M. Durigova, M.Sc., P.J. Roughley, Ph.D., J.S. Mort, Ph.D.

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Mechanism of proteoglycan aggregate degradation in cartilage stimulated with oncostatin M M. Durigova, M.Sc., P.J. Roughley, Ph.D., J.S. Mort, Ph.D. Osteoarthritis and Cartilage Volume 16, Issue 1, Pages 98-104 (January 2008) DOI: 10.1016/j.joca.2007.05.002 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 GAG release from cartilage cultures in response to cytokine stimulation. Bovine articular cartilage explants were cultured with the cytokines IL-1β (upper panel), IL-1α (middle panel), or TNFα (lower panel) with or without OSM. Results are shown for cytokine alone (hatched bars), OSM alone (gray bars), both factors (solid bars) or no factor (open bars). Culture media collected after each 2-day medium change were analyzed for GAG content by the DMMB dye-binding assay. *P<0.05. Osteoarthritis and Cartilage 2008 16, 98-104DOI: (10.1016/j.joca.2007.05.002) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Analysis of aggrecan G1 generation. Samples of cartilage extracts after 6 days of culture (upper panel) and pooled media collected at days 2, 4 and 6 of culture (lower panel) were treated with chondroitinase ABC and keratanase II, then analyzed by SDS/PAGE and immunoblotting using an antibody to the aggrecan G1 domain. Lane 1, control; lane 2, IL-1β; lane 3, IL-1β+IL-6; lane 4, IL-1β+IL-17; lane 5, IL-1β+OSM; lane 6, IL-1β+IL-6+IL-17+OSM; lane 7, OSM; lane 8, TNFα; lane 9, TNFα+OSM. The migration position of aggrecanase-generated free G1 is indicated on the right. Migration positions of molecular weight standards are indicated on the left. Results shown in lanes 1–6 and 7–9 represent two different experiments. Osteoarthritis and Cartilage 2008 16, 98-104DOI: (10.1016/j.joca.2007.05.002) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Analysis of LP. Samples of cartilage extracts (upper panel) and medium pooled from 6 days of culture (lower panel) were treated with chondroitinase ABC and keratanase II, then analyzed by SDS/PAGE and immunoblotting using a mouse monoclonal antibody to LP. Lane 1, control; lane 2, IL-1β; lane 3 IL-1β+IL-6; lane 4, IL-1β+IL-17; lane 5, IL-1β+OSM; lane 6, IL-1β+IL-6+IL-17+OSM; lane 7, OSM; lane 8, TNFα; lane 9, TNFα+OSM. Migration positions of molecular weight standards and LP are indicated. Additional bands seen in the medium samples are due to non-specific reaction of the second step antibody with contaminants in the BSA used as a carrier for the cytokines in the culture medium. Results shown in lanes 1–6 and 7–9 represent two different experiments. Osteoarthritis and Cartilage 2008 16, 98-104DOI: (10.1016/j.joca.2007.05.002) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Release of HA from cartilage explants. Medium samples were reduced and alkylated and HA content was measured using a competitive binding assay. Cytokine combinations used are indicated in the inset. *P<0.05. Osteoarthritis and Cartilage 2008 16, 98-104DOI: (10.1016/j.joca.2007.05.002) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Analysis of HA size. Medium samples taken at day 4 of culture were analyzed by agarose gel electrophoresis, followed by capillary blotting to CPC-treated nitrocellulose membranes and visualization using a biotinylated HABP. Lane 1, control; lane 2, OSM; lane 3, IL-1β; lane 4, IL-1β+OSM; lane 5, IL-1α; lane 6, IL-1α+OSM; lane 7, cartilage digest. Osteoarthritis and Cartilage 2008 16, 98-104DOI: (10.1016/j.joca.2007.05.002) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Inhibition of hyaluronidase activity. Bovine articular cartilage explants were cultured alone (open bar), in the presence of IL-1β+OSM (solid bar), or in the presence of IL-1β+OSM+apigenin (100μM, gray bar). Media from days 2 and 4 of culture were pooled, reduced and alkylated and analyzed for HA content. *P<0.05. Osteoarthritis and Cartilage 2008 16, 98-104DOI: (10.1016/j.joca.2007.05.002) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Analysis of aggrecan G3-containing components. Samples of cartilage extracts (upper panel) and medium pooled from 6 days of culture (lower panel) were treated with chondroitinase ABC and keratanase II, then analyzed by SDS/PAGE and immunoblotting using an antibody to the aggrecan G3 domain. Lane 1, control; lane 2, IL-1β; lane 3, IL-1β+IL-6; lane 4, IL-1β+IL-17; lane 5, IL-1β+OSM; lane 6, IL-1β+IL-6+IL-17+OSM; lane 7, OSM; lane 8, TNFα; lane 9, TNFα+OSM. Positions of molecular weight standards are indicated on the left and the position of aggrecanase-generated G3-containing components due to CS2 cleavage is indicated on the right. The additional band (∼81kDa) seen in lanes 7–9 is due to non-specific reaction of the second step antibody with contaminants in some BSA preparations used as a carrier for the cytokines in the culture medium. Results shown in lanes 1–6 and 7–9 represent two different experiments. Osteoarthritis and Cartilage 2008 16, 98-104DOI: (10.1016/j.joca.2007.05.002) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions