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Targeting Fibroblast growth factor-23 (FGF23) responsive pathways in the control of renal phosphate and vitamin D metabolism

ABSTRACT: FGF23 is a bone-derived hormone that mediates renal phosphate reabsorption and 1,25(OH)2 vitamin D metabolism via its required co-receptor alpha-Klotho (KL). The functional pathways guiding this hormone’s activity in kidney have not been studied extensively, and whether using other factors with overlapping signaling profiles to produce FGF23-like responses is unclear. To map FGF23-related genes, gene array and single-cell RNA sequencing were utilized on wild type mouse kidneys. After identifying Heparin-binding EGF-like growth factor (HBEGF) as an up-regulated gene in response to FGF23 delivery, KL-null and phosphate-deficient diet fed mouse models and in vitro experiments were utilized to further test HBEGF bioactivity in kidney. Gene array demonstrated that HBEGF was significantly up-regulated following FGF23 delivery to wild type (WT) mice. Next, mice injected with HBEGF had phenotypes consistent with partial FGF23-mimetic activity including robust induction of EGR1, and increased CYP24A1 mRNAs. Single cell RNA sequencing showed overlapping HBEGF and EGFR expression in the proximal tubule (PT), and KL expression in PT and distal tubule (DT) segments. In KL-null mice devoid of canonical FGF23 signaling, HBEGF injections significantly increased EGR1 and CYP24A, and correction of basally-elevated CYP27B1 was observed. In addition, mice placed on a phosphate deficient diet to suppress FGF23 had endogenously increased CYP27B1 mRNA, which was rescued in mice receiving HBEGF. In HEK293 renal epithelial cells, HBEGF and FGF23 increased CYP24A1 mRNA. Targeting pathways known to be downstream of FGF23 in kidney may help to control renal phosphate handling in diseases of altered FGF23 bioactivity.

ORGANISM(S): Mus musculus

PROVIDER: GSE138992 | GEO | 2020/10/01

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: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.

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.

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Targeting Fibroblast growth factor-23 (FGF23) responsive pathways in the control of renal phosphate and vitamin D metabolism

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