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A Parathyroid Hormone/Salt Inducible Kinase Signaling Axis Controls Renal Vitamin D Activation and Organismal Calcium Homeostasis [SIK]

ABSTRACT: The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms in renal epithelial cells downstream of the PTH receptor that control vitamin D metabolism remain unknown. Here we demonstrate that Salt Inducible Kinases (SIKs) control renal 1,25-vit D production downstream of PTH signaling via regulating Cyp27b1 expression. As PTH inhibits the cellular activity of SIKs by cAMP-dependent PKA phosphorylation in bone, we hypothesized that SIKs would also work as an essential mediator of PTH doownstream signaling in kidney, thus regulate vitamin D metabolism. Whole tissue and single cell transcriptomics in kidney demonstrates that both PTH and pharmacologic SIK inhibitors regulate a vitamin D gene module in specific proximal tubule cells. Moreover, small molecule SIK inhibitors directly increase 1,25-vit D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice show Cyp27b1 upregulation, elevated serum levels of 1,25-vit D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 shows PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which are also required for SIK inhibitors to increase Cyp27b1 in vivo. Lastly, in a podocyte injury mouse model of chronic kidney disease (CKD) characterized by low 1,25-vit D levels, SIK inhibitor treatment stimulates both renal Cyp27b1 expression and 1,25-vit D production.

ORGANISM(S): Mus musculus

PROVIDER: GSE207731 | GEO | 2023/03/01

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.

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A Parathyroid Hormone/Salt Inducible Kinase Signaling Axis Controls Renal Vitamin D Activation and Organismal Calcium Homeostasis [SIK]

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