Expression and regulation of Toll-like receptor 2 by IL-1β and fibronectin fragments in human articular chondrocytes S.-L. Su, M.S., C.-D. Tsai, Ph.D.,

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Expression and regulation of Toll-like receptor 2 by IL-1β and fibronectin fragments in human articular chondrocytes S.-L. Su, M.S., C.-D. Tsai, Ph.D., C.-H. Lee, M.D., D.M. Salter, M.D., H.-S. Lee, M.D., Ph.D. Osteoarthritis and Cartilage Volume 13, Issue 10, Pages 879-886 (October 2005) DOI: 10.1016/j.joca.2005.04.017 Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Toll-like receptor (TLR) 1–5 gene expression on (A) normal articular chondrocytes and (B) chrondrocytes from osteoarthritic cartilage by RT-PCR. Osteoarthritis and Cartilage 2005 13, 879-886DOI: (10.1016/j.joca.2005.04.017) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Expression of TLR2 in adult human articular cartilage. Normal (A) and osteoarthritic (B) human articular cartilage immunostained with antibody against TLR2. Very weak immunoreactivity was observed in normal chondrocytes, but positive immunoreactivity in the large majority of OA chondrocytes (original magnification ×400). Osteoarthritis and Cartilage 2005 13, 879-886DOI: (10.1016/j.joca.2005.04.017) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 Gene expression of TLR induced by IL-1β stimulation. OA chondrocytes were incubated with IL-1β for 0, 2 or 4h and subjected to total RNA isolation and RT-PCR. TLR2 was significantly upregulated following IL-1β stimulation for 2 and 4h. GAPDH served as internal control. Osteoarthritis and Cartilage 2005 13, 879-886DOI: (10.1016/j.joca.2005.04.017) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Effect of IL-1β on the TLR2 mRNA levels by OA primary chondrocytes. Total RNA was isolated and TLR2 mRNA was quantified by real-time RT-PCR as described in Materials and methods. Increased levels of TLR2 mRNA were demonstrated. Results were the mean±S.D. of five experiments performed with primary chondrocytes of different donors. Comparison of mean values was performed by ANOVA analysis (*P<0.05). Osteoarthritis and Cartilage 2005 13, 879-886DOI: (10.1016/j.joca.2005.04.017) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Effects of IL-1β, IL-1ra, and fibronectin fragments on the TLR2 gene expression. (A) TLR2 was upregulated by IL-1β and fibronectin (FN) 45, 29, and 70kDa fragments. Fibronectin 45kDa had the highest effect among the three fragments. (B) IL-1ra significantly inhibited the fibronectin-induced TLR2 gene upregulation. IL-1ra itself showed no effect on TLR2 gene expression. (C) The quantitative data is in triplicate for fibronectin 45kDa and IL-1ra. Osteoarthritis and Cartilage 2005 13, 879-886DOI: (10.1016/j.joca.2005.04.017) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 Western blot analysis of TLR2 in human articular chondrocytes. Cells were cultured for 12 and 24h in the presence of IL-1β (10ng/ml). The cells were lysed and protein extracts were analyzed by Western blot with a specific antibody for TLR2. Protein levels increased after IL-1β stimulation. Tubulin served as internal control. Osteoarthritis and Cartilage 2005 13, 879-886DOI: (10.1016/j.joca.2005.04.017) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 7 Survey of MyD88 activation. OA primary chondrocytes were incubated with (A) IL-1β for 0, 2, 4h or (B) 45kDa fibronectin fragment for 0, 6h. Cells were lysed and protein extracts were subjected to immunoprecipitation with tyrosine phosphorylation and immunodetection with MyD88. Increased tyrosine phosphorylation of MyD88 (p-MyD88) was identified. Osteoarthritis and Cartilage 2005 13, 879-886DOI: (10.1016/j.joca.2005.04.017) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 8 Electrophoresis mobility shift assay with NF-κB-binding site. Gel mobility shift assay was performed using a DIG-labeled synthetic oligonucleotides and nuclear extract from OA chondrocytes primary culture with (A) IL-1β stimulation for 0, 2, 4h or (B) 45kDa fibronectin fragment for 0, 6h. The competition assay used the same unlabeled oligonucleotide sequence with 10× concentration (A. left three lanes 0, 2, 4; B. left two lanes 0, 6). The arrows indicated the complex of NF-κB and the DIG-labeled oligonucleotides. NF-κB translocation was identified in OA chondrocyte incubation with IL-1β at 4h and 45kDa fibronectin fragment at 6h. C lane was free of oligonucleotides. Osteoarthritis and Cartilage 2005 13, 879-886DOI: (10.1016/j.joca.2005.04.017) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions