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Snail Is a Critical Mediator of Invadosome Formation and Joint Degradation in Arthritis

  • Annie Lauzier
    Affiliations
    Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada
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  • Roxane R. Lavoie
    Affiliations
    Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada
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  • Martine Charbonneau
    Affiliations
    Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada
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  • Béatrice Gouin-Boisvert
    Affiliations
    Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada
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  • Kelly Harper
    Affiliations
    Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada
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  • Claire M. Dubois
    Correspondence
    Address correspondence to Claire M. Dubois, Ph.D., Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12th Ave N, Sherbrooke, QC, Canada J1H 5N4.
    Affiliations
    Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada
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Open ArchivePublished:December 15, 2015DOI:https://doi.org/10.1016/j.ajpath.2015.10.021
      Progressive cartilage destruction, mediated by invasive fibroblast-like synoviocytes, is a central feature in the pathogenesis of rheumatoid arthritis (RA). Members of the Snail family of transcription factors are required for cell migration and invasion, but their role in joint destruction remains unknown. Herein, we demonstrate that Snail is essential for the formation of extracellular matrix–degrading invadosomal structures by synovial cells from collagen-induced arthritis (CIA) rats and RA patients. Mechanistically, Snail induces extracellular matrix degradation in synovial cells by repressing PTEN, resulting in increased phosphorylation of platelet-derived growth factor receptor and activation of the phosphatidylinositol 3-kinase/AKT pathway. Of significance, Snail is overexpressed in synovial cells and tissues of CIA rats and RA patients, whereas knockdown of Snail in CIA joints prevents cartilage invasion and joint damage. Furthermore, Snail expression is associated with an epithelial-mesenchymal transition gene signature characteristic of transglutaminase 2/transforming growth factor-β activation. Transforming growth factor-β and transglutaminase 2 stimulate Snail-dependent invadosome formation in rat and human synoviocytes. Our results identify the Snail-PTEN platelet-derived growth factor receptor/phosphatidylinositol 3-kinase axis as a novel regulator of the prodestructive invadosome-forming phenotype of synovial cells. New therapies for RA target inflammation, and are only partly effective in preventing joint damage. Blocking Snail and/or its associated gene expression program may provide an additional tool to improve the efficacy of treatments to prevent joint destruction.
      Rheumatoid arthritis (RA) is a systemic autoimmune disease that mainly affects the joints, leading to rapid and disabling erosive structural damages in a proportion of patients. The onset of RA causes important morphological changes in the joint lining, including the formation of an aggressive tumor-like synovial tissue called pannus that invades and erodes the cartilage and bone.
      • Meinecke I.
      • Rutkauskaite E.
      • Gay S.
      • Pap T.
      The role of synovial fibroblasts in mediating joint destruction in rheumatoid arthritis.
      Pannus is composed of many cell types, including a major cell population that possesses both epithelial and mesenchymal characteristics and are referred to as fibroblast-like synoviocytes (FLS). FLS of RA patients and animal models of arthritis (A-FLS) have been shown to play a major role in invasive joint destruction through mechanisms that remain to be fully established.
      • Muller-Ladner U.
      • Kriegsmann J.
      • Franklin B.N.
      • Matsumoto S.
      • Geiler T.
      • Gay R.E.
      • Gay S.
      Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice.
      • Firestein G.S.
      Invasive fibroblast-like synoviocytes in rheumatoid arthritis. Passive responders or transformed aggressors?.
      • Firestein G.S.
      • Paine M.M.
      Stromelysin and tissue inhibitor of metalloproteinases gene expression in rheumatoid arthritis synovium.
      Arthritic FLS resemble immature and transformed mesenchymal cells, which are highly invasive in vitro and in vivo. Such property is associated with massive production of inflammatory cytokines and matrix metalloproteinases (MMPs) that sustains inflammation and joint matrix degradation. Unlike normal synoviocytes, FLS from RA patients invade and degrade cartilage when cografted in immunocompromised mice, showing that these cells possess an imprinted invasive phenotype.
      • Muller-Ladner U.
      • Kriegsmann J.
      • Franklin B.N.
      • Matsumoto S.
      • Geiler T.
      • Gay R.E.
      • Gay S.
      Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice.
      • Pap T.
      • Muller-Ladner U.
      • Gay R.E.
      • Gay S.
      Fibroblast biology: role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis.
      Furthermore, the ex vivo invasive capability of FLS correlates with the rate of joint erosion in individual RA patients.
      • Tolboom T.C.
      • van der Helm-Van Mil A.H.
      • Nelissen R.G.
      • Breedveld F.C.
      • Toes R.E.
      • Huizinga T.W.
      Invasiveness of fibroblast-like synoviocytes is an individual patient characteristic associated with the rate of joint destruction in patients with rheumatoid arthritis.
      Because of the predominant role played by FLS in cartilage damage, it has been proposed that an improved understanding of the mechanisms underlying their invasive and prodestructive functions may help identify strategies to stop degeneration and potentially promote regeneration of synovial joints.
      • Bartok B.
      • Firestein G.S.
      Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis.
      Previous work has demonstrated that arthritic FLS degrade the extracellular matrix (ECM) through formation of actin-rich protrusions that resemble both podosomes produced by normal cells and invadopodia produced by tumor cells.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      • Linder S.
      Invadosomes at a glance.
      These degrading devices were called invadopodia-like structures or invadosomes.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      • Linder S.
      Invadosomes at a glance.
      In addition to actin and the actin-binding protein cortactin, FLS invadosomes were shown to contain signaling molecules, such as activated Src and the metalloproteinases MMP-3 and MMP-13, which were all associated with cartilage damage.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      In cell cultures, invadosome production by arthritic FLS can be maintained for many weeks, further suggesting that these cells have an imprinted prodestructive phenotype.
      • Muller-Ladner U.
      • Kriegsmann J.
      • Franklin B.N.
      • Matsumoto S.
      • Geiler T.
      • Gay R.E.
      • Gay S.
      Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice.
      In collagen-induced arthritis (CIA) joints, the invadosomal structures have been detected in synovial cells strategically located at the cartilage-synovial membrane interface, for ECM invasion and degradation.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      Interference with formation of invadosomes in arthritic FLS strongly inhibited ECM degradation in vitro and ex vivo as well as FLS invasion and destruction of the underlying cartilage in an experimental model of arthritis, further linking invadosomes to joint damage.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.
      In subsequent studies, transglutaminase 2 (TG2), an enzyme important for the regulation of epithelial-mesenchymal transition (EMT) and ECM composition/stiffness, was shown to play a key role in invadosome formation by A-FLS.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.
      TG2 activity was associated with its ability to cross-link ECM and to up-regulate the activity and signaling of the EMT inducer, transforming growth factor (TGF)-β. Considering that the invadosome structures produced by activated FLS are functionally important to the joint degradation process, we proposed that further studies on triggers and molecular components involving invadosome biogenesis may provide cues to specifically interfere with the destructive aspect of RA.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      EMT is a transcriptionally orchestrated and multistep developmental program that is reactivated during wound healing, chronic inflammation, and neoplasias.
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      • Huang R.Y.
      • Nieto M.A.
      Epithelial-mesenchymal transitions in development and disease.
      During EMT, cells change their cellular phenotype to one that is more aggressive with enhanced invasiveness and resistance to apoptosis.
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      • Christofori G.
      EMT, the cytoskeleton, and cancer cell invasion.
      Increased invasion has been associated with down-regulation of cell-cell adhesion and up-regulation of matrix metalloproteinases.
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      Transforming growth factor-beta 1 promotes matrix metalloproteinase-9-mediated oral cancer invasion through snail expression.
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      • Taki M.
      • Hosokawa H.
      • Nagayama M.
      Increased invasion and matrix metalloproteinase-2 expression by Snail-induced mesenchymal transition in squamous cell carcinomas.
      These alterations are reminiscent of metastatic processes in tumors, and of cartilage invasion by pannus in arthritis. EMT can be induced by various intrinsic (eg, gene mutations) and extrinsic (eg, growth factors and matrix composition/stiffness) signals.
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      • Christofori G.
      EMT, the cytoskeleton, and cancer cell invasion.
      In RA patients and in the CIA rat model, the synovial membrane or synovial fluid contains increased levels of EMT inducers, including the growth factors TGF-β, fibroblast growth factor, platelet-derived growth factor receptor (PDGFR), and TG2.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.
      • Taketazu F.
      • Kato M.
      • Gobl A.
      • Ichijo H.
      • ten Dijke P.
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      Enhanced expression of transforming growth factor-beta s and transforming growth factor-beta type II receptor in the synovial tissues of patients with rheumatoid arthritis.
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      Active and latent forms of transforming growth factor beta activity in synovial effusions.
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      Involvement of fibroblast growth factor-2 in joint destruction of rheumatoid arthritis patients.
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      • Miller G.G.
      Acidic fibroblast growth factor in synovial cells.
      Engagement of their cognate receptors or up-regulation of TG2 activity leads to activation of intracellular mediators, including Src, which we showed to be activated in arthritic FLS.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
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      • Slack R.
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      • Mueller S.C.
      Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells.
      • Frame M.C.
      Newest findings on the oldest oncogene; how activated src does it.
      All of these inducers/pathways lead to the activation of key regulators of EMT, such as the transcription factors Snail/Slug, Twist, Zeb, and E47 that orchestrate the molecular EMT program. The molecular events include interaction with the regulatory region of epithelial or mesenchymal markers or cooperation with components of the signal transduction cascade.
      • Thiery J.P.
      • Acloque H.
      • Huang R.Y.
      • Nieto M.A.
      Epithelial-mesenchymal transitions in development and disease.
      Besides their role in EMT, members of the Snail family of transcription factors, consisting of Snai1 (Snail), Snai2 (Slug), and the less-characterized Snail3 (Smug), have also been implicated in regulating cell proliferation,
      • Vega S.
      • Morales A.V.
      • Ocana O.H.
      • Valdes F.
      • Fabregat I.
      • Nieto M.A.
      Snail blocks the cell cycle and confers resistance to cell death.
      cell survival,
      • Vega S.
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      • Valdes F.
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      Snail blocks the cell cycle and confers resistance to cell death.
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      • Wade P.A.
      Aberrant expression of the transcription factors snail and slug alters the response to genotoxic stress.
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      • Mayor R.
      A balance between the anti-apoptotic activity of Slug and the apoptotic activity of msx1 is required for the proper development of the neural crest.
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      • Garcia de Herreros A.
      Repression of PTEN phosphatase by Snail1 transcriptional factor during gamma radiation-induced apoptosis.
      and expression of stem cell properties.
      • Kurrey N.K.
      • Jalgaonkar S.P.
      • Joglekar A.V.
      • Ghanate A.D.
      • Chaskar P.D.
      • Doiphode R.Y.
      • Bapat S.A.
      Snail and slug mediate radioresistance and chemoresistance by antagonizing p53-mediated apoptosis and acquiring a stem-like phenotype in ovarian cancer cells.
      • Southall T.D.
      • Brand A.H.
      Neural stem cell transcriptional networks highlight genes essential for nervous system development.
      Moreover, Snail and Slug have been convincingly shown to be associated with a more aggressive and invasive cell phenotype.
      • Yokoyama K.
      • Kamata N.
      • Fujimoto R.
      • Tsutsumi S.
      • Tomonari M.
      • Taki M.
      • Hosokawa H.
      • Nagayama M.
      Increased invasion and matrix metalloproteinase-2 expression by Snail-induced mesenchymal transition in squamous cell carcinomas.
      • Barrallo-Gimeno A.
      • Nieto M.A.
      The Snail genes as inducers of cell movement and survival: implications in development and cancer.
      • Fang R.
      • Zhang G.
      • Guo Q.
      • Ning F.
      • Wang H.
      • Cai S.
      • Du J.
      Nodal promotes aggressive phenotype via Snail-mediated epithelial-mesenchymal transition in murine melanoma.
      • Kim J.J.
      • Yin B.
      • Christudass C.S.
      • Terada N.
      • Rajagopalan K.
      • Fabry B.
      • Lee D.Y.
      • Shiraishi T.
      • Getzenberg R.H.
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      • An S.S.
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      Acquisition of paclitaxel resistance is associated with a more aggressive and invasive phenotype in prostate cancer.
      • Kashyap A.
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      • Galle P.R.
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      • Strand D.
      The human Lgl polarity gene, Hugl-2, induces MET and suppresses Snail tumorigenesis.
      • Liu S.
      • Kumar S.M.
      • Martin J.S.
      • Yang R.
      • Xu X.
      Snail1 mediates hypoxia-induced melanoma progression.
      Recent studies have shown that Snail activity is not confined to transdifferentiating epithelial cells. Activated fibroblasts localized in damaged or cancer tissues, and macrophages in wounded and inflamed tissues, reminiscent of the microenvironment found in arthritic joints, all express increased levels of Snail, leading to cell migration and invasion.
      • Franci C.
      • Takkunen M.
      • Dave N.
      • Alameda F.
      • Gomez S.
      • Rodriguez R.
      • Escriva M.
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      • Baro T.
      • Garrido M.
      • Bonilla F.
      • Virtanen I.
      • Garcia de Herreros A.
      Expression of Snail protein in tumor-stroma interface.
      • Rowe R.G.
      • Li X.Y.
      • Hu Y.
      • Saunders T.L.
      • Virtanen I.
      • Garcia de Herreros A.
      • Becker K.F.
      • Ingvarsen S.
      • Engelholm L.H.
      • Bommer G.T.
      • Fearon E.R.
      • Weiss S.J.
      Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs.
      • Hotz B.
      • Visekruna A.
      • Buhr H.J.
      • Hotz H.G.
      Beyond epithelial to mesenchymal transition: a novel role for the transcription factor Snail in inflammation and wound healing.
      In addition, a recent study indicates that Snail is overexpressed in synovium and cell lines from RA patients.
      • Chen S.Y.
      • Shiau A.L.
      • Li Y.T.
      • Lin C.C.
      • Jou I.M.
      • Liu M.F.
      • Wu C.L.
      • Wang C.R.
      Transcription factor snail regulates tumor necrosis factor alpha-mediated synovial fibroblast activation in the rheumatoid joint.
      In light of the above findings, we hypothesized that fibroblast-like synovial cells use components or pathways of the EMT program to promote invadosome formation, leading to arthritis progression toward a more destructive phenotype. Data shown herein provide evidence that the Snail transcriptional regulator is essential for ECM degradation by human and rat synovial cells and for cartilage degradation in a CIA model. We also show that Snail mediates TG2/TGF-β–induced invadosome formation by A-FLS through down-regulation of the tumor-suppressor phosphatase and tensin homolog (PTEN) that leads to enhanced PDGFR phosphorylation and signaling. This dependency on Snail or its associated gene expression program for synovial cell-driven joint damage may be exploited to improve diagnosis and treatment of RA patients.

      Materials and Methods

       Reagents

      LY364947 (Tocris Bioscience, Bristol, UK) and LY294002 (Cayman Chemical Co, Ann Arbor, MI) were prepared as stock solutions in dimethyl sulfoxide. AKT inhibitor XI (EMD Millipore, Billerica, MA), Bpv(pic) (Santa Cruz Biotechnology Inc., Dallas, TX), and PDGFR inhibitor V (EMD Millipore) were dissolved in water. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. Antibodies used were directed against cortactin (4F11; EMD Millipore), TKS5 (EMD Millipore), TG2 (TG100; Lab Vision/NeoMarkers, Fremont, CA), tubulin (Sigma-Aldrich), Snail, PTEN, phospho-PDGFRα (Tyr849)/PDGFRβ (Tyr857), total PDGFRα/β (phospho-S473), and total AKT (Cell Signaling Technology Inc., Danvers, MA), and the type II collagen cleavage product Col 2 3/4Cshort (Ibex Pharmaceuticals Inc., Montreal, QC, Canada).

       Induction and Clinical Evaluation of Arthritis

      Female Lewis/SsNHsd rats (100 to 124 g) were purchased from Charles River Laboratories (Sherbrooke, QC, Canada). Arthritis was induced by intradermal injection of type II collagen at the base of the tail, as previously described.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      In the case of in vivo Snail (Snai1) inhibition, rats received one intra-articular injection of 3 × 109 U of a lentivirus harboring Snail-shRNA or control (scrambled shRNA), 10 days after CIA induction. Animals were sacrificed on day 32.

       Histology

      Tissue sections from human joints or the left hind knee joints of rats were processed immediately after excision, after a standardized paraffin embedding protocol. Tissue sections were rehydrated and treated with 1% trypsin (rat tissues) or 0.01 mol/L citrate, pH 6.0 (human tissues), and immunohistochemical staining was performed according to the standard avidin-biotin immunoperoxidase complex technique using primary antibodies (1:50 dilution in 2% bovine serum albumin) or isotype-matched negative controls and diaminobenzidene (Vector Laboratories Inc., Burlingame, CA) as substrate. For quantitative immunohistochemistry, photomicrographs of three representative areas of the synovial membrane for each joint section were captured using an Axioskop 2 phase-contrast/epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY) equipped with a 10× objective and a Retiga SRV cooled color digital camera (Qimaging, Surrey, BC, Canada). Images were analyzed as previously described.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.

       Modified Mankin Grading

      Sections were stained using hematoxylin and eosin and safranin O/fast green staining protocols to allow visualization of cartilage structure. Pathology scores were evaluated for each tissue section by three observers (A.L., M.C., and C.M.D.) in a blind way using a modified Mankin scoring system, as previously described.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.
      • Pine P.R.
      • Chang B.
      • Schoettler N.
      • Banquerigo M.L.
      • Wang S.
      • Lau A.
      • Zhao F.
      • Grossbard E.B.
      • Payan D.G.
      • Brahn E.
      Inflammation and bone erosion are suppressed in models of rheumatoid arthritis following treatment with a novel Syk inhibitor.

       Cell Cultures

      Rat FLS were generated using standard cell isolation and culture procedures.
      • Blanchette F.
      • Day R.
      • Dong W.
      • Laprise M.H.
      • Dubois C.M.
      TGFbeta1 regulates gene expression of its own converting enzyme furin.
      Six human cell lines isolated from patients diagnosed with RA and undergoing arthroplasty (RA-FLS) and four cell lines isolated from control joints with no evidence of disease (C-FLS) were obtained from Asterand (Detroit, MI) and Cell Applications Inc. (San Diego, CA). Cells were cultured in Dulbecco’s modified Eagle’s medium–F12 medium supplemented with 10% fetal bovine serum and 40 μg/mL gentamicin. The cells were used between passages 3 and 8.

       Plasmids and Transfections

      A lentiviral construct driving the expression of green fluorescent protein (GFP)–tagged wt-TG2 was generated by subcloning the TG2 DNA fragment from pcDNA3.1 (generously provided by Dr. Gail V.W. Johnson, University of Rochester, Rochester, NY) using the following primers (5′-GGTGCTAGCGCCACCATGGCCGAGG-3′, forward, and 5′-TATGCTAGCCCGGCGGGGCCAATGA-3′, reverse) and NheI digestion, into the pLenti-V5/GFPtag plasmid. pTK-Snail was obtained from Bob Weinberg (addgene plasmid 36976)
      • Guo W.
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      • Donaher J.L.
      • Shibue T.
      • Tischler V.
      • Reinhardt F.
      • Itzkovitz S.
      • Noske A.
      • Zurrer-Hardi U.
      • Bell G.
      • Tam W.L.
      • Mani S.A.
      • van Oudenaarden A.
      • Weinberg R.A.
      Slug and Sox9 cooperatively determine the mammary stem cell state.
      and pGL3-wtPTEN from Joshua Mendell (addgene plasmid 21326).
      • O'Donnell K.A.
      • Wentzel E.A.
      • Zeller K.I.
      • Dang C.V.
      • Mendell J.T.
      c-Myc-regulated microRNAs modulate E2F1 expression.
      GFP-tagged pLenti-Snail was constructed by subcloning the Snail DNA fragment from pTK-Snail using the following primers (5′-TATGGATCCGCCACCATGCCGCGC-3′, forward, and 5′-TATACTAGTGGGCGAGGGCCTCCG-3′, reverse) and BamHI and SpeI digestion, into the pLenti-V5/GFPtag plasmid. GFP-lentiviral shRNA targeting rat TG2 and control (scrambled) shRNA plasmids were from EzBiolab (Carmel, IN). Control and rat/mouse Snail or PTEN targeting shRNA in pGIPZ plasmids was from Thermo Scientific (Montreal, QC, Canada). Viral particles were generated by transient transfection of 293T cells with the ViraPower lentiviral expression system (Invitrogen Thermo Fisher Scientific, Burlington, ON, Canada) or the psPAX2 (packaging plasmid) and pMD2G vectors (envelope plasmid). Experiments were conducted 24 hours after transient transfection using TransIT-LT1 (Mirus Bio LLC, Madison, WI) or 48 hours after lentivirus infection with 5 μg/mL Polybrene (EMD Millipore). pGIPZ transfected cells were selected by treatment with puromycin for 72 hours. pTK-Snail transfected cells were induced with 2 μg/mL doxycycline for 24 hours before use.

       Invadosome Assays

      Coverslips were covered with Alexa Fluor 488– or Oregon-green 488–conjugated gelatin (Thermo Fisher Scientific) at a final concentration of 1%, as previously described.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      Cells (30,000) were seeded onto each coverslip and incubated for 24 hours (rat cells) or 40 hours (human cells). Cells were fixed with 1% paraformaldehyde; nuclei and f-actin were stained with DAPI and Texas Red phalloidin, respectively; and coverslips were mounted in Vectashield (Vector Laboratories, Burlington, ON, Canada). Cells were visualized by fluorescence microscopy. Invadosomes were identified as f-actin–enriched areas of matrix degradation (loss of green fluorescence). Three fields of 100 cells were counted per coverslip. For quantification purposes of the areas of degradation, images of fluorescent matrix were analyzed using Image Pro Plus 6.0 software (MediaCybernetics, Rockville, MD), and degradation areas associated with each cell were calculated in pixels, for 10 to 15 cells per coverslip. To quantitate the number of invadosomes formed by cells, clusters of cortactin/actin were calculated for 10 to 15 cells per slide. In selected experiments, inhibitors or vehicle (control) was added 30 minutes after cell plating. When required, TGF-β or PDGF-BB (Peprotech, Rocky Hill, NJ) was added 30 minutes after addition of the inhibitors. To identify TG2-overexpressing cells, the cells were permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. Cells were washed and blocked with 2% bovine serum albumin for 30 minutes, and transfected cells were examined by immunofluorescence staining using antibodies against TG2 (1:50) for 1 hour, followed by appropriate Alexa-labeled secondary antibody. Invadosomes were counted in the case of TG2-positive cells only.

       Immunofluorescence and Confocal Microscopy

      To analyze invadosome structure formation, synoviocytes were cultured on coverslips for 4 to 6 hours and stained, as described above, using an antibody directed against cortactin (1:75), TKS5 (1:50), and Texas Red–conjugated phalloidin (Invitrogen; 1:200 or 1:50). Confocal images of the basal membrane were acquired using a Fluoview 1000 scanning confocal microscope (Olympus, Richmond Hill, ON, Canada) coupled to an inverted microscope equipped with a 60× oil immersion objective (Olympus). Color channels were scanned sequentially to avoid overlapping signals. For quantification of Snail staining, synoviocytes were cultured on coverslips for 4 to 6 hours and stained, as stated above, using an antibody directed against Snail (1:50) and TO-PRO3 iodide or DAPI (Life Technologies, Carlsbad, CA). Sets of 12 Z-stack images were collected through the thickness of the nuclei, and a merged Z-projection (maximum intensity) of the image volume (102.4 × 102.4 × 12 μm; 12 stacks) was reconstructed using the FluoView 10-ASW version 02.01 software (Olympus). A region of interest was generated around each nuclei, and intensity of Snail staining was calculated as follows: (intensity of fluorescence−background)/surface area (μm2), in a minimum of 25 cells.

       Transwell Invasion Assays

      Invasion assays were performed, as described by Tolboom et al,
      • Tolboom T.C.
      • van der Helm-Van Mil A.H.
      • Nelissen R.G.
      • Breedveld F.C.
      • Toes R.E.
      • Huizinga T.W.
      Invasiveness of fibroblast-like synoviocytes is an individual patient characteristic associated with the rate of joint destruction in patients with rheumatoid arthritis.
      native using 2.0 mg/mL type II collagen–coated transwells (Corning, New York, NY).

       RT-PCR and PCR Array Analysis of Gene Expression

      Cells were seeded at a density of 1 × 109 cells/mL, serum starved for 6 hours, and RNA extracted using the RNeasy mini kit (Qiagen, Toronto, ON, Canada) and in situ DNase treatment, according to the manufacturer's instructions. RNA was reverse transcribed using the QuantiTect Reverse transcription kit (Qiagen). Quantitative PCR was performed using the QuantiTect SYBR Green PCR master mix (Qiagen). Primers were as follows: rat snai1, 5′-GGAGCAGGAGAAAGGCTTC-3′ (forward) and 5′-CTCTGAAGATGCACATCCGAA-3′ (reverse); rat RPLP0, 5′-CCTTGTCTCCAGTCTTTATCAGC-3′ (forward) and 5′-GTCACAGTACCTGCTCAGAAC-3′ (reverse); human snai1, 5′-CCTTCGTCCTTCTCCTCTACTT-3′ (forward) and 5′-TTCGAGGCTGGAGATCCTT-3′ (reverse); human RPLP0, 5′-GATTACACCTTCCCACTTGC-3′ (forward) and 5′-CCAAATCCCATATCCTCGTCCG-3′ (reverse). Analysis of expression of 84 EMT-associated genes was performed on cDNA from three control and three A-FLS cell lines using the rat Epithelial to Mesenchymal Transition RT2 Profiler PCR Array (SABiosciences Qiagen, Toronto, ON, Canada). Results were normalized to expression of Actb, B2m, Hprt1, and Rplp1, using the manufacturer's PCR array data analysis web portal.

       Western Blot Analysis

      Whole cell extracts were prepared by lysis of overnight serum-starved cells (0.5 × 106) in radioimmunoprecipitation assay buffer. Proteins were immunoblotted, as previously described.
      • Charbonneau M.
      • Harper K.
      • Grondin F.
      • Pelmus M.
      • McDonald P.P.
      • Dubois C.M.
      Hypoxia-inducible factor mediates hypoxic and tumor necrosis factor alpha-induced increases in tumor necrosis factor-alpha converting enzyme/ADAM17 expression by synovial cells.
      Band intensities were analyzed using the Quantity One software version 4.6.5 (Bio-Rad Laboratories, Richmond, CA).

       Luciferase Assay

      Cells were transiently transfected with pGL3-PTEN or pGL3-Ctl (Promega, Madison, WI) using 1 mg/mL PEI (Polysciences, Warrington, PA). Cells were serum starved and treated overnight, as indicated later, 24 hours after transfection. Cell lysates were assayed for luciferase activity, as previously described.
      • Blanchette F.
      • Rivard N.
      • Rudd P.
      • Grondin F.
      • Attisano L.
      • Dubois C.M.
      Cross-talk between the p42/p44 MAP kinase and Smad pathways in transforming growth factor beta 1-induced furin gene transactivation.

       Statistical Analysis

      A paired or unpaired t-test or one-way analysis of variance test was used to assess statistical significance. P < 0.05 was considered significant.

       Study Approval

      All experimental procedures involving animals were conducted under protocols (protocol 110-12B) approved by the Ethics Committee on Animal Research of the University of Sherbrooke (Sherbrooke, QC, Canada), in accordance with regulations of the Canadian Council on Animal Care. Human synovial tissue samples were obtained from RA patients who fulfill the 2010 American College of Rheumatology/European League Against Rheumatism criteria
      • Aletaha D.
      • Neogi T.
      • Silman A.J.
      • Funovits J.
      • Felson D.T.
      • Bingham 3rd, C.O.
      • Birnbaum N.S.
      • Burmester G.R.
      • Bykerk V.P.
      • Cohen M.D.
      • Combe B.
      • Costenbader K.H.
      • Dougados M.
      • Emery P.
      • Ferraccioli G.
      • Hazes J.M.
      • Hobbs K.
      • Huizinga T.W.
      • Kavanaugh A.
      • Kay J.
      • Kvien T.K.
      • Laing T.
      • Mease P.
      • Menard H.A.
      • Moreland L.W.
      • Naden R.L.
      • Pincus T.
      • Smolen J.S.
      • Stanislawska-Biernat E.
      • Symmons D.
      • Tak P.P.
      • Upchurch K.S.
      • Vencovsky J.
      • Wolfe F.
      • Hawker G.
      2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative.
      and from patients with osteoarthritis (OA). All patients provided informed consent. The experimental protocol was approved by the Center Hospitalier Universitaire de Sherbrooke Ethics Committee (protocol 07-113).

      Results

       Snail and Snail Inducer TGF-β Are Overexpressed in Synoviocytes of Arthritic Joints

      We used a profiler PCR array consisting of 84 EMT-associated genes to assess the expression levels of EMT gene markers in synoviocytes from control and CIA rats. Comparative analysis indicated that, among the classic transcription factors involved in the EMT process (namely, Snail and Zeb family members as well as Twist1), only Snai1 (encoding Snail) was significantly overexpressed, in arthritis FLS (A-FLS) compared with C-FLS (Figure 1A and Supplemental Table S1). Snail overexpression was associated with up-regulation of a sub-group of genes that comprise the Snail/EMT inducer and growth factor Tgfb1, as well as Col1a2, fn1, Itga5, Timp1, and Il1ra, which are gene targets associated with TGF-β signaling.
      • Ranganathan P.
      • Agrawal A.
      • Bhushan R.
      • Chavalmane A.K.
      • Kalathur R.K.
      • Takahashi T.
      • Kondaiah P.
      Expression profiling of genes regulated by TGF-beta: differential regulation in normal and tumour cells.
      • Muzio M.
      • Sironi M.
      • Polentarutti N.
      • Mantovani A.
      • Colotta F.
      Induction by transforming growth factor-beta 1 of the interleukin-1 receptor antagonist and of its intracellular form in human polymorphonuclear cells.
      • Remst D.F.
      • Blom A.B.
      • Vitters E.L.
      • Bank R.A.
      • van den Berg W.B.
      • Blaney Davidson E.N.
      • van der Kraan P.M.
      Gene expression analysis of osteoarthritis synovium reveals elevation of transforming growth factor-beta responsive genes in osteoarthritis-related fibrosis.
      • Inagaki Y.
      • Truter S.
      • Ramirez F.
      Transforming growth factor-beta stimulates alpha 2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site.
      Validation of the data by PCR and quantitative PCR confirmed elevated Snai1 expression in most (>60%) of A-FLS compared with control cells (Figure 1, B and C). We have previously reported overexpression of TGF-β in A-FLS.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.
      Snail overexpression was maintained up to eight passages of the cells (Supplemental Figure S1), which is consistent with the known capacity of arthritic FLS to maintain their destructive phenotype under in vitro conditions.
      • Muller-Ladner U.
      • Kriegsmann J.
      • Franklin B.N.
      • Matsumoto S.
      • Geiler T.
      • Gay R.E.
      • Gay S.
      Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice.
      These results indicate that arthritic FLS displayed stable overexpression of the EMT component Snail and suggest that such up-regulation is under the control of TGF-β activity.
      Figure thumbnail gr1
      Figure 1Snail is overexpressed in cultured fibroblast-like synoviocytes of rat collagen-induced arthritis (CIA) joints (A-FLS), control joints (C-FLS), and CIA synovial membrane. A: Quantitative RT-PCR (RT-qPCR) array results showing relative changes in the expression of epithelial-mesenchymal transition (EMT)–associated genes in A-FLS compared with C-FLS. The mean level of expression for each gene was arbitrarily set to 1 (indicated by dotted line). The y axis represents fold expression, whereas the x axis corresponds to genes investigated. Data normalization was calculated using the average Ct value of the housekeeping genes Actb, B2m, Hprt1, and Rplp1. B: Total RNA from serum-starved C-FLS and A-FLS was analyzed for Snail by qPCR using Rplp0 as an internal control. Results are expressed as fold expression in A-FLS relative to C-FLS (arbitrarily set at 1). C: Representative levels of Snail1 and Rplp0 PCR amplification in individual cell lines. Data are given as means + SEM (A, analysis of variance, and B, unpaired t-test). n = 3 (A, A-FLS and C-FLS); n = 17 (B, A-FLS and C-FLS). *P < 0.05, ***P < 0.001.

       TG2/TGF-β–Induced Invadosome Formation Is Dependent on Snail

      We have shown that endogenous TGF-β activity, triggered by the cross-linking activity of TG2, was essential for expression of the aggressive invadosome-forming phenotype of arthritis FLS.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.
      On the basis of the fact that TG2 and TGF-β are known inducers of EMT, we determined their implication in Snail activation in A-FLS and whether this activation was necessary for the invadosome-promoting effects of TG2 and TGF-β. We first tested the ability of TG2 to modulate Snail expression in FLS from control rats. Quantification of immunofluorescence signal intensity indicated a 2.8-fold increase in Snail expression in TG2-overexpressing cells compared with control FLS (Figure 2A). Increase in Snail expression was markedly decreased by a selective inhibitor of TβR1 (Figure 2A). Conversely, FLS treated with TGF-β showed a 2.0-fold increase in Snail expression (Figure 2B). Quantitative PCR analysis of TGF-β–stimulated cell populations indicated that increased levels of Snail protein were associated with a 1.8-fold increase in Snail mRNA expression (Supplemental Figure S2). Furthermore, the expression of Snail in FLS from CIA rats returned to the levels of control cells after knockdown of endogenous TG2 or pharmacological inhibition of TGF-β signaling (Figure 2C). The efficiency of TG2 knockdown in FLS was confirmed in a previous study.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.
      These observations, therefore, show that TG2 and TGF-β increase Snail expression in FLS and that the TG2 induction of Snail is partly dependent on TGF-β signaling. Furthermore, the data indicate that the elevated levels of endogenous Snail in arthritic FLS are under the control of the TG2–TGF-β axis.
      Figure thumbnail gr2
      Figure 2Snail expression is induced by transglutaminase 2 (TG2) and transforming growth factor (TGF)-β and is essential for their invadosome-promoting activity. Control fibroblast-like synoviocytes (C-FLS) transfected with TG2 or control vector in the presence or absence of LY364947 (A) or C-FLS treated with 10 ng/mL TGF-β (B) were incubated for 6 hours. Representative confocal images of immunostaining for TG2, Snail (green), and nucleus (DAPI, blue) are shown. Graphs represent nuclear Snail fluorescence intensity of cells relative to C-FLS. C: C-FLS or arthritis (A)-FLS transduced with a LV encoding TG2 or scrambled [control (Ctl)] shRNA or treated with 500 nmol/L LY364947 were incubated for 6 hours. Graph shows nuclear Snail fluorescence intensity of cells relative to C-FLS. Statistical differences relative to A-FLS are shown. DJ: C-FLS expressing Snail-shRNA or Ctl-shRNA were transfected with TG2 or control vector (D) or were treated with TGF-β (F) and grown for 5 hours (confocal imaging) or 24 hours (invadosome assay) on Oregon Green 488–labeled gelatin. The percentage of cells forming invadosomes was counted for 100 transfected cells or 300 cells. Results are expressed as fold increase over control. E: Efficiency of Snail-shRNA. G: Quantification of extracellular matrix degradation area per cell. H: Number of F-actin– and cortactin-positive invadosomes per cell. I and J: Representative confocal images of TGF-β–treated cells showing actin-cortactin- (I) or actin-TKS5-rich (J) invadosomes that extend into the matrix substratum. Boxed area represents higher magnification of the corresponding panel. Arrows represent areas of actin, cortactin, or TKS5 localization at areas of matrix degradation (loss of green fluorescence). The associated graphs show the fluorescence intensity profile for F-actin (red), Oregon Green 488–labeled gelatin (green), and cortactin or TKS5 (blue) through the plane indicated by the solid line of the merged high magnification image. Data represent means + SEM (AF). n = 5 (AD); n = 3 (E); n = 4 (F); n = 10 to 15 cells (G and H). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, paired t-test (A, B, and DF) or analysis of variance (C, G, and H). Scale bar = 10 μm. Original magnification, ×60 (A and B).
      To determine the role of Snail in TG2- and TGF-β–induced invadosome formation, we evaluated the ability of FLS to produce invadosomes in response to TG2 overexpression or to addition of TGF-β after transduction of Snail-specific shRNA. Snail knockdown markedly diminished the percentage of cells that produce invadosomes in response to TG2 overexpression or TGF-β treatment (Figure 2, D–F). Furthermore, depletion of Snail in TGF-β–stimulated cells resulted in a significant decrease of the gelatin degradation area (Figure 2G) and the number of invadosome cores structures formed by cells identified by colocalization of actin and cortactin, two known markers of invadopodia (Figure 2H). To further characterize the invadosomal structures, we stained the cells for invadosome markers actin and cortactin or actin and TKs5. Confocal microscopy of the areas of degradation formed by TGF-β–treated FLS revealed cell membrane protrusions characterized by punctate actin-cortactin and actin-TKs5 cores that extended from the basal membrane of the cell into the underlying matrix, a feature typical of invadosomes (Figure 2, I and J). These results indicate that Snail is a mediator of TG2/TGF-β–induced invadosome formation and invadosome-induced ECM degradation and that up-regulation of Snail could be one of the ways by which the TG2/TGF-β axis stimulates FLS aggressiveness.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.

       Activated Arthritis FLS Require Snail for ECM Degradation and Cartilage Invasion

      We next measured the relative levels of Snail in ECM-degrading synoviocytes from CIA rats using a combination of invadosome assays and immunofluorescence staining. We observed that Snail was almost exclusively located in the nuclei of cells actively degrading the gelatin matrix (Figure 3A). In addition, invadosome-forming cells expressed an average of 3.2 times more Snail then degradation-inactive cells (Figure 3B), suggesting an interrelationship between the activation of Snail and ECM degradation. Furthermore, knockdown of Snail in A-FLS abolished their increased capacity to degrade ECM when compared with C-FLS (Figure 3C). These findings demonstrate that the ECM-degrading phenotype of A-FLS is dependent on Snail expression.
      Figure thumbnail gr3
      Figure 3Snail is associated with matrix degradation by fibroblast-like synoviocytes (FLS). A: Arthritis (A)-FLS grown on 1% labeled gelatin (green) for 24 hours were immunostained using an anti-Snail antibody. Representative confocal microscopy images showing Snail in A-FLS associated with areas of matrix degradation (arrows). Actin was stained with Alexa Fluor 405–labeled phalloidin (blue). B: Snail intensity for cells associated [invadosome (invado +)] or not (invado −) with areas of degradation. C: C-FLS, A-FLS, or A-FLS transduced with Snail-shRNA or (scrambled) control (Ctl)-shRNA were grown on labeled gelatin. Percentage of cells forming invadosomes (300 cells). D: Control [phosphate-buffered saline (PBS)] and collagen-induced arthritis (CIA) rat joint sections were immunostained with an antibody against Snail. Percentage of Snail-positive cells counted over the entire synovial membrane and mean Snail labeling intensity calculated in three random fields. Modified Mankin score of control (PBS), CIA, and CIA rats injected i.a. with a Snail-shRNA (CIA + Snail-shRNA) or Ctl-shRNA (CIA + Ctl-shRNA) expressing lentivirus 10 days after collagen or PBS injection (E) and number of zones invaded by the synovial membrane counted over the whole section and calculated mean depth of these invasion zones (F). Degraded collagen (Col2-3 short epitope) (G) and Snail labeling intensity (I) in the synovial membrane of control, CIA, and CIA rats injected i.a. with a Snail-shRNA– or Ctl-shRNA–expressing lentivirus. Representative images of degraded collagen (arrows represent degraded collagen at the synovial membrane/cartilage interface; H) and Snail immunostaining (arrows represent nuclear Snail staining; J). Results are expressed as means + SEM intensity relative to cells not forming invadosomes (B) or staining intensity per area calculated from three random fields for each tissue section (G and I). n = 3 (B); n = 4 (C); n = 7 (D, PBS and CIA); n = 13 (E, PBS); n = 15 (E, CIA and CIA + Ctl-shRNA); n = 16 (E, CIA + Snail-shRNA). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, paired t-test (B), analysis of variance (C and F), or unpaired t-test (D, E, G, and I).
      We next used a rat model of CIA that closely resembles RA pathology found in humans,
      • Kannan K.
      • Ortmann R.A.
      • Kimpel D.
      Animal models of rheumatoid arthritis and their relevance to human disease.
      to explore the role of Snail in cartilage invasion/degradation. Snail was detected by immunohistochemistry in cells of the synovial membrane and in chondrocytes of CIA tissues collected at the peak stage of the disease. There was a sixfold increase in Snail staining intensity in the synovial membrane of CIA joints relative to control synovial tissues (Figure 3D). This observation correlated with a higher number of Snail-positive cells (Figure 3D) within the synovial lining layer, further indicating that Snail is overexpressed in arthritis synovial membranes. We next examined whether silencing Snail influenced the progression of CIA. Snail-shRNA– or control-shRNA–expressing lentiviral vectors were injected intra-articularly into knee joints of rats at day 10 after arthritis induction. Hematoxylin and eosin– and toluidine-stained tissue sections from hind paws were evaluated according to a modified Mankin scoring system.
      • Pine P.R.
      • Chang B.
      • Schoettler N.
      • Banquerigo M.L.
      • Wang S.
      • Lau A.
      • Zhao F.
      • Grossbard E.B.
      • Payan D.G.
      • Brahn E.
      Inflammation and bone erosion are suppressed in models of rheumatoid arthritis following treatment with a novel Syk inhibitor.
      Results showed that Snail knockdown significantly reduced Mankin score from 9.1 in tissues of CIA animals to 5.1 in tissues of Snail-shRNA–treated arthritis joints (Figure 3E). Although a mild effect on synovial hyperplasia was noted (Supplemental Figure S3), most of the improvement was because of a reduction in the prevalence and depth of cartilage invasion by the synovial membrane (Figure 3F). In addition, immunostaining using an antibody directed against fragments of type II collagen showed that the extensive zones of collagen degradation observed in the joints of CIA rats were significantly reduced in tissues of CIA rats treated with Snail-shRNA (Figure 3, G and H). The efficiency of Snail knockdown in synovial lining was confirmed by immunohistochemistry. Data showed that the levels of Snail detected in the synovium of Snail knockdown-treated joints were comparable to those of control tissues (Figure 3, I and J). Thus, Snail expression is associated with the aggressive invadosome-forming phenotype of arthritis FLS, and inhibition of Snail in joints efficiently reduces cartilage invasion and joint damage.

       Snail-Induced Extracellular Invadosome Formation Involves PTEN Repression

      Although Snail can affect many downstream pathways, few direct targets have been identified. Snail binds to the promoter region of the tumor-suppressor gene PTEN in colorectal cancer cells and represses its activity.
      • Escriva M.
      • Peiro S.
      • Herranz N.
      • Villagrasa P.
      • Dave N.
      • Montserrat-Sentis B.
      • Murray S.A.
      • Franci C.
      • Gridley T.
      • Virtanen I.
      • Garcia de Herreros A.
      Repression of PTEN phosphatase by Snail1 transcriptional factor during gamma radiation-induced apoptosis.
      Furthermore, a loss of PTEN expression has been associated with zones of aggressive cartilage invasion by RA synoviocytes.
      • Pap T.
      • Franz J.K.
      • Hummel K.M.
      • Jeisy E.
      • Gay R.
      • Gay S.
      Activation of synovial fibroblasts in rheumatoid arthritis: lack of Expression of the tumour suppressor PTEN at sites of invasive growth and destruction.
      To examine the effect of PTEN modulation on ECM degradation by FLS, we treated C-FLS with bpV(pic), a potent protein tyrosine phosphatase inhibitor with selectivity for PTEN.
      • Lai J.P.
      • Dalton J.T.
      • Knoell D.L.
      Phosphatase and tensin homologue deleted on chromosome ten (PTEN) as a molecular target in lung epithelial wound repair.
      • Schmid A.C.
      • Byrne R.D.
      • Vilar R.
      • Woscholski R.
      Bisperoxovanadium compounds are potent PTEN inhibitors.
      BpV(pic) addition led to a dose-dependent increase in invadosome-forming ability of rat FLS (Figure 4A). PTEN inhibition also caused an increase in the number of invadosomes formed per cell, as identified by clusters of actin and cortactin (Figure 4B), two bona fide markers of invadopodia core formation.
      • Artym V.V.
      • Zhang Y.
      • Seillier-Moiseiwitsch F.
      • Yamada K.M.
      • Mueller S.C.
      Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function.
      Furthermore, enlargement of the areas of ECM degradation was observed in individual cells (Figure 4B). Similarly, knockdown of PTEN using shRNAs that targeted two independent regions of the mRNA also resulted in an increase in the number of invadosomes formed by cells and their ability to degrade the ECM (Figure 4C and Supplemental Figure S4).
      Figure thumbnail gr4
      Figure 4Snail-mediated matrix degradation involves PTEN repression. A: Control fibroblast-like synoviocytes (C-FLS) were cultured on gelatin for 24 hours with or without Bpv(pic) at the indicated concentrations. The percentage of cells forming invadosomes was calculated. Results are expressed relative to control cells treated with vehicle. B: Quantification of the number of invadosomes formed (actin/cortactin clusters) and the corresponding area of degradation per cell. C: C-FLS transduced with phosphatase and tensin homolog (PTEN)–shRNA or control (Ctl)–shRNA were evaluated in an invadosome assay. PTEN knockdown was assessed by immunoblotting. D: Luciferase reporter assay showing PTEN promoter activity in C-FLS transfected with an empty- or Snail-encoding vector and A-FLS transduced with Snail1-shRNA or Ctl-shRNA. E: Percentage of invadosome-forming cells in A-FLS transduced with Snail-shRNA or Ctl-shRNA. Results are expressed relative to control cells. F and G: Representative immunoblots of p-platelet-derived growth factor receptor (pPDGFR), PDGFR, pAKT, and AKT after incubation (1 to 60 minutes) of C-FLS with 1 of C- of bpV(pic) (F) or incubation (15 minutes) of C-FLS with 1 of C- of bpV(pic) with or without 50 nmol/L PDGFR inhibitor V (G). H: C-FLS were pretreated for 30 minutes with inhibitors before addition of 1 μmol/L bpV(pic) where indicated. Percentage of cells associated with invadosomes is shown. Data represent means + SEM (AE and H). n = 5 (A); n = 3 (BE); n = 4 (H). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, analysis of variance (A, B, C, E and H) or unpaired t-test (D). Inh., inhibitor; WB, Western blot.
      We next determined whether PTEN was a transcriptional target of Snail in A-FLS. Consistent with data indicating that Snail is overexpressed in A-FLS, luciferase assays using the PTEN promoter showed a significant decrease in promoter activity in A-FLS compared with C-FLS (Figure 4D). Overexpression of Snail in control FLS resulted in a decrease of PTEN promoter activity similar to that observed in A-FLS (Figure 4D). As expected, knockdown of Snail in arthritis FLS led to an increase in PTEN expression that was similar to the level found in control FLS (Figure 4D).
      Because Snail repressed PTEN promoter activity in A-FLS, we tested if PTEN repression could be a mechanism by which Snail promoted invadosome production in A-FLS. To this end, cells were transduced with a Snail-shRNA and incubated in the presence or absence of increasing concentrations of bpV(pic). Results showed that PTEN inhibition in A-FLS rescued the loss of invadosome formation caused by Snail depletion (Figure 4E), indicating that Snail induces matrix degradation by A-FLS through PTEN repression.

       Invadosome Formation in PTEN-Repressed Cells Depends on Enhanced PDGFR Activation and Signaling

      PDGFR activation is a key contributing factor in RA-FLS aggressiveness,
      • Pohlers D.
      • Huber R.
      • Ukena B.
      • Kinne R.W.
      Expression of platelet-derived growth factors C and D in the synovial membrane of patients with rheumatoid arthritis and osteoarthritis.
      • Butler D.M.
      • Leizer T.
      • Hamilton J.A.
      Stimulation of human synovial fibroblast DNA synthesis by platelet-derived growth factor and fibroblast growth factor: differences to the activation by IL-1.
      • Lafyatis R.
      • Remmers E.F.
      • Roberts A.B.
      • Yocum D.E.
      • Sporn M.B.
      • Wilder R.L.
      Anchorage-independent growth of synoviocytes from arthritic and normal joints: stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta and retinoids.
      and the phosphorylated/activated form of PDGFR might be a target of PTEN.
      • Mahimainathan L.
      • Choudhury G.G.
      Inactivation of platelet-derived growth factor receptor by the tumor suppressor PTEN provides a novel mechanism of action of the phosphatase.
      We, therefore, examined whether PTEN inhibition promoted PDGFR activation in FLS from control rats using phosphorylation-specific antibodies in Western blot analysis. The results showed that activation-associated phosphorylation of PDGFR was strongly up-regulated in FLS treated with the PTEN inhibitor, bpV(pic), compared with untreated cells (Figure 4F). One major effect of the PDGFR signaling pathway involves the recruitment of phosphatidylinositol 3-kinase (PI3K) and downstream activation of AKT.
      • Valius M.
      • Kazlauskas A.
      Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal.
      To investigate whether the increased PDGFR phosphorylation in PTEN-inhibited cells correlated with enhanced PDGFR signaling, we next assessed the activation-associated phosphorylation status of AKT. Results showed that up-regulation of PDGFR activity was associated with a significant increase in AKT phosphorylation (Figure 4F), an event that was partly inhibited using a PDGFR tyrosine kinase inhibitor V, an ATP-competitive inhibitor that blocks ligand-induced PDGFR phosphorylation and kinase activity
      • Furuta T.
      • Sakai T.
      • Senga T.
      • Osawa T.
      • Kubo K.
      • Shimizu T.
      • Suzuki R.
      • Yoshino T.
      • Endo M.
      • Miwa A.
      Identification of potent and selective inhibitors of PDGF receptor autophosphorylation.
      (Figure 4G). These results demonstrate that PDGFR activation in PTEN-inhibited synovial cells is associated with downstream PDGFR signaling. They also suggest that part of AKT activation in PTEN-inhibited cells lies downstream of PDGFR kinase activity.
      To next assess the role of the PTEN-PDGFR-PI3K/AKT axis in invadosome formation, we treated control FLS with bpV(pic) to remove PTEN repression, followed by the inhibition of PI3K, AKT, or PDGFR tyrosine kinase activity. Inhibition of PDGFR activity in bpV(pic)-treated cells resulted in strong, but partial, down-regulation of invadosome formation (Figure 4H). Similar results were observed in cells treated with the PI3K inhibitor, LY 294002, and in cells treated with AKT inhibitor XI to inhibit AKT kinase activity. Overall, the data demonstrate that Snail induces the aggressive invadosome-forming phenotype of arthritic FLS through PTEN repression, resulting in the activation of downstream signaling along the PDGFR-PI3K/AKT axis.

       Relevance of the Snail-PTEN Axis to Human RA Cells and Tissue Synoviocytes and Synovial Tissues from RA Patients

      Our results show that Snail activated through the TG2–TGF-β axis is an effector of invadosome formation and cartilage degradation in CIA. To explore the potential translation of these findings to human pathology, we first searched microarray databases for expression of Snail (encoded by the SNAI1 gene) and selected EMT markers in control (nonarthritic or OA patients) and RA synovial tissues and control and RA-FLS. Comparative analysis indicated that SNAI2, TWIST, and ZEB2 were significantly overexpressed in joint tissues and/or FLS of RA patients compared with control individuals (Figure 5A). In agreement with findings obtained from CIA samples, up-regulation of these EMT transcription factors was associated with a TGFB gene expression signature that included increased expression of TGFBR1, TG2, COL1A2, PDGFA, and MMP9, and down-regulation of PTEN.
      • Ranganathan P.
      • Agrawal A.
      • Bhushan R.
      • Chavalmane A.K.
      • Kalathur R.K.
      • Takahashi T.
      • Kondaiah P.
      Expression profiling of genes regulated by TGF-beta: differential regulation in normal and tumour cells.
      • Inagaki Y.
      • Truter S.
      • Ramirez F.
      Transforming growth factor-beta stimulates alpha 2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site.
      • Chou Y.T.
      • Wang H.
      • Chen Y.
      • Danielpour D.
      • Yang Y.C.
      Cited2 modulates TGF-beta-mediated upregulation of MMP9.
      • Ebert M.P.
      • Fei G.
      • Schandl L.
      • Mawrin C.
      • Dietzmann K.
      • Herrera P.
      • Friess H.
      • Gress T.M.
      • Malfertheiner P.
      Reduced PTEN expression in the pancreas overexpressing transforming growth factor-beta 1.
      • Chow J.Y.
      • Ban M.
      • Wu H.L.
      • Nguyen F.
      • Huang M.
      • Chung H.
      • Dong H.
      • Carethers J.M.
      TGF-beta downregulates PTEN via activation of NF-kappaB in pancreatic cancer cells.
      Because SNAI1 expression was not reported in the available databases, we assessed SNAI1 expression in six RA synovial cell lines using RT-PCR. Results indicated that SNAI1 was also significantly overexpressed in RA synovial cell lines compared with control synovial cells (Figure 5B). We also examined Snail expression levels in synovial tissues. Results showed that Snail is highly expressed in the rheumatoid synovium compared with OA tissues with prominent staining in the synovial intimal lining (Figure 5C). A similar pattern of expression was observed with the phosphorylated/activated form of Smad2/3. Quantification of staining intensity indicated that Snail expression is significantly elevated (P < 0.001) in RA compared with OA (Figure 5D).
      Figure thumbnail gr5
      Figure 5Snail is overexpressed in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and is essential for matrix degradation and invasion. A: Analysis of data extracted from public gene array databases [National Center for Biotechnology Information Gene Expression Omnibus (GEO); http://www.ncbi.nlm.nih.gov/geo] reveals increased expression of epithelial-mesenchymal transition (EMT)–related genes in synovial tissues and/or cultured synoviocytes of RA patients. Primary data were derived from data sets under the GEO accession numbers GSE7307 (ZEB2 and FN1), GSE1919 (SNAI2 in synovial tissues, TWIST1, TG2, MMP9, COL1A2, and PTEN), GSE7669 (SNAI2 in cultured FLS, TGFBR1, and PDGFA), and GSE2053 (PTEN). Results are shown as fold increases in RA gene expression relative to control [osteoarthritis (OA) for GSE1919 and GSE7669; normal tissue for GSE7307]. B: RT-qPCR analysis of Snai1 mRNA expression using Rplp0 as a housekeeping gene. Results are expressed as mean mRNA levels in RA-FLS relative to control (C)-FLS. C: Representative images of Snail, p-Smad2/3 immunostaining, and control rabbit IgG of synovial tissues of OA and RA patients. D: The associated graph shows relative labeling intensities in arbitrary units (AUs) for 10 OA and 10 RA patients. Data represent means ± SEM (unpaired t-test; A, B, and D). n = 6 (B, RA-FLS); n = 4 (B, C-FLS). P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. Original magnification, ×40 (C).
      To define the role of the Snail-PTEN axis in invadosome formation by human synoviocytes, we next analyzed the ability of PTEN inhibition to reverse the loss of invadosome formation because of Snail depletion. Consistent with results obtained from rat synoviocytes, Snail was overexpressed in invadosome-forming synoviocytes from RA (Figure 6A). Knockdown of Snail inhibited invadosome formation in RA-FLS, whereas inhibition of PTEN in Snail-depleted cells rescued the ability of RA-FLS to form invadosomes (Figure 6B). Similarly, Snail inhibition strongly inhibited the ability of RA synoviocytes to invade type II collagen matrices, whereas inhibition of PTEN in control FLS promoted cell invasion (Figure 6C). These results indicated that Snail is essential for the formation of matrix-degrading invadosomes and cell invasion in human RA synovial cells and suggest that PTEN inhibition is a mechanism involved in their Snail-induced prodestructive effector function.
      Figure thumbnail gr6
      Figure 6Snail is essential for matrix degradation and invasion by human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). A: RA-FLS were grown on 1% Alexa Fluor 488–labeled gelatin (green) and immunostained for Snail. Intensity was measured for cells associated [invadosome (invado +)] or not (invado −) with areas of matrix degradation. B: Control (C)-FLS or RA-FLS transduced with Snail-shRNA or control (Ctl)-shRNA treated with bpv(pic) for 40 hours in invadosome assay. C: Similar experiments performed for 72 hours in collagen type II–coated transwell invasion assay. For this assay, cells were also incubated in the presence or absence of 10 μg/mL GM6001. Results are expressed as means + SEM (A, paired t-test, intensity relative to cells not forming invadopodia for 100 cells; B, analysis of variance; and C, unpaired t-test, relative to control cells). n = 3 (A); n = 4 (B and C). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

      Discussion

       Synoviocytes Reactivate Snail to Gain Their Invadosome-Forming Prodestructive Phenotype

      Fibroblast-like synoviocytes are recognized as the primary effectors of cartilage degradation in RA through mechanisms that remain to be fully understood. Herein, we identified Snail as a critical regulator of the prodestructive invadosome-forming phenotype of FLS of rat and RA patients and cartilage degradation in experimental arthritis. Snail exerts its function through direct down-regulation of PTEN transcription, resulting in increased PDGFR phosphorylation and PI3K/AKT activation (Figure 7). Enhanced Snail gene expression was associated with a TG2–TGF-β/EMT gene signature in synovial cells and tissues from RA patients. Accordingly, TGF-β and TG2 stimulated Snail-induced invadosome production in rat and human synovial cells. More important, down-regulation of Snail in CIA joints prevented invasion of the articular cartilage by the hyperplastic synovium and cartilage degradation. Our findings not only reveal, for the first time, how Snail contributes to the prodestructive properties of FLS, but also provide a rationale to subvert cartilage erosion by synoviocytes using Snail-targeted therapy.
      Figure thumbnail gr7
      Figure 7Schematic model of the role of Snail in invadosome formation by arthritic synovial cells. Work presented herein indicates that Snail, up-regulated in arthritic synoviocytes by the transglutaminase 2 (TG2)/transforming growth factor (TGF)-β/Smad axis, directly suppresses PTEN transcription. Down-regulation of PTEN leads to an increase in platelet-derived growth factor receptor (PDGFR) phosphorylation/activation that induces the formation of invadosomes through phosphatidylinositol 3-kinase/AKT activation. Promising targets for chemical inhibition include the TG2/TGF-β axis, Snail, and PDGFR signaling. PTEN, phosphatase and tensin homolog; TGF-βR, TGF-β receptor.
      Snail is a transcription factor implicated in a broad spectrum of biological functions, including regulation of cell proliferation, differentiation, invasion, and survival in a variety of cells.
      • Vega S.
      • Morales A.V.
      • Ocana O.H.
      • Valdes F.
      • Fabregat I.
      • Nieto M.A.
      Snail blocks the cell cycle and confers resistance to cell death.
      • Kajita M.
      • McClinic K.N.
      • Wade P.A.
      Aberrant expression of the transcription factors snail and slug alters the response to genotoxic stress.
      • Escriva M.
      • Peiro S.
      • Herranz N.
      • Villagrasa P.
      • Dave N.
      • Montserrat-Sentis B.
      • Murray S.A.
      • Franci C.
      • Gridley T.
      • Virtanen I.
      • Garcia de Herreros A.
      Repression of PTEN phosphatase by Snail1 transcriptional factor during gamma radiation-induced apoptosis.
      • Barrallo-Gimeno A.
      • Nieto M.A.
      The Snail genes as inducers of cell movement and survival: implications in development and cancer.
      In a recent study, Snail overexpression was shown to increase cytokine expression and invasiveness of rat synovial cells, resulting in the exacerbation of CIA.
      • Chen S.Y.
      • Shiau A.L.
      • Li Y.T.
      • Lin C.C.
      • Jou I.M.
      • Liu M.F.
      • Wu C.L.
      • Wang C.R.
      Transcription factor snail regulates tumor necrosis factor alpha-mediated synovial fibroblast activation in the rheumatoid joint.
      In this study, we highlight the direct implication of Snail1 that acts as a key effector of ECM degradation in arthritic FLS by promoting invadosome formation. Snail was specifically associated with the FLS subpopulation responsible for active matrix degradation and overexpressed in synovial membranes of CIA joints. Accordingly, Snail-deficient A-FLS lose their ability to form ECM-degrading structures, whereas Snail knockdown in joints prevents cartilage invasion and degradation. Consistent with these findings, gene ontology analysis of Snail knockdown fibroblasts has demonstrated that Snail can orchestrate multiple processes, which are essential to fibroblast motile behavior, including adhesion, migration, and proteolysis. In particular, cortactin, RhoA, ROCK1, and MT1-MMP, important molecules in the biology of invadopodia, were found to be regulated in Snail-depleted cells.
      • Rowe R.G.
      • Li X.Y.
      • Hu Y.
      • Saunders T.L.
      • Virtanen I.
      • Garcia de Herreros A.
      • Becker K.F.
      • Ingvarsen S.
      • Engelholm L.H.
      • Bommer G.T.
      • Fearon E.R.
      • Weiss S.J.
      Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs.
      Snail has also been shown to up-regulate the expression and activity of MMP-2, MMP-3, MMP-9, and MT1-MMP in different carcinoma cell lines.
      • Sun L.
      • Diamond M.E.
      • Ottaviano A.J.
      • Joseph M.J.
      • Ananthanarayan V.
      • Munshi H.G.
      Transforming growth factor-beta 1 promotes matrix metalloproteinase-9-mediated oral cancer invasion through snail expression.
      • Yokoyama K.
      • Kamata N.
      • Fujimoto R.
      • Tsutsumi S.
      • Tomonari M.
      • Taki M.
      • Hosokawa H.
      • Nagayama M.
      Increased invasion and matrix metalloproteinase-2 expression by Snail-induced mesenchymal transition in squamous cell carcinomas.
      • Jiang W.
      • Zhang Y.
      • Kane K.T.
      • Collins M.A.
      • Simeone D.M.
      • di Magliano M.P.
      • Nguyen K.T.
      CD44 regulates pancreatic cancer invasion through MT1-MMP.
      Even though Snail overexpression or Snail depletion failed to regulate MT1-MMP expression in FLS (Supplemental Figure S5), high levels of Snai1 expression in these cells paralleled increases in metalloproteinase expression (MMP-9 and MMP-3) (Supplemental Table S1). MMP-9 was previously found to be enriched at the invadopodia of cancer cells,
      • Murphy D.A.
      • Courtneidge S.A.
      The “ins” and “outs” of podosomes and invadopodia: characteristics, formation and function.
      • Jacob A.
      • Jing J.
      • Lee J.
      • Schedin P.
      • Gilbert S.M.
      • Peden A.A.
      • Junutula J.R.
      • Prekeris R.
      Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells.
      and MMP-3 was associated with ECM degradation at sites of invadosome formation in A-FLS.
      • Lauzier A.
      • Charbonneau M.
      • Harper K.
      • Jilaveanu-Pelmus M.
      • Dubois C.M.
      Formation of invadopodia-like structures by synovial cells promotes cartilage breakdown in collagen-induced arthritis: involvement of the protein tyrosine kinase Src.
      These findings suggest that during the course of arthritis, synoviocytes reactivate the expression of the transcription factor Snail to gain their invasive and prodestructive phenotype through the formation of protease-enriched degrading devices, the invadosomes.

       The TG2/TGF-β Axis as a Trigger for Snail Expression

      Snail expression in FLS of CIA rats and RA patients was associated with an EMT gene signature characteristic of TGF-β/TG2 signaling. TGF-β is a major inducer of EMT in carcinogenesis.
      • Giannelli G.
      • Villa E.
      • Lahn M.
      Transforming growth factor-beta as a therapeutic target in hepatocellular carcinoma.
      Moreover, we have previously shown that endogenous TGF-β production and signaling, triggered by TG2 activity, play a critical role in the progression of experimental arthritis by promoting invadosome-induced cartilage breakdown.
      • Lauzier A.
      • Charbonneau M.
      • Paquette M.
      • Harper K.
      • Dubois C.M.
      Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.
      We, therefore, hypothesized that the presence of TG2 and/or TGF-β in synovial lining would serve as potent stimuli to increase FLS aggressiveness by controlling cues involved in the EMT program. We show herein that both TG2 and TGF-β increase Snail expression in FLS and that TG2-dependent induction of Snail is partly dependent on TGF-β signaling. Furthermore, knockdown of Snail markedly reduced the ability of A-FLS to produce invadosomes in response to TGF-β or TG2. Our findings point to the TG2/TGF-β axis as a potential trigger for Snail expression in synovial joints. However, we cannot rule out the participation of other potent EMT inducers, such as PDGF, which has been shown to be overexpressed in joints. In addition, tumor necrosis factor was recently shown to up-regulate Snail expression in synovial cells.
      • Chen S.Y.
      • Shiau A.L.
      • Li Y.T.
      • Lin C.C.
      • Jou I.M.
      • Liu M.F.
      • Wu C.L.
      • Wang C.R.
      Transcription factor snail regulates tumor necrosis factor alpha-mediated synovial fibroblast activation in the rheumatoid joint.
      The ability of PDGF-B and TGF-β to strongly potentiate the response of FLS to cytokines, such as tumor necrosis factor or IL-1, suggests that Snail overexpression in joints might rely on a complex network of cytokines and growth factors that include TGF-β.
      • Rosengren S.
      • Corr M.
      • Boyle D.L.
      Platelet-derived growth factor and transforming growth factor beta synergistically potentiate inflammatory mediator synthesis by fibroblast-like synoviocytes.

       Mechanisms of Snail-Induced Invadosome Formation Involve the PTEN-PDGFR/PI3K Axis

      Few direct targets of Snail have been identified. Among them, Snail has been shown to bind the PTEN promoter in cancer cells,
      • Escriva M.
      • Peiro S.
      • Herranz N.
      • Villagrasa P.
      • Dave N.
      • Montserrat-Sentis B.
      • Murray S.A.
      • Franci C.
      • Gridley T.
      • Virtanen I.
      • Garcia de Herreros A.
      Repression of PTEN phosphatase by Snail1 transcriptional factor during gamma radiation-induced apoptosis.
      repressing its activity. Herein, we found that Snail down-regulates the expression of PTEN in synoviocytes and that PTEN expression is reduced in synoviocytes derived from arthritic joints compared with control synoviocytes. Our findings confirm prior studies that showed that PTEN is not detected in the lining layer of RA synovium compared with normal tissue or in RA synoviocytes invading human cartilage in a severe combined immunodeficiency mouse coimplantation model.
      • Pap T.
      • Franz J.K.
      • Hummel K.M.
      • Jeisy E.
      • Gay R.
      • Gay S.
      Activation of synovial fibroblasts in rheumatoid arthritis: lack of Expression of the tumour suppressor PTEN at sites of invasive growth and destruction.
      We also observed that PTEN inhibition in arthritic FLS reversed the loss of invadoposome formation caused by Snail depletion, indicating that Snail induces ECM degradation through PTEN repression. Therefore, high expression of Snail with concomitant low expression of PTEN seems to be a discriminative feature of the prodestructive phenotype of FLS in arthritis.
      PTEN is known to facilitate cell migration and invasion through its action on multiple targets. Most of the PTEN suppressor functions have been attributed to its lipid phosphatase activity that antagonizes the action of PI3K by converting phosphatidylinositol-3,4,5-triphosphate (PIP3) to phosphatidylinositol-4,5-bisphosphate (PIP2). This action restricts the recruitment and activation of pleckstrin homology domain-harboring PI3K effector proteins, such as AKT and PDK1.
      • Song M.S.
      • Salmena L.
      • Pandolfi P.P.
      The functions and regulation of the PTEN tumour suppressor.
      Accordingly, our data indicated that PTEN inhibition in arthritic FLS results in a strong increase in AKT phosphorylation and that both PI3K and AKT activities were necessary for invadosome-associated ECM degradation. The formation of invadopodia in cancer cells or podosomes in osteoclasts has also been reported to require PI3K activity or PIP3 availability.
      • Hoshino D.
      • Jourquin J.
      • Emmons S.W.
      • Miller T.
      • Goldgof M.
      • Costello K.
      • Tyson D.R.
      • Brown B.
      • Lu Y.
      • Prasad N.K.
      • Zhang B.
      • Mills G.B.
      • Yarbrough W.G.
      • Quaranta V.
      • Seiki M.
      • Weaver A.M.
      Network analysis of the focal adhesion to invadopodia transition identifies a PI3K-PKCalpha invasive signaling axis.
      • Yamaguchi H.
      • Yoshida S.
      • Muroi E.
      • Yoshida N.
      • Kawamura M.
      • Kouchi Z.
      • Nakamura Y.
      • Sakai R.
      • Fukami K.
      Phosphoinositide 3-kinase signaling pathway mediated by p110alpha regulates invadopodia formation.
      • Chellaiah M.A.
      • Biswas R.S.
      • Yuen D.
      • Alvarez U.M.
      • Hruska K.A.
      Phosphatidylinositol 3,4,5-trisphosphate directs association of Src homology 2-containing signaling proteins with gelsolin.
      Although the molecular targets of the PI3K pathway in cell invasion have not been clearly identified, inhibition of this pathway was found to modulate MMP production and PDGF-induced F-actin rearrangement,
      • Brachmann S.M.
      • Yballe C.M.
      • Innocenti M.
      • Deane J.A.
      • Fruman D.A.
      • Thomas S.M.
      • Cantley L.C.
      Role of phosphoinositide 3-kinase regulatory isoforms in development and actin rearrangement.
      a process involved in podosome and invadopodia formation.
      • Burgstaller G.
      • Gimona M.
      Actin cytoskeleton remodelling via local inhibition of contractility at discrete microdomains.
      • Hoshino D.
      • Branch K.M.
      • Weaver A.M.
      Signaling inputs to invadopodia and podosomes.
      In addition, PIP3 may also serve as a precursor of PIP2, which has been shown to regulate invadopodia formation by recruiting Tks5, a scaffolding protein essential for F-actin core formation.
      • Yamaguchi H.
      • Yoshida S.
      • Muroi E.
      • Kawamura M.
      • Kouchi Z.
      • Nakamura Y.
      • Sakai R.
      • Fukami K.
      Phosphatidylinositol 4,5-bisphosphate and PIP5-kinase Ialpha are required for invadopodia formation in human breast cancer cells.
      It is, therefore, possible that PI3K inhibition suppresses invadosome-driven ECM degradation by impeding both invadosome core formation and MMP-mediated proteolysis.
      Another emerging target of PTEN is the receptor tyrosine kinase (RTK) PDGFR. Through its protein phosphatase activity, PTEN has been shown to bind and dephosphorylate the activated PDGFR in mesangial cells, impeding downstream PI3K/AKT signaling.
      • Mahimainathan L.
      • Choudhury G.G.
      Inactivation of platelet-derived growth factor receptor by the tumor suppressor PTEN provides a novel mechanism of action of the phosphatase.
      PDGFR family RTK, namely PDGFRα and PDGFRβ, and their ligands, PDGF A to D, have previously been implicated in RA-FLS aggressiveness. Increased levels of the PDGF receptors and ligands were detected in the synovial lining and in FLS cultures of RA patients.
      • Pohlers D.
      • Huber R.
      • Ukena B.
      • Kinne R.W.
      Expression of platelet-derived growth factors C and D in the synovial membrane of patients with rheumatoid arthritis and osteoarthritis.
      • Watanabe N.
      • Ando K.
      • Yoshida S.
      • Inuzuka S.
      • Kobayashi M.
      • Matsui N.
      • Okamoto T.
      Gene expression profile analysis of rheumatoid synovial fibroblast cultures revealing the overexpression of genes responsible for tumor-like growth of rheumatoid synovium.
      • Reuterdahl C.
      • Tingstrom A.
      • Terracio L.
      • Funa K.
      • Heldin C.H.
      • Rubin K.
      Characterization of platelet-derived growth factor beta-receptor expressing cells in the vasculature of human rheumatoid synovium.
      Furthermore, PDGF has been found to regulate synoviocyte proliferation, anchorage-independent growth, and collagenase transcription, which are features known to be part of the transformed and prodestructive phenotype of RA synoviocytes.
      • Butler D.M.
      • Leizer T.
      • Hamilton J.A.
      Stimulation of human synovial fibroblast DNA synthesis by platelet-derived growth factor and fibroblast growth factor: differences to the activation by IL-1.
      • Lafyatis R.
      • Remmers E.F.
      • Roberts A.B.
      • Yocum D.E.
      • Sporn M.B.
      • Wilder R.L.
      Anchorage-independent growth of synoviocytes from arthritic and normal joints: stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-beta and retinoids.
      • Kumkumian G.K.
      • Lafyatis R.
      • Remmers E.F.
      • Case J.P.
      • Kim S.J.
      • Wilder R.L.
      Platelet-derived growth factor and IL-1 interactions in rheumatoid arthritis. Regulation of synoviocyte proliferation, prostaglandin production, and collagenase transcription.
      However, the impact of PTEN regulation in synoviocytes on PDGFR activation and ensuing biological functions has not yet been demonstrated. We report herein that inhibition of PTEN activity in arthritic FLS resulted in increased phosphorylation of PDGFR that was required for the invadosome-driven ECM degradation capabilities of the cells. Enhanced activation of PDGFR was associated with a corresponding increase in AKT phosphorylation, an event that was partly dependent on PDGFR kinase activity. These findings suggest that invadosome formation in A-FLS depends on an autocrine RTK/PI3K/AKT activation loop that involves repression of the PTEN phosphatase. Although the involvement of other RTKs and phosphatases
      • Haj F.G.
      • Verveer P.J.
      • Squire A.
      • Neel B.G.
      • Bastiaens P.I.
      Imaging sites of receptor dephosphorylation by PTP1B on the surface of the endoplasmic reticulum.
      cannot be excluded, our studies suggest that PTEN is the primary PDGFR phosphatase causally involved in PTEN-driven PDGFR phosphorylation. Aberrant activation of RTKs has been linked to malignant progression in many human cancers,
      • Robertson S.C.
      • Tynan J.
      • Donoghue D.J.
      RTK mutations and human syndromes: when good receptors turn bad.
      and various studies have reported an up-regulation of tyrosine kinase activity in RA synoviocytes.
      • Krausz S.
      • Garcia S.
      • Ambarus C.A.
      • de Launay D.
      • Foster M.
      • Naiman B.
      • Iverson W.
      • Connor J.R.
      • Sleeman M.A.
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      • Hamann J.
      • Baeten D.
      • Tak P.P.
      • Reedquist K.A.
      Angiopoietin-2 promotes inflammatory activation of human macrophages and is essential for murine experimental arthritis.
      • Yamane S.
      • Ishida S.
      • Hanamoto Y.
      • Kumagai K.
      • Masuda R.
      • Tanaka K.
      • Shiobara N.
      • Yamane N.
      • Mori T.
      • Juji T.
      • Fukui N.
      • Itoh T.
      • Ochi T.
      • Suzuki R.
      Proinflammatory role of amphiregulin, an epidermal growth factor family member whose expression is augmented in rheumatoid arthritis patients.
      Furthermore, the balance between RTK and phosphatase activities is an emerging mechanism that influences cancer progression and resistance to treatment.
      • Sastry S.K.
      • Elferink L.A.
      Checks and balances: interplay of RTKs and PTPs in cancer progression.
      It is, therefore, likely that the reduction of PTEN expression observed in Snail-expressing cells within the RA synovium contributes to an imbalance of tyrosine kinases and phosphatases, hence promoting the tumor-like phenotype of synovial cells that involves enhanced invadosome production, cell invasion, and cartilage destruction.

       Components of the EMT Program as Potential Therapeutic Targets or Markers in RA

      Despite impressive developments in RA therapeutics during the past 20 years, results of clinical trials show that the various treatment strategies have only a moderate effect on joint destruction,
      • van der Heijde D.M.
      • van Riel P.L.
      • Nuver-Zwart I.H.
      • Gribnau F.W.
      • vad de Putte L.B.
      Effects of hydroxychloroquine and sulphasalazine on progression of joint damage in rheumatoid arthritis.
      • Wolfe F.
      • Sharp J.T.
      Radiographic outcome of recent-onset rheumatoid arthritis: a 19-year study of radiographic progression.
      • van Aken J.
      • Lard L.R.
      • le Cessie S.
      • Hazes J.M.
      • Breedveld F.C.
      • Huizinga T.W.
      Radiological outcome after four years of early versus delayed treatment strategy in patients with recent onset rheumatoid arthritis.
      • Smolen J.S.
      • Emery P.
      Efficacy and safety of leflunomide in active rheumatoid arthritis.
      although encouraging results have been reported using a combination of methotrexate with novel biological treatments, such as tumor necrosis factor inhibitors in subsets of patients.
      • Lipsky P.E.
      • van der Heijde D.M.
      • St Clair E.W.
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      • Weisman M.
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      • Harriman G.R.
      • Maini R.N.
      Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group: Infliximab and methotrexate in the treatment of rheumatoid arthritis.
      However, these biological agents are associated with several adverse effects and concerns, such as increased susceptibility to infection, potential risks of malignancies, and lack of response in a proportion of patients. Herein, we showed that cartilage invasion and degradation in CIA was efficiently prevented by blockade of Snail. Because beyond embryonic development, EMT components are only reactivated under stress or pathological conditions, such dependency on Snail or its associated gene expression program for synovial cell-driven joint damage could possibly be exploited to improve diagnosis and treatment of RA. In agreement with this possibility, recent data from cancer patients have indicated that reversal of EMT by apricoxib, which blocks downstream TGF-β–induced EMT, was associated with inhibition of cancer progression in several types of tumors.
      • Kirane A.
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      Epithelial-mesenchymal transition increases tumor sensitivity to COX-2 inhibition by apricoxib.
      • Kirane A.
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      • Zaknoen S.
      • Braunfeld J.
      • Schwarz R.E.
      • Burrows F.J.
      • Brekken R.A.
      Apricoxib, a novel inhibitor of COX-2, markedly improves standard therapy response in molecularly defined models of pancreatic cancer.
      • Rao P.N.
      • Grover R.K.
      Apricoxib, a COX-2 inhibitor for the potential treatment of pain and cancer.
      Similarly, retrospective analysis of tumor samples from clinical trials of erlotinib (an epidermal growth factor receptor antagonist) revealed an association between E-cadherin and vimentin expression and positive response to erlotinib in patients with refractory lung carcinoma.
      • Yauch R.L.
      • Januario T.
      • Eberhard D.A.
      • Cavet G.
      • Zhu W.
      • Fu L.
      • Pham T.Q.
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      Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients.
      These data indicate that EMT inducers or components of the EMT program are potential therapeutic targets, as well as predictive markers of response to treatment. Future investigation will be needed to evaluate the expression pattern and clinical outcome of Snail and its associated gene expression program in RA patients.
      In conclusion, our study provides direct evidence that Snail is a central component of the prodestructive, invadosome-forming phenotype of synoviocytes and identify a novel Snail-PTEN-PDGFR/PI3K pathway by which Snail exerts its effector function. Although our data suggest a link between components of the EMT program cartilage invasion and joint damage, further work is required to elucidate how these pathological changes evolve during the establishment and progression of the disease and whether they can be of clinical relevance in the stratification and/or treatment of patients experiencing rapid joint degradation and candidates in need of aggressive and timely treatment.

      Acknowledgments

      We thank Dr. Gilles Dupuis for critical reading of the manuscript; Dr. Leonid Volkov for assistance with confocal microscopy experiments; Dr. Gail V.W. Johnson (University of Rochester, Rochester, NY) for generously providing pcDNA3.1; Bob Weinberg for providing pTK-Snail (addgene plasmid 36976); and Joshua Mendell for providing pGL3-wtPTEN (addgene plasmid 21326).

      Supplemental Data

      • Supplemental Figure S1

        Snail overexpression is maintained during passages. Total RNA extracted from arthritis fibroblast-like synoviocytes (A-FLS) and control (C)-FLS at passages 4 to 9 was analyzed for Snai1 expression by real-time PCR using Rplp0 as internal control. Results are expressed as fold expression in A-FLS relative to C-FLS.

      • Supplemental Figure S2

        Transforming growth factor (TGF)-β induces Snail expression. Total RNA was extracted from control fibroblast-like synoviocytes (C-FLS) grown for 6 hours in the presence or absence of 10 ng/mL TGF-β and analyzed for Snai1 expression by real-time PCR using Rplp0 as internal control. Results are expressed as the mean of fold expression in TGF-β–treated C-FLS cells relative to vehicle-treated cells. Data are shown as the means ± SEM. n = 4 independent experiments. P < 0.05 (paired t-test).

      • Supplemental Figure S3

        Snail knockdown reduces synovial hyperplasia. Hind paw sections of control [phosphate-buffered saline (PBS)], collagen-induced arthritis (CIA), and CIA rat injected i.a. with a Snail-shRNA (CIA + Snail-shRNA) expressing lentivirus or scrambled control (Ctl)-shRNA (CIA + Ctl-shRNA) were stained with hematoxylin. The number of cell layers in the synovial membrane was assessed at six different locations. Values are expressed as the means ± SEM. n = 13 (PBS); n = 15 (CIA and CIA + Ctl-shRNA); n = 16 (CIA + Snail-shRNA). ∗∗P < 0.01 (unpaired t-test).

      • Supplemental Figure S4

        PTEN knockdown results in increased matrix degradation by fibroblast-like synoviocytes (FLS). Control (C)-FLS were transduced with PTEN-shRNA or scrambled (control)-shRNA, and the area of degradation per invadosome-positive cells was quantitated. Data are given as means + SEM. n = 2. P < 0.05 (paired t-test).

      • Supplemental Figure S5

        Snail fails to regulate MT1–matrix metalloproteinase (MMP) gene expression in synoviocytes. Total RNA was extracted from control fibroblast-like synoviocytes (C-FLS) transduced with pLenti-Snail or empty vector (A) or arthritis (A)-FLS transduced with pLenti-Snail-shRNA or (scrambled) pLenti-control (Ctl)-shRNA (B) and analyzed for Snai1 and MT1-MMP expression by real-time PCR using Rplp0 as internal control. Results are expressed as the mean of fold expression in untransduced C-FLS or A-FLS compared with transduced cells. Data are shown as the means ± SEM. n = 3 to 4 independent experiments. P < 0.05, ∗∗∗P < 0.001 (analysis of variance).

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