Phosphoproteomics reveals the BRAF-ERK1/2 axis as an important pathogenic signaling node in cartilage degeneration

Abstract

Objective: Osteoarthritis (OA) causes gradual cellular alterations, structural anomalies and joint dysfunction. Progressive decline of chondrocyte function plays a vital role on OA pathogenesis. Although protein phosphorylation controls cartilage metabolism, its regulation mechanism in OA remains unclear. Thus, proteomic methods were used to investigate phosphorylation changes in preserved and OA articular cartilage samples, and to explore the intervention targets of phosphorylated kinase.

Methods: Preserved (control) and lesioned (OA) cartilage samples from OA cases were assessed by phosphoproteomics. Immobilized metal affinity chromatography was performed for phosphopeptide enrichment. Quantitated phosphosites were comparatively assessed in the cartilage sample pair. Kinase-substrate enrichment analyses were carried out for identifying OA-related kinases. BRAF expression in cartilage tissues was assessed by immunohistochemical staining. The effects of BRAF inhibitor on cartilage degeneration were examined in mouse chondrocytes and OA mouse model.

Results: High-sensitivity mass spectrometry-based proteomics revealed 7,471 peptides and 4,375 phosphorylated peptides differing between preserved and lesioned cartilage samples, which represented the significant alteration of kinase hubs and transduction pathways. Phosphoproteomics identified BRAF may be involved in developing OA. BRAF regulated the downstream ERK signaling pathway. In addition, BRAF was upregulated in human OA cartilage as shown by immunohistochemistry. Remarkably, BRAF inhibition alleviated cartilage degradation in a mouse model of OA through its downstream of ERK pathway activation.

Conclusions: Jointly, these findings provide an overview of phosphoproteomic alterations occurring during cartilage degeneration, identifying the BRAF-ERK1/2 Axis signaling as a potential signaling pathway involved in OA.

Keywords: Cartilage degeneration; Kinome; Osteoarthritis; Phosphoproteomics.