Spingosine-1-phosphate stimulates proliferation and counteracts interleukin-1 induced nitric oxide formation in articular chondrocytes M.H. Stradner,

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Spingosine-1-phosphate stimulates proliferation and counteracts interleukin-1 induced nitric oxide formation in articular chondrocytes M.H. Stradner, M.D., J. Hermann, M.D., H. Angerer, M.Sc., D. Setznagl, B.M.L.Sc., I.-G. Sunk, M.D., R. Windhager, M.D., W.B. Graninger, M.D. Osteoarthritis and Cartilage Volume 16, Issue 3, Pages 305-311 (March 2008) DOI: 10.1016/j.joca.2007.06.018 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Expression of S1P receptors in bovine and human articular chondrocytes. RT-PCR of RNA isolated from bovine (A) and human (B) chondrocytes grown in monolayer. The data shown are representative of six independent samples. Osteoarthritis and Cartilage 2008 16, 305-311DOI: (10.1016/j.joca.2007.06.018) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effect of S1P on chondrocyte proliferation. Bovine articular chondrocytes (A) and human chondrocytes (B) were treated with the indicated concentrations of S1P for 24h. The appropriate volume of vehicle solution (0.4% fatty acid free BSA) was used as a control. Uptake of 3H-thymidine was measured and is expressed as cpm. Results are presented as mean±SD of two independent experiments, each performed in six replicates. *P<0.05 and **P<0.01. Osteoarthritis and Cartilage 2008 16, 305-311DOI: (10.1016/j.joca.2007.06.018) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 NO formation in the culture supernatant. Concentration of nitrite was measured after treatment of bovine chondrocytes with the indicated concentrations of S1P and 10ng/ml IL-1β for 24h. Chondrocytes treated with IL-1β only were supplemented with the appropriate volume of vehicle solution (0.4% fatty acid free BSA) as a control. Values are normalized to total protein. The data are presented as mean±SD of three independent experiments. *P<0.05 vs IL-1β treated vehicle control. Osteoarthritis and Cartilage 2008 16, 305-311DOI: (10.1016/j.joca.2007.06.018) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of S1P on iNOS expression of bovine chondrocytes. iNOS mRNA was quantified by real-time RT-PCR after 3h of treatment with the indicated concentrations of S1P alone (A) or in combination with 10ng/ml IL-1β (B). Chondrocytes not treated with S1P were supplemented with the appropriate amount of vehicle solution (0.4% fatty acid free BSA) as a control. Values were normalized to GAPDH and are presented as percentage of vehicle control. Data are means±SD of three independent experiments. *P<0.05 and **P<0.01 vs IL-1β treated vehicle control. (C) Representative Western blot from total protein isolated after 24h treatment with S1P and 10ng/ml IL-1β S1P alone (20μg protein/lane, 1min exposition time) or in combination with 10ng/ml IL-1β (7μg protein/lane, 10s exposition time). (D) Intensity of the Western blot was quantified and is expressed as a ratio of iNOS vs actin. Osteoarthritis and Cartilage 2008 16, 305-311DOI: (10.1016/j.joca.2007.06.018) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of S1P on GAG release. Bovine cartilage explants were treated for 72h with the indicated concentrations of S1P and 10ng/ml IL-1β. Explants not treated with S1P were supplemented with the appropriate volume of vehicle solution (0.4% fatty acid free BSA) as a control. GAG released to the culture supernatant (A) and GAG content of explants (B) was assessed using the DMB method. Values were normalized to cartilage weight. Results are presented as means (n=6)±SD. *P<0.05 and **P<0.01. Osteoarthritis and Cartilage 2008 16, 305-311DOI: (10.1016/j.joca.2007.06.018) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Effect of S1P on MMP-13 and ADAMTS-4 expression in IL-1β stimulated bovine chondrocytes. MMP-13 (A) and ADAMTS-4 (B) mRNA were quantified by real-time RT-PCR after 3h of treatment with the indicated concentrations of S1P and 10ng/ml IL-1β. Chondrocytes treated with IL-1β only were supplemented with the appropriate volume of vehicle solution (0.4% fatty acid free BSA) as a control. Values were normalized to GAPDH and are presented as percentage of the IL-1β treated vehicle control. Data are means±SD of three independent experiments. *P<0.05 and **P<0.01 vs IL-1β treated vehicle control. Osteoarthritis and Cartilage 2008 16, 305-311DOI: (10.1016/j.joca.2007.06.018) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions