Abstract
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 ezrin-mTOR 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.
Highlights
Rheumatoid arthritis (RA) is a common chronic autoimmune disease that affects approximately 1% of the population and is commonly associated with disability and deformities [1,2]
Rapamycin 10 μmol/L significantly reduced the median number of invading fibroblast-like synoviocytes (FLS) by 93% (n = 6; P = 0.002, Mann–Whitney test, Figure 1)
FLS were fixed with 4% formaldehyde (Ted Pella, Redding, CA, USA) for 15 min at room temperature followed by blocking with 5% nonfat milk for 30 min
Summary
Rheumatoid arthritis (RA) is a common chronic autoimmune disease that affects approximately 1% of the population and is commonly associated with disability and deformities [1,2]. The basic joint pathology in RA is characterized by pronounced synovial hyperplasia, called pannus, which produces several proinflammatory cytokines and proteases, and like a malignant tumor, invades and destroys cartilage and bone [2,3,4]. Called fibroblast-like synoviocytes (FLS), have a central role in the formation of pannus, and in cartilage and bone invasion and destruction [2,5]. The invasive properties of RA FLS have been studied in vitro, and correlate with radiographic evidence of damage in RA [6], which is a well-established severity and outcome parameter. The regulation of the invasive properties of FLS remains incompletely understood. We have previously reported our discovery that Cia5d is a genetic regulator of disease severity, bone and cartilage destruction, and FLS invasion in rat arthritis [7,8]. Microarray analyses of gene expression to compare highly invasive FLS from arthritis-susceptible
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