<i>In</i> <i>vitro </i>regulation of fibroblast growth factor 23 by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D synthesized by osteocyte-like MC3T3-E1 cells

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 toward 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 β (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 VDR. 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.</p

Organic phosphate but not inorganic phosphate regulates Fgf23 expression through MAPK and TGF-ꞵ signaling

One of the main regulators of phosphate homeostasis is fibroblast growth factor 23 (FGF23), secreted by osteocytes. The effects of organic versus inorganic dietary phosphate on this homeostasis are unclear. This study used MC3T3-E1 FGF23-producing cells to examine the transcriptomic responses to these phosphates. Most importantly, the expression and secretion of FGF23 were only increased in response to organic phosphate. Gene ontology terms related to a response to environmental change were only enriched in cells treated with organic phosphate while cells treated with inorganic phosphate were enriched for terms associated with regulation of cellular phosphate metabolism. Inhibition of MAPK signaling diminished the response of Fgf23 to organic phosphate, suggesting it activates FGF23. TGF-β signaling inhibition increased Fgf23 expression after the addition of organic phosphate, while the negative TGF-β regulator Skil decreased this response. In summary, the observed differential response of FGF23-producing to phosphate types may have consequences for phosphate homeostasis.</p