Okadaic acid-induced inhibition of protein phosphatase 2A enhances chondrogenesis in chicken limb bud micromass cell cultures

The role of major cellular serine/threonine-specific protein phosphatases, protein phosphatase 1 and 2A, was investigated during chicken cartilage dif ferentiation under in vitro conditions. Activity of protein phosphatase 2A decreased parallel to differentiation of chondrogenic cells, whereas activi ty of protein phosphatase 1 remained unchanged as assayed in the supernatan ts of the homogenised chicken limb bud micromass cell cultures. When okadai c acid, a potent inhibitor of protein phosphatase 1 and 2A was applied in 2 0 nM concentration for 1 h during the second and third culturing days, it s ignificantly increased the size of metachromatic cartilage areas measured i n 6-day-old colonies. Following okadaic acid treatments, a significant inhi bition in the activity of protein phosphatase 2A was found, while the activ ity of protein phosphatase 1 was unaffected as measured an days 2 and 3. TR ITC-phalloidin labelling demonstrated that okadaic acid disorganised actin filaments and induced rounding of chondrogenic cells. This deterioration of actin filaments was reversible. Electron microscopy and biochemical analys is of colonies revealed that the ultrastructure and major components of car tilage matrix remained unchanged under the effect of okadaic acid. Okadaic acid-treatment applied to cultures containing predominantly differentiated chondrocytes (after day 4) did not influence the cartilage formation. H-3-t hymidine and bromodeoxyuridine incorporation-assays demonstrated enhanced c ell proliferation in the okadaic acid-treated colonies compared to that of the untreated ones. Our results indicate, for the first time, that protein phosphatase 2A is involved in the regulation of chondrogenesis. Inhibition of protein phosphatase 2A with okadaic acid may result in increased chondro genesis via modulation of proliferation and cytoskeletal organisation, as w ell as via alteration of protein kinase A-signaling pathway of the chondrog enic cells.