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1.
Arthritis Rheumatol ; 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31738005

RESUMO

OBJECTIVE: In gout, autoinflammatory responses to urate crystals promote acute arthritis flares. Tophi, chronic synovitis, and erosion pathogeneses are less well understood. Defining epigenomic immunity training can reveal novel pathogenesis factors and biomarkers. Here, gout epigenome-wide analyses seminally probed differential DNA methylation. METHODS: San Diego gout cohort (n=16, n=14 healthy controls) peripheral blood mononuclear cell (PBMC) methylome data were processed with ChAMP package in R. Methods for Transcription Factor (TF)-gene network analyses used ENCODE data and Taiji. New Zealand Maori whole blood DNA provided an independent gout validation cohort (n=13, n=16 controls). RESULTS: Differentially methylated loci clearly separated gout from controls by hierarchical clustering and principal component analyses. Multiple differentially methylated gout risk genes included IL23R, which mediates granuloma formation and cell invasion. Differential methylome pathway enrichment was detected for B and T cell receptor signaling, Th17 differentiation and IL-17 signaling, and convergent longevity regulation, circadian entrainment, and AMP-activated kinase signaling that impact inflammation via IGF1R, PI3K-AKT, NF-κB, mTOR signaling, and autophagy. Cohorts overlapped for 37 of the 70 (52.9%) TFs with hypomethylated sequence enrichment, and 30 of 38 (78.9%) enriched KEGG pathways identified via TFs. Shared differentially methylated gout TF-gene networks, including NF-κB activation-limiting TFs MEF2C and NFATC2, identified osteoclast differentiation as the most strongly weighted differentially methylated pathway that overlapped in both gout cohorts. CONCLUSION: Seminal findings of differential DNA methylation of networked signaling, transcriptional, innate and adaptive immunity, and osteoclastogenesis genes and pathways suggest novel therapy targets for flares, tophi, chronic synovitis, and bone erosion in gout.

2.
Nat Immunol ; 20(7): 928-942, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31061532

RESUMO

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.


Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Perfilação da Expressão Gênica , Membrana Sinovial/metabolismo , Transcriptoma , Artrite Reumatoide/patologia , Autoimunidade/genética , Biomarcadores , Biologia Computacional/métodos , Estudos Transversais , Citocinas/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Membrana Sinovial/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fluxo de Trabalho
3.
Inflamm Res ; 68(4): 261-274, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30739130

RESUMO

OBJECTIVE/DESIGN: In a double-blind, placebo-controlled, multiple-dose study, we assessed the molecular mechanism of action of the selective histamine-4-receptor antagonist toreforant. PATIENTS/TREATMENT: Patients with active rheumatoid arthritis (RA) despite methotrexate were randomized (3:1) to toreforant 30 mg/day (weeks 0-52) or placebo (weeks 0-12) followed by toreforant 30 mg/day (weeks 12-52). METHODS: Primary biomarker analyses comprised 39 different proteins/mRNA transcripts measured in synovial biopsy (n = 39) and/or time-matched serum (n = 15) samples collected at baseline and week 6. Clinical response was assessed using C-reactive protein-based 28-joint disease activity scores. Data were summarized using descriptive statistics. RESULTS: Among 21 randomized, treated patients (toreforant-16, placebo-5), 18 (toreforant-13, placebo-5) completed the 12-week double-blind period (none completed open-label treatment) prior to the early study termination. Biomarker profiling indicated potential modest effects of toreforant on gene expression of histamine-1-receptor, tumor necrosis factor-alpha, and interleukin-8 in synovium. Potential trends between biomarkers and clinical response were observed with synovial monocyte chemoattractant protein-4 and phosphorylated extracellular-signal-regulated kinases and serum matrix metalloproteinase-3. Minimal synovial gene expression of interleukins-17A and 17F was detected. CONCLUSIONS: While clear biomarker signals associated with toreforant pharmacology in RA patients were not identified, modest associations between biomarkers and clinical response were noted. Synovial expression of interleukins-17A/17F was minimal. Limited sample size warrants cautious interpretation.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Benzimidazóis/uso terapêutico , Antagonistas dos Receptores Histamínicos/uso terapêutico , Piperidinas/uso terapêutico , Pirimidinas/uso terapêutico , Receptores Histamínicos H4/antagonistas & inibidores , Adolescente , Adulto , Idoso , Antirreumáticos/farmacologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Benzimidazóis/farmacologia , Método Duplo-Cego , Feminino , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Interleucina-17/imunologia , Masculino , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Piperidinas/farmacologia , Pirimidinas/farmacologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Resultado do Tratamento , Adulto Jovem
4.
Ann Rheum Dis ; 78(5): 600-609, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30808624

RESUMO

OBJECTIVE: We aimed to understand the role of the tyrosine phosphatase PTPN14-which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol-in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: Gene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis. RESULTS: RA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFß-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP-but not of a non-YAP-interacting PTPN14 mutant-enhanced SMAD reporter activity. YAP promoted TGFß-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity. CONCLUSION: In RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Tirosina Fosfatases não Receptoras/fisiologia , Transdução de Sinais/fisiologia , Sinoviócitos/metabolismo , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Artrite Reumatoide/metabolismo , Proteínas de Ciclo Celular/fisiologia , Humanos , Camundongos
5.
Arthritis Res Ther ; 20(1): 139, 2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-29996944

RESUMO

BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 µg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.


Assuntos
Artrite Reumatoide/patologia , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Membrana Sinovial/patologia , Criopreservação , Humanos
6.
Nat Commun ; 9(1): 1921, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29765031

RESUMO

Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with "Huntington's Disease Signaling" identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.


Assuntos
Artrite Reumatoide/genética , Epigênese Genética , Fibroblastos/metabolismo , Sinoviócitos/metabolismo , Adulto , Idoso , Artrite Reumatoide/metabolismo , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Feminino , Código das Histonas , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Metilação , Pessoa de Meia-Idade , Regiões Promotoras Genéticas
7.
Biochem Pharmacol ; 151: 282-290, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29408488

RESUMO

Despite improved therapy, rheumatoid arthritis (RA) remains an unmet medical need. Previous efforts to validate therapeutic targets in the mitogen-activated protein kinase (MAPK) family have had minimal success. Therefore, we evaluated the potential for targeting an upstream MAPK, namely apoptosis signal-regulating kinase 1 (ASK1), as an alternative approach. ASK1 protein and gene expression were observed in RA and osteoarthritis (OA) synovium as determined by immunohistochemistry (IHC) and qPCR, respectively, particularly in the synovial intimal lining. For RA, but not OA synovium, ASK1 correlated with IL-1ß and TNF gene expression. ASK1 was also expressed by cultured fibroblast-like synoviocytes (FLS), with significantly higher levels in RA compared with OA cells. IL-1ß and TNF stimulation significantly increased ASK1 expression in a time-and concentration-dependent manner in cultured FLS. ASK1 promoter activity was significantly increased by IL-1ß and TNF and was dependent on an upstream RelA binding motif. A selective small molecule ASK1 inhibitor reduced RA FLS invasion, migration and proliferation in vitro and decreased arthritis severity in the rat collagen-induced arthritis (CIA) model. In summary, our findings demonstrate that ASK1 modulates signaling pathways relevant to RA in vitro and in vivo. It is induced by inflammatory cytokines through the activation of NF-κB, which could provide some site- and event specificity. Thus, inhibitors of the upstream MAPK ASK1 could be a novel approach to treating inflammatory arthritis.


Assuntos
Artrite Reumatoide/enzimologia , MAP Quinase Quinase Quinase 5/metabolismo , Osteoartrite/enzimologia , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Células Cultivadas , Citocinas/imunologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Regulação da Expressão Gênica , Humanos , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase Quinase 5/genética , Terapia de Alvo Molecular , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , Ratos Endogâmicos Lew , Transdução de Sinais , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/metabolismo , Sinoviócitos/patologia
10.
J Immunol ; 199(7): 2316-2322, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28807995

RESUMO

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) display unique aggressive behavior, invading the articular cartilage and promoting inflammation. Using an integrative analysis of RA risk alleles, the transcriptome and methylome in RA FLS, we recently identified the limb bud and heart development (LBH) gene as a key dysregulated gene in RA and other autoimmune diseases. Although some evidence suggests that LBH could modulate the cell cycle, the precise mechanism is unknown and its impact on inflammation in vivo has not been defined. Our cell cycle analysis studies show that LBH deficiency in FLS leads to S-phase arrest and failure to progress through the cell cycle. LBH-deficient FLS had increased DNA damage and reduced expression of the catalytic subunit of DNA polymerase α. Decreased DNA polymerase α was followed by checkpoint arrest due to phosphorylation of checkpoint kinase 1. Because DNA fragments can increase arthritis severity in preclinical models, we then explored the effect of LBH deficiency in the K/BxN serum transfer model. Lbh knockout exacerbated disease severity, which is associated with elevated levels of IL-1ß and checkpoint kinase 1 phosphorylation. These studies indicate that LBH deficiency induces S-phase arrest that, in turn, exacerbates inflammation. Because LBH gene variants are associated with type I diabetes mellitus, systemic lupus erythematosus, RA, and celiac disease, these results suggest a general mechanism that could contribute to immune-mediated diseases.


Assuntos
Artrite Reumatoide/genética , Ciclo Celular/genética , Proteínas Nucleares/genética , Sinoviócitos/imunologia , Animais , Artrite Experimental , Artrite Reumatoide/imunologia , Artrite Reumatoide/fisiopatologia , Proteínas de Ciclo Celular , Células Cultivadas , Quinase 1 do Ponto de Checagem/genética , Dano ao DNA , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Regulação da Expressão Gênica , Genes cdc , Humanos , Interleucina-1beta/biossíntese , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Fosforilação , Transdução de Sinais
11.
Nat Rev Rheumatol ; 13(8): 463-475, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28701760

RESUMO

The synovium is the major target tissue of inflammatory arthritides such as rheumatoid arthritis. The study of synovial tissue has advanced considerably throughout the past few decades from arthroplasty and blind needle biopsy to the use of arthroscopic and ultrasonographic technologies that enable easier visualization and improve the reliability of synovial biopsies. Rapid progress has been made in using synovial tissue to study disease pathogenesis, to stratify patients, to discover biomarkers and novel targets, and to validate therapies, and this progress has been facilitated by increasingly diverse and sophisticated analytical and technological approaches. In this Review, we describe these approaches, and summarize how their use in synovial tissue research has improved our understanding of rheumatoid arthritis and identified candidate biomarkers that could be used in disease diagnosis and stratification, as well as in predicting disease course and treatment response.


Assuntos
Artrite Reumatoide/diagnóstico , Membrana Sinovial/patologia , Artrite Reumatoide/metabolismo , Biomarcadores/metabolismo , Pesquisa Biomédica , Humanos , Líquido Sinovial/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo
12.
JCI Insight ; 1(7)2016 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-27275015

RESUMO

The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) compared with osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here, we explored the molecular basis for PTPN11 overexpression in RA FLS and the role of SHP-2 in RA pathogenesis. Using computational methods, we identified a putative enhancer in PTPN11 intron 1, which contained a glucocorticoid receptor- binding (GR-binding) motif. This region displayed enhancer function in RA FLS and contained 2 hypermethylation sites in RA compared with OA FLS. RA FLS stimulation with the glucocorticoid dexamethasone induced GR binding to the enhancer and PTPN11 expression. Glucocorticoid responsiveness of PTPN11 was significantly higher in RA FLS than OA FLS and required the differentially methylated CpGs for full enhancer function. SHP-2 expression was enriched in the RA synovial lining, and heterozygous Ptpn11 deletion in radioresistant or innate immune cells attenuated K/BxN serum transfer arthritis in mice. Treatment with SHP-2 inhibitor 11a-1 reduced RA FLS migration and responsiveness to TNF and IL-1ß stimulation and reduced arthritis severity in mice. Our findings demonstrate how abnormal epigenetic regulation of a pathogenic gene determines FLS behavior and demonstrate that targeting SHP-2 or the SHP-2 pathway could be a therapeutic strategy for RA.

13.
Nat Commun ; 7: 11849, 2016 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-27282753

RESUMO

Stratifying patients on the basis of molecular signatures could facilitate development of therapeutics that target pathways specific to a particular disease or tissue location. Previous studies suggest that pathogenesis of rheumatoid arthritis (RA) is similar in all affected joints. Here we show that distinct DNA methylation and transcriptome signatures not only discriminate RA fibroblast-like synoviocytes (FLS) from osteoarthritis FLS, but also distinguish RA FLS isolated from knees and hips. Using genome-wide methods, we show differences between RA knee and hip FLS in the methylation of genes encoding biological pathways, such as IL-6 signalling via JAK-STAT pathway. Furthermore, differentially expressed genes are identified between knee and hip FLS using RNA-sequencing. Double-evidenced genes that are both differentially methylated and expressed include multiple HOX genes. Joint-specific DNA signatures suggest that RA disease mechanisms might vary from joint to joint, thus potentially explaining some of the diversity of drug responses in RA patients.


Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Metilação de DNA/genética , Perfilação da Expressão Gênica , Articulações/metabolismo , Articulações/patologia , Transcriptoma/genética , Análise por Conglomerados , Bases de Dados como Assunto , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Quadril/patologia , Humanos , Interleucina-6/metabolismo , Joelho/patologia , Pessoa de Meia-Idade , Especificidade de Órgãos/genética , Osteoartrite/genética , Osteoartrite/patologia , Análise de Componente Principal , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Sinoviócitos/metabolismo , Sinoviócitos/patologia
14.
Arthritis Rheumatol ; 68(11): 2637-2645, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27159840

RESUMO

OBJECTIVE: To identify nonobvious therapeutic targets for rheumatoid arthritis (RA), we performed an integrative analysis incorporating multiple "omics" data and the Encyclopedia of DNA Elements (ENCODE) database for potential regulatory regions. This analysis identified the limb bud and heart development (LBH) gene, which has risk alleles associated with RA/celiac disease and lupus, and can regulate cell proliferation in RA. We identified a novel LBH transcription enhancer with an RA risk allele (rs906868 G [Ref]/T) 6 kb upstream of the LBH gene with a differentially methylated locus. The confluence of 3 regulatory elements, rs906868, an RA differentially methylated locus, and a putative enhancer, led us to investigate their effects on LBH regulation in fibroblast-like synoviocytes (FLS). METHODS: We cloned the 1.4-kb putative enhancer with either the rs906868 Ref allele or single-nucleotide polymorphism (SNP) variant into reporter constructs. The constructs were methylated in vitro and transfected into cultured FLS by nucleofection. RESULTS: We found that both variants increased transcription, thereby confirming the region's enhancer function. Unexpectedly, the transcriptional activity of the Ref risk allele was significantly lower than that of the SNP variant and is consistent with low LBH levels as a risk factor for aggressive FLS behavior. Using RA FLS lines with a homozygous Ref or SNP allele, we confirmed that homozygous Ref lines expressed lower LBH messenger RNA levels than did the SNP lines. Methylation significantly reduced enhancer activity for both alleles, indicating that enhancer function is dependent on its methylation status. CONCLUSION: This study shows how the interplay between genetics and epigenetics can affect expression of LBH in RA.


Assuntos
Artrite Reumatoide/genética , Metilação de DNA/genética , RNA Mensageiro/metabolismo , Sinoviócitos/metabolismo , Transativadores/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Epigênese Genética , Homozigoto , Humanos , Isoantígenos , Mutação , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Proteínas de Plasma Seminal , Fatores de Transcrição
15.
Arthritis Rheumatol ; 68(2): 359-69, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26414708

RESUMO

OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis. METHODS: Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTPα-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation. RESULTS: RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y(527) and decreased phosphorylation of FAK on stimulatory Y(397) . Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTPα. RPTPα-KO mice were protected from arthritis development, which was due to radioresistant cells. CONCLUSION: By regulating the phosphorylation of Src and FAK, RPTPα mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA.


Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Quinases da Família src/metabolismo , Animais , Articulação do Tornozelo , Apoptose/efeitos dos fármacos , Apoptose/genética , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Western Blotting , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Interleucina-1/farmacologia , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Reação em Cadeia da Polimerase , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Membrana Sinovial/citologia , Fator de Necrose Tumoral alfa/farmacologia , Quinases da Família src/efeitos dos fármacos
16.
Ann Rheum Dis ; 75(1): 295-302, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25378349

RESUMO

OBJECTIVE: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) ß-target gene, PTPRK, promotes RA FLS invasiveness. METHODS: Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. RESULTS: PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. CONCLUSIONS: By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFß in RA FLS.


Assuntos
Artrite Reumatoide/patologia , Fibroblastos/patologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/fisiologia , Membrana Sinovial/patologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Artrite Reumatoide/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibroblastos/transplante , Regulação Enzimológica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos Nus , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , RNA Mensageiro/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Membrana Sinovial/metabolismo , Membrana Sinovial/transplante , Regulação para Cima
18.
Sci Transl Med ; 7(288): 288ra76, 2015 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-25995222

RESUMO

Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.


Assuntos
Antirreumáticos/farmacologia , Artrite Reumatoide/prevenção & controle , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Heparina/análogos & derivados , Proteoglicanas/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/antagonistas & inibidores , Membrana Sinovial/efeitos dos fármacos , Animais , Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Células HEK293 , Heparina/metabolismo , Humanos , Camundongos Knockout , Terapia de Alvo Molecular , Fosforilação , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/deficiência , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Sindecana-4/genética , Sindecana-4/metabolismo , Membrana Sinovial/enzimologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Fatores de Tempo , Transfecção
19.
PLoS One ; 10(4): e0124254, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25901943

RESUMO

Identifying novel therapeutic targets for the treatment of disease is challenging. To this end, we developed a genome-wide approach of candidate gene prioritization. We independently collocated sets of genes that were implicated in rheumatoid arthritis (RA) pathogenicity through three genome-wide assays: (i) genome-wide association studies (GWAS), (ii) differentially expression in RA fibroblast-like synoviocytes (FLS), and (iii) differentially methylation in RA FLS. Integrated analysis of these complementary data sets identified a significant enrichment of multi-evidence genes (MEGs) within pathways relating to RA pathogenicity. One MEG is Engulfment and Cell Motility Protein-1 (ELMO1), a gene not previously considered as a therapeutic target in RA FLS. We demonstrated in RA FLS that ELMO1 is: (i) expressed, (ii) promotes cell migration and invasion, and (iii) regulates Rac1 activity. Thus, we created links between ELMO1 and RA pathogenicity, which in turn validates ELMO1 as a potential RA therapeutic target. This study illustrated the power of MEG-based approaches for therapeutic target identification.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Artrite Reumatoide/genética , Fibroblastos/metabolismo , Membrana Sinovial/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Movimento Celular , Proliferação de Células , Metilação de DNA , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Humanos , Proteômica/métodos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Membrana Sinovial/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo
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