Toshio Okano

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 (K3), 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 K3 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-d7) 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-d7 in insect cells infected with UBIAD1 baculovirus. Converted MK-4-d7 was chemically identified by 2H-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.

Conversion of phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) in mice: Two possible routes for menaquinone-4 accumulation in cerebra of mice

There are two forms of naturally occurring vitamin K, phylloquinone and the menaquinones. Phylloquinone (vitamin K1) is a major type (>90%) of dietary vitamin K, but its concentrations in animal tissues are remarkably low compared with those of the menaquinones, especially menaquinone-4 (vitamin K2), the major form (>90%) of vitamin K in tissues. Despite this great difference, the origin of tissue menaquinone-4 has yet to be exclusively defined. It is postulated that phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. To clarify this, phylloquinone with a deuterium-labeled 2-methyl-1,4-naphthoquinone ring was given orally to mice, and cerebra were collected for D NMR and liquid chromatography-tandem mass spectrometry analyses. We identified the labeled menaquinone-4 that was converted from the given phylloquinone, and this conversion occurred following an oral or enteral administration, but not parenteral or intracerebroventricular administration. By the oral route, the phylloquinone with the deuterium-labeled side chain in addition to the labeled 2-methyl-1,4-naphthoquinone was clearly converted into a labeled menaquinone-4 with a non-deuterium-labeled side chain, implying that phylloquinone was converted into menaquinone-4 via integral side-chain removal. The conversion also occurred in cerebral slice cultures and primary cultures. Deuterium-labeled menadione was consistently converted into the labeled menaquinone-4 with all of the administration routes and the culture conditions tested. Our results suggest that cerebral menaquinone-4 originates from phylloquinone intake and that there are two routes of accumulation, one is the release of menadione from phylloquinone in the intestine followed by the prenylation of menadione into menaquinone-4 in tissues, and another is cleavage and prenylation within the cerebrum.

Low plasma phylloquinone concentration is associated with high incidence of vertebral fracture in Japanese women

It has been reported that vitamin K supplementation effectively prevents fractures and sustains bone mineral density in osteoporosis. However, there are only limited reported data concerning the association between vitamin K nutritional status and bone mineral density (BMD) or fractures in Japan. The objectives were to evaluate the association between plasma phylloquinone (K1) or menaquinone (MK-4 and MK-7) concentration and BMD or fracture in Japanese women prospectively. A total of 379 healthy women aged 30–88 years (mean age, 63.0 years) were consecutively enrolled. Plasma K1, MK-4, MK-7, and serum undercarboxylated osteocalcin (ucOC) concentrations, BMD, and incidence of vertebral fractures were evaluated. In stepwise multiple linear regression analyses, L2–4 BMD and a bone turnover marker, log K1, concentrations were independently correlated with vertebral fracture incidence. When subjects were divided into low and high K1 groups by plasma K1 concentration, the incidence of vertebral fracture in the low K1 group (14.4%) was significantly higher than that in the high K1 group (4.2%), and its age-adjusted RR was 3.58 (95% CI, 3.26–3.93). L2–4 BMD was not different between the two groups. These results suggest that subjects with vitamin K1 insufficiency in bone have increased susceptibility for vertebral fracture independently from BMD.

High prevalence of vitamin K and D deficiency and decreased BMD in inflammatory bowel disease.

SummaryVitamin K and D deficiency and decreased bone mineral density (BMD) were highly prevalent in patients with inflammatory bowel disease (IBD), especially Crohn’s disease (CD). Dietary intakes of these vitamins, however, were above the Japanese adequate intakes in IBD patients, suggesting that malabsorption is the basis for hypovitaminosis K and D and decreased BMD.IntroductionWe have studied the possible involvement of vitamin K and D deficiency in the pathogenesis of decreased BMD in IBD.MethodsSeventy patients with IBD were evaluated for their BMD; plasma levels of vitamin K; phylloquinone (PK), menaquinone-7 (MK-7), and 25OH-D; serum PTH, protein induced by vitamin K absence (PIVKA-II), and undercarboxylated osteocalcin (ucOC) levels; and their food intake.ResultsCompared with ulcerative colitis (UC) patients, CD patients had significantly lower plasma vitamin K and 25OH-D concentrations; significantly higher serum levels of PTH, PIVKA-II, and ucOC; and significantly lower BMD scores at almost all measurement sites. More IBD patients were vitamin K deficient in bone than in liver. Multiple regression analyses revealed that low plasma concentrations of vitamin K and 25OH-D were independent risk factors for low BMD and that they were associated with the patients’ fat intake, but not with their intake of these vitamins.ConclusionIBD patients have high prevalence of decreased BMD and vitamin K and D deficiency probably caused by malabsorption of these vitamins.

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 β2-adrenergic receptor (AR) agonists. While β2-AR agonists transiently increased mRNA expression of Vdr and its downstream gene, Rankl, 1α,25-dihydroxyvitamin-D(3) sustained the β2-AR-induced Rankl expression at high level by stabilizing VDR protein. These data suggest that VDR is essential for durable β2-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.

Elucidation of the mechanism producing menaquinone-4 in osteoblastic cells.

Vitamin K is an essential nutrient and a cofactor for the carboxylation of specific glutamyl residues of proteins to gamma-glutamyl residues, which activates osteocalcin related to bone formation. Among vitamin K homologues, menaquinone-4 (MK-4) is the most active biologically, up-regulating the gene expression of bone markers, and thus has been clinically used in the treatment of osteoporosis in Japan. Recently, we confirmed that MK-4 was converted from dietary phylloquinone (PK), and then accumulated in various tissues at high concentrations. This system should play an important role in biological functions including bone formation, however, the pathway by which MK-4 is converted remains unclear. In this study, we studied the mechanism of MK-4s conversion with chemical techniques using deuterated analogues.

Design and synthesis of biologically active analogues of vitamin K2 : Evaluation of their biological activities with cultured human cell lines

Novel omega-oxygenated vitamin K(2) analogues were efficiently synthesized and their biological activities were evaluated. Some were biologically active and the side-chain played an important role in gamma-carboxylation and apoptosis-inducing activity. The results provide useful information on the structure-activity relationship of vitamin K(2) analogues for the development of new 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.

Preparation and biological evaluation of 5-substituted retinoic acids

Various 5-substituted retinoic acids were prepared by a palladium-catalyzed cross coupling reactions of vinyl nonaflates and E- or Z-3-tributylstannyl-2-beten-1-ol as a key reaction. These coupling products were then converted to the corresponding all-E- and 9Z-retinoic acid analogs via Horner-Emmons reaction and subsequent basic hydrolysis, and their biological activities were evaluated. The all-E-derivatives, 5-butyl and isobutyl analogs exhibited stronger effects for anti-proliferative and differentiation-inducing activities in HL-60 cells. In contrast, in 9Z-derivatives, none of the analogs showed any activity.

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.