Increased expression of CCN4/WISP1 in osteoarthritic articular cartilage is epigenetically regulated and disrupts cartilage homeostasis

Abstract

Background Previously, we described increased expression of Wnt-1-induced signaling protein 1 (Wisp1) in murine synovium and cartilage after induction of two experimental osteoarthritis (OA) models. Wisp1 is a downstream target of the canonical Wnt signaling pathway that has been shown to play a pivotal role in the etiopathology of OA. In line with this, we have observed increased breakdown of the articular cartilage after overexpression of Wisp1 in naive mouse knee joints, whereas Wisp1-/- mice revealed decreased cartilage degeneration in three independent experimental OA models compared to wild type controls. Together, these data indicate a direct correlation between Wisp1 and OA in mice. Objectives In the current study we set out to characterize the relation between expression of WISP1 and human OA. Methods Articular cartilage from preserved and degenerated OA areas was collected from 39 Caucasian end-stage OA patients. Cartilage from non-OA-diagnosed individuals was collected after femoral neck fractures. Cartilage degeneration was classified according to the Mankin scoring system. DNA was isolated to determine correlation between WISP1 expression and methylation profiles using Generalized Linear Mixed Model (GLMM). RNA expression levels were determined with microarray analysis and RNA sequencing. Immunohistochemical staining was used to determine WISP1 protein expression. Recombinant WISP1 was added to human chondrocyte microparticles, and cartilage extracellular matrix deposition was determined by measuring cartilage microparticle size and Safranin O/Fast Green staining. Results We observed increased WISP1 expression in cartilage of OA patients compared to non-OA-diagnosed controls. Moreover, within OA patients, both WISP1 mRNA and protein expression were significantly increased in OA-affected cartilage compared to preserved regions of the same joint, and WISP1 expression significantly correlated with Mankin score. Interestingly, we found that positional CpG dinucleotides were hypomethylated in cartilage of OA-affected areas as compared to unaffected areas from the same joint, which correlated with increased RNA expression as determined with both microarray analysis and RNA sequencing analysis. Of note, methylation levels of a CpG affecting WISP1 transcription were found to highly significantly correlate to a single nucleotide polymorphism (SNP) at the WISP1 locus. Next, to investigate effects of increased WISP1 levels on chondrocyte microparticles, we added human recombinant WISP1. This resulted in a significantly decreased deposition of cartilage extracellular matrix as reflected by decreased microparticle circumference, and a strongly decreased proteoglycan content, suggesting that increased WISP1 levels are detrimental to cartilage. Conclusions The expression of WISP1, both RNA and protein, is increased in OA-affected as compared to preserved articular cartilage. This increased expression is inversely correlated with methylation levels of a positional CpG for WISP1, which was found to be under the influence of a SNP at the WISP1 locus. Together, our results suggest that tight regulation of WISP1 expression via methylation is essential to maintain cartilage homeostasis. Disclosure of Interest None declared