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P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture.

Rosenzweig DH, Ou SJ, Quinn TM - J. Cell. Mol. Med. (2013)

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.

View Article: PubMed Central - PubMed

Affiliation: Soft Tissue Biophysics Laboratory, Department of Chemical Engineering, McGill University, Montreal, QC H3A 2B2, Canada.

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Chondrocytes dedifferentiate in monolayer culture. (A) Phase images of primary chondrocytes in culture at indicated times post-seeding. Scale bar: 200 μm. (B) qPCR revealed an immediate pattern of dedifferentiation of chondrocytes cultured in monolayer. Light, grey and dark bars represent 3, 5 and 7 days culture, respectively, all normalized to day 1 cultures. Mean ± SEM (n = 3). (C) Western blot analysis of MAP kinase activity during 7 days of monolayer culture. α-tubulin was used as loading control.
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fig01: Chondrocytes dedifferentiate in monolayer culture. (A) Phase images of primary chondrocytes in culture at indicated times post-seeding. Scale bar: 200 μm. (B) qPCR revealed an immediate pattern of dedifferentiation of chondrocytes cultured in monolayer. Light, grey and dark bars represent 3, 5 and 7 days culture, respectively, all normalized to day 1 cultures. Mean ± SEM (n = 3). (C) Western blot analysis of MAP kinase activity during 7 days of monolayer culture. α-tubulin was used as loading control.

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.


P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture.

Rosenzweig DH, Ou SJ, Quinn TM - J. Cell. Mol. Med. (2013)

Chondrocytes dedifferentiate in monolayer culture. (A) Phase images of primary chondrocytes in culture at indicated times post-seeding. Scale bar: 200 μm. (B) qPCR revealed an immediate pattern of dedifferentiation of chondrocytes cultured in monolayer. Light, grey and dark bars represent 3, 5 and 7 days culture, respectively, all normalized to day 1 cultures. Mean ± SEM (n = 3). (C) Western blot analysis of MAP kinase activity during 7 days of monolayer culture. α-tubulin was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3822651&req=5

fig01: Chondrocytes dedifferentiate in monolayer culture. (A) Phase images of primary chondrocytes in culture at indicated times post-seeding. Scale bar: 200 μm. (B) qPCR revealed an immediate pattern of dedifferentiation of chondrocytes cultured in monolayer. Light, grey and dark bars represent 3, 5 and 7 days culture, respectively, all normalized to day 1 cultures. Mean ± SEM (n = 3). (C) Western blot analysis of MAP kinase activity during 7 days of monolayer culture. α-tubulin was used as loading control.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Soft Tissue Biophysics Laboratory, Department of Chemical Engineering, McGill University, Montreal, QC H3A 2B2, Canada.

Show MeSH
Related in: MedlinePlus