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. 2008 Aug;58(8):2296-306.
doi: 10.1002/art.23610.

The arthritis severity locus Cia5d is a novel genetic regulator of the invasive properties of synovial fibroblasts

Teresina Laragione  1 , Max Brenner, Adriana Mello, Marc Symons, Pércio S Gulko
Affiliations

The arthritis severity locus Cia5d is a novel genetic regulator of the invasive properties of synovial fibroblasts

Teresina Laragione et al. Arthritis Rheum. 2008 Aug.

Abstract

Objective: The synovial fibroblast, or fibroblast-like synoviocyte (FLS), has a central role in pannus invasion and destruction of cartilage and bone in rheumatoid arthritis (RA). However, regulation of the FLS remains incompletely understood. The aim of this study was to determine whether the invasive properties of FLS are genetically regulated by arthritis severity loci.

Methods: DA rats (arthritis susceptible) and rat strains congenic for arthritis-protective intervals were studied. Primary FLS cell lines were generated from each strain and used in a well-established FLS invasion model through a collagen-rich barrier. Cells or culture supernatants were analyzed for gene expression, activity of different matrix metalloproteinases (MMPs), cytoskeleton integrity, and cell proliferation.

Results: The median number of FLS from DA.F344(Cia5d) rats that invaded through the collagen-rich barrier was reduced 86.5% compared with the median number of invading FLS from DA rats. Histologic examination showed that DA.F344(Cia5d) rats preserved a normal joint without pannus, hyperplasia, or erosions. FLS from DA.F344(Cia5d) rats produced significantly lower levels of active MMP-2 compared with FLS from DA rats, but the levels of proMMP-2 and MMP-2 messenger RNA in DA.F344(Cia5d) rats were similar to those in DA rats. Treatment of FLS from DA rats with an MMP-2 inhibitor reduced cell invasion to a level similar to that in DA.F344(Cia5d) rats, demonstrating that MMP-2 activity accounted for the difference between FLS from these 2 strains. Analysis of MMP-2-activating pathways revealed increased levels of soluble membrane type 1 (MT1)-MMP in DA rats compared with DA.F344(Cia5d) rats.

Conclusion: These data represent the first evidence for a genetic component in the regulation of FLS invasion. A gene located within the Cia5d interval accounts for this effect and operates via the regulation of soluble MT1-MMP production and MMP-2 activation. These observations suggest novel potential pathways for prognostication and therapy.

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Figures

Figure 1
Figure 1
Map of the congenic intervals, and the effect of the Cia5d locus on fibroblast-like synoviocyte (FLS) invasion. A, Markers used in the breeding of DA.ACI(Cia10), DA.F344(Cia4), and DA.F344(Cia5d) rats are shown, along with their respective positions on chromosomes 2, 7, and 10, and genotypes. Numbers inside the bars represent the position in the chromosome (megabases). B, FLS from DA.F344(Cia5d) rats (passage range 6–9 [median 7.5]) had a significant reduction of 86.5% in the median number of invading cells, compared with FLS from DA rats (passage range 6–16 [median 8]). The median number of invading cells from DA.F344(Cia4) rats and that of invading cells from DA.ACI(Cia10) rats were not significantly lower than that for DA rats. (Experiments were performed with BD Matrigel invasion chambers.) * = P ≤ 0.001 versus DA rats, by Mann-Whitney test. C, The increased invasive properties of FLS from DA rats require exposure to pristane, as FLS from naive DA rats have invading properties similar to those of FLS from naive rats and DA.F344(Cia5d) rats with pristane-induced arthritis (PIA). Therefore, F344 alleles at the Cia5d interval render cells resistant to pristane-induced increased invasive properties. (Experiments were performed with Chemicon Matrigel invasion chambers. Samples were run on 2 separate days, and absorbance was normalized according to a set of controls/duplicates run on both days.) * = P = 0.018; # = P = 0.002 versus DA rats with PIA, by Mann-Whitney test. Bars in B and C are the 25th and 75th percentiles. D and E, Ankle joints from DA rats (D) and DA.F344(Cia5d) rats (E) were collected on day 32 after initiation of PIA. Synovial hyperplasia was extensive in DA rats, with cartilage and bone erosion, while synovium in DA.F344(Cia5d) rats was normal, with no erosions (arrows). (Hematoxylin and eosin stained; original magnification × 100.)
Figure 2
Figure 2
Effect of the matrix metalloproteinase inhibitor GM6001 on the invasive properties of fibroblast-like synoviocytes (FLS) derived from DA rats and FLS from DA.F344(Cia5d) rats. FLS from DA and DA.F344(Cia5d) were plated on Matrigel-coated invasion chambers, with or without 25 µM GM6001, for 24 hours. The absorbance of invading cells from DA (n = 12) was significantly reduced in the presence of GM6001. The absorbance of invading cells from DA.F344(Cia5d) without GM6001 (n = 7) was significantly lower than that of cells from DA without GM6001 and was similar to that of cells from DA treated with GM6001. Bars show the mean and SD. * = P = 0.018; # = P = 0.004 versus FLS from DA without GM6001, by t-test.
Figure 3
Figure 3
Levels of matrix metalloproteinase (MMP) mRNA, collagenase activity, and MMP-3 activity in fibroblast-like synoviocytes (FLS) from DA rats and DA.F344(Cia5d) rats. A, Messenger RNA levels of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, membrane type 1 (MT1)–MMP, and MT2-MMP were not significantly different in FLS cell lines from DA rats and FLS cell lines from DA.F344(Cia5d) rats, as determined by quantitative polymerase chain reaction. B, Collagenase activity was nearly identical in the supernatants of FLS cultures from DA rats (n=7) and DA.F344(Cia5d) rats (n = 7). Values in A and B are the mean and SD. C, Casein zymography revealed similar levels of MMP-3 in the supernatants of FLS cultures from DA rats (n = 4) and DA.F344(Cia5d) rats (n = 4). ΔCt = change in threshold cycle.
Figure 4
Figure 4
Reduced MMP-2 activity in DA.F344(Cia5d) explains the reduced invasive properties of FLS. A, Gelatin zymography showed significantly lower levels of active MMP-2 in supernatants of DA.F344(Cia5d) FLS (n = 6) compared with DA FLS (n = 7) after 24 hours in culture. The results are representative of experiments involving 11 DA and 8 DA.F344(Cia5d) rats, in which 4 µg of protein per FLS cell line supernatant per lane was loaded. B, Gelatin zymography of supernatants of DA, DA.F344(Cia5d), and F344 FLS showed reduced levels of activated MMP-2 in DA.F344(Cia5d) and F344 rats compared with DA rats (8 µg of protein per FLS cell line supernatant per lane was loaded). C, Gelatin zymography of supernatants of naive DA FLS (n = 5) and naive DA.F344(Cia5d) FLS (n = 4) demonstrated low levels of active MMP-2, similar to those in FLS from DA.F344(Cia5d) rats with pristane-induced arthritis (PIA) but significantly lower than those in FLS from DA rats with PIA (8 µg of total protein per cell line culture supernatant was loaded). D, Treatment with the MMP-2 inhibitor SB-3CT (compared with DMSO as control) significantly reduced the invasive properties of FLS from DA rats to levels similar to those of FLS from untreated DA.F344(Cia5d). DA.F344(Cia5d) FLS treated with SB-3CT also exhibited a modest reduction in invasive properties, consistent with low MMP-2 activation. Values are the mean and SD results for 5 different cell lines per group. * = P ≤ 0.001 versus untreated DA rats; # = P = 0.027 versus untreated DA rats; ¶ = P = 0.008 versus untreated DA.F344(Cia5d) rats, by t-test. See Figure 3 for other definitions.
Figure 5
Figure 5
Soluble and plasma membrane type 1 matrix metalloproteinase (MT1-MMP) in fibroblast-like synoviocytes (FLS). A, DA FLS cultured for 24 hours had reduced MT1-MMP activity in plasma membranes, compared with DA.F344(Cia5d) FLS. Values are the median and 25th and 75th percentiles. P = 0.052, by Mann-Whitney test. B, Western blotting showed increased levels of soluble MT1-MMP (sMT1-MMP) in supernatants of DA rat FLS (n = 3) compared with DA.F344(Cia5d) rat FLS (n = 3) cultured on Matrigel-coated Petri dishes (5 µg of protein loaded per lane). The data for sMT1-MMP were replicated in 3 additional FLS cell culture supernatants per strain.
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