Characterization of Rickets Type II Model Rats to Reveal Functions of Vitamin D and Vitamin D Receptor.

Vitamin D has been known to exert a wide range of physiological effects, including calcemic, osteogenic, anticancer, and immune responses. We previously generated genetically modified (GM) rats and performed a comparative analysis of their physiological properties to elucidate the roles of vitamin D and vitamin D receptor (VDR). In this study, our primary goal was to investigate the manifestations of type II rickets in rats with the VDR(H301Q) mutation, analogous to the human VDR(H305Q).

Synthesis of (22)-, (22)-22-Fluoro-, and 22,22-Difluoro-25-hydroxyvitamin D and Effects of Side-Chain Fluorination on Biological Activity and CYP24A1-Dependent Metabolism.

Three novel analogues of C22-fluoro-25-hydroxyvitamin D (-) were synthesized and evaluated to investigate the effects of side-chain fluorination on biological activity and metabolism of vitamin D. These novel analogues were constructed by convergent synthesis applying the Wittig-Horner coupling reaction between CD-ring ketones (,,) and A-ring phosphine oxide (). The introduction of C22-fluoro units was achieved by stereoselective deoxy-fluorination for synthesizing and or two-step cationic fluorination for .

Analysis of vitamin D metabolites in biological samples using a nanoluc-based vitamin D receptor ligand sensing system: NLucVDR.

Many assays are currently being developed to measure the levels of vitamin D metabolites in various samples (such as blood, urine, and saliva). This study focused on the measurement of vitamin D metabolites in serum and urine using the NLucVDR assay system, which consists of a split-type nanoluciferase and ligand-binding domain (LBD) of the human vitamin D receptor. Blood and urine samples were collected from 23 participants to validate the NLucVDR assay.

Synthesis of New 26,27-Difluoro- and 26,26,27,27-Tetrafluoro-25-hydroxyvitamin D: Effects of Terminal Fluorine Atoms on Biological Activity and Half-life.

As an extension of our research on providing a chemical library of side-chain fluorinated vitamin D analogues, we newly designed and synthesized 26,27-difluoro-25-hydroxyvitamin D (1) and 26,26,27,27-tetrafluoro-25-hydroxyvitamin D (2) using a convergent method applying the Wittig-Horner coupling reaction between CD-ring ketones (13, 14) and A-ring phosphine oxide (5). The basic biological activities of analogues, 1, 2, and 26,26,26,27,27,27-hexafluoro-25-hydroxyvitamin D [HF-25(OH)D] were examined. Although the tetrafluorinated new compound 2 exhibited higher binding affinity for vitamin D receptor (VDR) and resistance to CYP24A1-dependent metabolism compared with the difluorinated 1 and its non-fluorinated counterpart 25-hydroxyvitamin D [25(OH)D], HF-25(OH)D showed the highest activity among these compounds.

Functional analysis of vitamin D receptor (VDR) using adenovirus vector.

Recently, we generated type II rickets model rats, including Vdr(R270L), Vdr(H301Q), Vdr(R270L/H301Q), and Vdr-knockout (KO), by genome editing. All generated animals showed symptoms of rickets, including growth retardation and abnormal bone formation. Among these, only Vdr-KO rats exhibited abnormal skin formation and alopecia.

Development of nanoluciferase-based sensing system that can specifically detect 1α,25-dihydroxyvitamin D in living cells.

Previously, we reported a FLucN-LXXLL+LBD-FLucC system that detects VDR ligands using split firefly luciferase techniques, ligand binding domain (LBD) of VDR, and LXXLL sequences that interact with LBD after VDR ligand binding. In vivo, 25-hydroxyvitamin D (25(OH)D) and 1α,25-dihydroxyvitamin D (1α,25(OH)D) act as VDR ligands that bind to VDR, and regulate bone-related gene expression. Therefore, the amount of 25(OH)D and 1α,25(OH)D are indicators of bone-related diseases such as rickets and osteoporosis.

The First Convergent Synthesis of 23,23-Difluoro-25-hydroxyvitamin D and Its 24-Hydroxy Derivatives: Preliminary Assessment of Biological Activities.

In this paper, we report an efficient synthetic route for the 23,23-difluoro-25-hydroxyvitamin D () and its 24-hydroxylated analogues (,), which are candidates for the CYP24A1 main metabolites of . The key fragments, 23,23-difluoro-CD-ring precursors (-), were synthesized starting from Inhoffen-Lythgoe diol (), and introduction of the C23 difluoro unit to α-ketoester () was achieved using ,-diethylaminosulfur trifluoride (DAST). Preliminary biological evaluation revealed that 23,23-F-25(OH)D () showed approximately eight times higher resistance to CYP24A1 metabolism and 12 times lower VDR-binding affinity than its nonfluorinated counterpart 25(OH)D ().

Robust osteogenic efficacy of 2α-heteroarylalkyl vitamin D analogue AH-1 in VDR (R270L) hereditary vitamin D-dependent rickets model rats.

Active vitamin D form 1α,25-dihydroxtvitamin D (1,25(OH)D) plays pivotal roles in calcium homeostasis and osteogenesis via its transcription regulation effect via binding to vitamin D receptor (VDR). Mutated VDR often causes hereditary vitamin D-dependent rickets (VDDR) type II, and patients with VDDR-II are hardly responsive to physiological doses of 1,25(OH)D. Current therapeutic approaches, including high doses of oral calcium and supraphysiologic doses of 1,25(OH)D have limited success and fail to improve the quality of life of affected patients.

Stereoselective Synthesis of 24-Fluoro-25-Hydroxyvitamin D Analogues and Their Stability to hCYP24A1-Dependent Catabolism.

Two 24-fluoro-25-hydroxyvitamin D analogues (,) were synthesized in a convergent manner. The introduction of a stereocenter to the vitamin D side-chain C24 position was achieved via Sharpless dihydroxylation, and a deoxyfluorination reaction was utilized for the fluorination step. Comparison between (24)- and (24)-24-fluoro-25-hydroxyvitamin D revealed that the C24--configuration isomer was more resistant to CYP24A1-dependent metabolism than its 24-isomer .

Development of In Vitro and In Vivo Evaluation Systems for Vitamin D Derivatives and Their Application to Drug Discovery.

We have developed an in vitro system to easily examine the affinity for vitamin D receptor (VDR) and CYP24A1-mediated metabolism as two methods of assessing vitamin D derivatives. Vitamin D derivatives with high VDR affinity and resistance to CYP24A1-mediated metabolism could be good therapeutic agents. This system can effectively select vitamin D derivatives with these useful properties.

Evaluation of Anti-Adhesion Characteristics of Diamond-Like Carbon Film by Combining Friction and Wear Test with Step Loading and Weibull Analysis.

Anti-adhesion characteristics are important requirements for diamond-like carbon (DLC) films. The failure load corresponding to the anti-adhesion capacity varies greatly on three types of DLC film (hydrogen-free amorphous carbon film (a-C), hydrogenated amorphous carbon film (a-C:H), and tetrahedral hydrogen-free amorphous carbon film (ta-C)) in the friction and wear test with step loading using a high-frequency, linear-oscillation tribometer. Therefore, a new method that estimates a representative value of the failure load was developed in this study by performing a statistical analysis based on the Weibull distribution based on the assumption that the mechanism of delamination of a DLC film obeys the weakest link model.

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

CYP24A1-deficient (Cyp24a1 KO) rats were generated using the CRISPER/Cas9 system to investigate CYP24A1-dependent or -independent metabolism of 25(OH)D3, the prohormone of calcitriol. Plasma 25(OH)D3 concentrations in Cyp24a1 KO rats were approximately twofold higher than in wild-type rats. Wild-type rats showed five metabolites of 25(OH)D3 in plasma following oral administration of 25(OH)D3, and these metabolites were not detected in Cyp24a1 KO rats.

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

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D (1,25(OH)D) and 25-hydroxyvitamin D (25(OH)D) 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)D with an affinity equivalent to that for 25(OH)D, were also generated.

Novel biosensor using split-luciferase for detecting vitamin D receptor ligands based on the interaction between vitamin D receptor and coactivator.

Vitamin D receptor (VDR) ligands, such as 1α,25-dihydroxyvitamin D [1α,25(OH)D] and its analogs, have been investigated for their potential clinical use in the treatment of various diseases such as type I rickets, osteoporosis, psoriasis, leukemia, and cancer. Previously, we reported a split-luciferase-based biosensor that can detect VDR ligands and assess their affinity for the ligand binding domain (LBD) of the VDR in a short time. However, a further increase in its sensitivity was required to detect plasma levels of 1α,25(OH)D and its analogs.

Novel split luciferase-based biosensors for evaluation of vitamin D receptor ligands and their application to estimate CYP27B1 activity in living cells.

Recently, we successfully generated a novel detection system for vitamin D receptor (VDR) ligands in vivo and in vitro, using a split-luciferase technique called the LucN-LBD-LucC biosensor that is a chimeric fusion protein of firefly luciferase with the ligand binding domain (LBD) of VDR. In this system, the luciferase light intensity of the LucN-LBD-LucC biosensor was decreased by binding of VDR ligands. Although this system is quite useful for evaluation of VDR ligands in a short time, the sensitivity of the LucN-LBD-LucC biosensor is not high enough.

Mutant p53-Expressing Cells Undergo Necroptosis via Cell Competition with the Neighboring Normal Epithelial Cells.

p53 is a tumor suppressor protein, and its missense mutations are frequently found in human cancers. During the multi-step progression of cancer, p53 mutations generally accumulate at the mid or late stage, but not in the early stage, and the underlying mechanism is still unclear. In this study, using mammalian cell culture and mouse ex vivo systems, we demonstrate that when p53R273H- or p53R175H-expressing cells are surrounded by normal epithelial cells, mutant p53 cells undergo necroptosis and are basally extruded from the epithelial monolayer.

Development of a highly sensitive in vitro system to detect and discriminate between vitamin D receptor agonists and antagonists based on split-luciferase technique.

Split-luciferase techniques are widely used to detect protein-protein interaction and bioactive small molecules including some hormones and vitamins. Previously, we successfully expressed chimeric proteins of luciferase and the ligand binding domain (LBD) of the vitamin D receptor (VDR), LucC-LBD-LucN in COS-7 cells. The LucC-LBD-LucN biosensor was named split-luciferase vitamin D biosensor (SLDB).

Production of an active form of vitamin D by genetically engineered CYP105A1.

Our previous studies revealed that CYP105A1 can convert vitamin D (VD3) to its active form, 1α,25-dihydroxyvitamin D (1,25D3). Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity; the activity of double variants R73A/R84A and R73A/R84V was more than 100-fold higher than that of the wild type of CYP105A1. In contrast, these variants had a low ability to convert vitamin D (VD2) to 1α,25-dihydroxyvitamin D (1,25D2), whereas they catalyzed the sequential hydroxylation at positions C25 and C26 to produce 25,26D2.

Novel screening system for high-affinity ligand of heredity vitamin D-resistant rickets-associated vitamin D receptor mutant R274L using bioluminescent sensor.

Hereditary vitamin D-resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR) gene. Arg274 located in the ligand binding domain (LBD) of VDR is responsible for anchoring 1α,25-dihydroxyvitamin D3 (1α,25(OH)D) by forming a hydrogen bond with the 1α-hydroxyl group of 1α,25(OH)D The Arg274Leu (R274L) mutation identified in patients with HVDRR causes a 1000-fold decrease in the affinity for 1α,25(OH)D, and dramatically reduces vitamin D- related gene expression. Recently, we successfully constructed fusion proteins consisting of split-luciferase and LBD of the VDR.

Development of Novel Bioluminescent Sensor to Detect and Discriminate between Vitamin D Receptor Agonists and Antagonists in Living Cells.

Active forms of vitamin D regulate the expression of multiple genes that play essential roles in calcium and phosphate homeostasis, cell differentiation, and the immune system via the vitamin D receptor (VDR). Many vitamin D analogs have been synthesized for clinical use in the treatment of type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia, and breast cancer. We have constructed two fusion proteins containing split-luciferase and the ligand binding domain (LBD) of the VDR designated as LucN-LBD-LucC and LucC-LBD-LucN.

In vivo imaging of CREB phosphorylation in awake-mouse brain.

The cyclic adenosine monophosphate response element binding protein (CREB) is a phosphorylation-dependent transcription factor that plays important roles in memory consolidation and several neuropsychological disorders. Although analyzing the spatiotemporal pattern of CREB phosphorylation is required for elucidating the mechanism of memory consolidation, imaging of phosphorylation of a particular protein in the brain of live animals is impossible at present. Here, we developed a method for visualizing the CREB phosphorylation in the cerebral cortex of an awake mouse using a split luciferase technique.

Human hepatic metabolism of the anti-osteoporosis drug eldecalcitol involves sterol C4-methyl oxidase.

The metabolism of eldecalcitol (ED-71), a 2β-hydroxypropoxylated analog of the active form of vitamin D3 was investigated by using in vitro systems. ED-71 was metabolized to 1α,2β,25-trihydroxyvitamin D3 (1α,2β,25(OH)3D3) in human small intestine and liver microsomes. To identify the enzymes involved in this metabolism, we examined NADPH-dependent metabolism by recombinant P450 isoforms belonging to the CYP1, 2, and 3 families, and revealed that CYP3A4 had the activity.

Discovery of novel adenylyl cyclase inhibitor by cell-based screening.

We screened 2400 compounds to find novel inhibitors of the adenylyl cyclase (AC)-protein kinase A (PKA)-cAMP response-element-binding protein (CREB) signaling pathway (AC/PKA/CREB pathway). Using a multistep cell-based screening system employing split luciferase technique, we narrowed down the candidates effectively from 2400 chemical compounds and identified a novel AC inhibitor (compound 1). Since dysregulation of the AC/PKA/CREB pathway is known to cause diseases not only in the nervous system but also in other organs, compound 1 is expected to be developed as a medicine for these diseases.

Possible involvement of Hcn1 ion channel in learning and memory dysfunction in SAMP8 mice.

The senescence-accelerated mouse prone 8 (SAMP8) strain exhibits age-related learning and memory deficits (LMD) at 2 months of age. Combined linkage analysis of 264 F2 intercross SAMP8 × JF1 mice and RNA-seq analysis identified Hcn1 gene out of 29 genes in the LMD region on chromosome 13. Hcn1 in SAMP8 strain showed 15 times less polyglutamine repetition compared to Japanese fancy mouse 1 (JF1).

Measuring CREB activation using bioluminescent probes that detect KID-KIX interaction in living cells.

The cyclic adenosine monophosphate response element-binding protein (CREB) is a transcription factor that contributes to memory formation. The transcriptional activity of CREB is induced by its phosphorylation at Ser-133 and subsequent interaction with the CREB-binding protein (CBP)/p300. We designed and optimized firefly split luciferase probe proteins that detect the interaction of the kinase-inducible domain (KID) of CREB and the KIX domain of CBP/p300.