17beta-Estradiol inhibits chondrogenesis in the skull development of zebrafish embryos.

17beta-Estradiol (E2) plays important roles in the development and differentiation of the gonad and central nervous systems, but little is known regarding the effects of exogenous E2 on chondrogenesis in skeletal development. In the present study, we found that treatment with E2 1-5 days post-fertilization (dpf) at concentrations above 1.5x10(-5)M increased the mortality rate in zebrafish embryos. Morphological analysis showed that treatment with E2 1-5dpf caused abnormal cartilage formation in a dose-dependent manner at concentrations above 5x10(-6)M. E2 1-5dpf at 1.5x10(-5)M caused defects of the ethmoid plate, parallel cleft of the trabecular cartilage, and hypoplasia of Meckel's cartilage and the ceratohyal cartilage. The sensitivity of embryos to E2 depended on the developmental stage. In early chondrogenesis (1-2dpf), the embryos were highly sensitive to E2, leading to hypoplasia of the cartilage. In situ hybridization studies showed that expression levels of patched1 (ptc1) and patched2 (ptc2) receptor mRNAs were markedly decreased by exposure to 2x10(-5)M E2 1-2dpf. However, the expression levels of sonic hedgehog (shh) and tiggywinkle hedgehog (twhh) mRNAs were constant in the E2-treated embryos. In addition, the estrogen receptor antagonist ICI 182,780 did not completely abolish the effects of E2, suggesting that E2 may not inhibit chondrogenesis through its nuclear estrogen receptor. These results suggest that exposure to exogenous E2 possibly inhibits chondrogenesis via inhibition of the hedgehog (Hh) signal transduction system.

Association between serotonin transporter gene polymorphisms and depressed mood caused by job stress in Japanese workers.

To estimate the genetic factors influencing depressed mood caused by job stress, a total of 243 employees at a manufacturing company and a local hospital in Japan (mean age 40.8+/-10.3 years) were recruited with informed consent. The Brief Job Stress Questionnaire was used to assess the present status of stress. Alcohol consumption and smoking were assessed as lifestyle factors. DNA samples were prepared to detect gene polymorphisms of serotonin transporter (5HTT), aldehyde dehydrogenase 2, D2 dopamine receptor, and cytochrome p450 2A6. The relationship between job stress, lifestyle factors and these polymorphisms was assessed for each gender. The level of depressed mood for female subjects was significantly higher among the carriers of two short (s/s) alleles of the 5HTT regulatory region compared with the carriers of one (s/l) or two (l/l) long alleles (Mann-Whitney U test, p<0.05). The odds ratio of depressed mood also confirmed this relationship for the female subjects, whereas there was no relationship for the male subjects. When social support was taken into consideration, the depressed mood score for those who had high support was significantly lower than for those who had low support, irrespective of 5HTT polymorphisms and gender. Job stress may elicit biological responses that contribute to depressed mood in relation to 5HTT polymorphisms, and social support may reduce depressed mood irrespective of 5HTT polymorphisms.

Menaquinone-7 regulates gene expression in osteoblastic MC3T3E1 cells.

Previous study has shown that the vitamin K2 analog menaquinone-7 (MK-7) induces expression of the osteoblast-specific genes osteocalcin, osteoprotegerin, receptor activator of NFkappaB, and its ligand. Since MK-7 may also regulate osteoblast cell function, we examined the expression of osteoblast genes regulated by MK-7 administration. Differences between gene expression in control and MK-7-administered MC3T3E1 cells were analyzed using the suppression subtractive hybridization method. After 24 h of MK-7 administration, genes upregulated by MK-7 included tenascin C and BMP2. Genes downregulated by MK-7 administration included biglycan and butyrophilin. Real-time PCR showed a marked increase in tenascin C. When the protein level was examined using Western blot analysis, tenascin C was higher in MK-7-administered cells than in control cells. These results indicated that MK-7 affected the cellular function of osteoblastic MC3T3E1 cells. Considering BMP2 mRNA expression was higher in MK-7-administered cells than in control cells, the effect of MK-7 administration on the signal transduction system was examined. Western blot analysis showed that cells administered MK-7 displayed a higher phosphorylated Smad1 level than control cells. Because MC3T3E1 cells have a nuclear binding receptor for MK-7, this result might indicate an indirect effect of MK-7 through BMP2 production.

[Influence of nutrients intake on bone turnover markers].

Bone strength is determined by bone mineral density and bone quality. Bone quality can be assessed by only bone turnover markers. Nutrients that reduce bone resorption markers are calcium and isoflavone, nutrients that increase bone formation markers are vitamin C, vitamin D and vitamin K. These nutrients affect bone turnover and, as a result, improve bone density. These nutrients might contribute to prevent the incidence of osteoporosis when they are taken from adolescence.

Menaquinone-7 regulates the expressions of osteocalcin, OPG, RANKL and RANK in osteoblastic MC3T3E1 cells.

Epidemiological studies show that dietary intake of natto, which contains significant amount of vitamin K(2), reduces the risk of bone formation loss. However, many confounding factors, such as calcium and isoflavone, are found in natto, because it is made from soybeans. In this study, the direct effects of MK-7, a vitamin K(2) analogue, were assessed in osteoblasts. Osteoblastic MC3T3E1 cells were cultured with or without MK-7 for 10 days and the number of cells was calculated. The cell count was not different between MK-7 treated cells and control cells for 1, 2, and 4 days. However, it was significantly suppressed in MK-7 treated cells at 10 days, suggesting that MK-7 suppressed cell proliferation. Real-time PCR analysis showed that mRNAs of osteocalcin (OC), osteoprotegerin (OPG), and the receptor activator of the NFkappaB ligand (RANKL) were induced after MK-7 administration to the culture medium. RANK mRNA expression was also enhanced by MK-7 administration. Immunocytochemical analysis showed that MK-7 increased the protein levels of OC and RANKL. RANK protein was also enhanced, but this induction was suppressed by anti-RANK antibody administration. This suppression was recovered when anti-RANK antibody and MK-7 were administered. These observations suggest that MK-7 may directly affect MC3T3E1 cells and stimulate osteoblastic differentiation, not proliferation.

Promotion of bone formation by fermented soybean (Natto) intake in premenopausal women.

A therapeutic agent of vitamin K2 is approved for the treatment of osteoporosis in Japan. However, little is known about the efficacy of dietary intake of vitamin K2 for bone health. We compared the effects of various levels of fermented soybeans (Natto) intake, which contains plenty of vitamin K2, on bone stiffness and bone turnover markers in healthy premenopausal women. Seventy-three healthy premenopausal women were randomly divided into four groups matched for age and parity categories. Natto was supplied as follows: Group 1 (no intake), Group 2 (once per month), Group 3 (once per week) and Group 4 (three times per week). Subjects took Natto at a lunch for 1 y, and the stiffness index by quantitative ultrasound and bone turnover markers were assessed at baseline, 6 mo and 1 y. There was no statistical difference in the stiffness index during the 1 y observation. However, bone specific alkaline phosphatase (BAP) in Group 4 was higher than that in Group 3 at 1 y and undercarboxylated osteocalcin (Glu) in Group 4 was significantly lower than those in Groups 1, 2 and 3 at 6 mo. Logistic regression analysis showed that the risk of reduction of bone formation markers declined to 0.07 in Group 4 based on that in Group 1. In premenopausal women who had to keep the stiffness index as high as possible before menopause, Natto intake may have contributed to the promotion of bone formation.

Usual dietary intake of fermented soybeans (Natto) is associated with bone mineral density in premenopausal women.

Fermented soybeans (Natto), a traditional Japanese food, contain more than 100 times as much vitamin K2 as various cheeses and are considered to promote gamma-carboxylation. Thus it is conceivable that Natto may play a preventive role in the development of osteoporosis. In this study, the relationships between the bone stiffness index measured by ultrasound, bone turnover markers, and lifestyle factors, including Natto intake, were examined in relation to vitamin D receptor (VDR) polymorphism. Among 117 premenopausal volunteers, approximately 75% were bb homozygotes, 20% were Bb heterozygotes, and only 5% were BB homozygotes. The B allele group and the bb group were subdivided according to Natto intake. In a monovariate analysis, no significant differences in indices for dietary intake, including Ca and vitamin D intake, were observed. The stiffness index in the B allele group, however, was slightly lower than in the bb groups when there was no Natto intake. There were no significant differences in serum ALP and Gla-osteocalcin, bone formation markers, or NTx and Ca in urine, bone resorption markers. A logistic regression test, including the interactional effect of Natto intake and VDR RFLP, indicated that the B allele group was a risk factor of bone mineral loss and that Natto was effective in maintaining bone stiffness in this group. Although the present study was cross sectional and requires longitudinal investigation, Natto may improve the bone health of people who have a low affinity receptor for vitamin D.