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Tsuyuki S.,Meiji University | Takabayashi M.,Meiji University | Takabayashi M.,Taisho Toyama Pharmaceutical Co. | Kawazu M.,Meiji University | And 8 more authors.
Autophagy | Year: 2014

Autophagy is a cellular bulk degradation system for long-lived proteins and organelles that operates during nutrient starvation and is thus a type of recycling system. In recent years, a series of mammalian orthologs of yeast autophagyrelated (ATG) genes have been identified; however, the importance of the transcriptional regulation of ATG genes underlying autophagosome formation is poorly understood. In this study, we identified several ATG genes, including the genes ULK1, MAP1LC3B, GABARAPL1, ATG13, WIPI1, and WDR45/WIPI4, with elevated mRNA levels in thapsigargin-, C2-ceramide-, and rapamycin-treated as well as amino acid-depleted HeLa cells except for MAP1LC3B mRNA in rapamycin-treated HeLa cells. Rapamycin had a weaker effect on the expressions of ATG genes. The increase in WIPI1 and MAP1LC3B mRNA was induced prior to the accumulation of the autophagy marker protein MAP1LC3 in the thapsigargin-and C2-ceramidetreated A549 cells. By counting the puncta marked with MAP1LC3B in HeLa cells treated with different autophagy inducers, we revealed that the time-dependent mRNA elevation of a specific set of ATG genes was similar to that of autophagosome accumulation. The transcriptional attenuation of WIPI1 mRNA using RNA interference inhibited the puncta number in thapsigargin-treated HeLa cells. Remarkably, increases in the abundance of WIPI1 mRNA were also manifested in thapsigargin-and C2-ceramide-treated human fibroblasts (WI-38 and TIG-1), human cancer cells (U-2 OS , Saos-2, and MCF7), and rodent fibroblasts (Rat-1). Taken together, these results suggest that the detection of WIPI1 mRNA is likely to be a convenient method of monitoring autophagosome formation in a wide range of cell types. © 2014 Landes Bioscience.

Sugimoto M.,Taisho Pharmaceutical Co. | Futaki N.,Taisho Toyama Pharmaceutical Co. | Harada M.,Taisho Toyama Pharmaceutical Co. | Kaku S.,Taisho Pharmaceutical Co.
Bone | Year: 2013

Eldecalcitol (ELD), a 2β-hydroxypropyloxy derivative of 1α,25 (OH) 2D3, inhibits bone resorption more potently than alfacalcidol (ALF) while maintaining osteoblastic function in an ovariectomized (OVX) osteoporosis rat model. Alendronate (ALN), which is the most common bisphosphonate used for the treatment of osteoporosis, increases the bone mineral density (BMD) by suppressing bone resorption. In this study, we investigated the effects of combination treatments with ELD and ALN or with ALF and ALN on bone mass and strength in OVX rats. Seventy female rats, 32. weeks old, were assigned to seven groups: (1) a sham-operated control group; (2) an OVX-control group; (3) an ELD group; (4) an ALF group; (5) an ALN group; (6) an ELD. +. ALN group; and (7) an ALF. +. ALN group. OVX rats were orally treated with ELD (0.015 μg/kg), ALF (0.0375 μg/kg), or ALN (0.2. mg/kg) daily for 12. weeks. In both the lumbar spine and the femur, ELD and ALF monotherapy significantly increased the BMD, and ELD. +. ALN and ALF. +. ALN significantly increased the BMD, compared with ALN monotherapy, as an additive effect. In particular, ELD. +. ALN resulted in a significantly higher BMD than ALF. +. ALN in the femur. On mechanical testing of the lumbar spine, ELD and ALF monotherapy significantly increased the ultimate load, and ELD. +. ALN and ALF. +. ALN significantly increased the ultimate load compared with ALN monotherapy. In the femur, ELD, ELD. +. ALN, and ALF. +. ALN treatment significantly increased the ultimate load, compared with the OVX-control group, and ELD. +. ALN resulted in a significantly higher ultimate load than ALN monotherapy. A histomorphometric analysis showed that ELD monotherapy and ELD. +. ALN combination therapy had a potent inhibitory effect on bone resorption parameters (osteoclast surface and eroded surface), while maintaining bone formation parameters (osteoblast surface and osteoid surface). By contrast, ALF and ALF. +. ALN significantly lowered the histological parameters of both bone resorption and formation. These results suggested that ELD or ALF used in combination with ALN has therapeutic advantages over ALN monotherapy, with ELD. +. ALN combination treatment producing an especially beneficial anti-osteoporotic effect by inhibiting osteoclastic bone resorption and maintaining osteoblastic function, compared with ALF. +. ALN combination treatment. © 2012 Elsevier Inc.

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