KCa1.1 potassium channels regulate key proinflammatory and invasive properties of fibroblast-like synoviocytes in rheumatoid arthritis.
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ABSTRACT:
SUBMITTER: Hu X
PROVIDER: S-EPMC3281680 | biostudies-literature | 2012 Feb
REPOSITORIES: biostudies-literature
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KCa1.1 potassium channels regulate key proinflammatory and invasive properties of fibroblast-like synoviocytes in rheumatoid arthritis.
Hu Xueyou X Laragione Teresina T Sun Liang L Koshy Shyny S Jones Karlie R KR Ismailov Iskander I II Yotnda Patricia P Horrigan Frank T FT Gulko Pércio S PS Beeton Christine C
The Journal of biological chemistry 20111110 6
Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA). Potassium channels have regulatory roles in many cell functions. We have identified the calcium- and voltage-gated KCa1.1 channel (BK, Maxi-K, Slo1, KCNMA1) as the major potassium channel expressed at the plasma membrane of FLS isolated from patients with RA (RA-FLS). We further show that blocking this channel perturbs the calcium homeostasis of the cells and inhibits the proliferation, prod ...[more]
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Project description:BackgroundFibroblast-like synoviocytes (FLS) and CCR7- effector memory T (TEM) cells are two of the major cell types implicated in the progression of rheumatoid arthritis (RA). In particular, FLS become highly invasive, whereas TEM cells proliferate and secrete proinflammatory cytokines, during RA. FLS and T cells may also interact and influence each other's phenotypes. Inhibition of the pathogenic phenotypes of both FLS and TEM cells can be accomplished by selectively blocking the predominant potassium channels that they upregulate during RA: KCa1.1 (BK, Slo1, MaxiK, KCNMA1) upregulated by FLS and Kv1.3 (KCNA3) upregulated by activated TEM cells. In this study, we investigated the roles of KCa1.1 and Kv1.3 in regulating the interactions between FLS and TEM cells and determined if combination therapies of KCa1.1- and Kv1.3-selective blockers are more efficacious than monotherapies in ameliorating disease in rat models of RA.MethodsWe used in vitro functional assays to assess the effects of selective KCa1.1 and Kv1.3 channel inhibitors on the interactions of FLS isolated from rats with collagen-induced arthritis (CIA) with syngeneic TEM cells. We also used flow cytometric analyses to determine the effects of KCa1.1 blockers on the expression of proteins used for antigen presentation on CIA-FLS. Finally, we used the CIA and pristane-induced arthritis models to determine the efficacy of combinatorial therapies of KCa1.1 and Kv1.3 blockers in reducing disease severity compared with monotherapies.ResultsWe show that the interactions of FLS from rats with CIA and of rat TEM cells are regulated by KCa1.1 and Kv1.3. Inhibiting KCa1.1 on FLS reduces the ability of FLS to stimulate TEM cell proliferation and migration, and inhibiting Kv1.3 on TEM cells reduces TEM cells' ability to enhance FLS expression of KCa1.1 and major histocompatibility complex class II protein, as well as stimulates their invasion. Furthermore, we show that combination therapies of selective KCa1.1 and Kv1.3 blockers are more efficacious than monotherapies at reducing signs of disease in two rat models of RA.ConclusionsOur results demonstrate the importance of KCa1.1 and Kv1.3 in regulating FLS and TEM cells during RA, as well as the value of combined therapies targeting both of these cell types to treat RA.
| S-EPMC6322314 | biostudies-literature
Project description:Large-conductance calcium-activated potassium channel (KCa1.1; BK, Slo1, MaxiK, KCNMA1) is the predominant potassium channel expressed at the plasma membrane of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) isolated from the synovium of patients with RA. It is a critical regulator of RA-FLS migration and invasion and therefore represents an attractive target for the therapy of RA. However, the molecular mechanisms by which KCa1.1 regulates RA-FLS invasiveness have remained largely unknown. Here, we demonstrate that KCa1.1 regulates RA-FLS adhesion through controlling the plasma membrane expression and activation of β1 integrins, but not α4, α5, or α6 integrins. Blocking KCa1.1 disturbs calcium homeostasis, leading to the sustained phosphorylation of Akt and the recruitment of talin to β1 integrins. Interestingly, the pore-forming α subunit of KCa1.1 coimmunoprecipitates with β1 integrins, suggesting that this physical association underlies the functional interaction between these molecules. Together, these data outline a new signaling mechanism by which KCa1.1 regulates β1-integrin function and therefore invasiveness of RA-FLSs.-Tanner, M. R., Pennington, M. W., Laragione, T., Gulko, P. S., Beeton, C. KCa1.1 channels regulate β1-integrin function and cell adhesion in rheumatoid arthritis fibroblast-like synoviocytes.
| S-EPMC5503717 | biostudies-literature
Project description:Rheumatoid arthritis (RA) is a worldwide chronic autoimmune inflammatory disease which is affecting approximately 1% of the total population. It is characterized by abnormal proliferation of fibroblast-like synoviocytes (FLS) and increased production of proinflammatory cytokines. In the current study, we were aiming to investigate the role of ubiquitin-specific protease 5 (USP5) in the inflammatory process in RA-FLS. Expression of USP5 was found upregulated in RA-FLS compared with that in osteoarthritis- (OA-) FLS, and IL-1? stimulation increased USP5 expression in a time-dependent manner. Furthermore, we found that USP5 overexpression significantly aggravated proinflammatory cytokine production and related nuclear factor ?B (NF-?B) signaling activation. Consistently, silencing of USP5 decreased the release of cytokines and inhibited the activation of NF-?B. In addition, USP5 was found to interact with tumor necrosis factor receptor-associated factor 6 (TRAF6) and remove its K48-linked polyubiquitination chains therefore stabilizing TRAF6. Our data showed that a USP5-positive cell regulates inflammatory processes in RA-FLS and suggested USP5 as a potential target for RA treatment.
| S-EPMC7085372 | biostudies-literature
Project description:Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA-FLS) contribute to joint inflammation and damage characteristic of the disease. RA-FLS express KCa1.1 (BK, Slo1, MaxiK, KCNMA1) as their major plasma membrane potassium channel. Blocking KCa1.1 reduces the invasive phenotype of RA-FLS and attenuates disease severity in animal models of RA. This channel has therefore emerged as a promising therapeutic target in RA. However, the pore-forming ? subunit of KCa1.1 is widely distributed in the body, and blocking it induces severe side effects, thus limiting its value as a therapeutic target. On the other hand, KCa1.1 channels can also contain different accessory subunits with restricted tissue distribution that regulate channel kinetics and pharmacology. Identification of the regulatory subunits of KCa1.1 expressed by RA-FLS may therefore provide the opportunity for generating a selective target for RA treatment.Highly invasive RA-FLS were isolated from patients with RA, and FLS from patients with osteoarthritis (OA) were used as minimally invasive controls. The ? subunit expression by FLS was assessed by quantitative reverse transcription polymerase chain reactions, Western blotting, and patch-clamp electrophysiology combined with pharmacological agents. FLS were sorted by flow cytometry on the basis of their CD44 expression level for comparison of their invasiveness and with their expression of KCa1.1 ? and ? subunits. ?1 and ?3 subunit expression was reduced with small interfering RNA (siRNA) to assess their specific role in KCa1.1? expression and function and in FLS invasiveness.We identified functional ?1 and ?3b regulatory subunits in RA-FLS. KCa1.1 ?3b subunits were expressed by 70 % of the cells and were associated with highly invasive CD44(high) RA-FLS, whereas minimally invasive CD44(low) RA-FLS and OA-FLS expressed either ?1 subunit. Furthermore, we found that silencing the ?3 but not the ?1 subunit with siRNA reduced KCa1.1 channel density at the plasma membrane of RA-FLS and inhibited RA-FLS invasiveness.Our findings suggest the KCa1.1 channel composed of ? and ?3b subunits as an attractive target for the therapy of RA.
| S-EPMC4863321 | biostudies-literature
Project description: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.
| S-EPMC5953939 | biostudies-literature
Project description:Despite advances in the treatment of rheumatoid arthritis (RA), currently approved medications can have significant side effects due to their direct immunosuppressive activities. Additionally, current therapies do not address residual synovial inflammation. In this study, we evaluated the role of integrin ?9 and its ligand, tenascin-C (Tn-C), on the proliferative and inflammatory response of fibroblast-like synoviocytes (FLSs) from RA patients grown in three-dimensional (3D)-micromass culture. FLSs from osteoarthritis patients, when grown in the 3D-culture system, formed self-directed lining-like structures, whereas FLSs from RA tissues (RA-FLSs) developed an abnormal structure of condensed cellular accumulation reflective of the pathogenic features of RA synovial tissues. Additionally, RA-FLSs grown in 3D culture showed autonomous production of proinflammatory mediators. Predominant expression of ?9 and Tn-C was observed in the condensed lining, and knockdown of these molecules abrogated the abnormal lining-like structure formation and suppressed the spontaneous expression of matrix metalloproteinases, IL-6, TNFSF11/RANKL, and cadherin-11. Disruption of ?9 also inhibited expression of Tn-C, suggesting existence of a positive feedback loop in which the engagement of ?9 with Tn-C self-amplifies its own signaling and promotes progression of synovial hyperplasia. Depletion of ?9 also suppressed the platelet-derived growth factor-induced hyperplastic response of RA-FLSs and blunted the TNF-?-induced expression of matrix metalloproteinases and IL-6. Finally, ?9-blocking Ab also suppressed the formation of the condensed cellular lining by RA-FLSs in 3D cultures in a concentration-related manner. This study demonstrates the central role of ?9 in pathogenic behaviors of RA-FLSs and highlights the potential of ?9-blocking agents as a nonimmunosuppressive treatment for RA-associated synovitis.
| S-EPMC5672818 | biostudies-other
Project description:ObjectiveSince previous studies indicate that metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), we undertook this study to determine if changes in the genome-wide chromatin and DNA states in genes associated with nutrient transporters could help to identify activated metabolic pathways in RA FLS.MethodsData from a previous comprehensive epigenomic study in FLS were analyzed to identify differences in genome-wide states and gene transcription between RA and osteoarthritis. We utilized the single nearest genes to regions of interest for pathway analyses. Homer promoter analysis was used to identify enriched motifs for transcription factors. The role of solute carrier transporters and glutamine metabolism dependence in RA FLS was determined by small interfacing RNA knockdown, functional assays, and incubation with CB-839, a glutaminase inhibitor. We performed 1 H nuclear magnetic resonance to quantify metabolites.ResultsThe unbiased pathway analysis demonstrated that solute carrier-mediated transmembrane transport was one pathway associated with differences in at least 4 genome-wide states or gene transcription. Thirty-four transporters of amino acids and other nutrients were associated with a change in at least 4 epigenetic marks. Functional assays revealed that solute carrier family 4 member 4 (SLC4A4) was critical for invasion, and glutamine was sufficient as an alternate source of energy to glucose. Experiments with CB-839 demonstrated decreased RA FLS invasion and proliferation. Finally, we found enrichment of motifs for c-Myc in several nutrient transporters.ConclusionOur findings demonstrate that changes in the epigenetic landscape of genes are related to nutrient transporters, and metabolic pathways can be used to identify RA-specific targets, including critical solute carrier transporters, enzymes, and transcription factors, to develop novel therapeutic agents.
| S-EPMC9246826 | biostudies-literature
Project description:ObjectiveUp-regulation of glucose metabolism has been implicated not only in tumor cell growth but also in immune cells upon activation. However, little is known about the metabolite profile in rheumatoid arthritis (RA), particularly in fibroblast-like synoviocytes (FLS). This study was undertaken to evaluate whether changes in glucose metabolism in RA FLS could play a role in inflammation and joint damage.MethodsSynovium and FLS were obtained from patients with RA and patients with osteoarthritis (OA). The rate of glycolysis after stimulation of FLS with lipopolysaccharide and platelet-derived growth factor BB was measured using glycolysis stress test technology. FLS function was evaluated using a glycolysis inhibitor, 2-deoxy-d-glucose (2-DG). After stimulation of the FLS, a migration scratch assay, MTT assay, and enzyme-linked immunosorbent assay were performed to measure the effect of 2-DG on FLS migration, viability of the FLS, and cytokine secretion, respectively. IRDye 800CW 2-DG was used to assess glucose uptake in the arthritic joints and stromal cells of mice after K/BxN mouse serum transfer. The mice were injected daily, intraperitoneally, with 3-bromopyruvate (BrPa; 5 mg/kg) to assess the effect of inhibition of glycolysis in vivo.ResultsCompared to human OA FLS, the balance between glycolysis and oxidative phosphorylation was shifted toward glycolysis in RA FLS. Glucose transporter 1 (GLUT1) messenger RNA (mRNA) expression correlated with baseline functions of the RA FLS. Glucose deprivation or incubation of the FLS with glycolytic inhibitors impaired cytokine secretion and decreased the rate of proliferation and migration of the cells. In a mouse model of inflammatory arthritis, GLUT1 mRNA expression in the synovial lining cells was observed, and increased levels of glucose uptake and glycolytic gene expression were detected in the stromal compartment of the arthritic mouse joints. Inhibition of glycolysis by BrPa, administered in vivo, significantly decreased the severity of arthritis in this mouse model.ConclusionTargeting metabolic pathways is a novel approach to understanding the mechanisms of disease. Inhibition of glycolysis may directly modulate synoviocyte-mediated inflammatory functions and could be an effective treatment strategy for arthritis.
| S-EPMC4963240 | biostudies-literature
Project description:The pathway of Janus tyrosine kinases (JAKs) has a central role in the pathogenesis of Rheumatoid Arthritis (RA) by regulating multiple immune functions and cytokine production. The JAK inhibitor tofacitinib is effective in RA patients not responding to methotrexate or TNF-inhibitors. Since hyperactive autophagy has been associated with impaired apoptosis of RA fibroblast-like synoviocytes (FLS), we aimed to investigate the role of tofacitinib in modulating autophagy and apoptosis in these cells. FLS isolated from RA biopsies were cultured with tofacitinib in presence of autophagy inducer rapamycin and in serum deprivation condition. Levels of autophagy, apoptosis, and citrullinated proteins were analyzed by western blot, flow cytometry, immunocytofluorescence, and Real-Time PCR. Rapamycin induced an increase in RA-FLS autophagy while the levels of autophagy marker LC3-II were reduced after in vitro treatment with tofacitinib. The analysis of autophagic flux by specific fluorescence dye confirmed the reduction of autophagy in RA FLS. The treatment with tofacitinib did not influence apoptosis of RA FLS. Modulation of the autophagic process by tofacitinib did not significantly change citrullination. The results of this study demonstrate that tofacitinib is able to modulate autophagy of FLS contributing to its effectiveness in RA patients.
| S-EPMC8928732 | biostudies-literature
Project description:Fibroblast-like synoviocytes (FLS) were cultured from synovial tissues from 19 RA patients. RNA was isolated using the Fast Track 2.0 kit (Invitrogen, Carlsbad, California)from the FLS cultures. Fluorescent cDNA probes were prepared from 1 ug experimental mRNA by oligo dT-primed polymerization using Superscript II reverse transcriptase in the presence of Cy5 labeled dCTP. A common reference mRNA sample (CRE) that consists of a mixture of mRNAs isolated from 11 different cell-lines, was labeled with Cy3. The Cy5 and Cy3 labeled cDNAs were pooled and hybridized to a 24,000 cDNA microarray. Set of arrays that are part of repeated experiments Biological Replicate Using regression correlation
2006-01-18 | E-GEOD-4061 | biostudies-arrayexpress