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1α,25-Dihydroxyvitamin D3 Regulates Packaging of microRNAs within 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. Chondrocytes in the growth plate’s growth zone (GC cells) produce and export matrix vesicles (MVs) under the regulation of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. 1α,25(OH)2D3 secreted by the cells acts on the MV membrane, releasing its contents. This study examined the regulatory role 1α,25(OH)2D3 has over the production and packaging of microRNA into MVs by GC cells and the ability to release microRNA from MVs once produced. We treated GC cells with 1α,25(OH)2D3 and then sequenced the microRNA in the cells and MVs. We also treated MVs with 1α,25(OH)2D3 and determined if the microRNA 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 the production and packaging of microRNA into matrix vesicles. MVs did not release microRNA when treated with 1α,25(OH)2D3, indicating a heterogeneous MV population or a protective factor. 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 microRNAs.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE190020 | GEO | 2021/12/06

REPOSITORIES: GEO

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Project description:Fibroblast growth factor 23 (FGF23) is produced and secreted by osteocytes and is essential for maintaining phosphate homeostasis. One of the main regulators of FGF23, 1,25-dihydroxyvitamin D (1,25(OH)2D3), is primarily synthesized in the kidney from 25-hydroxyvitamin D (25(OH)D) by 1α-hydroxylase (encoded by CYP27B1). Hitherto, it is unclear whether osteocytes can convert 25(OH)D and thereby allow for 1,25(OH)2D3 to induce FGF23 production and secretion locally. Here, we differentiated MC3T3-E1 cells towards osteocyte-like cells expressing and secreting FGF23. Treatment with 10-6 M 25(OH)D resulted in conversion of 25(OH)D to 150 pmol/L 1,25(OH)2D3 and increased FGF23 expression and secretion but the converted amount of 1,25(OH)2D3 was insufficient to trigger an FGF23 response, so the effect on FGF23 was most likely directly caused by 25(OH)D. Interestingly, combining phosphate with 25(OH)D resulted in a synergistic increase in FGF23 expression and secretion, likely due to activation of additional signaling pathways by phosphate. Blockage of the vitamin D receptor (VDR) only partially abolished the effects of 25(OH)D or 25(OH)D combined with phosphate on Fgf23, while completely inhibiting the upregulation of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1), encoding for 24-hydroxylase. RNA sequencing and in silico analyses showed that this could potentially be mediated by the nuclear receptors Retinoic Acid Receptor b (RARB) and Estrogen Receptor 2 (ESR2). Taken together, we demonstrate that osteocytes are able to convert 25(OH)D to 1,25(OH)2D3, but this is insufficient for FGF23 activation, implicating a direct effect of 25(OH)D in the regulation of FGF23, which occurs at least partially independent from its cognate vitamin D receptor Moreover, phosphate and 25(OH)D synergistically increase expression and secretion of FGF23, which warrants investigating consequences in patients receiving a combination of vitamin D analogues and phosphate supplements. These observations help us to further understand the complex relations between, phosphate, vitamin D and FGF23.

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

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