IGF-1 regulation of type II collagen and MMP-13 expression in rat endplate chondrocytes via distinct signaling pathways M. Zhang, Ph.D., Q. Zhou, M.D.,

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IGF-1 regulation of type II collagen and MMP-13 expression in rat endplate chondrocytes via distinct signaling pathways M. Zhang, Ph.D., Q. Zhou, M.D., Ph.D., Q.-Q. Liang, Ph.D., C.-G. Li, M.D., J.D. Holz, M.S., D. Tang, M.D., T.-J. Sheu, Ph.D., T.-F. Li, M.D., Ph.D., Q. Shi, M.D., Y.-J. Wang, M.D., Ph.D. Osteoarthritis and Cartilage Volume 17, Issue 1, Pages 100-106 (January 2009) DOI: 10.1016/j.joca.2008.05.007 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Monolayer culture of the endplate chondrocytes isolated from cervical spine of rats was synchronized in serum-free medium for 12h and treated with IGF-1 for additional 12h. After RNA extraction and reverse transcription, Col2a1 mRNA expression was assessed by real time polymerase chain reaction (RT-PCR). The bars represent the mean±SE of three independent experiments. *P<0.05, **P<0.01 vs control. (A) IGF-1 stimulated Col2a1 mRNA expression in a dose-dependent manner. (B) IGF-1 stimulated Col2a1 mRNA expression in a time-dependent manner. (C) Confluent endplate chondrocytes in monolayer culture were switched to serum-free media, cultured 12h and then treated with IGF-1 (0, 50 and 100ng/ml) for 24h. Protein samples were collected and western blot was performed with an antibody against col2. β-actin was used as a loading control. Osteoarthritis and Cartilage 2009 17, 100-106DOI: (10.1016/j.joca.2008.05.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Subconfluent endplate chondrocytes in monolayer culture were synchronized in serum-free medium for 12h and stimulated by IGF-1 (100ng/ml) alone or in combination either with actinomycin D (A) or cycloheximide (B). After 12-h incubation, RNA was extracted and Col2a1 mRNA expression was assessed by real time RT-PCR. The bars represent the mean±SE of three independent experiments. *P<0.05 vs control; #P<0.05 vs IGF-1. A-D, actinomycin D; CHX, cycloheximide. Osteoarthritis and Cartilage 2009 17, 100-106DOI: (10.1016/j.joca.2008.05.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Subconfluent endplate chondrocytes in monolayer culture were synchronized in serum-free medium for 12h and stimulated by IGF-1 for additional 12h. RNA was extracted and MMP-13 mRNA expression was assessed by real time RT-PCR. The bars represent the mean±SE of three independent experiments. *P<0.05, **P<0.01 vs control. (A) IGF-1 inhibited MMP-13 mRNA expression in a dose-dependent manner. (B) IGF-1 inhibited MMP-13 mRNA expression in a time-dependent manner. (C) Confluent endplate chondrocytes in monolayer culture were switched to serum-free media for 12h and then treated with IGF-1 (0, 50 and 100ng/ml) for 24h. Protein samples were collected and western blot was performed with an antibody against MMP-13. β-actin was used as a loading control. Osteoarthritis and Cartilage 2009 17, 100-106DOI: (10.1016/j.joca.2008.05.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 (A) Kinetic assay of the MMP activity showed that IGF-1 repressed MMP activity. (B) Normalized results with MMP-13 specific inhibitor demonstrated that IGF-1 specifically inhibits MMP-13 enzymatic activity. *P<0.05. Osteoarthritis and Cartilage 2009 17, 100-106DOI: (10.1016/j.joca.2008.05.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Subconfluent endplate chondrocytes in monolayer cultures were switched to serum-free media for 12h and then treated with IGF-1 (0, 50 and 100ng/ml) for 15min. Cell lysates were prepared and western blot was done with an antibody against either phospho ERK1/2 (A) or Akt (C). Chondrocytes were pretreated with either MEK inhibitor PD98059 (25μM, B) or PI3K inhibitor Wartmannin (50nM, D) for 60min, followed by stimulation with IGF-1 (50ng/ml) for 15min. Cell lysates were prepared and western blot was done with an antibody against either phospho ERK1/2 or Akt. (E) The cell viability assay using the Celltiter-Blue reagent expressed as fluorescence intensity. Osteoarthritis and Cartilage 2009 17, 100-106DOI: (10.1016/j.joca.2008.05.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Subonfluent chondrocytes were cultured in serum-free medium for 12h and stimulated by IGF-1 (100ng/ml) alone or in combination either with PD98059 (25μM) or Wartmannin (50nM). After 24-h incubation, RNA was extracted and Col2a1 mRNA expression (A) and MMP-13 mRNA expression (B) were assessed by real time RT-PCR. The columns represent the mean±SE of three independent experiments. *P<0.05 vs control. #P<0.05 vs IGF-1 treatment. Bar 1: control, 2: IGF-1 alone, 3: IGF-1 plus PD98059, 4: IGF-1 plus Wartmannin. Osteoarthritis and Cartilage 2009 17, 100-106DOI: (10.1016/j.joca.2008.05.007) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions