Molecular Medicine

, Volume 16, Issue 9–10, pp 352–358 | Cite as

mTOR Regulates the Invasive Properties of Synovial Fibroblasts in Rheumatoid Arthritis

  • Teresina Laragione
  • Pércio S. Gulko
Research Article


The invasive properties of fibroblast-like synoviocytes (FLS) correlate with radiographic and histologic damage in rheumatoid arthritis (RA) and pristane-induced arthritis (PIA). We previously determined that highly invasive FLS obtained from PIA-susceptible DA (blood type D, Agouti) rats have increased expression of genes associated with invasive cancers, including Villin-2/ezrin. Villin-2/ezrin mediates invasion via mTOR. In the present study we used the mTOR inhibitor rapamycin to assess the role of the ezrinmTOR pathway on the invasive properties of FLS. FLS were isolated from synovial tissues from arthritic DA rats, and from RA patients. FLS were treated with rapamycin or dimethyl sulfoxide (DMSO) for 24 h and then studied in a Matrigel-invasion assay. Supernatants were assayed for matrix metalloproteinase (MMP) activity, and cell lysates were used for quantification of mTOR, p70S6K1, 4EBP1 and FAK, as well as their respective phosphorylated subsets. Actin filament and FAK localization were determined by immunofluorescence. Rapamycin decreased FLS invasion in DA and RA tissues by 93% and 82%, respectively. Rapamycin treatment reduced the phosphorylation of mTOR and its substrates, p70S6K1 and 4EBP1, confirming mTOR inhibition. In conclusion, rapamycin prevented actin reorganization in both DA and RA FLS, and inhibited the directional formation of lamellipodia. Phosphorylation of the lamellipodia marker FAK was also reduced by rapamycin. MMPs were not significantly affected by rapamycin. Rapamycin significantly reduced RA and DA rat FLS invasion via the suppression of the mTOR signaling pathway. This discovery suggests that rapamycin could have a role in RA therapy aimed at reducing the articular damage and erosive changes mediated by FLS.



This study was funded by National Institutes of Health grants R01-AR46213, R01-AR052439 (NIAMS) and R01-AI54348 (NIAID) to P Gulko.


  1. 1.
    Gregersen PK, Plenge RM, Gulko PS. (2006) Genetics of Rheumatoid Arthritis. In: Rheumatoid Arthritis. Firestein G, Panayi G, Wollheim FA (eds.). Oxford University Press, New York, pp. 3–14.Google Scholar
  2. 2.
    Gulko PS, Winchester RJ. (2001) Rheumatoid Arthritis. In: Samter’s Immunologic Diseases. Austen KF, Frank MM, Atkinson JP, Cantor H (eds.). Lippincott, Williams & Wilkins, Baltimore, pp. 427–63.Google Scholar
  3. 3.
    Okada Y, et al. (1990) Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts: purification and activation of the precursor and enzymic properties. Eur. Biochem. 194:721–30.CrossRefGoogle Scholar
  4. 4.
    Hanemaaijer R, et al. (1997) Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells: regulation by tumor necrosis factor-alpha and doxycycline. J. Biol. Chem. 272:31504–9.CrossRefGoogle Scholar
  5. 5.
    Muller-Ladner U, et al. (1996) Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice. Am. J. Pathol. 149:1607–15.PubMedPubMedCentralGoogle Scholar
  6. 6.
    Tolboom TC, et al. (2005) Invasiveness of fibroblast-like synoviocytes is an individual patient characteristic associated with the rate of joint destruction in patients with rheumatoid arthritis. Arthritis Rheum. 52:1999–2002.CrossRefGoogle Scholar
  7. 7.
    Brenner M, et al. (2005) The non-MHC quantitative trait locus Cia5 contains three major arthritis genes that differentially regulate disease severity, pannus formation, and joint damage in collagen-and pristane-induced arthritis. J. Immunol. 174:7894–903.CrossRefGoogle Scholar
  8. 8.
    Laragione T, Brenner M, Mello A, Symons M, Gulko PS. (2008) The arthritis severity locus Cia5d is a novel genetic regulator of the invasive properties of synovial fibroblasts Arthritis Rheum. 58:2296–306.CrossRefGoogle Scholar
  9. 9.
    Laragione T, Brenner M, Li W, Gulko PS. (2008) Cia5d regulates a new fibroblast-like synoviocyte invasion-associated gene expression signature. Arthritis Res. Ther. 10: R92.CrossRefGoogle Scholar
  10. 10.
    Zipin-Roitman A, et al. (2007) CXCL10 promotes invasion-related properties in human colorectal carcinoma cells. Cancer Res. 67:3396–405.CrossRefGoogle Scholar
  11. 11.
    Alami J, Williams BR, Yeger H. (2003) Derivation and characterization of a Wilms’ tumour cell line, WiT 49. Int. J. Cancer. 107:365–74.CrossRefGoogle Scholar
  12. 12.
    Sizemore S, Cicek M, Sizemore N, Ng KP, Casey G. (2007) Podocalyxin increases the aggressive phenotype of breast and prostate cancer cells in vitro through its interaction with ezrin. Cancer Res. 67:6183–91.CrossRefGoogle Scholar
  13. 13.
    Khanna C, et al. (2004) The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nature Med. 10:182–6.CrossRefGoogle Scholar
  14. 14.
    Eskandarpour M, Huang F, Reeves KA, Clark E, Hansson J. (2009) Oncogenic NRAS has multiple effects on the malignant phenotype of human melanoma cells cultured in vitro. Int. J. Cancer. 124:16–26.CrossRefGoogle Scholar
  15. 15.
    Kim MS, Cho WH, Song WS, Lee SY, Jeon DG. (2007) Prognostic significance of ezrin expression in pleomorphic malignant fibrous histiocytoma. Anticancer Res. 27:1171–8.PubMedGoogle Scholar
  16. 16.
    Tynninen O, Carpen O, Jaaskelainen J, Paavonen T, Paetau A. (2004) Ezrin expression in tissue microarray of primary and recurrent gliomas. Neuropathol. Appl Neurobiol. 30:472–7.CrossRefGoogle Scholar
  17. 17.
    Yu Y, et al. (2004) Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nature Med. 10:175–81.CrossRefGoogle Scholar
  18. 18.
    Wick W, et al. (2001) Ezrin-dependent promotion of glioma cell clonogenicity, motility, and invasion mediated by BCL-2 and transforming growth factor-beta2. J. Neurosci. 21:3360–8.CrossRefGoogle Scholar
  19. 19.
    Wan X, Mendoza A, Khanna C, Helman LJ. (2005) Rapamycin inhibits ezrin-mediated metastatic behavior in a murine model of osteosarcoma. Cancer Res. 65:2406–11.CrossRefGoogle Scholar
  20. 20.
    Krishnan K, et al. (2006) Ezrin mediates growth and survival in Ewing’s sarcoma through the AKT/mTOR, but not the MAPK, signaling pathway. Clin. Exp. Metastasis. 23:227–36.CrossRefGoogle Scholar
  21. 21.
    Vingsbo C, et al. (1996) Pristane-induced arthritis in rats: a new model for rheumatoid arthritis with a chronic disease course influenced by both major histocompatibility complex and non-major histocompatibility complex genes. Am. J. Pathol. 149:1675–83.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Brenner M, et al. (2005) The non-MHC quantitative trait locus Cia10 contains a major arthritis gene and regulates disease severity, pannus formation and joint damage. Arthritis Rheum. 52:322–32.CrossRefGoogle Scholar
  23. 23.
    Arnett FC, et al. (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31:315–24.CrossRefGoogle Scholar
  24. 24.
    Chan A, et al. (2007) The GTPase rac regulates the proliferation and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients. Mol. Med. 13:297–304.CrossRefGoogle Scholar
  25. 25.
    Tolboom TC, et al. (2002) Invasive properties of fibroblast-like synoviocytes: correlation with growth characteristics and expression of MMP-1, MMP-3, and MMP-10. Ann. Rheum. Dis. 61:975–80.CrossRefGoogle Scholar
  26. 26.
    Michael IP, et al. (2005) Biochemical and enzymatic characterization of human kallikrein 5 (hK5), a novel serine protease potentially involved in cancer progression. J. Biol. Chem. 280:14628–35.CrossRefGoogle Scholar
  27. 27.
    Ghosh MC, Grass L, Soosaipillai A, Sotiropoulou G, Diamandis EP. (2004) Human kallikrein 6 degrades extracellular matrix proteins and may enhance the metastatic potential of tumour cells. Tumour Biol. 25:193–9.CrossRefGoogle Scholar
  28. 28.
    Ballou LM, Lin RZ. (2008) Rapamycin and mTOR kinase inhibitors. J. Chem. Biol. 1:27–36.CrossRefGoogle Scholar
  29. 29.
    Saemann MD, Haidinger M, Hecking M, Horl WH, Weichhart T. (2009) The multifunctional role of mTOR in innate immunity: implications for transplant immunity. Am. J. Transplant. 9:2655–61.CrossRefGoogle Scholar
  30. 30.
    An JY, et al. (2009) Prognostic role of p-mTOR expression in cancer tissues and metastatic lymph nodes in pT2b gastric cancer. Int. J. Cancer. 126:2904–13.Google Scholar
  31. 31.
    Zhou F, He X, Iwakura Y, Horai R, Stuart JM. (2005) Arthritis in mice that are deficient in interleukin-1 receptor antagonist is dependent on genetic background. Arthritis Rheum. 52:3731–8.CrossRefGoogle Scholar
  32. 32.
    Meng Q, Xia C, Fang J, Rojanasakul Y, Jiang BH. (2006) Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway. Cell. Signal. 18:2262–71.CrossRefGoogle Scholar
  33. 33.
    Zhou X, et al. (2004) Activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers. Clin. Cancer Res. 10:6779–88.CrossRefGoogle Scholar
  34. 34.
    Zhou L, Huang Y, Li J, Wang Z. (2010) The mTOR pathway is associated with the poor prognosis of human hepatocellular carcinoma. Med. Oncol. 27:255–61.CrossRefGoogle Scholar
  35. 35.
    Pantuck AJ, et al. (2007) Prognostic relevance of the mTOR pathway in renal cell carcinoma: implications for molecular patient selection for targeted therapy. Cancer. 109:2257–67.CrossRefGoogle Scholar
  36. 36.
    Machesky LM. (2008) Lamellipodia and filopodia in metastasis and invasion. FEBS Lett. 582:2102–11.CrossRefGoogle Scholar
  37. 37.
    Small JV, et al. (2008) Unravelling the structure of the lamellipodium. J. Microsc. 231:479–85.CrossRefGoogle Scholar
  38. 38.
    Owen KA, et al. (2007) Regulation of lamellipodial persistence, adhesion turnover, and motility in macrophages by focal adhesion kinase. J. Cell Biol. 179:1275–87.CrossRefGoogle Scholar
  39. 39.
    Tilghman RW, et al. (2005) Focal adhesion kinase is required for the spatial organization of the leading edge in migrating cells. J. Cell Sci. 118:2613–23.CrossRefGoogle Scholar
  40. 40.
    Carlson RP, et al. (1993) Rapamycin, a potential disease-modifying antiarthritic drug. J. Pharmacol. Exp. Ther. 266:1125–38.PubMedGoogle Scholar
  41. 41.
    Bruyn GA, et al. (2008) Everolimus in patients with rheumatoid arthritis receiving concomitant methotrexate: a 3-month, double-blind, randomised, placebo-controlled, parallel-group, proof-of-concept study. Ann. Rheum. Dis. 67:1090–5.CrossRefGoogle Scholar
  42. 42.
    Lefevre S, et al. (2009) Synovial fibroblasts spread rheumatoid arthritis to unaffected joints. Nat Med. 15:1414–20.CrossRefGoogle Scholar

Copyright information

© The Feinstein Institute for Medical Research 2010

Authors and Affiliations

  1. 1.Laboratory of Experimental Rheumatology, Center for Genomics and Human GeneticsFeinstein Institute for Medical ResearchManhassetUSA
  2. 2.Elmezzi Graduate School of Molecular MedicineManhassetUSA

Personalised recommendations