P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture.
Bottom Line: Inhibition of p38 MAPK (p38) caused a significant up-regulation of collagen type II while suppressing collagen type I expression.P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces.ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I.
Affiliation: Soft Tissue Biophysics Laboratory, Department of Chemical Engineering, McGill University, Montreal, QC H3A 2B2, Canada.Show MeSH
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Mentions: To test for changes in chondrogenic phenotype, 105 primary bovine chondrocytes were seeded on standard polystyrene culture dishes and cultured for 7 days. As expected, cell morphology changed from rounded to fibroblast-like as cells attached and expanded (Fig. 1A). Quantitative real-time PCR analysis revealed significant declines in expression of the chondrogenic genes Col2a1, Aggrecan, COMP and Sox9 after 3, 5 and 7 days of culture, as compared to day 1 controls (Fig. 1B). The fibrotic marker Col1a2 was below detection in all samples tested (data not shown). To assess MAPK activity, Western blot probing for phosphorylated ERK1/2, p38 and JNK/SAPK was performed on cell lysates from freshly isolated chondrocytes (day 0), and cells cultured for 1, 3, 5 and 7 days. Compared with freshly isolated cells, MAPK activity sharply declined upon starting the monolayer culture and then gradually returned to near-control levels over 7 days (Fig. 1C). The gradual increase in MAPK activity from day 1 to 7 correlated with a steady decline in chondrogenic marker expression.
Affiliation: Soft Tissue Biophysics Laboratory, Department of Chemical Engineering, McGill University, Montreal, QC H3A 2B2, Canada.