Insights into the association of Gla-rich protein and osteoarthritis, novel splice variants and γ-carboxylation status

Scope: Gla-rich protein (GRP) is a vitamin K dependent protein, characterized by a high density of gamma-carboxylated Glu residues, shown to accumulate in mouse and sturgeon cartilage and at sites of skin and vascular calcification in humans. Therefore, we investigated the involvement of GRP in pathological calcification in osteoarthritis (OA). Methods and results: Comparative analysis of GRP patterning at transcriptional and translational levels was performed between controls and OA patients. Using a RT-PCR strategy we unveiled two novel splice variants in human-GRP-F5 and F6-potentially characterized by the loss of full gamma-carboxylation and secretion functional motifs. GRP-F1 is shown to be the predominant splice variant expressed in mouse and human adult tissues, particularly in OA cartilage, while an overexpressing human cell model points it as the major gamma-carboxylated isoform. Using validated conformational antibodies detecting carboxylated or undercarboxylated GRP (c/uc GRP), we have demonstrated cGRP accumulation in controls, whereas ucGRP was the predominant form in OA-affected tissues, colocalizing at sites of ectopic calcification. Conclusion: Overall, our results indicate the predominance of GRP-F1, and a clear association of ucGRP with OA cartilage and synovial membrane. Levels of vitamin K should be further assessed in these patients to determine its potential therapeutic use as a supplement in OA treatment.