Abstract
ABSTRACTFibroblast growth factor 23 (FGF23) production is regulated by both calciotropic hormones and inflammation. Consistent with this, elevated FGF23 levels are associated with inflammatory markers as well as parathyroid hormone (PTH) in various disease states, including chronic kidney disease (CKD). However, the molecular mechanisms underpinning Fgf23 transcription in response to these regulators are largely unknown. We therefore utilized chromatin immunoprecipitation followed by DNA sequencing (ChIP‐seq) data from an osteocyte cell line to identify potential regulatory regions of the Fgf23 gene. Based on ChIP‐seq analysis of enhancer‐associated histone modifications, including H3K4 methylation and H3K9 acetylation, we discovered several potential enhancers for Fgf23, one of which was located 16kb upstream of the gene's transcriptional start site. Deletion of this putative enhancer from the mouse genome using CRISPR‐Cas9 technology led to lower bone, thymus, and spleen expression of Fgf23 mRNA without altering circulating levels of the intact hormone, although as previously reported, only bone displayed significant basal expression. Nevertheless, lack of the −16kb enhancer blunted FGF23 upregulation in a tissue‐specific manner by the acute inflammatory inducers lipopolysaccharide (LPS), interleukin‐1‐beta (IL‐1β), and tumor necrosis factor‐alpha (TNFα) in bone, non‐osseous tissues, and in circulation. Lack of the −16kb enhancer also inhibited PTH‐induced bone Fgf23 mRNA. Moreover, the absence of this Fgf23 enhancer in an oxalate diet‐induced murine CKD model prevented the early onset induction of osseous, renal, and thymic Fgf23 mRNA levels and led to a significant blunting of elevated circulating intact FGF23 levels. These results suggest that −16kb enhancer mediates the induction of Fgf23 by inflammation and PTH and facilitates the increase in FGF23 expression in a murine model of CKD. As exemplified herein, these Fgf23 enhancer‐deleted mice will provide a unique model in which to study the role of FGF23 expression in inflammatory diseases. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.
Highlights
Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that regulates phosphate homeostasis and vitamin D metabolism
FGF23 protein is synthesized as a full-length, biologically active 32kDa glycoprotein that is biologically inactivated through cleavage by furin and furin-like convertases.[1,2,3,4] Under physiological conditions, increased serum phosphate levels function to upregulate FGF23, which acts in turn at the kidney through both fibroblast growth factor receptors (FGFRs) and the co-receptor alpha Klotho to decrease levels and activity of the sodium phosphate cotransporters NPT2a and NPT2c.(5) This action inhibits renal phosphate reabsorption[6] and alters the metabolism of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3).(2)
In line with the Fgf23 mRNA levels, IL-1b and tumor necrosis factor-alpha (TNFa) increased circulating intact FGF23 (iFGF23) levels that were lower than those upregulated in response to LPS (Fig. 1I)
Summary
Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that regulates phosphate homeostasis and vitamin D metabolism. FGF23 is expressed in many cell types[21,22,24,25] and is regulated by hormones such as 1,25(OH)2D3(26–28) and cytokines such as IL1b.(18) Studies to date that have explored the underlying mechanisms of this regulation have utilized a combination of (i) transient transfection techniques that are limited to an evaluation of the activity of promoter-proximal regions attached to a reporter and/or (ii) the use of selected pathway inhibitors These methods have revealed roles for nuclear factor kappa b (NFkB)(18,21) and hypoxia-inducible factor 1a (HIF1a)(19,29) binding at the Fgf promoter region, suggesting that they might mediate inflammation-induction of Fgf transcription. Inhibitors of these transcriptional activators failed to prevent the induction in Fgf mRNA levels by inflammation and do not provide any in vivo evidence for the gene’s direct regulation, highlighting the need for unbiased identification of Fgf transcriptional regulatory regions
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