yeast. We demonstrated that MRDI indeed has methylthioribose-1-phosphate isomerase activity and is required for methionine salvage in human cells. We further demonstrated that the catalytic activity of MRDI is independent of its role in cell invasion. Thus, MRDI is a dual function protein with promiscuous roles both as a metabolic enzyme and as an effector of signaling and cancer cell invasion.     

CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase

The proper coordination between DNA replication and mitosis during cell-cycle progression is crucial for genomic stability. During G2 and mitosis, Set8 catalyzes monomethylation of histone H4 on lysine 20 (H4K20me1), which promotes chromatin compaction. Set8 levels decline in S phase, but why and how this occurs is unclear. Here, we show that Set8 is targeted for proteolysis in S phase and in response to DNA damage by the E3 ubiquitin ligase, CRL4(Cdt2). Set8 ubiquitylation occurs on chromatin and is coupled to DNA replication via a specific degron in Set8 that binds PCNA. Inactivation of CRL4(Cdt2) leads to Set8 stabilization and aberrant H4K20me1 accumulation in replicating cells. Transient S phase expression of a Set8 mutant lacking the degron promotes premature H4K20me1 accumulation and chromatin compaction, and triggers a checkpoint-mediated G2 arrest. Thus, CRL4(Cdt2)-dependent destruction of Set8 in S phase preserves genome stability by preventing aberrant chromatin compaction during DNA synthesis.     

Developmental and tissue-specific expression of NITRs

Novel immune-type receptors (NITRs) are encoded by large multi-gene families and share structural and signaling similarities to mammalian natural killer receptors (NKRs). NITRs have been identified in multiple bony fish species, including zebrafish, and may be restricted to this large taxonomic group. Thirty-nine NITR genes that can be classified into 14 families are encoded on zebrafish chromosomes 7 and 14. Herein, we demonstrate the expression of multiple NITR genes in the zebrafish ovary and during embryogenesis. All 14 families of zebrafish NITRs are expressed in hematopoietic kidney, spleen and intestine as are immunoglobulin and T cell antigen receptors. Furthermore, all 14 families of NITRs are shown to be expressed in the lymphocyte lineage, but not in the myeloid lineage, consistent with the hypothesis that NITRs function as NKRs. Sequence analyses of NITR amplicons identify known alleles and reveal additional alleles within the nitr1, nitr2, nitr3, and nitr5 families, reflecting the recent evolution of this gene family.     

Biochemical Basis of the Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Immunoglobulin-like Repeats of Filamin

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to β-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.     

ATR: a master conductor of cellular responses to DNA replication stress

The integrity of the genome is constantly challenged by intrinsic and extrinsic genotoxic stresses that damage DNA. The cellular responses to DNA damage are orchestrated by DNA damage signaling pathways, also known as DNA damage checkpoints. These signaling pathways play crucial roles in detecting DNA damage, regulating DNA repair and coordinating DNA repair with other cellular processes. In vertebrates, the ATM- and Rad3-related (ATR) kinase plays a key role in the response to a broad spectrum of DNA damage and DNA replication stress. Here, we will discuss the recent findings on how ATR is activated by DNA damage and how it protects the genome against interference with DNA replication.     

Why do leafcutter bees cut leaves? New insights into the early evolution of bees

Stark contrasts in clade species diversity are reported across the tree of life and are especially conspicuous when observed in closely related lineages. The explanation for such disparity has often been attributed to the evolution of key innovations that facilitate colonization of new ecological niches. The factors underlying diversification in bees remain poorly explored. Bees are thought to have originated from apoid wasps during the Mid-Cretaceous, a period that coincides with the appearance of angiosperm eudicot pollen grains in the fossil record. The reliance of bees on angiosperm pollen and their fundamental role as angiosperm pollinators have contributed to the idea that both groups may have undergone simultaneous radiations. We demonstrate that one key innovation--the inclusion of foreign material in nest construction--underlies both a massive range expansion and a significant increase in the rate of diversification within the second largest bee family, Megachilidae. Basal clades within the family are restricted to deserts and exhibit plesiomorphic features rarely observed among modern bees, but prevalent among apoid wasps. Our results suggest that early bees inherited a suite of behavioural traits that acted as powerful evolutionary constraints. While the transition to pollen as a larval food source opened an enormous ecological niche for the early bees, the exploitation of this niche and the subsequent diversification of bees only became possible after bees had evolved adaptations to overcome these constraints.