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 γ-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-4’s conversion with chemical techniques using deuterated analogues.     

Bone markers during a 6-month space flight: effects of vitamin K supplementation

Rapid bone loss is a serious health problem for astronauts during long lasting missions in space. We have recorded the changes of biochemical markers for bone metabolism in one of the astronauts during the 6-month space flight of the EUROMIR-95 mission. Immediately after launch both bone resorption markers and urinary calcium excretion increased about two fold, whereas bone formation markers remained unchanged. After 12 1/2 weeks the astronaut received vitamin K1 (10 mg/day for 6 weeks). Vitamin K is known to be involved in the formation of gamma-carboxyglutamate (Gla) in proteins, such as the calcium-binding bone Gla-proteins osteocalcin and matrix Gla-protein. Concomitant with the start of vitamin K treatment, the calcium-binding capacity of osteocalcin increased, and so did the urinary excretion of free Gla. This is suggestive for a subclinical vitamin K-deficiency in the astronaut before vitamin K-supplementation. During periods of high vitamin K status markers for bone formation (osteocalcin and bone alkaline phosphatase) had increased as compared to the first part of the flight. The mean increases were 14 and 23%, respectively. Our data suggest that increased intake of vitamin K may contribute to counteracting microgravity-induced loss of bone mass during long lasting space missions, but need confirmation in more astronauts.     

Pregnane X Receptor Knockout Mice Display Aging-Dependent Wearing of Articular Cartilage

Steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR), are nuclear receptors that are expressed at high levels in the liver and the intestine where they function as xenobiotic sensors that induce expression of genes involved in detoxification and drug excretion. Recent evidence showed that SXR and PXR are also expressed in bone tissue where they mediate bone metabolism. Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints. Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice. We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2. Lastly, we demonstrated the biological significance of Fam20a expression in chondrocytes by evaluating osteoarthritis-related gene expression of primary articular chondrocytes. Consistent with epidemiological findings, our results indicate that SXR/PXR protects against aging-dependent wearing of articular cartilage and that ligands for SXR/PXR have potential role in preventing osteoarthritis caused by aging.     

Vitamin K in combination with other biochemical markers to diagnose osteoporosis.

The significance of a multiparametric classification approach of vitamin K is analysed to differentiate premenopausal (CTRL), postmenopausal non-osteoporotic (nOSP) and osteoporotic (OSP) women. Data records of women between 28 and 74 years of age were used for evaluation. Bone mineral density was determined by quantitative computed tomography of the lumbar spine using the T-score to diagnose osteoporosis. Vitamin K and biochemical markers of bone formation and resorption--alkaline phosphatase (AP), bone alkaline phosphatase (bAP), osteocalcin (OC), undercarboxylated osteocalcin (ucOC), procollagen type I carboxyterminal propeptide (PICP), pyridinoline (PYD), deoxypyridinoline (DPD), N-terminal cross-linked telopeptide of type I collagen (NTx) and bone sialo protein (BSP)--were analysed in all women on days 1 and 42. Vitamin K was significantly lower in the OSP group versus nOSP and CTRL. The odds ratio results revealed the following: vitamin K, 16.7; PYD, 7.5; NTx, 6.0; DPD, 2.7; and ucOC, 2.7. Vitamin K represented a sensitivity rate of 64% and a specificity rate of 82%. In the receiver operating curve analysis, vitamin K reached the highest area under curve (AUC) score. The combination of vitamin K and AP, bAP and PYD resulted in increased AUC scores (>0.9). The parameter combination of vitamin K/PYD and vitamin K/bAP demonstrated a sensitivity rate of 75-88%, with a specificity rate of more than 82%. The data suggests that a combination of vitamin K with other biochemical bone indices might be a useful tool for assessing bone metabolism, especially in metabolic bone diseases such as osteoporosis.     

Bone effects of vitamin D - Discrepancies between in vivo and in vitro studies

Vitamin D was discovered as an anti-rachitic agent, but even at present, there is no direct evidence to support the concept that vitamin D directly stimulates osteoblastic bone formation and mineralization. It appears to be paradoxical, but vitamin D functions in the process of osteoclastic bone resorption. In 1952, Carlsson reported that administration of vitamin D(3) to rats fed a vitamin D-deficient, low calcium diet raised serum calcium levels. Since the diet did not contain appreciable amounts of calcium, the rise in serum calcium was considered to be derived from bone. Since then, this assay has been used as a standard bioassay for vitamin D compounds. Osteoclasts, the cells responsible for bone resorption, develop from hematopoietic cells of the monocyte-macrophage lineage. Several lines of evidence have shown that the active form of vitamin D(3), 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] is one of the most potent inducers of receptor activator of NF-κB ligand (RANKL), a key molecule for osteoclastogenesis, in vitro. In fact, 1α,25(OH)(2)D(3) strongly induced osteoclast formation and bone resorption in vitro. Nevertheless, 1α,25(OH)(2)D(3) and its prodrug, Alfacalcidol (1α-hydroxyvitamin D(3)) have been used as therapeutic agents for osteoporosis since 1983, because they increase bone mineral density and reduce the incidence of bone fracture in vivo. Furthermore, a new vitamin D analog, Eldecalcitol [2β-(3-hydroxypropoxy)-1α,25(OH)(2)D(3)], has been approved as a new drug for osteoporosis in Japan in January 2011. Interestingly, these beneficial effects of in vivo administration of vitamin D compounds are caused by the suppression of osteoclastic bone resorption. The present review article describes the mechanism of the discrepancy of vitamin D compounds in osteoclastic bone resorption between in vivo and in vitro.     

Vitamin D and aging: old concepts and new insights

Aging is a complex biological process driven by a selective class of molecules and pathways that affect overall deterioration of physiological functions to increase the risk of age-related diseases. A role of vitamin D in mammalian aging is well documented. Since vitamin D has an essential role in bone formation and mineralization, its deficiency results in impaired bone mineralization, such as rickets in children, osteomalacia in adults and osteoporosis in the aged population. Vitamin D replacement therapy therefore is one of the most commonly prescribed treatments for the elderly. Recent studies using genetically altered mouse models, such as in Fgf-23^−/− and klotho mutant mice, that exhibit altered mineral ion metabolism due to high vitamin D activities showed features of premature aging that include atherosclerosis, emphysema, osteopenia/osteoporosis, hypogonadism, soft tissue calcifications and generalized atrophy of organs; the pathologic effects of vitamin D in these mouse models are obvious, as diminution or genetic ablation of the vitamin D pathway ameliorated most of the above-mentioned phenotypes, by reversing mineral ion metabolism, and the resultant effect being prolonged survival of the mutant mice. These in vivo mouse studies, although subject to further molecular characterization, add new insights into the role of vitamin D in aging.     

The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration

An excess of osteoclastic bone resorption relative to osteoblastic bone formation results in progressive bone loss, characteristic of osteoporosis. Understanding the mechanisms of osteoclast differentiation is essential to develop novel therapeutic approaches to prevent and treat osteoporosis. We showed previously that Wrch1/RhoU is the only RhoGTPase whose expression is induced by RANKL during osteoclastogenesis. It associates with podosomes and the suppression of Wrch1 in osteoclast precursors leads to defective multinucleated cell formation. Here we further explore the functions of this RhoGTPase in osteoclasts, using RAW264.7 cells and bone marrow macrophages as osteoclast precursors. Suppression of Wrch1 did not prevent induction of classical osteoclastic markers such as NFATc1, Src, TRAP (Tartrate-Resistant Acid Phosphatase) or cathepsin K. ATP6v0d2 and DC-STAMP, which are essential for fusion, were also expressed normally. Similar to the effect of RANKL, we observed that Wrch1 expression increased osteoclast precursor aggregation and reduced their adhesion onto vitronectin but not onto fibronectin. We further found that Wrch1 could bind integrin ß3 cytoplasmic domain and interfered with adhesion-induced Pyk2 and paxillin phosphorylation. Wrch1 also acted as an inhibitor of M-CSF-induced prefusion osteoclast migration. In mature osteoclasts, high Wrch1 activity inhibited podosome belt formation. Nevertheless, it had no effect on mineralized matrix resorption. Our observations suggest that during osteoclastogenesis, Wrch1 potentially acts through the modulation of αvß3 signaling to regulate osteoclast precursor adhesion and migration and allow fusion. As an essential actor of osteoclast differentiation, the atypical RhoGTPase Wrch1/RhoU could be an interesting target for the development of novel antiresorptive drugs.     

维生素D与PTH相关性研究进展

维生素D是人体必需的一种脂溶性营养素,随着科学技术不断进步,维生素D对人类健康的作用逐渐被发现。已有研究表明,维生素D不仅与多种骨代谢相关疾病有关,并与心血管疾病、代谢综合征、感染、肿瘤、自身免疫疾病等关系密切。在骨代谢方面,维生素D的缺乏可能会导致软骨病、佝偻病、骨质疏松症,甚至会导致急性跌倒事件的发生和骨折的形成,而甲状旁腺激素(PTH)是骨代谢过程中的关键分子。本文综述了维生素D代谢过程及维生素D受体多样性及维生素D与甲状旁腺激素(PTH)相关性,以便有助于探究维生素D与骨代谢之间的关系。     

Biochemical bone marrow markers and their significance in postmenopausal osteoporosis--a new method in the diagnosis of osteoporosis?]

This study analyzes the qualification of biochemical markers in the diagnosis of osteoporosis and evaluates the potential of a multiparametric classification of premenopausal and non-osteoporotic as well as osteoporotic postmenopausal women, which is based on biochemical marker profiles. For this evaluation data of 29 women in the age between 28-74 years were used. The classification of osteoporosis was done by the trabecular density of the lumbar spine using qCT-measurements. The biochemical markers of formation and resorption AP, bAP, OC, ucOC, PICP, PYD, DPD, NTX, BSP and vitamin K were analyzed on day 1 and 42 in all patients. For vitamin K we found significant distribution differences between non-osteoporotic and osteoporotic women (p<0.005). The crosslinks PYD and DPD showed weakly significant differences. All other parameters exhibited non-significant results. Vitamin K acted with a sensitivity of 64% and a specificity of 82%. The used multiparameter classification process improved sensitivity and specificity considerably. The parameter profiles of OC/PYD, vitamin K/PYD and vitamin K/bAP revealed the highest sensitivities with specificities of more than 82%.     

Vitamin K deficiency inhibits mineralization and enhances deformity in vertebrae of haddock (Melanogrammus aeglefinus L.)

Vitamin K has been known to regulate bone formation through osteocalcin synthesis by osteoblasts, which is important for mineralization and bone structure. The mechanism underlying the relationship of vitamin K with the changes of microanatomy is not fully understood, and our goal is to test whether bone deformities develop in association with vitamin K deficiency. Fish were fed a semi-purified diet containing either devoid (0.00 mg/kg diet) or adequate (40.0 mg/kg diet supplemented but 20.8 mg/kg analyzed) levels of vitamin K (menadione sodium bisulphite) for 20 weeks. At the end of 8 and 20 weeks, fish were subjected to gross examination and X-ray, and mineral content of the vertebrae was measured. The vertebrae were also subjected to histological, histomorphometric and enzyme histochemical examinations to determine the bone formation and resorption. Vitamin K deficiency primarily decreased bone mineralization and subsequently a decrease in bone mass thus resulted in an increased susceptibility to bone deformity. The occurrence of bone deformities coincided with an increased amount of osteoid tissue and decreased bone mineral content. Number of osteoblasts and osteoclasts were not affected by dietary vitamin K. In conclusion, vitamin K deficiency can impair bone mineralization and enhances bone deformities.     

Synthesis and biological activities of a new series of secosteroids: vitamin D phosphonate hybrids

By a structural combination of phosphonate and bisphosphonate moieties with the vitamin D skeleton a series of new vitamin D analogs was synthesized. Derivatives with 24beta-hydroxy- or 24-keto groups exerted considerable vitamin D activities in vitro while the hypercalcemic potentials were significantly reduced as compared to 1alpha,25-dihydroxyvitamin D(3) (calcitriol). Whereas the 24-hydroxy analogs did not influence bone formation in vivo in dosages below the hypercalcemic threshold, the 24-ketones were found to induce synthesis of new bone matrix in non-hypercalcemic doses. Vitamin D bisphosphonate hybrids, on the other hand, which did not elicit substantial vitamin D activities in vitro and tend to decrease serum calcium levels in vivo clearly induced osteoid formation in rats, indicating a mechanism of action different to calcitriol.     

Vitamin K suppresses the lipopolysaccharide-induced expression of inflammatory cytokines in cultured macrophage-like cells via the inhibition of the activation of nuclear factor κB through the repression of IKKα/β phosphorylation

Vitamin K is essential for blood coagulation and bone metabolism in mammals. This vitamin functions as a cofactor in the posttranslational synthesis of γ-carboxyglutamic acid (Gla) from glutamic acid residues. However, other functions of vitamin K have been reported recently. We previously found that vitamin K suppresses the inflammatory reaction induced by lipopolysaccharide (LPS) in rats and human macrophage-like THP-1 cells. In this study, we further investigated the mechanism underlying the anti-inflammatory effect of vitamin K by using cultures of LPS-treated human- and mouse-derived cells. All the vitamin K analogues analyzed in our study exhibited varied levels of anti-inflammatory activity. The isoprenyl side chain structures, except geranylgeraniol, of these analogues did not show such activity; warfarin did not interfere with this activity. The results of our study suggest that the 2-methyl-1,4-naphtoquinone ring structure contributes to express the anti-inflammatory activity, which is independent of the Gla formation activity of vitamin K. Furthermore, menaquinone-4, a form of vitamin K₂, reduced the activation of nuclear factor κB (NFκB) and inhibited the phosphorylation of IKKα/β after treatment of cells with LPS. These results clearly show that the anti-inflammatory activity of vitamin K is mediated via the inactivation of the NFκB signaling pathway.     

In vivo anti-osteoporotic activity of isotaxiresinol, a lignan from wood of Taxus yunnanensis

Isotaxiresinol, the main lignan isolated from the water extract of wood of Taxus yunnanensis, was investigated for its effect on bone loss, on serum biochemical markers for bone remodeling and on uterine tissue, using ovariectomized (OVX) rats as the model of postmenopausal osteoporosis. After oral administration of isotaxiresinol (50 and 100mg/kg/d) for 6 weeks, bone mineral content (BMC) and bone mineral density (BMD) in total and cortical bones were increased as compared to those of OVX control rats, and decreases of three bone strength indexes induced by OVX surgery were prevented. Serum biochemical markers for bone remodeling revealed that isotaxiresinol slightly increased bone formation and significantly inhibited bone resorption without side effect on uterine tissue. These results suggest that isotaxiresinol may be useful for treatment of postmenopausal osteoporosis, especially for prevention of bone fracture induced by estrogen deficiency.     

Anti-oxidant/pro-oxidant reactions of vitamin K

Experiments were designed to measure O2 consumption caused by the oxidation of linoleic acid. These experiments show that vitamin K has antioxidant activity and that the reduction in linoleic acid oxidation is directly dependent upon vitamin K concentration. Conversely, vitamin K hydroquinone enhances linoleic acid oxidation in the absence of iron catalyst, again in a concentration dependent manner. At equilmolar concentrations vitamin K is about 80% as effective as vitamin E as an antioxidant. Vitamin E inhibits the oxidation of linoleic acid catalyzed by vitamin K hydroquinone. Vitamin E also strongly inhibits vitamin K dependent formation of both vitamin K epoxide and gamma-carboxyglutamic acid (gla). The significance of these observations to vitamin K action in vivo is discussed.