FIGURE 1.

Effects of HA depletion on chondrocyte metabolism. Following treatment without or with Streptomyces hyaluronidase (−/+) for 24 h, high density monolayers of bovine chondrocytes (A) or human OA chondrocytes (B) were lysed and analyzed by real time qRT-PCR. The fold changes (y axes) in mRNA copy number for MMP13, ADAMTS4, and TSG6 (A and B) were compared with control-untreated cultures (black bars) and quantified using the ΔΔCt approach with normalization to GAPDH. Values represent the fold increase and the average ± S.D. of data derived from three independent experiments. A two-tailed unpaired Student's t test was used for comparison of treatment group to control; *, p < 0.05; **, p < 0.01. Cell lysates and serum-free media from other human OA chondrocyte cultures were also processed for Western blotting analysis of MMP13, TSG6, and ADAMTS4 protein (C). Shown is a representative example of three replicated, independent experiments. Aliquots of equal protein were loaded onto gels from the cell lysate fractions, and the medium samples represent aliquots of equal volume. D–F, human OA chondrocytes were treated without or with 0.5 to 2.0 mm 4-MU for 24 h. Serum-free media were collected and processed for HA ELISA (D, values represent percent of control shown as 100%) or Western blot analysis of MMP13 protein (F). Cell lysates were collected either for quantitative real time RT-PCR analysis of MMP13 mRNA (E) or Western blot analysis of MMP13 protein (F); both using a similar approach as detailed for B and C. All blots with cell lysates were stripped and re-probed for β-actin. For statistical analysis of data shown in D and E, a one-way ANOVA followed by Dunnett's test was used; *, p < 0.05; **, p < 0.01.