Anti-apoptotic effect of transforming growth factor-β1 on human articular chondrocytes: role of protein phosphatase 2A

Objective: To study whether transforming growth factor-beta 1 (TGF-beta 1) is able to protect human chondrocytes from apoptosis and to analyze the role of phosphatases in the possible anti-apoptotic effect of TGF-beta 1. Methods: Cartilage was obtained from patients with osteoarthritis (OA) who were undergoing joint replacement; normal cartilage was obtained from cadavers who had no history of joint disease. Chondrocytes stimulated with tumor necrosis factor-alpha (TNF-alpha) plus Ro 31-8220 (a specific inhibitor of mitogen-activated kinase phosphatase-1 - MKP-1) were employed as an in vitro model of apoptosis. Apoptosis was assessed by flow cytometry and a cell death immunoassay. Protein phosphatase 2A (PP2A) activity was estimated by measuring the absorbance of a molybdate: malachite green:phosphate reaction complex. MKP-1, bcl-2 and bax expressions were quantified by western blot. Results: In CA cells, TGF-beta 1 significantly reduced the percentage of hypo-diploid chondrocytes, as well as the percentage of intern ucleosomal DNA breakage, However, in normal chondrocytes, TGF-beta 1 did not reduce apoptosis, as assessed by both the percentage of hypo-diploid chondrocytes and internucleosomal DNA breakage. MKP-1 expression did not show significant modulation in OA or normal chondrocytes. However. PP2A activity was differentially modulated in normal and OA chondrocytes. In OA chondrocytes. PP2A activity was not altered by TGF-beta 1 stimulation; however in normal chondrocytes PP2A activity was significantly activated by TGF-beta 1. The preincubation of normal chondrocytes with TGF-beta 1 plus the PP2A inhibitor protein, IPP2A, reduced internucleosomal DNA breakage when compared with TGF-beta 1 stimulation alone. The bcl-2/bax protein ratio was significantly higher in TGF-beta 1 plus IPP2A preincubated normal chondrocytes than in cells stimulated with TGF-beta 1 alone. Conclusion: By manipulating the degree of PP2A activity, these results show the major role that PP2A plays in the outcome of TGF-beta 1 signal transcluction. These data suggest that PP2A could be a pivotal regulator of anti-apoptotic TGF-beta 1-induced effects. (C) 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.