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Am J Physiol Cell Physiol 297: C898-C906, 2009. First published August 5, 2009; doi:10.1152/ajpcell.00571.2008 Free Article
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RECEPTORS AND SIGNAL TRANSDUCTION

Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes

Simon R. Tew,1 Mandy J. Peffers,1 Tristan R. McKay,2 Emma T. Lowe,3 Wasim S. Khan,3 Timothy E. Hardingham,3 and Peter D. Clegg1

1Veterinary Clinical Sciences, University of Liverpool Veterinary Teaching Hospital, Leahurst, Neston, Cheshire, United Kingdom; 2North West Embryonic Stem Cell Centre and 3Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

Submitted 6 November 2008 ; accepted in final form 4 August 2009

The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471–39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24–48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

osmolarity; p38 mitogen-activated protein kinase; mRNA stability; actin; COL2A1


Address for reprint requests and other correspondence: S. R. Tew, Veterinary Clinical Sciences, Univ. of Liverpool Veterinary Teaching Hospital, Leahurst, Neston, Cheshire, CH64 7TE, United Kingdom (e-mail: tew{at}liverpool.ac.uk).






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