Article: In vivo determination of vitamin d function using transgenic mice carrying a human osteocalcin luciferase reporter gene.Tomoko Nakanishi, Rumiko Saito, Makoto Taniguchi, Haruka Oda, Atsumi Soma, Mayu Yasunaga, Mariko Yamane, Kenzo Sato[show abstract] [hide abstract]
ABSTRACT: Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1 α ,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening.BioMed research international. 01/2013; 2013:895706.
Article: Regulation of autophagy and its associated cell death by sphingolipid rheostat: reciprocal role of ceramide and sphingosine-1-phosphate in the mTOR pathway.Makoto Taniguchi, Kazuyuki Kitatani, Tadakazu Kondo, Mayumi Hashimoto-Nishimura, Satoshi Asano, Akira Hayashi, Susumu Mitsutake, Yasuyuki Igarashi, Hisanori Umehara, Hiroyuki Takeya, Junzo Kigawa, Toshiro Okazaki[show abstract] [hide abstract]
ABSTRACT: The role of sphingolipid rheostat by ceramide and sphingosine-1-phosphate (S1P) in the regulation of autophagy remains unclear. In human leukemia HL-60 cells, amino acid deprivation (AA(-)) caused autophagy with an increase in acid sphingomyleinase (SMase) activity and ceramide, which serves as an autophagy inducing lipid. Knockdown of acid SMase significantly suppressed the autophagy induction. S1P treatment counteracted autophagy induction by AA(-) or C(2)-ceramide. AA(-) treatment promoted mTOR dephosphorylation/inactivation, inducing autophagy. S1P treatment suppressed mTOR inactivation and autophagy induction by AA(-). S1P exerts biological actions via cell surface receptors, and S1P(3) among five S1P receptors was predominantly expressed in HL-60 cells. We evaluated the involvement of S1P(3) in suppressing autophagy induction. S1P treatment of CHO cells had no effects on mTOR inactivation and autophagy induction by AA(-) or C(2)-ceramide. Whereas S1P treatment of S1P(3) overexpressing CHO cells resulted in activation of the mTOR pathway, preventing cells from undergoing autophagy induced by AA(-) or C(2)-ceramide. These results indicate that S1P-S1P(3) plays a role in counteracting ceramide signals that mediate mTOR-controlled autophagy. In addition, we evaluated the involvement of ceramide-activated protein phosphatases (CAPPs) in ceramide-dependent inactivation of the mTOR pathway. Inhibition of CAPP by okadaic acid in AA(-)- or C(2)-ceramide- treated cells suppressed dephosphorylation/inactivation of mTOR, autophagy induction, and autophagy-associated cell death, indicating a novel role of ceramide-CAPPs in autophagy induction. Moreover, S1P(3) engagement by S1P counteracted cell death. Taken together, these results indicated that sphingolipid rheostat in ceramide-CAPPs and S1P-S1P(3) signaling modulates autophagy and its associated cell death through regulation of the mTOR pathway.Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
Article: Bioluminescence imaging of bone formation using hairless osteocalcin-luciferase transgenic mice.Tomoko Nakanishi, Kazuo Kokubun, Haruka Oda, Mika Aoki, Atsumi Soma, Makoto Taniguchi, Yasuhiro Kazuki, Mitsuo Oshimura, Kenzo Sato[show abstract] [hide abstract]
ABSTRACT: Osteocalcin is a major noncollagenous protein component of bone extracellular matrix, synthesized and secreted exclusively by osteoblastic cells during the late stage of maturation. We introduced a 10kb human osteocalcin enhancer/promoter (OC)-luciferase (Luc) construct into a hairless mouse line. Examination of tissue RNAs from these transgenic mice showed a predominant restriction of Luc mRNA expression to bone-associated tissues. Immunohistochemical staining of calvaria tissue sections revealed the localization of Luc protein to osteoblasts. Utilizing in vivo bioluminescence imaging, supplementation of 1α,25-dihydroxyvitamin D(3) increased Luc activity throughout the skeleton, consistent with in vitro transient transfection studies in osteoblast-like cells. Moreover, we observed an abrupt decrease in bioluminescence activity as the mice reached puberty, and a further decrease gradually thereafter. Using a radius skeletal repair model, we observed enhanced bioluminescence at the fracture site in both young (14-22 weeks old) and aged (50-66 weeks old) mice. However, peak bioluminescence was delayed in aged mice compared with young mice, suggesting retarded osteocalcin expression with aging. Our in vivo imaging system may contribute to the therapy and prevention of various bone metabolic disorders through its effective monitoring of the bone formation process.Bone 06/2012; 51(3):369-75. · 4.02 Impact Factor
Article: Regulation of cell migration by sphingomyelin synthases: sphingomyelin in lipid rafts decreases responsiveness to signaling by the CXCL12/CXCR4 pathway.Satoshi Asano, Kazuyuki Kitatani, Makoto Taniguchi, Mayumi Hashimoto, Kota Zama, Susumu Mitsutake, Yasuyuki Igarashi, Hiroyuki Takeya, Junzo Kigawa, Akira Hayashi, Hisanori Umehara, Toshiro Okazaki[show abstract] [hide abstract]
ABSTRACT: Sphingomyelin synthase (SMS) catalyzes the formation of sphingomyelin, a major component of the plasma membrane and lipid rafts. To investigate the role of SMS in cell signaling and migration induced by binding of the chemokine CXCL12 to CXCR4, we used mouse embryonic fibroblasts deficient in SMS1 and/or SMS2 and examined the effects of SMS deficiency on cell migration. SMS deficiency promoted cell migration through a CXCL12/CXCR4-dependent signaling pathway involving extracellular signal-regulated kinase (ERK) activation. In addition, SMS1/SMS2 double-knockout cells had heightened sensitivity to CXCL12, which was significantly suppressed upon transfection with the SMS1 or SMS2 gene or when they were treated with exogenous sphingomyelin but not when they were treated with the SMS substrate ceramide. Notably, SMS deficiency facilitated relocalization of CXCR4 to lipid rafts, which form platforms for the regulation and transduction of receptor-mediated signaling. Furthermore, we found that SMS deficiency potentiated CXCR4 dimerization, which is required for signal transduction. This dimerization was significantly repressed by sphingomyelin treatment. Collectively, our data indicate that SMS-derived sphingomyelin lowers responsiveness to CXCL12, thereby reducing migration induced by this chemokine. Our findings provide the first direct evidence for an involvement of SMS-generated sphingomyelin in the regulation of cell migration.Molecular and cellular biology 06/2012; 32(16):3242-52. · 6.06 Impact Factor
Article: Skewed Production of IL-6 and TGFβ by Cultured Salivary Gland Epithelial Cells from Patients with Sjögren's Syndrome.Takafumi Kawanami, Toshioki Sawaki, Tomoyuki Sakai, Miyuki Miki, Haruka Iwao, Akio Nakajima, Takuji Nakamura, Tomomi Sato, Yoshimasa Fujita, Masao Tanaka, Yasufumi Masaki, Toshihiro Fukushima, Yuko Hirose, Makoto Taniguchi, Naotoshi Sugimoto, Toshiro Okazaki, Hisanori Umehara[show abstract] [hide abstract]
ABSTRACT: To determine the cytokine production profile of cultured salivary gland epithelial (SGE) cells obtained from patients with Sjögren's syndrome (SS). SGE cells obtained from 9 SS patients and 6 normal controls were cultured in the presence of exogenous IFNγ. Cell proliferation and apoptosis in response to IFNγ were determined by WST1 assay and by FACS analysis. The concentrations of IL-6 and TGFβ secreted into culture supernatants were analyzed by ELISA. IFNγ did not significantly affect the proliferation or apoptosis of SGE cells. However, IL-6 concentrations were higher, and TGFβ concentrations were lower, in culture supernatants of SGE cells from SS patients than from normal controls. Cytokine production by SGE cells from SS patients showed a skewed balance compared with normal controls, with increased IL-6 and decreased TGFβ secretion. This imbalance may be critical in the regulation of Treg/Th17 cells and may foster a pathogenic milieu that may be causative and predictive in SS.PLoS ONE 01/2012; 7(10):e45689. · 4.09 Impact Factor
Interested in membrane and intracellular sphingolipids such as ceramide or sphingomyelin on trafficking, signal transduction, viral infection and gene expression.