Identification of new microRNAs targeting genes regulating the Pi/PPi balance in chondrocytes.

Abstract

Osteoarthritis (OA) is a whole-joint disease characterized by cartilage degradation and mineralization associated with chondrocyte phenotype changes, subchondral bone sclerosis and mild synovial inflammation. The extracellular levels of inorganic phosphate (ePi) and pyrophosphate (ePPi) are major regulators of the mineralization process but also play a role in the maintenance of the differentiated chondrocyte phenotype. Four membrane proteins control the balance between ePi and ePPi: the transporters ANK (exporting PPi outside the cells) and PiT-1 (importing ePi into the cells), and the enzymes PC-1 (generating ePPi from nucleotides) and Tissue Non-specific Alkaline Phosphatase (TNAP, hydrolyzing ePPi into ePi). In the present work, we investigated the ability of specific microRNAs (miR) to modulate activity and level of the mRNA coding for the regulatory proteins of the ePi/ePPi balance in chondrocytes. The 4 following microRNAs, let7e, miR-9, miR-188 and miR-219, were selected by bioinformatics for their ability to putatively target the mRNA 3'UTRs of these regulators. In IL-1β-stimulated human chondrocytes, chosen as a model of differentiated phenotype loss implicating the PPi transporter ANK, miR-9, miR-188 and let7e levels increased. However, luciferase reporter assays and transient miR overexpression in the ATDC5 chondrogenic cell line only support that miR-9 was a negative post-transcriptional regulator of PC-1, Pit-1 and TNAP mRNAs. This suggests that miR-9 could contribute to the acquirement of an altered chondrocyte phenotype by disrupting the ePi/ePPi balance.