Effects of Wnt3A and mechanical load on cartilage chondrocyte homeostasis

Abstract

Introduction: Articular cartilage functions in withstanding mechanical loads and provides a lubricating surface for frictionless movement of joints. Osteoarthritis, characterised by cartilage degeneration, develops due to the progressive erosion of structural integrity and eventual loss of functional performance. Osteoarthritis is a multi-factorial disorder; two important risk factors are abnormal mechanical load and genetic predisposition. A single nucleotide polymorphism analysis demonstrated an association of hip osteoarthritis with an Arg324Gly substitution mutation in FrzB, a Wnt antagonist. The purpose of this study was two-fold: to assess whether mechanical stimulation modulates β-catenin signalling and catabolic gene expression in articular chondrocytes, and further to investigate whether there is an interplay of mechanical load and Wnt signalling in mediating a catabolic response.

Methods: Chondrocytes were pre-stimulated with recombinant Wnt3A for 24 hours prior to the application of tensile strain (7.5%, 1 Hz) for 30 minutes. Activation of Wnt signalling, via β-catenin nuclear translocation and downstream effects including the transcriptional activation of c-jun, c-fos and Lef1, markers of chondrocyte phenotype (type II collagen (col2a1), aggrecan (acan), SOX9) and catabolic genes (MMP3, MMP13, ADAMTS-4, ADAMTS-5) were assessed.

Results: Physiological tensile strain induced col2a1, acan and SOX9 transcription. Load-induced acan and SOX9 expression were repressed in the presence of Wnt3A. Load induced partial β-catenin nuclear translocation; there was an additive effect of load and Wnt3A on β-catenin distribution, with both extensive localisation in the nucleus and cytoplasm. Immediate early response (c-jun) and catabolic genes (MMP3, ADAMTS-4) were up-regulated in Wnt3A stimulated chondrocytes. With load and Wnt3A there was an additive up-regulation of c-fos, MMP3 and ADAMTS-4 transcription, whereas there was a synergistic interplay on c-jun, Lef1 and ADAMTS-5 transcription.

Conclusion: Our data suggest that load and Wnt, in combination, can repress transcription of chondrocyte matrix genes, whilst enhancing expression of catabolic mediators. Future studies will investigate the respective roles of abnormal loading and genetic predisposition in mediating cartilage degeneration.

Figure 1

β-catenin levels in chondrocytes subjected to tensile strain, separately or in combination with Wnt3A. Untreated chondrocytes served as controls. (A) Representative Western blot depicting β-catenin protein expression detected in cells processed immediately (direct); equivalent protein levels were confirmed by probing for the housekeeping protein GAPDH. Densitometric data are presented as β-catenin levels expressed as a percentage of the untreated controls after normalisation to GAPDH; statistical analysis was performed on Western blots from three independent experiments. (B) β-catenin localisation, immediately following loading (i - iv) or after a four-hour recovery period (v - x) as detected using confocal microscopy. β-catenin was detected using a FITC-conjugated secondary antibody (green) and nuclei counterstained with DAPI (blue): representative 3D-reconstructions are presented (scale bar = 6 μm).

Figure 2

Effects of tensile strain and Wnt3A, separately or combined, on chondrocyte early response gene transcription. Untreated chondrocytes served as controls. (A) c-fos, (B) c-jun and (C) Lef1 gene expression levels analysed immediately or (D), (E) and (F) following a four-hour recovery period respectively. Gene expression levels were assessed using quantitative PCR, normalised to the housekeeping gene 18s and relative to the untreated cells. Data are presented as mean ± S.E.M. (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

Effects of tensile strain and Wnt3A, separately or combined, on chondrocyte marker gene expression. Untreated chondrocytes served as controls. (A) type II collagen (col2a1) and (B) aggrecan (acan) gene expression levels analysed immediately or (C) and (D) following a four-hour recovery period respectively (refer to Figure 2 for calculations and statistical significance).

Figure 4

Effects of tensile strain and Wnt3A, separately or combined, on catabolic proteinase transcription in chondrocytes. Untreated chondrocytes served as controls. (A) MMP3, (B) MMP13, (C) ADAMTS-4 and (D) ADAMTS-5 gene expression levels analysed immediately or (E), (F), (G) and (H) following a four-hour recovery period respectively (refer to Figure 2 for calculations and statistical significance).