1α,25-Dihydroxyvitamin D3 Regulates microRNA Packaging in Extracellular Matrix Vesicles and Their Release in the Matrix

Abstract

Growth plate chondrocytes are regulated by numerous factors and hormones as they mature during endochondral bone formation, including transforming growth factor beta-1 (TGFb1), bone morphogenetic protein 2 (BMP2), insulin-like growth factor-1 (IFG1), parathyroid hormone and parathyroid hormone related peptide (PTH, PTHrP), and Indian hedgehog (IHH). Chondrocytes in the growth plate's growth zone (GC) produce and export matrix vesicles (MVs) under the regulation of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. 1α,25(OH)₂D₃ regulates MV enzyme composition genomically and 1α,25(OH)₂D₃ secreted by the cells acts on the MV membrane nongenomically, destabilizing it and releasing MV enzymes. This study examined the regulatory role 1α,25(OH)₂D₃ has over production and packaging of microRNA (miRNA) into MVs by GC cells and the release of miRNA by direct action on MVs. Costochondral cartilage GC cells were treated with 1α,25(OH)₂D₃ and the miRNA in the cells and MVs sequenced. We also treated MVs with 1α,25(OH)₂D₃ and determined if the miRNA was released. To assess whether MVs can act directly with chondrocytes and if this is regulated by 1α,25(OH)₂D₃, we stained MVs with a membrane dye and treated GC cells with them. 1α,25(OH)₂D₃ regulated production and packaging of a unique population of miRNA into MVs compared to the vehicle control population. 1α,25(OH)₂D₃ treatment of MVs did not release miRNA. Stained MVs were endocytosed by GC cells and this was increased with 1α,25(OH)₂D₃ treatment. This study adds new regulatory roles for 1α,25(OH)₂D₃ with respect to packaging and transport of MV miRNAs.

Keywords: 1α,25-dihydroxyvitamin D3; Chondrocytes; Endocytosis; Growth plate cartilage; Matrix vesicles; microRNA.