title: Regulation of SOX9 in normal and osteoarthritic equine articular chondrocytes by hyperosmotic loading.
abstract: OBJECTIVE
  SOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9.
  
  
  METHODS
  Equine chondrocytes harvested from normal or OA joints were subjected to different osmotic loading patterns as either primary (P0) or passaged (P2) cells. The involvement of MEK-ERK signalling was demonstrated by using pharmacological inhibitors. In addition SOX9 gene stability was determined. Levels of transcripts encoding SOX9, Col2A1 and aggrecan were measured using qRT-PCR. De novo glycosaminoglycan synthesis of explants was determined with (35)S sulphate during static hyperosmolar loading.
  
  
  RESULTS
  MEK-ERK signalling increases glycosaminoglycans (GAG) synthesis in explants. Static hyperosmotic conditions significantly reduced SOX9 mRNA in normal P2 and OA P0 but not normal P0 chondrocytes. SOX9 mRNA was stabilised by hyperosmotic conditions. Cyclical loading of normal P2 and OA P0 but not normal P0 cells led to an increase in SOX9 gene expression and this was prevented by MEK1/2 inhibition.
  
  
  CONCLUSIONS
  The response to osmotic loading of SOX9 mRNA is dependent on the nature of the osmotic stimulation and the chondrocyte phenotype. This variation may be important in disease progression.
authors.affiliation: Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst, Neston, Wirral , CH64 7TE, UK. peffs@liverpool.ac.uk
authors.forename: M J
authors.initials: MJ
authors.lastname: Peffers
authors.forename: P I
authors.initials: PI
authors.lastname: Milner
authors.forename: S R
authors.initials: SR
authors.lastname: Tew
authors.forename: P D
authors.initials: PD
authors.lastname: Clegg
mesh_terms: D053674:Aggrecans / Q000378:metabolism; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism*; D019902:Chondrocytes / Q000378:metabolism*; D005786:Gene Expression Regulation; D006025:Glycosaminoglycans / Q000378:metabolism; D006736:Horses; D009994:Osmolar Concentration; D010003:Osteoarthritis / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D053674:Aggrecans; D006025:Glycosaminoglycans; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor
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