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In vitro regulation of fibroblast growth factor 23 by 25(OH)D and 1,25(OH)2D3 synthesized by osteocyte-like MC3T3-E1 cells

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE239761 | GEO | 2024/08/01

REPOSITORIES: GEO

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Project description:Human kidney was obtained from the University of Wisconsin Madison Organ Procurement Program from a deceased 69 year old female patient and the cortex was dissected from 3 different cortex locations. Associated publication abstract: Targeted genomic deletions identify diverse enhancer functions and generate a kidney-specific, endocrine-deficient Cyp27b1 pseudo-null mouse Vitamin D3 is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) via cytochrome P450 family 27 subfamily B member 1 (CYP27B1), whose gene is regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3. Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase like 1 (Mettl1) and Mettl21b that mediate basal and PTH induced expression of Cyp27b1 and FGF23- and 1,25(OH)2D3-mediated repression. Gross deletion of these segments in mice has severe consequences on Cyp27b1 regulation and skeletal phenotype, but does not affect Cyp27b1 expression in non-renal target cells (NRTCs). Here, we report a bimodal activity in the Mettl1 intronic enhancer with components responsible for PTH-mediated Cyp27b1 induction and 1,25(OH)2D3-mediated repression and additional activities, including FGF23 repression, within the Mettl21b enhancers. Deletion of both submodules eliminated basal Cyp27b1 expression and regulation in the kidney, leading to systemic and skeletal phenotypes similar to those of Cyp27b1-null mice. However, basal expression and lipopolysaccharide-induced regulation of Cyp27b1 in NRTCs was unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescued the skeletal phenotype of this mutant mouse, creating an ideal in vivo model to study non-renal 1,25(OH)2D3 production in health and disease. Finally, we confirmed a conserved chromatin landscape in human kidney that is similar to that in mouse. These findings define a finely balanced homeostatic mechanism involving PTH and FGF23 together with protection from 1,25(OH)2D3 toxicity that is responsible for both adaptive vitamin D metabolism and mineral regulation.

Project description:CYP24A1 is a multifunctional, P450 mitochondrial 24-hydroxylase enzyme that is responsible for catabolism of the most active vitamin D hormone (calcitriol, 1,25(OH)2D3), its precursor (calcifediol, 25(OH)D3), and numerous other vitamin D metabolites at the 23- and 24-carbon positions. Cyp24a1 gene expression is induced by 1,25(OH)2D3 in all tissues that contain the vitamin D receptor in a feedback mechanism. In the kidney, Cyp24a1 is induced by 1,25(OH)2D3, induced by FGF23, and potently suppressed by PTH to tightly control the circulating blood levels of 1,25(OH)2D3. This gene is known to be under the control of a pair of classic promoter proximal (PRO) vitamin D response elements (VDREs) that are aided by distal, downstream (DS) containing enhancers that we identified more recently. The DS1 enhancer collection was found to respond to PTH and FGF23 actions in a kidney-specific manner. The DS2 enhancers were found to assist in the response of 1,25(OH)2D3 in kidney, as well as other target tissues. Despite this knowledge, there remain several outstanding questions for the regulation of Cyp24a1 such as the in vivo contribution of the PRO VDREs to expression and the chromatin architecture of the locus, what drives Cyp24a1 basal expression in the kidney, how FGF23 activates Cyp24a1, and importantly, how PTH suppresses Cyp24a1. Here in this study, we utilize homology directed CRISPR to mutate one or both VDREs in the PRO region of the Cyp24a1 gene in vivo in the mouse to address these questions. We found that the more proximal VDRE (VDRE1) to the transcriptional start site (TSS) is the dominant VDRE of the pair and mutation of both VDREs leads to a dramatic loss of VDR, a reduction of Cyp24a1 gene expression in the kidney, and a near elimination of 1,25(OH)2D3 induction in the intestine. FGF23 induction of Cyp24a1 was reduced with mutation of the PRO VDREs, however, co-treatment of 1,25(OH)2D3 and FGF23 synergistically increased Cyp24a1 expression even with the loss of the PRO VDREs. PTH suppression of Cyp24a1 expression was unchanged with PRO VDRE mutations, despite a minor reduction in total pCREB occupancy that remained unaffected by PTH treatment. Finally, VDR occupancy was dramatically reduced across the DS enhancers in the Cyp24a1 locus after the PRO VDREs mutation. Taken together, our data suggest a cooperative relationship between the DS and PRO enhancers in the regulation of Cyp24a1 by 1,25(OH)2D3 and FGF23, and despite the overall reduction of CREB on the genome it was unchanged with PTH treatment indicating that suppression either does not rely on CREB or that the PRO VDREs are unconnected to PTH suppression altogether. These studies help further define the interconnected genomic control of these hormones on vitamin D catabolism.

Project description:Fibroblast growth factor 23 (FGF23), a hormone, mainly produced by osteocytes, regulates phosphate and vitamin D metabolism. By contrast, 1,25-dihydroxyvitamin D3, the active form of vitamin D, has been shown to enhance FGF23 production. While it is likely that osteocytes are heterogenous in terms of gene expression profiles, specific subpopulations of Fgf23-expressing osteocytes have not been identified. Single-cell RNA sequencing (scRNA-seq) technology can characterize the transcriptome of an individual cell. Recently, scRNA-seq has been used for bone tissue analysis. However, owing to technical difficulties associated with isolation of osteocytes, studies using scRNA-seq analysis to characterize FGF23-producing osteocytes are lacking. In this study, we characterized osteocytes secreting FGF23 from murine femurs in response to calcitriol (1,25-dihydroxyvitamin D3) using scRNA-seq. We first detected Dmp1, Mepe, and Phex expression in murine osteocytes by in situ hybridization and used these as marker genes of osteocytes. After decalcification, enzyme digestion, and removal of CD45+ cells, femoral bone cells were subjected to scRNA-seq. We identified cell clusters containing osteocytes using marker gene expression. While Fgf23 expression was observed in some osteocytes isolated from femurs of calcitriol-injected mice, no Fgf23 expression was detected in untreated mice. In addition, the expression of several genes which are known to be changed after 1,25-dihydroxyvitamin D3 treatment such as Ccnd2, Fn1, Igfbp7, Pdgfa, and Timp1 was also affected by calcitriol treatment in Fgf23-expressing osteocytes, but not in those lacking Fgf23 expression, even after calcitriol administration. Furthermore, box-and-whisker plots indicated that Fgf23 expression was observed in osteocytes with higher expression levels of the Fam20c, Dmp1, and Phex genes, whose inactivating mutations have been shown to cause FGF23-related hypophosphatemic diseases. These results indicate that osteocytes are heterogeneous with respect to their responsiveness to 1,25-dihydroxyvitamin D3, and sensitivity to 1,25-dihydroxyvitamin D3 is one of the characteristics of osteocytes with Fgf23 expression. It is likely that there is a subpopulation of osteocytes expressing several genes, including Fgf23, involved in phosphate metabolism.

Project description:Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-seq analyses, we show that while substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast-like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by ChIP-seq, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)2D3 that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by RXR and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)2D3-regulated genes, the expression of others adjacent to VDR binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)2D3 was found to induce the Boc and Cdon co-receptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)2D3. ChIP-seq was performed on differentiated IDGSW3 cells at day 35 for VDR and RXR following 3 hour vehicle or 1,25(OH)2d3 treatment in biological replicate. ChIP-seq was also performed for H3K4me1, H3K4me2, H3K4me3, H3K27Ac, H4K5Ac, H3K9Ac, H4K20me1, H3K36me3, or H3K9me3 at day 3 and day 35 of differentiation in the basal condition.

Project description:Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-seq analyses, we show that while substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast-like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by ChIP-seq, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)2D3 that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by RXR and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)2D3-regulated genes, the expression of others adjacent to VDR binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)2D3 was found to induce the Boc and Cdon co-receptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)2D3. Two sets of IDG-SW3 differentitiation mRNA-seq experiments are presented here in biological triplicate. One a differentiation time course in the basal condition at days 3, 7, 14, 21, 28, 35. The second experiment is IDG-SW3 cells differentiated to day 3 or day 35 and treated with vehicle or 100nM 1,25(OH)2D3 for 24 hours prior to mRNA isolation and sequencing.

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In vitro regulation of fibroblast growth factor 23 by 25(OH)D and 1,25(OH)2D3 synthesized by osteocyte-like MC3T3-E1 cells

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