N Burton-Wurster, Ph. D. , W Liu, M. D. , Ph. D. , G. L Matthews, D. V

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TGF beta 1 and biglycan, decorin, and fibromodulin metabolism in canine cartilage N Burton-Wurster, Ph.D., W Liu, M.D., Ph.D., G.L Matthews, D.V.M., Ph.D., G Lust, Ph.D., P.J Roughley, Ph.D., T.T Glant, M.D., Ph.D., G Cs-Szabó, Ph.D. Osteoarthritis and Cartilage Volume 11, Issue 3, Pages 167-176 (March 2003) DOI: 10.1053/S1063-4584(02)00349-7

Fig. 1 Autoradiograph of small PGs in control and TGF beta 1-treated cartilage explants. This figure contrasts newly synthesized small PGs in control cultures after 2 days with small PGs in TGF beta 1-treated cultures after 16 days. Small PGs were purified by DEAE anion exchange chromatography and analyzed by SDS/PAGE and autoradiography. From left to right, lanes 1–4 show anion exchange fractions of proteins labeled with 35SO4-GAG and lanes 5–8 show anion exchange fractions of proteins labeled with a combination of 3H-leucine and 35S-methionine. The numbers between lanes 1–4 and 5–8 indicate the positions of molecular weight markers. Fractions in lanes 1, 3, 5, and 7 were digested with chondroitinase ABC prior to electrophoresis (+C). Fractions in lanes 2, 4, 6, and 8 were intact (−C). Lanes 1, 2, 7, and 8 are 1.0M NaCl DEAE fractions from 16-day TGF beta 1-treated cultures (TGF d16). Lanes 3, 4, 5, and 6 are 0.3M NaCl DEAE fractions from 2-day control cultures (Contr d2). Increased synthesis of small PG in response to TGF beta 1 is apparent (compare lane 2 with lane 4, and lane 8 with lane 6). The data also show that both small PGs in day 2 control cultures, and the highest molecular weight small PGs in day 16 TGF beta-treated cultures were completely digested by chondroitinase ABC (see lanes 1 and 3, and lanes 5 and 7), leaving core proteins of identical molecular weight (compare lane 5 with lane 7). The open arrow points to intact decorin. The closed arrow points to intact, high molecular weight decorin synthesized in the presence of TGF beta 1. Osteoarthritis and Cartilage 2003 11, 167-176DOI: (10.1053/S1063-4584(02)00349-7)

Fig. 2 Western analysis of decorin in control and TGF beta 1-treated cartilage. Decorin present in cartilage extract was analyzed by SDS/PAGE and immunoblotting as the native molecule (panel 1) and after removal of GAG chains by chondroitinase ABC digestion (panel 2). Lanes 1 and 2 (duplicates) analyze 16-day control cultures. Lanes 3 and 4 (duplicates) analyze samples from TGF beta 1-treated samples after 16 days in culture. Native samples were blotted to a positively charged membrane (Hybond–N+, panel 1). Core proteins, resulting from digestion with chondroitinase ABC, were blotted to nitrocellulose (panel 2). The blots were probed with a rabbit polyclonal antibody raised against decorin C-terminal peptide sequences. The arrowheads in panel 1 point to a modest accumulation of HMW decorin in TGF beta 1-treated culture. Osteoarthritis and Cartilage 2003 11, 167-176DOI: (10.1053/S1063-4584(02)00349-7)

Fig. 3 Western analysis of fibromodulin in control and TGF beta 1-treated cartilage. Fibromodulin present in cartilage extracts was analyzed with a series of enzymatic digestions as described in section Methods, in order to evaluate the extent of substitution with KS and non-sulfated oligosaccharides. Analysis was done by SDS/PAGE and immunoblotting. Lanes 1, 2 (duplicates) and 5, 6 (duplicates) are controls. Lanes 3, 4 (duplicates) and 7, 8 (duplicates) are TGF beta-treated. Lanes 1–4 analyze 2-day cultures. Lanes 5–8 analyze 16-day cultures. Note: the band that appears in all blots above 100kD is non-specific. Native PGs were blotted to positively charged nylon (panel 1) or nitrocellulose (panel 2). Nitrocellulose was also used for blotting following digestion of native PGs with chondroitinase ABC and keratanase II (panel 3) plus N-glycanase (panel 4). KS-substituted GAGs (wide bands) can be seen in panel 1, and more faintly in panel 2. Fibromodulin, after removal of KS, has a molecular weight of 66kDa (panel 3), indicating substitution with N-linked constituents, which can be removed by N-glycanase, leaving the core proteins (panel 4). Fibromodulin is present in partially KS-, partially non-sulfated oligosaccharide-substituted forms, but not as a free-core protein. Osteoarthritis and Cartilage 2003 11, 167-176DOI: (10.1053/S1063-4584(02)00349-7)

Fig. 4 Effect of explant culture on content of small PGs in articular cartilage. The figure represents a quantitative comparison of each of the small PGs in control cultures between 2 days (white bars) and 16 days (black bars) of incubation. At days 2 and 16, cartilage was collected and extracted with guanidinium chloride. Extracts were digested with chondroitinase ABC, or chondroitinase ABC followed by keratanase II, prior to electrophoresis on 12% SDS gels, and blotting to nitrocellulose membranes. The blots were probed with rabbit polyclonal antibodies and immunoreactive material was detected with ECL. Results of quantitation of the signal on the film are presented graphically. The average of all replicates for day 2 control cultures was set to 100. The data are presented as mean±standard deviation. (n=4 for decorin and biglycan; n=2 for fibromodulin). No significant change with time was noted. Osteoarthritis and Cartilage 2003 11, 167-176DOI: (10.1053/S1063-4584(02)00349-7)

Fig. 5 Accumulation of decorin, biglycan, and fibromodulin in articular cartilage in response to TGF beta 1. The figure represents a quantitative comparison of each of the small PGs after 16 days in culture in the absence (white bars) and in the presence (black bars) of TGF beta 1. Core proteins were analyzed by Western blotting as described inFig. 4. Results of quantitation are presented graphically for day 16 samples. Data were obtained from two different experiments for decorin and biglycan and from three experiments for fibromodulin. The average of all replicates for day 16 control cultures was set equal to 100 in each experiment. The data are presented as mean±standard deviation (n=7 for decorin and biglycan; n=8 and 7, respectively, for fibromodulin). ∗indicates differences that were significant at P<0.05. Osteoarthritis and Cartilage 2003 11, 167-176DOI: (10.1053/S1063-4584(02)00349-7)