Anti-apoptotic effect of transforming growth factor-beta1 on human articular chondrocytes: role of protein phosphatase 2A.
OBJECTIVE
To study whether transforming growth factor-beta1 (TGF-beta1) is able to protect human chondrocytes from apoptosis and to analyze the role of phosphatases in the possible anti-apoptotic effect of TGF-beta1.


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 OA cells, TGF-beta1 significantly reduced the percentage of hypo-diploid chondrocytes, as well as the percentage of internucleosomal DNA breakage. However, in normal chondrocytes, TGF-beta1 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-beta1 stimulation; however in normal chondrocytes PP2A activity was significantly activated by TGF-beta1. The preincubation of normal chondrocytes with TGF-beta1 plus the PP2A inhibitor protein, IPP2A, reduced internucleosomal DNA breakage when compared with TGF-beta1 stimulation alone. The bcl-2/bax protein ratio was significantly higher in TGF-beta1 plus IPP2A preincubated normal chondrocytes than in cells stimulated with TGF-beta1 alone.


CONCLUSIONS
By manipulating the degree of PP2A activity, these results show the major role that PP2A plays in the outcome of TGF-beta1 signal transduction. These data suggest that PP2A could be a pivotal regulator of anti-apoptotic TGF-beta1-induced effects.
Lires-Deán
M
M
Osteoarticular and Aging Research Laboratory, Biomedical Research Center, Rheumatology Division, CH Universitario Juan Canalejo, Coruña, Spain.
Caramés
B
B
Cillero-Pastor
B
B
Galdo
F
F
López-Armada
M J
MJ
Blanco
F J
FJ
D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000187:drug effects*; D002358:Cartilage, Articular / Q000187:drug effects* / Q000378:metabolism; D016022:Case-Control Studies; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D010003:Osteoarthritis / Q000473:pathology*; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
D053773:Transforming Growth Factor beta1; D054648:Protein Phosphatase 2