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MacKinnon E.S.,St. Michaels Hospital | Venket Rao A.,University of Toronto | Rao L.G.,St. Michaels Hospital | Rao L.G.,Calcium Research Laboratory
Journal of Nutrition, Health and Aging | Year: 2011

Background and Objectives: Lycopene is a carotenoid commonly found in tomatoes and tomato products which acts as an antioxidant to decrease oxidative stress and osteoporosis risk. We wanted to determine the effects of a lycopene-restricted diet on oxidative stress parameters and bone turnover markers in postmenopausal women. Setting: St. Michael's Hospital, Toronto, ON, Canada. Participants and Study Design: 23 healthy postmenopausal women, 50-60 years old, provided blood samples at baseline and following a one-month lycopene-depletion period. Measurements: Serum samples were analyzed for carotenoids; the oxidative stress parameters protein thiols and thiobarbituric-malondialdehyde reactive substances; the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and the bone turnover markers bone alkaline phosphatase and crosslinked N-telopeptide of type I collagen (NTx). A paired t-test was used to test for significant differences in bone turnover markers, oxidative stress parameters and antioxidant status after lycopene restriction. Results: Dietary lycopene restriction resulted in significantly decreased serum lycopene (p<0.0001), lutein/zeaxanthin (p<0.01), and α-/β-carotene (p<0.05). GPx (p<0.01), lipid and protein oxidation increased (not significant), while CAT and SOD were significantly depressed (p<0.05 and p<0.005, respectively). These changes coincided with significantly increased NTx (p<0.05). Conclusions: These findings suggest that the daily consumption of lycopene may be important as it acts as an antioxidant to decrease bone resorption in postmenopausal women and may therefore be beneficial in reducing the risk of osteoporosis. © 2011 Serdi and Springer Verlag France.

Tanaka K.-I.,Kobe University | Tanaka K.-I.,The University of Shimane | Inoue Y.,Kobe University | Hendy G.N.,Nagasaki University | And 13 more authors.
Bone | Year: 2012

Bone morphogenetic proteins (BMPs) are critical for bone regeneration and induce ectopic bone formation in vivo. The constitutively activating mutation (R206H) of the BMP type 1 receptor, activin A type 1 receptor/activin-like kinase 2 (ACVR1/ALK2), underlies the molecular pathogenesis of fibrodysplasia ossificans progressiva (FOP) in which heterotopic ossification occurs in muscle tissue. In the present study, we performed a comparative DNA microarray analysis between stable empty vector- and ALK2(R206H)-transfected mouse myoblastic C2C12 cells. Forty genes were identified whose expression was increased > 3.5 times in the experimental group versus the control. The bone formation-related factor, Tmem119, was included in this group. Osteoblast differentiation markers and mineralization were enhanced in C2C12 cells stably expressing Tmem119. Differentiation of myoblastic cells into myotubes was suppressed but differentiation into chondrocytes was little affected. Transcriptional activity of the BMP-2 signaling molecules, Smad1/5, was increased even in the absence of exogenous BMP-2. Endogenous BMP-2 levels positively correlated with Tmem119 levels. A BMP-2/4 neutralizing antibody and dorsomorphin, an ALK2 inhibitor, antagonized Tmem119-enhanced alkaline phosphatase (ALP) levels. Tmem119 siRNA antagonized the BMP-2-induced ALP and osteocalcin, but not Runx2 and Osterix, mRNAs, in C2C12 cells. In conclusion, Tmem119 levels were increased by the FOP-associated constitutively activating ALK2 mutation in myoblasts. The data show that Tmem119 promotes the differentiation of myoblasts into osteoblasts and the interaction with the BMP signaling pathway likely occurs downstream of Runx2 and Osterix in myoblasts. Tmem119 may play a critical role in the commitment of myoprogenitor cells to the osteoblast lineage. © 2012 Elsevier Inc.

Hisa I.,Kobe University | Inoue Y.,Kobe University | Hendy G.N.,McGill University | Hendy G.N.,Calcium Research Laboratory | And 8 more authors.
Journal of Biological Chemistry | Year: 2011

The mechanisms whereby the parathyroid hormone (PTH) exerts its anabolic action on bone are incompletely understood. We previously showed that inhibition of ERK1/2 enhanced Smad3-induced bone anabolic action in osteoblasts. These findings suggested the hypothesis that changes in gene expression associated with the altered Smad3-induced signaling brought about by an ERK1/2 inhibitor would identify novel bone anabolic factors in osteoblasts. We therefore performed a comparative DNA microarray analysis between empty vector-transfected mouse osteoblastic MC3T3-E1 cells and PD98059-treated stable Smad3-overexpressing MC3T3-E1 cells. Among the novel factors, Tmem119 was selected on the basis of its rapid induction by PTH independent of later increases in endogenous TGF-β. The levels of Tmem119 increased with time in cultures of MC3T3-E1 cells and mouse mesenchymal ST-2 cells committed to the osteoblast lineage by BMP-2. PTH stimulated Tmem119 levels within 1 h as determined by Western blot analysis and immunocytochemistry in MC3T3-E1 cells. MC3T3-E1 cells stably overexpressing Tmem119 exhibited elevated levels of Runx2, osteocalcin, alkaline phosphatase, and β-catenin, whereas Tmem119 augmented BMP-2-induced Runx2 levels in mesenchymal cells. Tmem119 interacted with Runx2, Smad1, and Smad5 in C2C12 cells. In conclusion, we identified a Smad3-related factor, Tmem119, that is induced by PTH and promotes differentiation in mouse osteoblastic cells. Tmem119 is an important molecule in the pathway downstream of PTH and Smad3 signaling in osteoblasts. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

MacKinnon E.S.,St. Michaels Hospital | Rao A.V.,University of Toronto | Josse R.G.,St. Michaels Hospital | Rao L.G.,St. Michaels Hospital | Rao L.G.,Calcium Research Laboratory
Osteoporosis International | Year: 2011

Summary: To date, no intervention studies have been published demonstrating the effect of the antioxidant lycopene on bone. Postmenopausal women supplemented with lycopene had significantly increased antioxidant capacity and decreased oxidative stress and the bone resorption marker N-telopeptide (NTx). Lycopene decreases bone resorption markers and may reduce the risk of osteoporosis. Introduction: We have previously shown in vitro and in vivo that lycopene from tomato is associated with a protective effect on bone, but lycopene intervention studies have not been reported. Our aim was to carry out a randomized controlled intervention study to determine whether lycopene would act as an antioxidant to decrease oxidative stress parameters, resulting in decreased bone turnover markers, thus reducing the risk of osteoporosis in postmenopausal women. Methods: Sixty postmenopausal women, 50-60 years old, were recruited. Following a 1-month washout without lycopene consumption, participants consumed either (N=15/group): (1) regular tomato juice, (2) lycopene-rich tomato juice, (3) tomato Lyc-O-Mato® lycopene capsules, or (4) placebo capsules, twice daily for total lycopene intakes of 30, 70, 30, and 0 mg/day respectively for 4 months. Serum collected after the washout, 2 and 4 months of supplementation, was assayed for cross-linked aminoterminal N-telopeptide, carotenoid content, total antioxidant capacity (TAC), lipid, and protein oxidation. Results: Participants who consumed juice or lycopene capsules were analyzed in one group designated "LYCOPENE-supplemented". Repeated measures ANOVA showed that LYCOPENE-supplementation for 4 months significantly increased serum lycopene compared to placebo (p<0.001). LYCOPENE-supplementation for 4 months resulted in significantly increased TAC (p<0.05) and decreased lipid peroxidation (p<0.001), protein oxidation (p<0.001), and NTx (p<0.001). These decreases in lipid peroxidation, protein oxidation, and NTx were significantly different from the corresponding changes resulting from placebo supplementation (p<0.05, p<0.005, and p<0.02, respectively). Conclusions: Our findings suggest that the antioxidant lycopene is beneficial in reducing oxidative stress parameters and the bone resorption marker NTx. © 2010 International Osteoporosis Foundation and National Osteoporosis Foundation.

Tanaka K.-I.,The University of Shimane | Kaji H.,Kinki University | Yamaguchi T.,The University of Shimane | Kanazawa I.,The University of Shimane | And 5 more authors.
Calcified Tissue International | Year: 2014

The osteoinductive factors BMP-2 and Tmem119 that promote the differentiation of myoblasts into osteoblasts, each increase the levels of the other. However, the relative contributions of BMP-2 and Tmem119 to the osteogenic differentiation and the mechanisms involved are incompletely understood. In the present study, we examined the relationship among BMP-2, Tmem119, and the PERK-eIF2α-ATF4 endoplasmic reticulum (ER) stress response pathway in the differentiation of C2C12 myoblasts into osteoblastic cells. Both BMP-2 and Tmem119 induced levels of the osteoblast markers Runx2, Osterix, Col1a1, ALP, and osteocalcin, as well as mineralization. BMP-2 activation of the ER stress sensor PERK stimulated phosphorylation of eIF2α and led to increased biosynthesis of the osteoblast differentiation factor ATF4. When dephosphorylation of eIF2α was blocked by the selective inhibitor salubrinal, the osteogenic effects of BMP-2 and Tmem119 were enhanced further. Although BMP-2 stimulated both P-eIF2α and ATF4 levels, Tmem119 had no effect on P-eIF2α but stimulated ATF4 only. Reduction in endogenous Tmem119 levels by siRNA reduced both basal and BMP-2-stimulated levels of the ATF4 protein. In conclusion, BMP-2 stimulates differentiation of myoblasts into osteoblasts via the PERK-eIF2α-ATF4 pathway but in addition stimulates Tmem119, which itself increases ATF4. Hence, BMP-2 stimulates ATF4 both dependently and independently of the PERK-eIF2α ER stress response pathway. © 2013 Springer Science+Business Media.

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