Osteoarthritis (OA) is a whole-joint disease characterized by articular cartilage loss, tissue inflammation, abnormal bone formation and extracellular matrix (ECM) mineralization. Disease-modifying treatments are not yet available and a better understanding of osteoarthritis pathophysiology should lead to the discovery of more effective treatments. Gla-rich protein (GRP) has been proposed to act as a mineralization inhibitor and was recently shown to be associated with OA in vivo. Here, we further investigated the association of GRP with OA mineralization-inflammation processes. Using a synoviocyte and chondrocyte OA cell system, we showed that GRP expression was up-regulated following cell differentiation throughout ECM calcification, and that inflammatory stimulation with IL-1 beta results in an increased expression of COX2 and MMP13 and up-regulation of GRP. Importantly, while treatment of articular cells with gamma-carboxylated GRP inhibited ECM calcification, treatment with either GRP or GRP-coated basic calcium phosphate (BCP) crystals resulted in the down-regulation of inflammatory cytokines and mediators of inflammation, independently of its gamma-carboxylation status. Our results strengthen the calcification inhibitory function of GRP and strongly suggest GRP as a novel anti-inflammatory agent, with potential beneficial effects on the main processes responsible for osteoarthritis progression. In conclusion, GRP is a strong candidate target to develop new therapeutic approaches.

Gla-rich protein (GRP, also known as UCMA for upper zone of growth plate and cartilage matrix associated protein), a novel vitamin K-dependent (VKD) protein, has been identified and isolated from sturgeon by our group and found to have orthologs in nearly all taxonomic groups of vertebrates (named GRP1) and a paralog in bony fish (named GRP2). Additional evidence supporting this hypothesis was recently obtained in zebrafish through analysis of the genomic environment of its 2 grp genes compared to that of the mouse gene. Data confirmed that fish and mammalian GRP genes are true orthologs and that the two zebrafish genes result from ancestral genomic fragment duplication now located in zebrafish chromosomes 4 and 25. In sturgeon, as well as during mouse development, cartilaginous tissues or their precursors are primary sites of GRP expression. Recently, our laboratory has shown that GRP is a circulating protein also expressed and accumulated in some soft tissues and clearly associated with ectopic calcification events. Since this protein has the highest Gla density of any known VKD protein, it was proposed that GRP might be a potent physiological modulator of soft tissue calcification. Contradictory functional studies have recently been published for grp. While in zebrafish recent data indicated a role of Grp-1 (Ucmaa) during zebrafish skeletal development, another recent genetic study using a knockout strategy failed to identify a relevant function for grp during mouse development. Therefore, further studies are required in order to understand this discrepancy and provide insight into grp function and the molecular players involved in its regulation. In the present work we have taken a computational approach to identify cis-regulatory transcription factor (TF) binding motifs in both grp1/ucmaa and grp2/ucmab genes from two model fish with compact and well characterized genomes, the fugu (Takifugu rubripes) and the tetraodon (Tetraodon nigroviridis). By comparing promoters from ortholog genes we expected to define conserved transcriptional motifs indicative of regulatory networks affecting these genes. Accordingly, our computational methods identified several TFs, whose binding profiles are available in the TRANSFAC database, which can be important for the regulation of these two genes.