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.

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.

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25-D(3)) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, its calcemic effect in vivo limits its therapeutic applications. Here, we report the efficacy of 22-oxa-1alpha,25-dihydroxyvitamin D(3) (22-oxa-1alpha,25-D(3)), a low calcemic analog of vitamin D, against the development of metastatic lung carcinoma after an intravenous injection of green fluorescent protein-transfected Lewis lung carcinoma (LLC-GFP) cells in C57BL/6 mice. The mice injected with tumor cells were implanted simultaneously with osmotic minipumps containing either 1alpha,25-D(3), 22-oxa-1alpha,25-D(3) or vehicle. The 1alpha,25-D(3) treatment group had been hypercalcemic, but the 22-oxa-1alpha,25-D(3) and vehicle treatment groups remained normocalcemic for the duration of the experiment. The total number of lung metastases, lung weight and the expression of GFP mRNA in the lung were markedly decreased in 1alpha,25-D(3) and 22-oxa-1alpha,25-D(3)-treated mice. In the in vitro experiment, 1alpha,25-D(3) and 22-oxa-1alpha,25-D(3) reduced the expression of matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor and parathyroid hormone-related protein in LLC-GFP cells. Furthermore, in the angiogenesis assay, the number of tumor cells or basic fibroblast growth factor-induced angiogenesis was reduced in 1alpha,25-D(3) and 22-oxa-1alpha,25-D(3)-treated mice. Moreover, using a new experimental model of vitamin D receptor (VDR) null mutant (VDR(-/-)) mice with corrected hypocalcemia and hypervitaminosis D, we examine the anti-cancer effect of 22-oxa-1alpha,25-D(3) without other functions induced by 22-oxa-1alpha,25-D(3) in the host. In the VDR(-/-) mice, 22-oxa-1alpha,25-D(3) directly inhibited the metastatic activity of LLC-GFP cells in a dose-dependent manner without exerting a direct influence on the calcemic activity or other actions regulated by 22-oxa-1alpha,25-D(3) in the host. These results indicate that the inhibition of metastasis and angiogenesis-inducing activity in cancer cells seemed to be a major mechanism responsible for the anti-cancer effects of 22-oxa-1alpha,25-D(3). Our findings show that 22-oxa-1alpha,25-D(3) is beneficial for the prevention of metastasis in lung carcinoma.

Background:Menadione is an intermediate in phylloquinone to menaquinone-4 conversion in mammals. Results: Menadione is released from phylloquinone in the intestine and converted to menaquinone-4 in tissues after being reduced. Conclusion: Menadione is a catabolic product of phylloquinone and circulating precursor of tissue menaquinone-4. Significance: Determining how phylloquinone is metabolized in the body is crucial for understanding vitamin K biology.

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the major regulator of calcium homeostasis, has potent antiproliferative and anti-invasive properties in vitro in cancer cells. Studies in vivo demonstrated that 1alpha,25(OH)(2)D(3) slows the progression of breast, prostate and other carcinomas. A key question is whether 1alpha,25(OH)(2)D(3) exerts its anticarcinogenic effects in vivo by a mechanism that is dependent on its capacity to limit the proliferation and invasiveness of cancer cells in vitro. It has not been clear whether the calcemic activity and regulation of the host defenses by 1alpha,25(OH)(2)D(3) contribute to the effect on cancer cells. In this study we have focused on the influence of 1alpha,25(OH)(2)D(3) on the metastasis of lung cancer, without involvement of the calcemic activity and other effects of 1alpha,25(OH)(2)D(3) in the host. We used metastatic Lewis lung carcinoma cells expressing green fluorescent protein (LLC-GFP cells) and examined metastatic activity in vitamin D receptor (VDR) null mutant (VDR(-/-)) mice and their wild-type counterparts (VDR(+/+) mice). VDR(-/-) mice exhibit hypocalcemia and extremely high serum levels of 1alpha,25(OH)(2)D(3). We expected that serum 1alpha,25(OH)(2)D(3) would act in vivo to directly inhibit the metastatic growth of VDR-positive LLC-GFP cells in VDR(-/-) mice. The metastatic activities of LLC-GFP cells were remarkably reduced in VDR(-/-) mice compared with VDR(+/+) mice. To test the hypothesis that serum 1alpha,25(OH)(2)D(3) is an intrinsic factor that inhibits metastatic growth of lung cancer cells, we corrected hypocalcemia and/or hypervitaminosis D in VDR(-/-) mice by dietary manipulation. The metastatic growth of LLC-GFP cells was remarkably reduced in response to serum levels of 1alpha,25(OH)(2)D(3), but not to serum calcium levels. Furthermore, we found that VDR(+/+) mice fed the manipulated diets displayed an apparent inverse relationship between the physiological levels of serum 1alpha,25(OH)(2)D(3) (8-15 pg/ml) and tumorigenesis. Here we show that 1alpha,25(OH)(2)D(3) inhibits the metastatic growth of lung cancer cells in a defined animal model.