Upregulated ank expression in osteoarthritis can promote both chondrocyte MMP-13 expression and calcification via chondrocyte extracellular PPi excess

AbstractObjective: In idiopathic chondrocalcinosis and in osteoarthritis (OA), increased extracellular PPi (ecPPi) promotes calcification. In chromosome 5p-associated familial chondrocalcinotic degenerative arthropathy, certain mutations in the membrane protein ANK may chronically raise ecPPi via enhanced PPi channeling. Therefore, we assessed if dysregulated wild-type ANK expression could contribute to pathogenesis of idiopathic degenerative arthropathy through elevated ecPPi.Design: Using cells with genetic alterations in expression of ANK and the PPi-generating nucleotide pyrophosphatase phosphodiestrase (NPP) PC-1, we examined how increased ANK expression elevates ecPPI, testing for codependent effects with PC-1. We also evaluated the effects of ANK expression on chondrocyte growth, matrix synthesis, and MMP-13 expression and we immunohistochemically examined ANK expression in situ in human knee OA cartilages.Results: Using cells expressing defective ANK, as well as PC-1 knockout cells, we demonstrated that ANK required PC-1 (and vice versa) to raise ecPPi and that the major ecPPi regulator TGFβ required both ANK and PC-1 to elevate ecPPi. Upregulation of wild-type ANK by transfection in normal chondrocytes not only raised ecPPi 5-fold to ∼100nM but also directly stimulated matrix calcification and inhibited collagen and sulfated proteoglycans synthesis. In addition, upregulated ANK induced chondrocyte MMP-13, an effect that also was stimulated within 2h by treatment of chondrocytes with 100nM PPi alone. Finally, ANK expression was upregulated in situ in human knee OA cartilages.Conclusion: Elevation of ecPPi by ANK critically requires the fraction of cellular PPi generated by PC-1. The upregulation of ANK expression in OA cartilage and the capacity of increased ANK expression to induce MMP-13 and to promote matrix loss suggest that increased ANK expression and ecPPi exert noxious effects in degenerative arthropathies beyond stimulation of calcification