Materials and Methods
Reagents
Induction and Clinical Evaluation of Arthritis
Histology
Modified Mankin Grading
Cell Cultures
Plasmids and Transfections
Invadosome Assays
Immunofluorescence and Confocal Microscopy
Transwell Invasion Assays
RT-PCR and PCR Array Analysis of Gene Expression
Western Blot Analysis
Luciferase Assay
Statistical Analysis
Study Approval
- 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.
Results
Snail and Snail Inducer TGF-β Are Overexpressed in Synoviocytes of Arthritic Joints

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

Activated Arthritis FLS Require Snail for ECM Degradation and Cartilage Invasion

Snail-Induced Extracellular Invadosome Formation Involves PTEN Repression

Invadosome Formation in PTEN-Repressed Cells Depends on Enhanced PDGFR Activation and Signaling
- Lafyatis R.
- Remmers E.F.
- Roberts A.B.
- Yocum D.E.
- Sporn M.B.
- Wilder R.L.
Relevance of the Snail-PTEN Axis to Human RA Cells and Tissue Synoviocytes and Synovial Tissues from RA Patients


Discussion
Synoviocytes Reactivate Snail to Gain Their Invadosome-Forming Prodestructive Phenotype

The TG2/TGF-β Axis as a Trigger for Snail Expression
Mechanisms of Snail-Induced Invadosome Formation Involve the PTEN-PDGFR/PI3K Axis
- 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.
- Lafyatis R.
- Remmers E.F.
- Roberts A.B.
- Yocum D.E.
- Sporn M.B.
- Wilder R.L.
- Krausz S.
- Garcia S.
- Ambarus C.A.
- de Launay D.
- Foster M.
- Naiman B.
- Iverson W.
- Connor J.R.
- Sleeman M.A.
- Coyle A.J.
- Hamann J.
- Baeten D.
- Tak P.P.
- Reedquist K.A.
- 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.
Components of the EMT Program as Potential Therapeutic Targets or Markers in RA
- Lipsky P.E.
- van der Heijde D.M.
- St Clair E.W.
- Furst D.E.
- Breedveld F.C.
- Kalden J.R.
- Smolen J.S.
- Weisman M.
- Emery P.
- Feldmann M.
- Harriman G.R.
- Maini R.N.
- Yauch R.L.
- Januario T.
- Eberhard D.A.
- Cavet G.
- Zhu W.
- Fu L.
- Pham T.Q.
- Soriano R.
- Stinson J.
- Seshagiri S.
- Modrusan Z.
- Lin C.Y.
- O'Neill V.
- Amler L.C.
Acknowledgments
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).
- Supplemental Table S1
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Article info
Publication history
Footnotes
Supported by the Canadian Institutes for Health Research (CIHR) grant MOP-86634 (C.M.D.), membership in the Fonds de la Recherche su Québec-Santé–funded Center de Recherche du Centre Hospitalier Universitaire de Sherbrooke (C.M.D.), and a CIHR scholarship (K.H.).
Disclosures: None declared.
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