title: Cyclical hyperosmolar loading of chondrocytes enhances extracellular matrix production and occurs as a consequence of p38 MAPK/ERK signalling amd increased mRNA half life
Creators: name: Peffers, Mandy
Publisher: name: University of Liverpool Repository
Year: 2012

description: It was hypothesised that the application of cyclical hyperosmolar loading on chondrocytes enhanced extracellular matrix (ECM) production and occurred as a consequence of p38 mitogen activated protein kinase (MAPK) and extracellular signal-regulated kinase signalling and increased SOX9 mRNA half life. SOX9 is a transcription factor required for cartilage formation and is essential for cartilage ECM formation. Osteoarthritis (OA) is characterized by a loss of ECM. In human SOX9 gene expression is regulated by osmotic loading. COX-2 is a bifunctional enzyme with both oxygenase and peroxidase activities, responsible for the formation of prostanoids. Aberrant expression of COX-2 protein in articular tissues is a feature of arthritis. SOX9 and COX-2 mRNA regulation was characterised through static and cyclical application of hyperosmotic conditions in human articular cartilage (HAC) derived from OA joints and in addition normal and osteoarthritic of equine chondrocytes in monolayer culture. The roles of ERK1/2 and p38 MAPK pathways were investigated using pharmacological inhibition to identify mechanisms of SOX9 regulation. SOX9 and COX-2 half lives (t1/2) were determined in HAC (SOX9 and COX-2) and equine articular cartilage (SOX9). ECM production was measured by De novo glycosaminoglycan (GAG) synthesis of equine articular during static hyperosmolar loading and the expression of the ECM genes aggrecan and COL2A1was identified. Decay curves generated in freshly isolated HAC for SOX9 and COX-2 showed that hyperosmolarity increased the mRNA stability of both genes. HAC exposed to static and dynamic hyperosmotic loading showed a significant increase in SOX9 and COX-2 mRNA. There was a significant increase in SOX9 and COX-2 mRNA following treatment with the MEK1/2 inhibitor, U0126 during static loading. Equine articular chondrocytes in contrast demonstrated that static hyperosmotic conditions significantly reduced SOX9 mRNA but increased COX-2 mRNA in normal P2 and OA P0 but not normal P0. Cyclical loading of equine derived normal P2 and OA P0 but not normal P0 cells led to an increase in SOX9 gene expression and this was prevented by both p38 MAPK and MEK1/2 inhibition. Furthermore in equine articular chondrocytes there was no effect on COX-2 mRNA of cyclic hyperosmolar loading, although p38 MAPK signalling reduced COX-2 expression. Hyperosmotic loading in HAC increased the activation of p44/42 MAPK and p38 MAPK. In HAC the presence of either the MEK1/2 inhibitor U0126 or the p38 MAPK inhibitor SB202190 in conjunction with cyclical hyperosmotic loading reduced the induction of SOX9 mRNA. Only the presence of the p38 MAPK inhibitor SB202190 effected a reduction in COX-2 under these conditions. In equine articular explants static hyperosmolar loading increases GAG synthesis and this was reduced by ERK inhibition. The response to osmotic loading of SOX9 and COX-2 mRNA is dependent on the nature of the osmotic stimulation and the chondrocyte phenotype and the species. MEK-ERK signalling was not required for the stabilisation of SOX9 in static hyperosmotic conditions. The p38 MAPK and ERK1/2 pathways were involved in the induction of SOX9 under cyclical hyperosmotic loading in most chondrocytes. Additionally COX-2 is regulated by hyperosmotic conditions post transcriptionally. These findings suggest that the response of chondrocytes from OA cartilage is significantly different from that of normal chondrocytes. The altering sensing of the osmotic environment and inappropriate responses of the resident cell population may be important in the progression of OA.