Figure 7. Expression of pro-inflammatory and anti-inflamma- tory cytokines in human OA cartilage. Total RNA was isolated from human cartilage samples obtained from early OA and late OA regions and the expression of target genes were analyzed by qPCR. Target genes in early OA were compared to late OA and expressed as fold induction. Values are mean 6 SD of 3 experiments per parameter per joint per patient. doi:10.1371/journal.pone.0034020.g007 XT-I gene expression is regulated by IL-1b and TGF-b1 in cartilage Beside its ability to induce degradation of articular cartilage, the proinflammatory cytokine IL-1b has been shown to suppress the synthesis of PGs by chondrocytes [28,29]. In contrast the growth factor TGF-b1 is a potent inducer of chondrocyte matrix deposition [30–32]. We investigated whether XT-I gene expres- sion is regulated by IL-1b and TGF-b1. Cartilage explants were treated or not with IL-1b and TGF-b1, respectively and XT-I expression was analyzed using quantitative PCR. Our result showed that IL-1b down-regulated the expression of XT-I by about 50% (Fig. 8A). In contrast, TGF-b1 induced the expression by about 3-fold (Fig. 8B). These results clearly indicated that XT-I gene expression is regulated by the two main cytokines involved in cartilage metabolism. Figure 8. Effect of IL-1b (A) and TGF-b1 (B) on XT-I gene expression in normal articular cartilage. Cartilage samples isolated from normal regions of human femoral condyle was exposed to IL-1b or TGF-b1 and then XT-I gene expression was analyzed by qPCR. Measurements were normalized to control (non treated). Each value represents the mean 6 SD of 3 experiments per parameter per joint per patient. *Significantly (P,0.05) lower than control group; #significantly (P,0.01) higher than control group. doi:10.1371/journal.pone.0034020.g008 XT-I and Proteoglycan Synthesis in Osteoarthritis Discussion Our present findings demonstrate that XT-I is likely an important factor in the regulation of susceptibility of humans to cartilage degeneration during OA development and progression. Recently, we showed that loss of XT-I expression increases propensity to loss of sulfated-GAG synthesis and deposition, leading to cartilage destruction in antigen-induced arthritis in rats [13]. Conversely, interruption with XT-I expression disrupts cartilage repair in papain-injected knee joints of rats, providing evidence to date that XT-I plays a key role in replacement of sulfated-GAGs in joint diseases [13]. In order to get more insights, we explore whether XT-I is an important player in regulating sulfated-GAG synthesis and content in human cartilage. Based on the recognition of wide variation in the degenerative changes in articular cartilages of OA, the use of grading system pioneered by Mankin et al [15] provides a critical assessment of the severity of the OA lesion and the metabolic activity of articular cartilage in OA. There are numerous studies which attempted to correlate biosynthesis changes of aggrecan in OA with patholog- ical findings. The studies reported by Thompson and Oegema [33] reported a decrease in the content of GAG as the histological grade of arthritis increased. In addition studies by Rizkalla et al [34] also reported that in early disease (low grade) there was an increase in GAG synthesis, whereas in late disease it was decreased. Interestingly, using cartilage obtained from different disease areas of femoral condyle from the same OA subject, we describe here, for the first time, the identification of pathology- related alterations in the gene expression and activity of XT-I. The levels of XT-I expression correlated with the severity of degeneration in OA joints. This finding strongly indicated that cartilage close to lesion expressed higher levels of XT-I and increased GAG content, which would explain the high synthesis and content of GAGs in early stages of degeneration. We also showed the association of low expression of the XT-I gene in diseased cartilage where the GAG content was demonstrably reduced. Thus, loss of XT-I expression may set the stage for gradual reduction in sulfated-GAG synthesizing ability of chondrocytes, displaying abnormalities in compensating for the increased degradation of PG-GAGs during OA development. Collectively, our present findings allowed us to extract clinically meaningful information from XT-I gene expression profile and suggest that the pathologic changes involving PG-GAGs in OA may relate to the degree of alteration of XT-I expression and activity. A striking feature of this study is the upregulation of XT-I gene expression, which becomes significant in view of the present results on human studies indicating that mRNA levels of aggrecan core protein are also increased in early OA. Increased availability of core protein, in the presence of increased XT-I activity, would be expected to increase the transfer of xylose onto core protein acceptor and facilitate the initiation of new CS-GAG chains. This might explain the increase in sulfated-GAG synthesis observations and could offer an explanation for the increase in chondrocyte’s capacity to synthesize CS-GAG in early degeneration in OA. PG-GAG turnover in the cartilage is regulated by a class of proinflammatory and anti-inflammatory cytokines [4,5]. The proinflammatory cytokine IL-1b was found to induce cartilage loss by reducing PG synthesis [28,29,35] and its effects are mediated through a high-affinity cell surface IL-1 receptor type I (IL-1RI) [36–38]. Interestingly, our data showed that IL-1RI gene expression was down-regulated in cartilage ‘‘next to lesion’’ compared to late-OA cartilage, indicating a mechanism protecting cartilage against IL-1b-induced effects. In parallel to this PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e34020