Identification of novel modulators of WNT/β-catenin signalling in articular chondrocytes by large scale siRNA screening

Abstract

Purpose: Tight control of canonical Wnt/β-catenin signalling is essential for cartilage health. Both hyper-activation and absence of Wnt/β-catenin signalling in articular cartilage are linked with the development of osteoarthritis (OA). Interest in Wnt biology within this field has therefore strongly grown. Wnt/β-catenin signalling is an extraordinarily complex pathway for which transcriptional responses vary in a context-, cell- and tissue-specific manner. Thus, understanding the tissue-specific regulation of Wnt signalling in cartilage is of great relevance. Here, we aim to identify specific genes that modulate Wnt/β-catenin signalling in articular chondrocytes. Comprehensive identification of cartilage-specific Wnt/β-catenin signalling modulators, could help to understand the pathophysiology of OA and contribute to the discovery of new therapeutic targets. Methods: For large scale identification of genes that modulate the Wnt/β-catenin signalling pathway, we set up, conducted and validated a siRNA library-based screening in the C28/I2 human articular chondrocyte cell line. First, cells were transfected with a reporter for Wnt signalling activation, a luciferase based-assay system that provides a quantitative and sensitive measure of Wnt/β-catenin signalling pathway activation. After Wnt-reporter transfection, Wnt signalling was activated with CHIR99021 (a GSK3β inhibitor). Cell seeding densities and well-formats were optimized using positive and negative controls. Subsequently, cells were screened against the human siGENOME/ON-TARGETplus SMARTpool siRNA library interrogating 1577 different genes, grouped in kinases, phosphatases and proteases. To validate the hits obtained as regulators of Wnt/β-catenin signalling, selected genes were individually tested for luciferase assay in C28/I2 cells with the specific siRNA. Silencing for β-catenin (siCTNNB1) was used as a positive control. Further, prioritization of genes of interest was performed by bioinformatics and literature analysis. Results: After successful optimization of the high-throughput screening assay, a library of 1577 of genes was screened. Of all genes that were screened, 241 were considered as potential Wnt modulator genes using a cut-off of log2(Fold Change in luciferase activity) > 2 and < 0.5. Based on literature review, 134 genes were not previously linked to Wnt signalling in any cell-type, from which 45 corresponded to potential Wnt activator genes as their silencing led to reduced luciferase activity. In addition, bibliographic searches and analysis of available transcriptome datasets of the 45 potential Wnt activators resulted in 12 genes that may be related to chondrocyte biology or osteoarthritis. Conclusions: With our large-scale screening technology, we identified novel specific genes that modulate the canonical Wnt/β-catenin signalling pathway in the articular chondrocyte. We have discovered 45 novel potential Wnt activator genes, and among these, 12 genes are considered highly promising as they are linked to chondrocyte biology or OA.