IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION IMBALANCED OSTEOARTHRITIS ASSOCIATED FIBRONECTIN SPLICE VARIANTS

Purpose: Transcriptome-wide analyses demonstrated FN1 is the highest expressed gene in articular cartilage and is significantly upregulated with ongoing OA pathophysiology. FN1 encodes fibronectin, a dimeric glycoprotein, particularly localized in the pericellular matrix directly surrounding the chondrocytes. Fibronectin mediates a wide variety of cellular interactions by binding to extra cellular matrix proteins and to chondrocytes via integrins. The main integrin-binding domain of fibronectin is the RGD motif, which binds multiple integrin heterodimers, including the classic fibronectin receptor integrin α5β1. FN1 can give rise to 27 different splice variants, which can occur at three major sites, called extra domain A (A), extra domain B (B), and variable region (V). Such splicing variants provides cells capacity to generate protein isoforms with different binding properties hence functions. Among the FN1 splice variants is the intact 70 kDa N-terminus of the full length protein, also known as migration-stimulating factor (MSF), which has been associated with tumor progression in multiple cancers. Despite being prominent in articular cartilage, it is unknown whether FN1 transcripts are involved in the OA pathophysiology. We, therefore, aimed to identify FN1 transcripts associated with OA pathophysiology and investigated downstream effects upon modulation of transcripts relative to full length FN1 expression in our 3D in vitro organoid cartilage model with human primary chondrocytes.

Methods: FN1 transcriptomic data was obtained from our previously assessed RNA-seq dataset of lesioned and preserved OA cartilage samples (N=35, RAAK study). Differential transcript expression analysis was performed on all 27 FN1 transcripts annotated in Ensembl database. Pearson’s correlations were calculated to generate a co-expression network between FN1 and our previously reported differentially expressed genes in OA cartilage. Human primary chondrocytes obtained from the RAAK study were transduced with lentiviral particles containing shRNA targeting full length FN1 transcripts or non-targeting shRNA. Subsequently, matrix deposition was induced in our 3D in vitro neo-cartilage model. Effects of changes in FN1 transcript ratio on sulphated glycosaminoglycan deposition were investigated by Alcian blue staining and dimethylmethylene blue assay. Moreover, gene expression levels of eight cartilage relevant markers and three highly correlating genes with FN1 were determined by RT-qPCR.

Results: We identified 22 FN1 transcripts to be robustly expressed in OA cartilage. In line with previous findings, the highest expressed protein-coding transcripts were EDA^- variants, while EDA⁺ variants were less abundantly expressed. To identify specific FN1 transcripts associates with the OA process, differential expression analysis was performed on these 22 transcripts between lesioned and preserved OA cartilage, resulting in 16 FDR significantly upregulated transcripts. Of these 16 transcripts, 5 were protein-coding and 11 non-protein coding, suggesting these non-protein coding transcripts may play a role in OA pathophysiology. Notably, FN1-208, encoding Migrating Stimulating Factor (MSF), was the most significantly upregulated protein-coding transcript. Upon downregulation of full length FN1 in our 3D in vitro neo-cartilage model, we generated an increased ratio of FN1-208 relative to full length FN1 transcript, as such mimicking cartilage in OA affected state. This resulted in decreased cartilage sulfated glycosaminoglycan deposition (Figure 1), as well as significantly decreased ACAN and COL2A1 and increased ADAMTS-5, ITGB1, and ITGB5 gene expression levels, implying a detrimental effect on neo-cartilage deposition. Moreover, NT5E expression was downregulated as a result of FN1 downregulation, indicating NT5E expression is regulated downstream of FN1 signaling.

Conclusions: We identified FN1-208 as the most significantly upregulated protein-coding transcripts, which has not been previously associated with OA. We show that full length FN1 downregulation and concomitant relative FN1-208 upregulation was unbeneficial for deposition of neo-cartilage matrix, likely due to decreased availability of the classical RGD integrin-binding site of fibronectin. We identified NT5E as previously unknown FN1 signaling downstream gene in cartilage. Together, our data highlight the importance of proper balance of FN1 transcripts for healthy cartilage homeostasis.