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FGF23 and PTH Directly Control Kidney 1,25(OH)2 Vitamin D3 Metabolism Through the ETS Transcription Factor ETV1 Via Coupled but Reciprocal Mechanisms

ABSTRACT: The balanced synthesis of active vitamin D (1,25(OH)2D3, 1,25D) is directed in the kidney proximal tubule (PT) by the catabolic enzyme CYP24A1 and anabolic CYP27B1 which are both controlled by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25D itself. FGF23 and 1,25D lower 1,25D by increasing CYP24A1 and suppressing CYP27B1, in a directly converse manner to the actions of PTH. PTH and 1,25D act through CREB and VDR, respectively, however the transcription factors through which FGF23 signals and the mechanisms of PTH suppression are unknown. Using scRNAseq after temporal FGF23 injections into normal mice, we found that the E twenty-six (ETS) family member ETV1 was stimulated specifically in PT S1-S2 cells in parallel with increased Cyp24a1. Importantly, ETV1 was rapidly recruited to genomic enhancers within the Cyp24a1 locus responsible for both FGF23 and PTH activities that overlapped with VDR binding. In vitro, ETV1 protein was upregulated by FGF23 and ETV1 cDNA drove CYP24A1 mRNA in HEK-mKL cells. ETV1-VDR co-precipitation was greatly enhanced by 1,25D and FGF23+1,25D co-treatment synergistically increased CYP24A1 expression in vitro, and in vivo. Conditional targeting of kidney epithelial Etv1 in mice resulted in ‘FGF23 resistance’ with elevated FGF23 and 1,25D, due to a blockade of Cyp24a1 and altered 1,25D metabolism. FGF23 was shown to inhibit E-3 Ubiquitin ligase Rfwd2 (COP1) nuclear localization, enhancing ETV1 protein stability. In vivo, PTH injections suppressed nuclear ETV1 protein and opposed FGF23 increases by removing ETV1 from the genome (4,790 sites to 22 sites). In vitro, a ‘PTH mimetic’ SIK inhibitor (SIKi) abolished ETV1 production by enhancing COP1-mediated degradation, whereas COP1 shRNA rescued SIKi-mediated ETV1 suppression. In sum, our findings demonstrate that ETV1 acts as a novel ‘unifying’ TF positively regulating FGF23/KL bioactivity with VDR on Cyp24a1 and suppressive PTH actions, thus opening novel pathways critically needed to address severe diseases involving FGF23 and vitamin D.

ORGANISM(S): Mus musculus

PROVIDER: GSE337338 | GEO | 2026/08/26

REPOSITORIES: GEO

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Project description:The active form of vitamin D, 1,25(OH)2 vitamin D3 (‘1,25D’), is required for maintaining normal immune responses, cancer resistance, vascular function, and musculoskeletal structure and function. The balanced synthesis of 1,25D from liver-produced 25(OH)D3 is exquisitely directed in final metabolizing steps solely in the kidney proximal tubule (PT) by the catabolic enzyme vitamin D 24-hydroxylase (CYP24A1) and anabolic vitamin D 1α-hydroxylase (CYP27B1). FGF23 and 1,25D itself, synergistically lower 1,25D by increasing CYP24A1 and suppressing CYP27B1, in a directly converse manner to the actions of PTH. We previously found that the necessary balance of maintaining vitamin D metabolism relies more on catabolic Cyp24a1 expression as the critical factor controlling circulating 1,25D. Using scRNAseq after temporal FGF23 injections into normal mice, we found that the E twenty-six (ETS) family member ETV1 was stimulated specifically in PT S1-S2 cells in parallel with increased Cyp24a1, and a 10-fold induction of ETS occupancy across the genome. In vivo, FGF23 induced localization of ETV1 to megalin+ PT nuclei. Importantly, ETV1 was rapidly recruited to genomic enhancers within the Cyp24a1 promoter and a kidney-specific distal enhancer (DS1) responsible for both FGF23 and PTH activities that overlapped with VDR binding. In a converse manner, PTH suppressed nuclear ETV1 protein in vivo, and opposed FGF23 increases by completely removing ETV1 from the Cyp24a1 genomic enhancers (xx sites down to xx sites). In vitro, a human ETV1 cDNA drove CYP24A1 mRNA in HEK-mKL cells, and ETV1 protein was upregulated by FGF23, but not by 1,25D. However 1,25D strongly enhanced co-precipitated ETV1-VDR complexes. Conditional targeting of kidney epithelial Etv1 in mice resulted in ‘FGF23 resistance’ with elevated FGF23 and 1,25D, and almost complete blockade of Cyp24a1 mRNA responses, which altered 1,25D metabolism as determined by MS. FGF23 was shown to inhibit an ancient system of E-3 Ubiquitin ligase Rfwd2 (COP1) nuclear localization, enhancing ETV1 protein stability. Further, a ‘PTH mimetic’ SIK inhibitor (SIKi) abolished ETV1 production by enhancing COP1-mediated degradation, whereas COP1 shRNA rescued SIKi-mediated ETV1 suppression. In sum, our findings demonstrate that ETV1 acts as a novel ‘unifying’ TF positively regulating FGF23/KL bioactivity with VDR on Cyp24a1 and opposing PTH actions, thus opening novel pathways critically needed to address severe diseases involving FGF23 and vitamin D.

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:Introduction: Biologically active vitamin D (1,25-dihydroxyvitamin D or 1,25D) has emerged as a key regulator of human innate immunity. 1,25D signaling in macrophages strongly induces the expression of neutrophil chemoattractants, such as IL-8/CXCL8. Meta-analysis of vitamin D-regulated expression profiles has suggested that 1,25D may regulate granule formation in granulocytic cells. Here, we have examined the effects of 1,25D signaling on human neutrophil gene expression, alone and in combination with the inflammatory signal lipopolysaccharide (LPS). These studies are of interest because, whereas 1,25D signaling boosts innate immunity, it is anti-inflammatory. Methods and results: We determined the effects of 1,25D alone and in combination with LPS on gene expression of primary human neutrophils by RNAseq. LPS did not affect or slightly enhanced the expression of several well-characterized 1,25D-target genes, but strongly suppressed that encoding the 1,25D catabolic enzyme CYP24A1. Chromatin immunoprecipitation (ChIP) assays revealed that 1,25D- dependent vitamin D receptor (VDR) binding to the major CYP24A1 enhancer was eliminated in neutrophils treated with LPS, whereas binding to other 1,25D-target genes was unaffected. Notably, LPS induced binding of transcriptional repressors MAFF and BACH1 to the major CYP24A1 enhancer region. In other studies, pathway analyses revealed that 1,25D suppressed LPS-induced genes encoding inflammatory proteins. In addition, RNAseq and confirmatory RT/qPCR studies revealed that 1,25D, both on its own and in combination with LPS, increased mRNA expression of genes encoding antimicrobial components of secretory granules, including that encoding cathelicidin antimicrobial peptide (CAMP). Consistently, exposure of neutrophils to 1,25D enhanced bacterial killing, as revealed by a 20-25% reduction in E. coli colonies incubated with 1,25D-treated neutrophil conditioned media. The increased bacterial killing by 1,25D is mediated by 1,25D- induced secretion of cathelicidin, as an antibody against LL-37, the active form of cathelicidin, blocked antimicrobial activity. Discussion: Collectively, the data suggest that LPS prolongs vitamin D signaling by suppressing expression of the 1,25D catabolic enzyme CYP24A1. 1,25D signaling in the presence of LPS attenuates the expression of several genes associated with LPS inflammatory responses, whereas 1,25D in the absence or presence of LPS enhances the release of antibacterial proteins secreted by neutrophils in response to infection.

Project description:The proto-oncogenes ETV1, ETV4, and ETV5 encode members of the E26 transformation-specific (ETS) transcription factor family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COnstitutive Photomorphogenic-1 (COP1, also called RFWD2) as a tumor suppressor that negatively regulates ETV1, ETV4, and ETV5. ETV1, which is the member mutated more frequently in prostate cancer, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs (degrons) and was 50-fold more stable than wild-type ETV1. Almost all patient translocations eliminate these ETV1 degrons, implying that translocations rendering ETV1 insensitive to COP1 confer a significant selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 levels and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. The combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, relatively rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein expression, and abnormally elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a bona fide tumor suppressor whose down-regulation promotes prostatic epithelial cell proliferation and tumorigenesis. LNCap prostate cancer cell line were treated with 5 different sets of siRNAs: (1) control siRNA; (2) COP1 (RFWD2) siRNA; (3) COP1 siRNA + ETV1 siRNA; (4) COP1 siRNA + c-JUN siRNA; (5) COP1 siRNA + ETV1 siRNA + c-JUN siRNA. The experiments were conducted in two batches; each batch has its own control siRNA group, so that the batch effect can be properly modelled. Each group has 4-6 replicates; there are 31 samples in total.

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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FGF23 and PTH Directly Control Kidney 1,25(OH)2 Vitamin D3 Metabolism Through the ETS Transcription Factor ETV1 Via Coupled but Reciprocal Mechanisms

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