Different expression of [béta] subunits of the KCa1.1 channel by invasive and non-invasive human fibroblast-like synoviocytes

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Comprehensive epigenetic landscape of rheumatoid arthritis fibroblast-like synoviocytes.

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

Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes

BackgroundGlucose metabolism, specifically, hexokinase 2 (HK2), has a critical role in rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) phenotype. HK2 localizes not only in the cytosol but also in the mitochondria, where it protects mitochondria against stress. We hypothesize that mitochondria-bound HK2 is a key regulator of RA FLS phenotype.MethodsHK2 localization was evaluated by confocal microscopy after FLS stimulation. RA FLSs were infected with Green fluorescent protein (GFP), full-length (FL)-HK2, or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (Ad). RA FLS was also incubated with methyl jasmonate (MJ; 2.5 mM), tofacitinib (1 µM), or methotrexate (1 µM). RA FLS was tested for migration and invasion and gene expression. Gene associations with HK2 expression were identified by examining single-cell RNA sequencing (scRNA-seq) data from murine models of arthritis. Mice were injected with K/BxN serum and given MJ. Ad-FLHK2 or Ad-HK2ΔN was injected into the knee of wild-type mice.ResultsCobalt chloride (CoCl2) and platelet-derived growth factor (PDGF) stimulation induced HK2 mitochondrial translocation. Overexpression of the HK2 mutant and MJ incubation reversed the invasive and migrative phenotype induced by FL-HK2 after PDGF stimulation, and MJ also decreased the expression of C-X-C Motif Chemokine Ligand 1 (CXCL1) and Collagen Type I Alpha 1 Chain (COL1A1). Of interest, tofacitinib but not methotrexate had an effect on HK2 dissociation from the mitochondria. In murine models, MJ treatment significantly decreased arthritis severity, whereas HK2FL was able to induce synovial hypertrophy as opposed to HK2ΔN.ConclusionOur results suggest that mitochondrial HK2 regulates the aggressive phenotype of RA FLS. New therapeutic approaches to dissociate HK2 from mitochondria offer a safer approach than global glycolysis inhibition

Image_6_KIF1C and new Huntingtin-interacting protein 1 binding proteins regulate rheumatoid arthritis fibroblast-like synoviocytes’ phenotypes.tif

BackgroundHuntingtin-interacting protein-1 (HIP1) is a new arthritis severity gene implicated in the regulation of the invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). These invasive properties of FLS strongly correlate with radiographic and histology damage in patients with RA and rodent models of arthritis. While HIP1 has several intracellular functions, little is known about its binding proteins, and identifying them has the potential to expand our understanding of its role in cell invasion and other disease-contributing phenotypes, and potentially identify new targets for therapy.MethodsFLS cell lines from arthritic DA (highly invasive) and from arthritis-protected congenic rats R6 (minimally invasive), which differ in an amino-acid changing HIP1 SNP, were cultured and lysed, and proteins were immunoprecipitated with an anti-HIP1 antibody. Immunoprecipitates were analyzed by mass spectrometry. Differentially detected (bound) proteins were selected for functional experiments using siRNA knockdown in human RA FLS to examine their effect in cell invasiveness, adhesion, cell migration and proliferation, and immunofluorescence microscopy.ResultsProteins detected included a few known HIP1-binding proteins and several new ones. Forty-five proteins differed in levels detected in the DA versus R6 congenic mass spectrometry analyses. Thirty-two of these proteins were knocked down and studied in vitro, with 10 inducing significant changes in RA FLS phenotypes. Specifically, knockdown of five HIP1-binding protein genes (CHMP4BL1, COPE, KIF1C, YWHAG, and YWHAH) significantly decreased FLS invasiveness. Knockdown of KIF1C also reduced RA FLS migration. The binding of four selected proteins to human HIP1 was confirmed. KIF1C colocalized with lamellipodia, and its knockdown prevented RA FLS from developing an elongated morphology with thick linearized actin fibers or forming polarized lamellipodia, all required for cell mobility and invasion. Unlike HIP1, KIF1C knockdown did not affect Rac1 signaling.ConclusionWe have identified new HIP1-binding proteins and demonstrate that 10 of them regulate key FLS phenotypes. These HIP1-binding proteins have the potential to become new therapeutic targets and help better understand the RA FLS pathogenic behavior. KIF1C knockdown recapitulated the morphologic changes previously seen in the absence of HIP1, but did not affect the same cell signaling pathway, suggesting involvement in the regulation of different processes.</p