Objective. Autophagy is a process for turnover of intracellular organelles and molecules that protects cells during stress responses. We undertook this study to evaluate the potential roles of Unc-51-like kinase 1 (ULK1), an inducer of autophagy, Beclin1, a regulator of autophagy, and microtubule-associated protein 1 light chain 3 (LC3), which executes autophagy, in the development of osteoarthritis (OA) and in cartilage cell death.Methods. Expression of ULK1, Beclin1, and LC3 was analyzed in normal and OA human articular cartilage and in knee joints of mice with aging-related and surgically induced OA, using immunohistochemistry and Western blotting. Poly(ADP-ribose) polymerase (PARP) p85 expression was used to determine the correlation between cell death and autophagy.Results. ULK1, Beclin1, and LC3 were constitutively expressed in normal human articular cartilage. ULK1, Beclin1, and LC3 protein expression was reduced in OA chondrocytes and cartilage, but these 3 proteins were strongly expressed in the OA cell clusters. In mouse knee joints, loss of glycosaminoglycans (GAGs) was observed at ages 9 months and 12 months and in the surgical OA model, 8 weeks after knee destabilization. Expression of ULK1, Beclin1, and LC3 decreased together with GAG loss, while PARP p85 expression was increased.Conclusion. Autophagy may be a protective or homeostatic mechanism in normal cartilage. In contrast, human OA and aging-related and surgically induced OA in mice are associated with a reduction and loss of ULK1, Beclin1, and LC3 expression and a related increase in apoptosis. These results suggest that compromised autophagy represents a novel mechanism in the development of OA.

Objectives Osteoarthritis (OA) is associated with cell death and extracellular matrix degradation in articular cartilage. Autophagy is an essential cellular homeostasis mechanism that was found to be deficient in aging and OA cartilage. This study determined whether pharmacological inhibition of the mammalian target of rapamycin (mTOR), a key inhibitor of autophagy, has disease-modifying activity in experimental OA. Methods Experimental OA was induced by transection of the medial meniscotibial ligament and the medial collateral ligament in 2-month old C57Bl/6 mice (n=36). Rapamycin (1 mg/kg weight/day) (n=18 mice) or DMSO vehicle control (n=18 mice) was administered intraperitoneally for 10 weeks. Histopathological changes in articular cartilage and synovium were examined by using semiquantitative scoring systems. Rapamycin effects on mTOR signaling, autophagy, cartilage homeostasis and inflammation were analyzed by immunohistochemistry and immunofluorescence staining. Results Rapamycin affected the mTOR signaling pathway in mouse knee joints as indicated by inhibition of ribosomal protein S6 phosphorylation, a target of mTOR and activation of LC3, a main marker of autophagy. The severity of cartilage degradation was significantly (P < 0.01) reduced in the rapamycin treated group compared to the control group and this was associated with a significant (P < 0.05) decrease in synovitis. Rapamycin treatment also maintained cartilage cellularity, and decreased ADAMTS-5 and IL-1β expression in articular cartilage. Conclusions These results suggest that rapamycin, at least in part by autophagy activation, reduces the severity of experimental OA. Pharmacological activation of autophagy may be an effective therapeutic approach for OA.

Objective. High mobility group box chromosomal protein 1 (HMGB-1), a nuclear DNA binding protein, was recently rediscovered as a new proinflammatory cytokine. The purpose of this study was to demonstrate HMGB-1 expression in vivo and to identify the role of HMGB-1 in the pathogenesis of rheumatoid arthritis (RA).Methods. HMGB-1 concentrations in synovial fluid (SF) and serum from RA and osteoarthritis (OA) patients were measured by immunoblot analysis. The protein's specific receptor, receptor for advanced glycation end products (RAGE), was examined in SF macrophages (SFMs). We measured levels of proinflammatory cytokines released by SFMs treated with HMGB-1 via enzyme-linked immunosorbent assay and used soluble RAGE (sRAGE) to block the release of tumor necrosis factor ␣ (TNF␣). Immunohistochemical analysis and immunofluorescence assay were employed to examine localization of HMGB-1 in RA synovium and its translocation in SFMs after TNF␣ stimulation.Results. HMGB-1 concentrations were significantly higher in SF of RA patients than in that of OA patients. SFMs expressed RAGE and released TNF␣, interleukin-1␤ (IL-1␤), and IL-6 upon stimulation with HMGB-1. HMGB-1 was found in CD68-positive cells of RA synovium, and TNF␣ stimulation translocated HMGB-1 from the nucleus to the cytosol in SFMs. Blockade by sRAGE inhibited the release of TNF␣ from SFMs.Conclusion. HMGB-1 was more strongly expressed in SF of RA patients than in that of OA patients, inducing the release of proinflammatory cytokines from SFMs. HMGB-1 plays a pivotal role in the pathogenesis of RA and may be an original target of therapy as a novel cytokine.High mobility group box chromosomal protein 1 (HMGB-1) has 219 residues in its primary amino acid sequence, and there is Ͼ98% sequence identity between the HMGB-1 of rodents and that of humans (1-6). In most cells, HMGB-1 is located in the nucleus. It is an abundant, highly conserved cellular protein and is widely known as a nuclear DNA binding protein that stabilizes nucleosome formation (7,8), facilitates gene transcription, and regulates the activity of steroid hormone receptors (9,10). However, it has been reported that HMGB-1 might be translocated from the nucleus to the cytosol and then released extracellularly.A previous study demonstrated that extracellular HMGB-1 induces the production of proinflammatory cytokines in macrophages (11). When released by activated monocytes, it participates in the development of lethality and activates downstream cytokine release. Furthermore, like other cytokine mediators of endotoxemia, HMGB-1 activates proinflammatory cytokine re-

The transcriptional activation by SRY-type high mobility group box 9 (SOX9) and the transforming growth factor ␤ (TGF-␤) signals are necessary for chondrogenic differentiation. We have previously shown that CREB-binding protein (CBP/p300) act as an important SOX9 co-activator during chondrogenesis. In the present study, we investigated the relationship between TGF-␤-dependent Smad2/3 signaling pathways and the SOX9-CBP/p300 transcriptional complex at the early stage of chondrogenesis. Overexpressed Smad3 strongly induced the primary chondrogenesis of human mesenchymal stem cells. In addition, Smad3 enhanced the transcriptional activity of SOX9 and the expression of ␣1(II) collagen gene (COL2A1), and small interference RNA against Smad3 (si-Smad3) inhibited them. We observed that Smad2/3 associated with Sox9 in a TGF-␤-dependent manner and formed the transcriptional complexes with SOX9 on the enhancer region of COL2A1. Interestingly, the association between Sox9 and CBP/p300 was increased by Smad3 overexpression and was suppressed by si-Smad3. Our findings indicate that Smad3 has a stronger potential to stimulate the SOX9-dependent transcriptional activity by modulating the interaction between SOX9 and CBP/ p300, rather than Smad2. This study suggests that the Smad3 pathway presents a key role for the SOX9-dependent transcriptional activation in primary chondrogenesis.

Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated formation of activated protein C (APC). We have found that the N-terminal lectin-like domain (D1) of TM has unique antiinflammatory properties. TM, via D1, binds high-mobility group-B1 DNA-binding protein (HMGB1), a factor closely associated with necrotic cell damage following its release from the nucleus, thereby preventing in vitro leukocyte activation, in vivo UV irradiation-induced cutaneous inflammation, and in vivo lipopolysaccharide-induced lethality. Our data also demonstrate antiinflammatory properties of a peptide spanning D1 of TM and suggest its therapeutic potential. These findings highlight a novel mechanism, i.e., sequestration of mediators, through which an endothelial cofactor, TM, suppresses inflammation quite distinctly from its anticoagulant cofactor activity, thereby preventing the interaction of these mediators with cell surface receptors on effector cells in the vasculature.