Anti-apoptotic effect of transforming growth factor-β1 on human articular chondrocytes: role of protein phosphatase 2A M. Lires-Deán, B.S., B. Caramés,

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Anti-apoptotic effect of transforming growth factor-β1 on human articular chondrocytes: role of protein phosphatase 2A M. Lires-Deán, B.S., B. Caramés, Ph.D., B. Cillero-Pastor, B.S., F. Galdo, M.D., M.J. López-Armada, Ph.D., F.J. Blanco, M.D., Ph.D. Osteoarthritis and Cartilage Volume 16, Issue 11, Pages 1370-1378 (November 2008) DOI: 10.1016/j.joca.2008.04.001 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Overview of experimental design. Human knee articular chondrocytes isolated from cadavers or patients with OA were preincubated with TGF-β1 (12.5ng/ml) or simultaneously with TGF-β1 and the PP2A inhibitor protein IPP2A (0.1nM; normal for 120h). The chondrocytes were then induced to undergo apoptosis with simultaneous treatment with TNF-α (10ng/ml) and Ro 31-8220 (Ro 31; 5mM) for 16h, after which apoptosis was evaluated by several methods. Osteoarthritis and Cartilage 2008 16, 1370-1378DOI: (10.1016/j.joca.2008.04.001) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Characterization of TGF-β1 protection by PI flow cytometry. Panel A: Quantification of apoptotic cells among human OA chondrocytes stimulated simultaneously for 16h with TNF-α and Ro 31, with or without pre-incubation for 120h with TGF-β1. OA chondrocytes showed significantly increased levels of induced apoptosis after TNF-α and Ro 31 treatment, when compared to the basal level. OA chondrocytes pre-incubated with TGF-β1 cells showed a significant reduction in apoptosis after exposure to TNF-α and Ro 31 treatment. Panel B: Quantification of apoptotic cells among human normal chondrocytes stimulated for 16h with TNF-α and Ro 31. Normal chondrocytes showed significantly increased levels of induced apoptosis, when compared to basal levels. In normal chondrocytes, the TGF-β1 pre-incubated cells did not show a significant reduction of apoptosis (*p<0.05). The results are from five different donors of chondrocytes and five independent experiments performed in duplicate. Osteoarthritis and Cartilage 2008 16, 1370-1378DOI: (10.1016/j.joca.2008.04.001) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Characterization of the protective effect of TGF-β1 by DNA fragmentation analysis. Panel A: Human OA chondrocytes were simultaneously stimulated for 16h with TNF-α (10ng/ml) plus Ro 31-8220 (Ro 31; 5mM) with or without pre-incubation with TGF-β1 (12.5ng/ml) for 120h. OA chondrocytes treated only with TNF-α plus Ro 31 showed a significantly increased level of DNA fragmentation, when compared to the basal level. However, OA chondrocytes pre-incubated with TGF-β1 before exposure to TNF-α plus Ro 31 showed a significant reduction in DNA fragmentation when compared to the TNF-α plus Ro 31-treated cells without pre-incubation with TGF-β1. Panel B: Normal human chondrocytes were stimulated for 16h with TNF-α plus Ro 31, with or without preincubation with TGF-β1 for 120h. Normal chondrocytes treated only with TNF-α plus Ro 31 showed a significantly increased level of DNA fragmentation, when compared to the basal level. However, normal chondrocytes pre-incubated with TGF-β1 before exposure to TNF-α plus Ro 31 TGF-β1 did not show a significant reduction in DNA fragmentation (**p<0.01; *p<0.05). The results are from five different donors of chondrocytes and five independent experiments performed in duplicate and are expressed as the mean fold-increase in DNA fragmentation. Osteoarthritis and Cartilage 2008 16, 1370-1378DOI: (10.1016/j.joca.2008.04.001) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effect of TGF-β1 on phosphatases. Panel A: MKP-1. The quantification of levels of MKP-1 by western blot analysis in normal and OA chondrocytes simultaneously stimulated for 16h with TNF-α (10ng/ml) plus Ro 31-8220 (Ro 31; 5mM) after pre-incubation with TGF-β1 (12.5ng/ml) for 120h. The levels of MKP-1 were not altered at any of the times evaluated in either normal or OA chondrocytes. The levels of MKP-1 were also quantified after a second administration of TGF-β1 (72h; data not shown) with similar results. The data are representative from three experiments. Panel B: PP2A. The PP2A activity was measured using a Ser/Thr phosphatase assay in OA and normal chondrocytes after pre-incubation with TGF-β1 (12.5ng/ml). The histograms show that TGF-β1 stimulated PP2A activity over time (30min and 3h) in normal cells, but not in OA cells. *p<0.05 when compared to the basal level. PP2A activity was also measured after the second administration of TGF-β1 (72h; data not shown) with similar results. The results are from five different donors of chondrocytes and five independent experiments performed in triplicate. Osteoarthritis and Cartilage 2008 16, 1370-1378DOI: (10.1016/j.joca.2008.04.001) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effect of the inhibition of PP2A on normal chondrocytes. (A) The specific inhibition of PP2A with IPP2A induced significant levels of apoptosis in normal chondrocytes, similar to those induced by treatment with TNF-α (10ng/ml) plus Ro 31-8220 (Ro 31; 5mM) alone. (B) Preincubation with TGF-β1 (12.5ng/ml) for 120h blocked the IPP2A induced apoptosis. The simultaneous preincubation of normal chondrocytes with TGF-β1 and IPP2A followed by treatment with TNF-α plus Ro 31 significantly reduced the apoptosis induced by the combination of TNF-α plus Ro 31 alone (*p<0.01). The results are from five different donors of chondrocytes and five independent experiments performed at least in duplicate and are expressed as the mean fold-increase in DNA fragmentation. Osteoarthritis and Cartilage 2008 16, 1370-1378DOI: (10.1016/j.joca.2008.04.001) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Effect of TGF-β1 and PP2A inhibitory protein (IPP2A) on the bcl-2/bax ratio. Quantification of the protein levels of bcl-2 (Panel A) and bax (Panel B) detected by western blot analysis in normal chondrocytes preincubated for 120h with TGF-β1-alone or with TGF-β1+IPP2A, and then stimulated with TNF-α (10ng/ml) plus Ro 31-8220 (Ro 31; 5mM) for 16h. The expressions of bax was significantly upregulated by preincubation with TGF-β1 (p<0.05 vs control), whereas simultaneous stimulation with TGF-β1 and IPP2A upregulated bcl-2 expression (p<0.05 vs control). Panel C: The ratio of bcl-2/bax proteins is significantly higher in normal chondrocytes simultaneously preincubated with TGF-β1 and IPP2A, when compared to preincubation with TGF-β1 alone. The values are the mean of three samples from three different donors of chondrocytes (*p<0.05; **p<0.01). Osteoarthritis and Cartilage 2008 16, 1370-1378DOI: (10.1016/j.joca.2008.04.001) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Flowchart showing the hypothesized pivotal role of phosphatase 2A (PP2A) in the signal pathway of TGF-β1. In normal chondrocytes, TGF-β1 upregulates PP2A activity, decreasing the bcl-2/bax ratio. Importantly, the specific inhibition of PP2A with IPP2A, simultaneous with TGF-β1 stimulation, produced an increase in the ratio of bcl-2/bax proteins, contributing to cell protection. In OA chondrocytes, TGF-β1 does not upregulate the level of PP2A activity; thus the bcl-2/bax ratio is increased (previously reporting), contributing to cell protection. Osteoarthritis and Cartilage 2008 16, 1370-1378DOI: (10.1016/j.joca.2008.04.001) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions