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

Abstract

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3) and 25-hydroxyvitamin D 3 (25(OH)D 3) 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) 2 D 3 with an affinity equivalent to that for 25(OH)D 3 , 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)D 3 reversed rickets symptoms in Cyp27b1-KO and Vdr (R270L) rats. Interestingly, 1,25(OH) 2 D 3 was synthesized in Cyp27b1-KO rats, probably by Cyp27a1. In contrast, the effects of 25(OH)D 3 on Vdr (R270L) rats strongly suggested a direct action of 25(OH)D 3 via VDR-genomic pathways. These results convincingly suggest the usefulness of our in vivo system. The active form of vitamin D 3 , 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3), plays important roles in osteogenesis, calcium homeostasis, cellular differentiation, and immune responses 1. 1,25(OH) 2 D 3 is generated by two hydroxylation steps from vitamin D 3 : C-25 hydroxylation by hepatic CYP2R1 and CYP27A1 and subsequent 1α-hydroxylation by renal 1α-hydroxylase (CYP27B1) 2. The vitamin D receptor (VDR) mediates the genomic action of active vitamin D 3. Binding of active vitamin D 3 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. CYP24A1, one of the well-known vitamin D target genes, is involved in inactivating 1,25(OH) 2 D 3 through sequential metabolism that starts with C-24 or C-23 hydroxylation of 1,25(OH) 2 D 3 4. 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. However, as with 1,25(OH) 2 D 3 , they might also have non-VDR-mediated actions. Thus, pharmacological action studies of vitamin D derivatives are essential for future drug discovery.

Rat models of human diseases and related phenotypes: a systematic inventory of the causative genes

“…The F344 and SD mutants display hypercholesterolemia, hypertriglyceridemia, atherosclerosis, xanthomatosis; hepatic steatosis was also found in the SD mutant [195,198,199] Hypercholesterol-emia (diet-induced: ExHc rat) The human neurobehavioral manifestations are due to mutations in SHANK3; one of these mutations (a deletion) was introduced in rats, which exhibited disabilities related to those seen in the human patients; these deficits were attenuated by oxytocin treatment [42] Pinked The Wistar KO mutant shows growth failure and rickets, with alopecia; a mutant containing the human mutation R270L was also isolated: it also shows rickets symptoms, reversed by 25-Hydroxyvitamin D3 administration; with the above mutant, these 2 mutants could be useful to study the effects of vitamin D derivatives [286] Sitosterolemia Abcg5** 6q12, 7.94…”

Elucidation of metabolic pathways of 25-hydroxyvitamin D3 mediated by CYP24A1 and CYP3A using Cyp24a1 knockout rats generated by CRISPR/Cas9 system

“…Cyp24a1 KO rats were generated by CRISPR/Cas9 genome editing system as well as our previous study ( 17 ). The target site was selected to delete the cysteine at position 462 in Exon 10, which is the fifth ligand of heme iron and an active center of CYP24A1 ( Fig.…”

“…Daily administration of 25(OH)D3 to Cyp24a1 KO rats 25(OH)D3 was also daily dietary administrated to Cyp24a1 KO rats to clarify effects of long-term administration. 25(OH) D3 containing food was prepared as described in our previous study (17). Briefly, pellet diet containing 1.5 mg 25(OH)D3 per 1 kg F-2 normal diet was prepared by Oriental Yeast Co. Normal F-2 diet was fed prior to 9-week age, and after that 25(OH)D3-containing diet was fed.…”

“…Supporting information-This article contains supporting information (17). Study on metabolic pathway of 25(OH)D3 using Cyp24a1 KO rats…”

“…Recently, we successfully generated genetically modified (GM) rats deficient in Cyp27b1 or Vdr genes ( 17 ). Type II rickets model rats with a mutant Vdr (R270L), which recognizes 1,25(OH) 2 D3 with an affinity equivalent to that of 25(OH)D3, were also generated.…”