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1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium Regulating Components (RNA-seq)

ABSTRACT: 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney and skeleton. Interestingly, while several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine this issue, Cyp27b1 null mice on either a normal or a high calcium/phosphate-containing rescue diet were treated with either vehicle or 1,25(OH)2D3 and evaluated 6h later. RNA samples from duodena were then subjected to RNA-seq analysis and the data analyzed bioinformatically. 1,25(OH)2D3 altered expression of large collections of genes raised under either dietary condition. 45 genes were found common to both 1,25(OH)2D3-treated groups, and were comprised of genes previously linked to intestinal calcium uptake including S100g, Trpv6, Atp2b1 and Cldn2 as well as others. An additional distinct network of 56 genes was regulated exclusively by diet. This study revealed a global network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.

ORGANISM(S): Mus musculus

PROVIDER: GSE69170 | GEO | 2015/06/03

SECONDARY ACCESSION(S): PRJNA284754

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:The proximal tubule is the sensing site of sodium and phosphorus and the main place for the synthesis and metabolism of 1,25 (OH)2D3. Sodium may share the sensing mechanism with phosphate in proximal tubule epithelial cells; whether sodium cooperates with phosphate or plays an independent role in the regulation of phosphorus homeostasis remains unknown. In this in vitro study, we investigated the effects of high sodium on the synthesis and function of active vitamin D and local phosphorus regulation in proximal tubular epithelial cells. Human proximal tubule epithelial (HK-2) cells were treated with different concentrations of sodium/phosphorus. The expression of 1α-OHase (Cyp27b1) and 24-OHase (Cyp24a1) was determined by RT-PCR and Western Blot. Liquid chromatography/mass spectrometry (LC/MS) and enzyme-linked immunosorbent assay (ELISA) were used to detect the levels of 1,25 (OH)2D3. Intracellular Ca2+ ([Ca2+]i) was detected with the Ca2+ indicator dye Fura-4. Chromatin samples were immunoprecipitated with antibodies against parathyroid receptor 1 (PTH1R) and Klotho. High sodium decreased the expression of 1,25 (OH)2D3 by reducing 1α-OHase and 24-OHase in HK-2 cells, reduced the expression of PTH1R and Klotho, and increased the intracellular calcium concentration. These effects were reversed by sodium phosphorus transporter inhibitor (PFA), sodium hydrogen transporter inhibitor (Caliporide), and a chelator of the extracellular calcium, whereas enhanced by ouabain. Vitamin D receptor agonists significantly increased the recruitment of VDR to the Vitamin D response element (VDRE) of PTH1R and Klotho promoter, thus increasing the expression of PTH1R and Klotho. High extracellular sodium can decrease the synthesis of active vitamin D in the proximal tubules, affect the gene regulation of 1,25 (OH)2D3/VDR, and significantly reduce the expression of PTH1R and Klotho. The data reveal the influence of a high-sodium diet on mineral metabolism and the core role of vitamin D in kidney mineral metabolism.

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1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium Regulating Components (RNA-seq)

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