Vitamin K converting enzyme UBIAD1 plays an important role in osteogenesis and chondrogenesis in mice.

Dietary vitamin K1 (phylloquinone: PK) and menaquinone (MK-n) are converted to menadione (MD) in the small intestine and then translocated to various tissues where they are converted to vitamin K2 (menaquinone-4: MK-4) by UbiA prenyltransferase domain containing protein 1 (UBIAD1). MK-4 is effective in bone formation and is used to treat osteoporosis in Japan. UBIAD1 is expressed in bone and osteoblasts and shows conversion to MK-4, but the role of UBIAD1 in osteogenesis is unknown. In this study, we investigated the function of UBIAD1 in osteogenesis using a tamoxifen-dependent UBIAD1-deficient mouse model. When UBIAD1 deficiency was induced from the first week of life, the femur was significantly shortened, and bone mineral density (BMD) was reduced. In addition, the expression of bone and chondrocyte matrix proteins and chondrocyte differentiation factors was significantly decreased. In primary cultured chondrocytes, chondrocyte differentiation was significantly reduced by UBIAD1 deficiency. These results suggest that UBIAD1 is an important factor for the regulation of chondrocyte proliferation and differentiation during osteogenesis.

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. Among these metabolites, 25(OH)D3-26,23-lactone was identified as the second major metabolite with a significantly higher Tmax value than others. When 23S,25(OH)2D3 was administered to Cyp24a1 KO rats, neither 23,25,26(OH)3D3 nor 25(OH)D3-26,23-lactone was observed. However, when 23S,25R,26(OH)3D3 was administered to Cyp24a1 KO rats, plasma 25(OH)D3-26,23-lactone was detected. These results suggested that CYP24A1 is responsible for the conversion of 25(OH)D3 to 23,25,26(OH)3D3 via 23,25(OH)2D3, but enzyme(s) other than CYP24A1 may be involved in the conversion of 23,25,26(OH)3D3 to 25(OH)D3-26,23-lactone. Enzymatic studies using recombinant human CYP species and the inhibitory effects of ketoconazole suggested that CYP3A plays an essential role in the conversion of 23,25,26(OH)3D3 into 25(OH)D3-26,23-lactone in both rats and humans. Taken together, our data indicate that Cyp24a1 KO rats are valuable for metabolic studies of vitamin D and its analogs. In addition, long-term administration of 25(OH)D3 to Cyp24a1 KO rats at 110 µg/kg body weight/day resulted in significant weight loss and ectopic calcification. Thus, Cyp24a1 KO rats could represent an important model for studying renal diseases originating from CYP24A1 dysfunction.

Characterization of Astrocytes in the Minocycline-Administered Mouse Photothrombotic Ischemic Stroke Model.

Astrocytes, together with microglia, play important roles in the non-infectious inflammation and scar formation at the brain infarct during ischemic stroke. After ischemia occurs, these become highly reactive, accumulate at the infarction, and release various inflammatory signaling molecules. The regulation of astrocyte reactivity and function surrounding the infarction largely depends on intercellular communication with microglia. However, the mechanisms involved remain unclear. Furthermore, recent molecular biological studies have revealed that astrocytes are highly divergent under both resting and reactive states, whereas it has not been well reported how the communication between microglia and astrocytes affects astrocyte divergency during ischemic stroke. Minocycline, an antibiotic that reduces microglial activity, has been used to examine the functional roles of microglia in mice. In this study, we used a mouse photothrombotic ischemic stroke model to examine the characteristics of astrocytes after the administration of minocycline during ischemic stroke. Minocycline increased astrocyte reactivity and affected the localization of astrocytes in the penumbra region. Molecular characterization revealed that the induced expression of mRNA encoding the fatty acid binding protein 7 (FABP7) by photothrombosis was enhanced by the minocycline administration. Meanwhile, minocycline did not significantly affect the phenotype or class of astrocytes. The expression of Fabp7 mRNA was well correlated with that of tumor-necrosis factor α (TNFα)-encoding Tnf mRNA, indicating that a correlated expression of FABP7 from astrocytes and TNFα is suppressed by microglial activity.

Induced Prostanoid Synthesis Regulates the Balance between Th1- and Th2-Producing Inflammatory Cytokines in the Thymus of Diet-Restricted Mice.

Multiple external and internal factors have been reported to induce thymic involution. Involution involves dramatic reduction in size and function of the thymus, leading to various immunodeficiency-related disorders. Therefore, clarifying and manipulating molecular mechanisms governing thymic involution are clinically important, although only a few studies have dealt with this issue. In the present study, we investigated the molecular mechanisms underlying thymic involution using a murine acute diet-restriction model. Gene expression analyses indicated that the expression of T helper 1 (Th1)-producing cytokines, namely interferon-γ and interleukin (IL)-2, was down-regulated, while that of Th2-producing IL-5, IL-6, IL-10 and IL-13 was up-regulated, suggesting that acute diet-restriction regulates the polarization of naïve T cells to a Th2-like phenotype during thymic involution. mRNAs for prostanoid biosynthetic enzymes were up-regulated by acute diet-restriction. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses detected the increased production of prostanoids, particularly prostaglandin D2 and thromboxane B2, a metabolite of thromboxane A2, in the diet-restricted thymus. Administration of non-steroidal anti-inflammatory drugs, namely aspirin and etodolac, to inhibit prostanoid synthesis suppressed the biased expression of Th1- and Th2-cytokines as well as molecular markers of Th1 and Th2 cells in the diet-restricted thymus, without affecting the reduction of thymus size. In vitro stimulation of thymocytes with phorbol myristate acetate (PMA)/ionomycin confirmed the polarization of thymocytes from diet-restricted mice toward Th2 cells. These results indicated that the induced production of prostanoids during diet-restriction-induced thymic involution is involved in the polarization of naïve T cells in the thymus.

UBIAD1 Plays an Essential Role in the Survival of Pancreatic Acinar Cells.

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) is a vitamin K2 biosynthetic enzyme. We previously showed the lethality of this enzyme in UBIAD1 knockout mice during the embryonic stage. However, the biological effects of UBIAD1 deficiency after birth remain unclear. In the present study, we used a tamoxifen-inducible systemic UBIAD1 knockout mouse model to determine the role of UBIAD1 in adult mice. UBIAD1 knockout resulted in the death of the mice within about 60 days of administration of tamoxifen. The pancreas presented with the most prominent abnormality in the tamoxifen-induced UBIAD1 knockout mice. The pancreas was reduced remarkably in size; furthermore, the pancreatic acinar cells disappeared and were replaced by vacuoles. Further analysis revealed that the vacuoles were adipocytes. UBIAD1 deficiency in the pancreatic acinar cells caused an increase in oxidative stress and autophagy, leading to apoptotic cell death in the tamoxifen-induced UBIAD 1 knockout mice. These results indicate that UBIAD1 is essential for maintaining the survival of pancreatic acinar cells in the pancreas.

Nongenomic effects of 1α,25-dihydroxyvitamin D3 on cartilage formation deduced from comparisons between Cyp27b1 and Vdr knockout mice.

The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25D3), plays an important role in the maintenance of calcium (Ca) homeostasis, bone formation, and cell proliferation and differentiation via nuclear vitamin D receptor (VDR). It is formed by the hydroxylation of vitamin D at the 1α position by 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1) in the kidney. However, Cyp27b1-/- mice, deficient in CYP27B1, and VDR-deficient mice (Vdr-/-) have not been extensively examined, particularly in a comparative framework. To clarify the physiological significance of 1α,25D3 and VDR, we produced Cyp27b1-/- mice and compared their phenotypes with those of Vdr-/- mice. Cyp27b1-/- mice exhibited hypocalcemia, growth defects, and skeletogenesis dysfunction, similar to Vdr-/- mice. However, unlike Cyp27b1-/- mice, Vdr-/- mice developed alopecia. Cyp27b1-/- mice exhibited cartilage mass formation and had difficulty walking on hindlimbs. Furthermore, a phenotypic analysis was performed on Cyp27b1-/- mice provided a high Ca diet to correct for the Ca metabolic abnormality. In addition, the effects of 1α,25D3 that are not mediated by Ca metabolic regulatory activity were investigated. Even when the blood Ca concentration was corrected, abnormalities in growth and cartilage tissue formation did not improve in Cyp27b1-/- mice. These results suggested that 1α,25D3 directly controls chondrocyte proliferation and differentiation. Using Cyp27b1-/- mice produced in this study, we can analyze the physiological effects of novel vitamin D derivatives in the absence of endogenous 1α,25D3. Accordingly, this study provides a useful animal model for the development of novel vitamin D formulations that are effective for the treatment and prevention of osteoporosis.

Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme.

Vitamin K occurs in the natural world in several forms, including a plant form, phylloquinone (PK), and a bacterial form, menaquinones (MKs). In many species, including humans, PK is a minor constituent of hepatic vitamin K content, with most hepatic vitamin K content comprising long-chain MKs. Menaquinone-4 (MK-4) is ubiquitously present in extrahepatic tissues, with particularly high concentrations in the brain, kidney and pancreas of humans and rats. It has consistently been shown that PK is endogenously converted to MK-4 (refs 4-8). This occurs either directly within certain tissues or by interconversion to menadione (K(3)), followed by prenylation to MK-4 (refs 9-12). No previous study has sought to identify the human enzyme responsible for MK-4 biosynthesis. Previously we provided evidence for the conversion of PK and K(3) into MK-4 in mouse cerebra. However, the molecular mechanisms for these conversion reactions are unclear. Here we identify a human MK-4 biosynthetic enzyme. We screened the human genome database for prenylation enzymes and found UbiA prenyltransferase containing 1 (UBIAD1), a human homologue of Escherichia coli prenyltransferase menA. We found that short interfering RNA against the UBIAD1 gene inhibited the conversion of deuterium-labelled vitamin K derivatives into deuterium-labelled-MK-4 (MK-4-d(7)) in human cells. We confirmed that the UBIAD1 gene encodes an MK-4 biosynthetic enzyme through its expression and conversion of deuterium-labelled vitamin K derivatives into MK-4-d(7) in insect cells infected with UBIAD1 baculovirus. Converted MK-4-d(7) was chemically identified by (2)H-NMR analysis. MK-4 biosynthesis by UBIAD1 was not affected by the vitamin K antagonist warfarin. UBIAD1 was localized in endoplasmic reticulum and ubiquitously expressed in several tissues of mice. Our results show that UBIAD1 is a human MK-4 biosynthetic enzyme; this identification will permit more effective decisions to be made about vitamin K intake and bone health.

YY1 positively regulates human UBIAD1 expression.

Vitamin K is involved in bone formation and blood coagulation. Natural vitamin K compounds are composed of the plant form phylloquinone (vitamin K1) and a series of bacterial menaquionones (MK-n; vitamin K2). Menadione (vitamin K3) is an artificial vitamin K compound. MK-4 contains 4-isoprenyl as a side group in the 2-methyl-1,4-naphthoquinone common structure and has various bioactivities. UbiA prenyltransferase domain containing 1 (UBIAD1 or TERE1) is the menaquinone-4 biosynthetic enzyme. UBIAD1 transcript expression significantly decreases in patients with prostate carcinoma and overexpressing UBIAD1 inhibits proliferation of a tumour cell line. UBIAD1 mRNA expression is ubiquitous in mouse tissues, and higher UBIAD1 mRNA expression levels are detected in the brain, heart, kidneys and pancreas. Several functions of UBIAD1 have been reported; however, regulation of the human UBIAD1 gene has not been elucidated. Here we report cloning and characterisation of the human UBIAD1 promoter. A 5' rapid amplification of cDNA ends analysis revealed that the main transcriptional start site was 306 nucleotides upstream of the translation initiation codon. Deletion and mutation analyses revealed the functional importance of the YY1 consensus motif. Electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that YY1 binds the UBIAD1 promoter in vitro and in vivo. In addition, YY1 small interfering RNA decreased endogenous UBIAD1 mRNA expression and UBIAD1 conversion activity. These results suggest that YY1 up-regulates UBIAD1 expression and UBIAD1 conversion activity through the UBIAD1 promoter.

Menadione (vitamin K3) is a catabolic product of oral phylloquinone (vitamin K1) in the intestine and a circulating precursor of tissue menaquinone-4 (vitamin K2) in rats.

Mice have the ability to convert dietary phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) and store the latter in tissues. A prenyltransferase enzyme, UbiA prenyltransferase domain-containing 1 (UBIAD1), is involved in this conversion. There is evidence that UBIAD1 has a weak side chain cleavage activity for phylloquinone but a strong prenylation activity for menadione (vitamin K3), which has long been postulated as an intermediate in this conversion. Further evidence indicates that when intravenously administered in mice phylloquinone can enter into tissues but is not converted further to menaquinone-4. These findings raise the question whether phylloquinone is absorbed and delivered to tissues in its original form and converted to menaquinone-4 or whether it is converted to menadione in the intestine followed by delivery of menadione to tissues and subsequent conversion to menaquinone-4. To answer this question, we conducted cannulation experiments using stable isotope tracer technology in rats. We confirmed that the second pathway is correct on the basis of structural assignments and measurements of phylloquinone-derived menadione using high resolution MS analysis and a bioassay using recombinant UBIAD1 protein. Furthermore, high resolution MS and (1)H NMR analyses of the product generated from the incubation of menadione with recombinant UBIAD1 revealed that the hydroquinone, but not the quinone form of menadione, was an intermediate of the conversion. Taken together, these results provide unequivocal evidence that menadione is a catabolic product of oral phylloquinone and a major source of tissue menaquinone-4.

Role for vitamin D receptor in the neuronal control of the hematopoietic stem cell niche.

Hematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow to the circulation by the cytokine, granulocyte colony-stimulating factor, via sympathetic nervous system (SNS)-mediated osteoblast suppression. Because the orientation of HSPCs in their osteoblastic niche is reported to be guided by [Ca(2+)], we speculated on a cooperation between the calcium-regulating hormones and SNS in the regulation of HSPC trafficking. Here, we present the severe impairment of granulocyte colony-stimulating factor-induced osteoblast suppression and subsequent HSPC mobilization in vitamin D receptor (VDR)-deficient mice. In osteoblasts, functional VDR possessing, at least in part, a transcriptional activity, was specifically induced by ß2-adrenergic receptor (AR) agonists. While ß2-AR agonists transiently increased mRNA expression of Vdr and its downstream gene, Rankl, 1α,25-dihydroxyvitamin-D(3) sustained the ß2-AR-induced Rankl expression at high level by stabilizing VDR protein. These data suggest that VDR is essential for durable ß2-AR signaling in the stem cell niche. Our study demonstrates not only a novel function of VDR as a critical modulator of HSPC trafficking, but also the presence of a SNS-mediated, bone-remodeling mechanism through VDR. VDR contributes to brain-bone-blood integration in an unanticipated way distinct from other classical calcium-regulating hormones.

Vitamin D Status in Japanese Young Women in 2016-2017 and 2020: Seasonal Variation and the Effect of Lifestyle Including Changes Caused by the COVID-19 Pandemic.

Avoidance of sunlight and self-restraint due to the COVID-19 pandemic may contribute to reduced vitamin D status. This study provides comparable data on vitamin D status in Japanese young women and assesses the effect of lifestyle, including changes caused by the COVID-19 pandemic, on vitamin D status. In study 1, 39 young healthy Japanese women aged 21-25 y were recruited from May 2016-June 2017. Serum 25-hydroxyvitamin D (25OHD) concentration and diet and lifestyle information were obtained from participants each month (n=124). In study 2, using the same parameters as study 1, young women aged 21-23 y (n=10) were recruited in September 2020. In the results of study 1, we found the frequencies of vitamin D deficiency (25OHD<20 ng/mL) in spring, summer, fall, and winter were 90.5%, 62.5%, 81.5%, and 91.3%, respectively. The substantial difference of serum 25OHD concentration was obtained in spring (Δ3.6 ng/mL) and summer (Δ5.1 ng/mL) depending on the frequency of sunscreen use (0-2 d/wk, 3-7 d/wk). In study 2, serum 25OHD concentration in September 2020 was extremely lower than in September 2016 (13.2 ng/mL vs. 21.7 ng/mL). The number of days spent outside in 2020 decreased drastically compared with 2019. In conclusion, vitamin D deficiency was highly common in Japanese women in their early 20s, and frequent sunscreen use contributed to low vitamin D status. Moreover, because the decrease in days outside due to the COVID-19 pandemic obviously resulted in a decline in vitamin D status, both appropriate sunbathing and increased dietary vitamin D intake are recommended to young women.

Replacement of the hydrophobic part of 9-cis-retinoic acid with cyclic terpenoid moiety results in RXR-selective agonistic activity.

Retinoid X receptor (RXR) agonists are interesting candidates for the treatment of metabolic syndrome. 9-Cis-retinoic acid (9cRA: 1) is a natural RXR agonist, that also works as a retinoic acid receptor (RAR) agonist. This fact prompted us to study the structure-activity relationship (SAR) of RXR agonists derived from 1. Though 3 and 4, in which the cyclohexene part of 1 is replaced with bulkier hydrophobic moieties, show RXR-selective agonistic activity, some analogs containing other ring structures show RAR agonistic activity. Thus, we were interested in establishing what kind of ring skeleton is required for RXR-selective agonistic activity. In this study, we systematically prepared 5 and 6, in which the cyclohexene ring of 1 is replaced with various cyclic terpenoid moieties, and evaluated their RXR and RAR agonistic activities. Our previously reported CsF-promoted Stille coupling reaction was employed as a key step for the comprehensive synthesis of 5 and 6. The results of transcriptional assay showed that compounds 5b-f, which possess a menthane skeleton, exhibit RXR-selective agonistic activity. These results should be helpful for the design of superior RXR-selective agonists based on the structure of 1.

Inhibitory effect of 1α-hydroxyvitamin D3 on N-nitrosobis(2-oxopropyl)amine-induced cholangiocarcinogenesis in Syrian hamsters.

Sixty-three male 5-week-old Syrian hamsters received the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) s.c. in 5 weekly injections (the first, 70 mg/kg body, and the remaining, 20mg/kg each). The hamsters that received BOP were given intragastric administration of 0.2 ml of medium chain triglyceride (MCT) with or without 0.04 µg of 1α-hydroxyvitamin D3 [1α(OH)D3] through a feeding tube for 12 weeks. Thus, 3 groups were assigned:Group 1;BOP alone (n＝20), Group 2;BOP＋MCT (n＝18) and Group 3;BOP＋1α(OH)D3 (n＝25). The mean body weight of Group 3 was lower than those of Groups 1 and 2 at the end of the experiment (p＜0.001,Tukey-Kramer HSD test). At the end of week 12, all surviving hamsters were put to sleep. The incidences of liver tumors were 80%, 72% and 32% in Groups 1, 2 and 3, respectively. The incidence of tumors in Group 3 was significantly lower than in Group 1 and Group 2 (p＜0.05, χ2-test). All tumors were cholangiocarcinoma. These results indicated that BOP-induced cholangiocarcinogenesis was suppressed by the supplemental administration of 1α(OH)D3.

[Diseases Due to the Deficiency in Vitamin K Conversion System and Its Prevention].

Vitamin K is a fat-soluble vitamin that plays an important role in blood coagulation and bone formation. Vitamin K has homologues due to differences in the side chain structure, phylloquinone (abbreviated as vitamin K1, PK) having a phytyl side chain and menaquinones (MK-n, n=1 to 14) having an isoprenoid side chain structure. The main vitamin K that we take from our daily diet is PK, and a fermented food, natto, contains MK-7 produced by Bacillus subtilis natto. However, the majority of vitamin K present in the tissues of mammals, including humans, is menaquinone-4 (abbreviated as vitamin K2, MK-4) having a geranylgeranyl side chain. This reason is that PK or MK-n obtained in the diet is converted into MK-4 in the body. We identified that the UbiA prenyltransferase domain containing protein 1 (UBIAD1) is the conversion enzyme of PK and MK-n to MK-4. The physiological roles of MK-4 in all tissues of the whole body and the physiological significance of MK-4 converted from PK and MK-n by UBIAD1 have not been sufficiently elucidated yet. To investigate the function of UBIAD1 in vivo, we generated UBIAD1 systemic knockout mice and tissue-specific UBIAD1 knockout mice. In this paper, we introduce the usefulness of vitamin K for diseases that may involve vitamin K and UBIAD1.

Depressed expression of Klotho and FGF receptor 1 in hyperplastic parathyroid glands from uremic patients.

Fibroblast growth factor 23 (FGF23) exerts its effect by binding to its cognate FGF receptor 1 (FGFR1) in the presence of its co-receptor Klotho. Parathyroid glands express both FGFR1 and Klotho, and FGF23 decreases parathyroid hormone gene expression and hormone secretion directly. In uremic patients with secondary hyperparathyroidism (SHPT), however, parathyroid hormone secretion remains elevated despite extremely high FGF23 levels. To determine the mechanism of this resistance, we measured the expression of Klotho, FGFR1, and the proliferative marker Ki67 in 7 normal and 80 hyperplastic parathyroid glands from uremic patients by immunohistochemistry. All uremic patients had severe SHPT along with markedly high FGF23 levels. Quantitative real-time reverse transcription PCR showed that the mRNA levels for Klotho and FGFR1correlated significantly with their semi-quantitative immunohistochemical intensity. Compared with normal tissue, the immunohistochemical expression of Klotho and FGFR1 decreased, but Ki67 expression increased significantly in hyperplastic parathyroid glands, particularly in glands with nodular hyperplasia. These results suggest that the depressed expression of the Klotho-FGFR1 complex in hyperplastic glands underlies the pathogenesis of SHPT and its resistance to extremely high FGF23 levels in uremic patients.

Efficient synthesis and biological evaluation of demethyl geranylgeranoic acid derivatives.

Synthetic retinoids have generated in the fields of dermatology and oncology due to their potent anti-proliferative and differentiation activities. We efficiently synthesized different demethyl geranylgeranoic acid (GGA) analogs, and evaluated their biological activities. Among the demethyl analogs synthesized, 3-demethyl derivative exhibited the highest anti-proliferative activity in HL-60 cells. In addition, a 3-demethyl derivative induced apoptosis more potently than 9Z-retinoic acid. These activities were due to the high binding affinity of 3-demethyl derivative for retinoid receptors. We found that, in a conjugated polyene system combined with a methyl substituent, the position of the methyl played an important role in the regulation of gene transcription and apoptosis-inducing activity. These results provided useful information on the structure-activity relationships of GGA derivatives that function as acyclic retinoic acid analogs. This information is likely to be useful in the development of new anti-cancer drugs.

Structure-activity relationships in the conversion of vitamin K analogues into menaquinone-4. Substrates essential to the synthesis of menaquinone-4 in cultured human cell lines.

To reveal an essential biological role of menaquinone-4, we have clarified that dietary PK was converted to menaquinone-4 (MK-4) in animal tissues using deuterated vitamin K analogues. However, the kinds of analogue converted into MK-4 have not been elucidated. In this study, we examined structure-activity relationships in the conversion of several vitamin K analogues, with a substituted side chain, into MK-4 using cultured human cell lines. The results differed with the side chain of the analogues, that is, (1) the length of the isoprene unit and (2) the number of double bonds in the side chain. These findings would be useful for clarifying the mechanism of conversion of other vitamin K homologs into MK-4 as well as related enzymes.

Synthesis and biological evaluation of 9Z-retinoic acid analogs having 2-substituted benzo[b]furan.

Palladium-catalyzed cross-coupling reactions of a 2-substituted 3-iodobenzo[b]furans and stannanyl ester afforded the stereoselective production of 9Z-retinoic acid ester analogs in good yields. These esters were then converted to the corresponding acids via basic hydrolysis in excellent yields, and their biological activities were evaluated. The analog changed the connected position of polyene side chain from 2-position to 3-position of benzo[b]furan decreased the biological activities dramatically, and the introduction of various substituents at 2-position afforded almost no effect on the activities.

Induced mRNA expression of matrix metalloproteinases Mmp-3, Mmp-12, and Mmp-13 in the infarct cerebral cortex of photothrombosis model mice.

A strong therapeutic target of ischemic stroke is controlling brain inflammation. Recent studies have implicated the critical role of C-C chemokine receptor 5 (CCR5) in neuroinflammation during ischemic stroke. It has been reported that the expression of the matrix metalloproteinases, MMP-3, MMP-12, and MMP-13, is controlled by CCR5; however, their expressional regulation in the infarct brain has not been clearly understood. This study investigated the mRNA expression of Mmp-3, -12, and -13 in the ischemic cerebral cortex of photothrombosis mouse model. The three Mmps were highly upregulated in the early stages of ischemic stroke and were expressed in different types of cells. Mmp-3 and Mmp-13 were expressed in blood vessel endothelial cells after ischemia-induction, whereas Mmp-12 was expressed in activated microglia. The expression of Mmp-13 in resting microglia and in neurons of uninjured cerebral cortex was lost in the infarct region. Therefore, the MMPs responding to CCR5 are differentially regulated during ischemic stroke.

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 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.