Generation of novel genetically modified rats to reveal the molecular mechanisms of vitamin D actions

Miyu Nishikawa,Hiroki Mano,Masashi Takamatsu,Yoshihisa Hirota,Kairi Okamoto,Shinichi Ikushiro,Naoko Tsugawa,Toshiyuki Sakaki,Kimie Nakagawa,Eiichi Hinoi,Kyohei Horibe,Tetsuhiro Horie,Keisuke Abe,Toshio Okano,Kaori Yasuda

doi:10.1038/s41598-020-62048-1

Abstract

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 25-hydroxyvitamin D3 (25(OH)D3) and ligand-independent effects of the VDR. Here, we describe a novel in vivo system using genetically modified rats deficient in the Cyp27b1 or Vdr genes. Type II rickets model rats with a mutant Vdr (R270L), which recognizes 1,25(OH)2D3 with an affinity equivalent to that for 25(OH)D3, were also generated. Although Cyp27b1-knockout (KO), Vdr-KO, and Vdr (R270L) rats each showed rickets symptoms, including abnormal bone formation, they were significantly different from each other. Administration of 25(OH)D3 reversed rickets symptoms in Cyp27b1-KO and Vdr (R270L) rats. Interestingly, 1,25(OH)2D3 was synthesized in Cyp27b1-KO rats, probably by Cyp27a1. In contrast, the effects of 25(OH)D3 on Vdr (R270L) rats strongly suggested a direct action of 25(OH)D3 via VDR-genomic pathways. These results convincingly suggest the usefulness of our in vivo system.

Highlights

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 25-hydroxyvitamin D3 (25(OH)D3) and ligand-independent effects of the VDR
The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), plays important roles in osteogenesis, calcium homeostasis, cellular differentiation, and immune responses1. 1,25(OH)2D3 is generated by two hydroxylation steps from vitamin D3: C-25 hydroxylation by hepatic CYP2R1 and CYP27A1 and subsequent 1α-hydroxylation by renal 1α-hydroxylase (CYP27B1)[2]
To confirm the direct action of 25(OH)D3 in vivo, we previously examined its effect on osteogenesis in Cyp27b1 knockout (KO) mice

Summary

Introduction

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 25-hydroxyvitamin D3 (25(OH)D3) and ligand-independent effects of the VDR. Binding of active vitamin D3 to the VDR triggers its heterodimerization to the retinoid X receptor and subsequent translocation to the nucleus This translocation results in regulating target gene expression by formation of the VDR complex on vitamin D-responsive elements in the promoter regions of target genes, such as osteocalcin and osteopontin in bones and the calcium channels and calbindins in intestines[3]. A variety of vitamin D derivatives have been developed as drugs for rickets, osteoporosis, psoriasis, secondary hyperparathyroidism, and chronic kidney disease Because all of these compounds show high affinity for the VDR, these pharmacological actions are considered to be VDR mediated. To confirm the direct action of 25(OH)D3 in vivo, we previously examined its effect on osteogenesis in Cyp27b1 knockout (KO) mice These mice have no detectable 1,25(OH)2D3 in their plasma and exhibit all the hallmarks of type I rickets, such as reduced bone mineral density and hypocalcemia. Based on the activity of 1α-hydroxylation toward 25(OH)D3 in liver mitochondrial fractions prepared from Cyp27b1-KO mice, we assumed that Cyp27a1 converted 25(OH)D3 to 1,25(OH)2D312

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Conclusion