protection mechanism, cartilage ‘‘next to lesion’’ produced more TGF-b1, a potent growth factor for cartilage metabolism [39], than late-OA cartilage. Accordingly, previous studies reported that TGF-b1 levels are increased in the first stage of the disease [40]. Although these studies indicated that alterations in PG-GAG anabolism can be influenced by these cytokines, the molecular basis for these diverse actions are poorly understood. Along these lines, our study found that IL-1b may promote cartilage loss by decreasing the expression of XT-I gene with a concomitant reduction in sulfated-GAG synthesis and content in cartilage explants. In this regard, previous studies [41–43], including work from our laboratory [13] have shown IL-1b to down-regulate aggrecan core protein, XT-I gene and GAG synthesis in cartilage. In contrast, TGF-b1 treatment results in activation of XT-I gene expression followed by increased sulfated-GAG levels in cartilage explants. Interestingly, regulation of XT-I by TGF-b1 has been also demonstrated in other cell types such as astrocytes [44] and cardiac fibroblasts [45]. It can thus be postulated that the anabolic differences exhibited by IL-1b and TGF-b1 in cartilage explants could originate from their ability to alter XT-I as well as aggrecan core protein expression, with obvious antagonistic relationship to one another in terms of cartilage destruction or repair, respectively. Based on our data, it seems likely that up-regulation of XT-I and sulfated-GAG anabolism in cartilage ‘‘next to lesion’’ was a consequence of combined effects of a protective mechanism against IL-1b and stimulatory effects of TGF-b1. To unmask the role of XT-I in sulfated-GAG formation and cartilage homeostasis, we studied the effects of targeted suppres- sion of XT-I with gene silencing strategy in chondrocytes and cartilage explants. Our data demonstrates that transfection of chondrocytes with XT-I shRNA reduced their ability to synthesize sulfated-GAGs compared to normal controls. We also provide evidence that cartilage explants in which XT-I expression was silenced by shRNA synthesized lesser amount of sulfated-GAGs compared to non-transfected controls. These results confirm our prediction implicating XT-I as an important enzymatic source of sulfated-GAG production. From the clinical perspective, development of a targeted therapy with potential cartilage reparative properties is highly significant. Therefore, we wished to determine whether XT-I overexpression represented a valuable therapeutic option to stimulate sulfated-GAG synthesis in OA. We found that forced References 1. Heinegard D, Saxne T (2011) The role of the cartilage matrix in osteoarthritis. Nature Reviews Rheumatology 7: 50–56. 2. Mankin HJ, Lippiello L (1970) Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. J Bone Joint Surg Am 52: 424–434. 3. Pratta MA, Yao W, Decicco C, Tortorella MD, Liu RQ, et al. (2003) Aggrecan protects cartilage collagen from proteolytic cleavage. J Biol Chem 278: 45539–45545. 4. Kapoor M, Johanne Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H (2011) Role of proinflammatory cytokines in the pathophysiology of osteoar- thritis. osteoarthritis. Nature Reviews Rheumatology 7: 33–42. 5. Goldring MB, Otero M, Plumb DA, Dragomir C, Favero M, et al. (2011) Roles of inflammatory and anabolic cytokines in cartilage metabolism: signals and multiple effectors converge upon MMP-13 regulation in osteoarthritis. Eur Cell Mater 21: 202–220. 6. Hardingham TE, Bayliss MT, Rayan V, Noble DP (1992) Effects of growth factors and cytokines on proteoglycan turnover in articular cartilage. Br J Rheumatol 31 Suppl 1: 1–6. 7. Roughley PJ (2006) The structure and function of cartilage proteoglycans. Eur Cell Mater 12: 92–101. 8. Iozzo RV (1998) Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 67: 609–652. 9. Hermansson M, Sawaji Y, Bolton M, Alexander S, Wallace A, et al. (2004) Proteomic analysis of articular cartilage shows increased type II collagen XT-I and Proteoglycan Synthesis in Osteoarthritis expression of XT-I strongly upregulated GAG synthesis in chondrocytes and cartilage explants. These in vitro cell culture and cartilage explant data can be used to predict the beneficial effects that XT-I would have on OA chondrocytes. Next, in the hope of achieving clinical benefit in patients with advance OA disease, we determined if we could rescue the low sulfated-GAG synthesis activity of chondrocytes from diseased regions of OA cartilage with over-expression of the XT-I gene. The results demonstrated that forced expression of XT-I significantly improved sulfated-GAG synthesis rate in diseased cartilage. These novel experiments indicate that when XT-I gene is overexpressed in humans with OA these individuals may be capable of presenting increased GAG synthesis activity and provide support in pursuing the demanding goal of cartilage repair. Clinical implications and conclusions Although the patient population examined in this study is not large, nevertheless, the changes in XT-I gene expression we perceive could contribute to many of the observed biochemical characteristics of the disease, particularly for the pathological changes in CS-GAG synthesis and content. Currently, there are no effective therapies to stimulate sulfated-GAG synthesis in cartilage repair, and this deficiency is in part a reflection of a previous lack of understanding of the mechanisms involved in the loss of GAG synthesis in arthritic diseases. Our findings reported here record the protective function of the endogenous XT-I and its role in the maintenance of cartilage GAG homeostasis. Signifi- cantly, our results show that the quantitative changes in the expression and activity of XT-I might dictate the disease-related variations in sulfated-GAG synthesis and content. Therefore, pharmacological upregulation of XT-I gene expression along with aggrecan core protein might be expected to increase the amount of functional PG being synthesized and have a therapeutic potential for cartilage repair in OA and also in other degenerative joint diseases. Author Contributions Conceived and designed the experiments: MO NV. Performed the experiments: NV LB MB. Analyzed the data: NV MO SFG JM DM PN. Contributed reagents/materials/analysis tools: NV LB. Wrote the paper: MO NV. synthesis in osteoarthritis and expression of inhibin betaA (activin A), a regulatory molecule for chondrocytes. J Biol Chem 279: 43514–43521. 10. Young AA, Smith MM, Smith SM, Cake MA, Ghosh P, et al. (2005) Regional assessment of articular cartilage gene expression and small proteoglycan metabolism in an animal model of osteoarthritis. Arthritis Res Ther 7: R852–R861. 11. Aigner T, Zien A, Gehrsitz A, Gebhard PM, McKenna L (2001) Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology. Arthritis Rheum 44: 2777–2789. 12. Nelson F, Dahlberg L, Laverty S, Reiner A, Pidoux I, et al. (1998) Evidence for altered synthesis of type II collagen in patients with osteoarthritis. J Clin Invest 102: 2115–2125. 13. Venkatesan N, Barre´ L, Magdalou J, Mainard D, Netter P, et al. (2009) Modulation of xylosyltransferase I expression provides a mechanism regulating glycosaminoglycan chain synthesis during cartilage destruction and repair. FASEB J 23: 813–822. 14. Altman R, Asch E, Bloch D, Bole G, Borenstein D, et al. (1986) Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Arthritis Rheum. 29: 1039–1049. 15. Mankin HJ, Dorfman H, Lippiello L, Zarins A (1971) Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am 53: 523–537. PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e34020