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Abstract
Objective
βig-h3 is an extracellular matrix protein, which is overexpressed in synovial tissues of rheumatoid arthritis (RA) similar to adhesive glycoproteins. We sought to evaluate the compensatory role of βig-h3 with adhesive glycoproteins in mediating the adhesion of fibroblast-like synoviocytes (FLS) and to confirm the inhibitory effect of YH18 peptide of the 2nd fas-1 domain in βig-h3-mediated adhesion.
Methods
The adhesion of FLS isolated from synovial tissues of RA, was evaluated in 96 well microtiter plate coated with matrix proteins. Inhibitory effect of YH18 peptides from the 2nd and 4th fas-1 domains was estimated in βig-h3-mediated adhesion of FLS.
Results
The adhesion of FLS on βig-h3 was weaker than that of fibronectin and vitronectin. The βig-h3-mediated adhesion was enhanced by the stimulation with phorbol myristate acetate (PMA), but not by cytokines and growth factors. Combination of fibronectin with βig-h3 synergistically enhanced the adhesion of FLS, in contrast to the additive effect of vitronectin combined with βig-h3. YH18 peptide of the 2nd fas-1 domain did not block the βig-h3-mediated adhesion of FLS.
References
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Fig. 1.
Comparison of βig-h3, vitronectin, and fibronectin in adhesion of human FLS. FLS were seeded onto surfaces of 96-well culture plates which were precoated with different concentrations (0.1 ug/mL, 1 ug/mL and 10 ug/mL) of βig-h3, vitronectin, or fibronectin and then were incubated for 2 hours at 37oC. After seeding and incubation, FLS attached to the surfaces were quantified by hexosaminidase assay. Values are presented as the mean±SEM of triplicate experiments.
Fig. 2.
Regulation of βig-h3-mediated adhesion of FLS by cytokines, growth factors, and nonspecific stimulants. FLS were treated with different cytokines (recombinant human TNF-α, IL-1β, IL-6, IL-4, IL-10, IFN-γ, and TGF-β1), growth factors (VEGF, IGF-1, EGF, PDGF-BB, and FGF-basic), and nonspecific cell stimulants (PMA and LPS) for 30 minutes at 37oC. Pretreated FLS were added to the coated wells with 5 ug/mL of βig-h3 and were incubated for 2 hours at 37°C. After seeding and incubation, FLS attached to the surfaces were quantified by hexosaminidase assay. TGF-β1, 5 ng/mL; TNF-α, IL-1β, IL-4, IL-6, IL-10, VEGF, IGF-1 and EGF, 10 ng/mL; PDGF-BB, FGF-basic, and IFN-γ, 100 ng/mL; PMA, 60 ng/mL; and LPS 5 μg/mL. Values are presented as the mean±SEM of triplicate experiments.
Fig. 3.
Compensatory role of βig-h3 with matrix proteins in mediating adhesion of FLS. FLS were added to the precoated 96-well culture plates coated with (A) βig-h3 and fibronectin or (B) βig-h3 and vitronectin, and were incubated for 2 hours at 37oC. Low concentration (0.1 ug/mL) of fibronectin, 1 ug/mL of vitronectin, and different concentrations (0.1 ug/mL, 1 ug/mL, and 10 ug/mL) of βig-h3 were used. After incubation, FLS attached to the surfaces were quantified. Values are presented as the mean±SEM of, at least, 3 independent experiments.
Fig. 4.
Identification of the motif mediating FLS adhesion to βig-h3. The 2nd YH18 peptide (spanning amino acids 301∼318 in the 2nd fas-1 domain), 4th YH18 peptide (spanning amino acids 563∼580 in the 4th fas-1 domain), and YH18 control peptide (containing the amino acids of the 4th YH18 peptide but in a scrambled order) were prepared. FLS were preincubated with the YH18 control, 2nd YH18, or 4th YH18 peptides at different concentrations (0, 10, 50, 100, and 500 uM) for 30 minutes at 37oC and then added to the βig-h3-coated wells. Values are presented as the mean±SEM of at least 3 independent experiments. ∗p＜0.05, 4th YH18 peptide versus YH18 control peptide.
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