Altered Expression of REST and Associated Corepressors and their Contribution to the Osteoarthritic Phenotype

Abstract

Osteoarthritis is a common disease that is expected to continue to rise in prevalence in the future. There is currently no disease modifying treatment, which adds a disability burden and a financial burden to manage this disease on society. To help develop useful treatments, further information is needed about the pathophysiology of osteoarthritis. In a recent study, GluN2B was found in osteoarthritic chondrocytes but not healthy ones. Additionally, CamKII is known to be increased in osteoarthritis compared to healthy chondrocytes. Since there is evidence GluN2B and CamKII are regulated by REST and its corepressors RCOR1 and RCOR2 in the brain, this thesis investigates if REST, RCOR1 or RCOR2 are altered in osteoarthritis. If so, how it could contribute to the expression of GluN2B, CamKII and osteoarthritic phenotypic markers. Primary human chondrocytes, sourced from knee joint replacements, were used to examine the amounts of REST, RCOR1 and RCOR2 in cells from macroscopically undamaged versus macroscopically damaged cartilage. Viral transduction was used to knock-in REST, RCOR1 or RCOR2 into damaged chondrocytes; Investigating if restoring their levels restored the chondrocyte phenotype to a more normal one. siRNA technology was used to knockdown REST, RCOR1 and RCOR2 in undamaged chondrocytes to see if reducing these caused chondrocytes to display an osteoarthritic phenotype. RT-qPCR was used to measure all RNA levels and western blot or ELISA to measure protein levels. Results show REST, RCOR1 and RCOR2 are all significantly decreased in osteoarthritic chondrocytes compared to normal chondrocytes at the RNA level. GluN2B and its RNA was unable to be detected so this must be further investigated. There were some phenotype changes with siRNA knockdown, mainly a reduction of TIMP3 with all, and reduction of CaMKIIδ with REST knockdown. Viral knock-ins of REST, RCOR1 or RCOR2 in damaged chondrocytes resulted in reduced ACAN, COL2A, ADAMTS5, and CaMKIIγ. There was also a reduction of CaMKIIδ with RCOR knock-in. Our results show REST, RCOR1 and RCOR2 are decreased in osteoarthritic chondrocytes and suggest they have a role in osteoarthritis, potentially through the CamKII pathway.