Furthermore, analysis of sulfated GAG synthesis showed about 2- fold increase of 35S-sulfate incorporation in XT-I-transfected chondrocytes compared to empty vector-transfected cells (Fig. 5B) indicating that overexpression of XT-I significantly enhanced sulfated GAG synthesis in chondrocytes (probably by increasing the number of GAG chains that are initiated on a core protein). These results suggested that XT-I enzyme catalyzes a rate-limiting step in sulfated GAG synthesis pathway. Interestingly, transfection of cartilage explants from different patient with pCMV-XT-I vector produced a 2- to 3.5-fold increase in sulfated GAG synthesis compared to control (Fig. 5C). These results collectively show that XT-I regulates GAG anabolism in cartilage. Expression of IL-1b receptor was down-regulated while TGF-b1 was up-regulated in early OA cartilage To determine the link between alteration of XT-I expression and the pathologic state of the disease in OA cartilage potential molecular mechanisms involved were investigated. Proinflamma- tory cytokine IL-1b and growth factor TGF-b1 regulate GAG anabolism and expression of various factors in chondrocytes. Indeed, treatment of cartilage explants with IL-1b reduced GAG anabolism by about 70% (Fig. 6A). In contrast, TGF-b1 treatment XT-I and Proteoglycan Synthesis in Osteoarthritis led to an increase of GAG synthesis of about 2-fold (Fig. 6B). FACE analysis confirmed that TGF-b1 stimulated cartilage CS- GAG synthesis whereas IL-1b treatment inhibited this process (Fig. 6C). Interestingly, gene expression analysis indicated significant downregulation of IL-1b receptor (IL-1R1) (3-fold) in early OA cartilage compared to late-stage OA cartilage (Fig. 7). This was associated with an increase of about 2.5-fold in the expression of the anabolic factor TGF-b1, respectively (Fig. 7). These results suggested that cartilage next to lesion not only triggers a mechanism counteracting the effect IL-1b but also stimulates the anabolic activity of chondrocytes by inducing the expression of TGF-b1. Figure 5. Effect of XT-I overexpression on GAG anabolism in chondrocytes and cartilage. (A) Immunoblot of XT-I protein in pCMV- or pCMV-XT-I-transfected chondrocytes 48 h after transfection. XT-I recombinant protein was indicated by the arrow. (B) GAG synthesis in primary human chondrocytes or (C) in cartilage explants from late OA region of human femoral condyle transfected with pCMV- (control) or pCMV-XT-I vector. XT-I- or mock-transfected cells or cartilage were labeled with 35S-sulfate, and the amount of 35S-labeled GAGs was measured 48 h posttransfection. Data are mean 6 SD of 3 experiments per parameter, per joint, per patient. **,*Significantly (P,0.01, P,0.05, respectively) higher than controls. doi:10.1371/journal.pone.0034020.g005 Figure 6. Analysis of the effect of IL-1b and TGF-b1 on GAG synthesis and GAG chain composition. Cartilage explants from normal regions of human femoral condyle were exposed to IL-1b (A) or TGF-b1 (B) for 24 h and then GAG synthesis was measured by 35S- sulfate incorporation. Measurements were normalized to control (non treated). Data are mean 6 SD of 3 experiments per parameter, per joint, per patient. **Significantly (P,0.0018) lower than controls; $significantly (P,0.05) higher than controls. (C) Effect of IL-1b and TGF-b1 on GAG chain composition in human cartilage. Cartilage samples derived from normal regions of human femoral condyle were treated with cytokines for 12 h and analyzed by FACE. Gels are representative images from 3 experiments per parameter, per joint, per patient. Markers are DDi0S, nonsulfated chondroitin-unsaturated disaccharide; DDi4S, chondroitin- 4-sulfated unsaturated disaccharide; DDi6S, chondroitin-6-sulfated unsaturated disaccharide. doi:10.1371/journal.pone.0034020.g006 PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e34020