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Fujiidera, Japan

Higashimura Y.,Osaka Prefecture University | Nakajima Y.,Osaka Prefecture University | Yamaji R.,Osaka Prefecture University | Harada N.,Osaka Prefecture University | And 3 more authors.
Archives of Biochemistry and Biophysics | Year: 2011

Hypoxia up-regulates the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a cell type-specific manner. It is unknown whether this occurs in breast cancer. Here, we report that hypoxia up-regulates the GAPDH gene expression through breast cancer-specific molecular mechanisms in MCF-7 cells. Mutation analysis identified a novel hypoxia response element (HRE), in addition to the HRE found previously in prostate cancer LNCaP cells. Knockdown and overexpression of hypoxia-inducible factor (HIF)-1α indicated that HIF-1 contributed to the up-regulation of GAPDH gene expression by hypoxia. Although chromatin immunoprecipitation (ChIP) and plasmid immunoprecipitation analyses revealed the presence of HIF-1α on the novel HRE in both hypoxic cell lines, a mutation in either the novel HRE or its 3′-flanking GC-box resulted in a reduction of hypoxia-increased GAPDH promoter activity only in MCF-7 cells. ChIP analysis showed that Sp1 bound to the GC-box in MCF-7 cells, but not in LNCaP cells, in normoxia and hypoxia. Knockdown of Sp1 reduced hypoxia-increased promoter activity and expression level of GAPDH in MCF-7 cells. These results indicate that in MCF-7 cells, the activation of HIF-1 on the novel HRE contributes to the breast cancer-specific hypoxic induction of GAPDH gene expression and absolutely depends on the presence of Sp1 on the GC-box. © 2011 Elsevier Inc. All rights reserved. Source

Mitani T.,Osaka Prefecture University | Harada N.,Osaka Prefecture University | Nakano Y.,Osaka Womens Junior College | Inui H.,Osaka Prefecture University | Yamaji R.,Osaka Prefecture University
Journal of Biological Chemistry | Year: 2012

The androgen receptor (AR) acts as a ligand-dependent transcriptional factor and plays a critical role in the development and progression of androgen-dependent and castration-resistant prostate cancer. Castration results in hypoxia in prostate cancer cells, and hypoxia enhances transcriptional activity of AR through hypoxia-inducible factor (HIF)-1α at low serum androgen levels mimicking the castration-resistant stage. However, HIF-1α is necessary but not sufficient for hypoxia-activated AR transactivation, and the molecular mechanism that regulates AR function in castration-resistant prostate cancer remains unclear. Here, we report that β-catenin is required for HIF-1α- mediated AR transactivation in hypoxic LNCaP prostate cancer cells under low androgen conditions. HIF-1α and β-catenin coordinately enhanced AR N-terminal and C-terminal interaction. β-Catenin accumulated in the nucleus in the HIF-1α protein- positive cells of LNCaP xenografts in castrated mice. In LNCaP cells, when HIF-1α was knocked down or was exogenously expressed in the cytoplasm, hypoxia-induced nuclear localization of β-catenin was inhibited. β-Catenin formed a complex with HIF-1β both in the nucleus and in the cytoplasm. Hypoxia increased the amount of a complex composed of AR and β-catenin, and knockdown of HIF-1α attenuated the recruitment of AR and β-catenin to the androgen response elements (AREs) of androgen-responsive genes. Furthermore, together with β-catenin, HIF-1α bound to the AREs in the presence of androgen. These results demonstrate that (i) HIF-1α and β-catenin coordinately enhance AR transactivation by accelerating N-terminal and C-terminal interaction; (ii) HIF-1α promotes nuclear translocation of β-catenin in hypoxia; and (iii) AR, HIF-1α, and β-catenin form a ternary complex on AREs. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Ogawa M.,Osaka Prefecture University | Kariya Y.,Osaka Prefecture University | Kitakaze T.,Osaka Prefecture University | Yamaji R.,Osaka Prefecture University | And 5 more authors.
British Journal of Nutrition | Year: 2013

Muscle atrophy increases the production of reactive oxygen species and the expression of atrophy-related genes, which are involved in the ubiquitin-proteasome system. In the present study, we investigated the effects of β-carotene on oxidative stress (100 μm-H2O2)-induced muscle atrophy in murine C2C12 myotubes. β-Carotene (10 μm) restored the H2O2-induced decreased levels of myosin heavy chain and tropomyosin (P< 0·05, n 3) and decreased the H2O2-induced increased levels of ubiquitin conjugates. β-Carotene reduced the H2O2-induced increased expression levels of E3 ubiquitin ligases (Atrogin-1 and MuRF1) and deubiquitinating enzymes (USP14 and USP19) (P< 0·05, n 3) and attenuated the H2O2-induced nuclear localisation of FOXO3a. Furthermore, we determined the effects of β-carotene on denervation-induced muscle atrophy. Male ddY mice (8 weeks old, n 30) were divided into two groups and orally pre-administered micelle with or without β-carotene (0·5 mg once daily) for 2 weeks, followed by denervation in the right hindlimb. β-Carotene was further administered once daily until the end of the experiment. At day 3 after denervation, the ratio of soleus muscle mass in the denervated leg to that in the sham leg was significantly higher in β-carotene-administered mice than in control vehicle-administered ones (P< 0·05, n 5). In the denervated soleus muscle, β-carotene administration significantly decreased the expression levels of Atrogin-1, MuRF1, USP14 and USP19 (P< 0·05, n 5) and the levels of ubiquitin conjugates. These results indicate that β-carotene attenuates soleus muscle loss, perhaps by repressing the expressions of Atrogin-1, MuRF1, USP14 and USP19, at the early stage of soleus muscle atrophy. © 2012 The Authors. Source

Harada N.,Osaka Prefecture University | Atarashi K.,Osaka Prefecture University | Murata Y.,Osaka Prefecture University | Yamaji R.,Osaka Prefecture University | And 2 more authors.
Journal of Steroid Biochemistry and Molecular Biology | Year: 2011

Androgen receptor (AR) is a ligand-dependent transcription factor and plays a key role in the development of prostate cancer. Resveratrol, a polyphenolic compound, inhibits AR function and reduces the level of prostate-specific antigen (PSA), a notable target gene of AR. Here, we investigated the mechanisms by which resveratrol inhibits AR function. Although the protein levels of AR were decreased by resveratrol treatment for 24 h, the decrease could not fully account for the suppression of AR function. The total and the nuclear AR levels were not affected after incubation with 10 μM resveratrol for 3 h, whereas resveratrol inhibited the binding of AR to the enhancer region of PSA and decreased the acetylation of AR even at this early phase. Inhibition of transcription by resveratrol was weaker in the AR acetylation site mutant than in the wild-type. In later phase (24 h) after incubation with resveratrol, the ligand-induced nuclear accumulation of AR was markedly decreased by resveratrol. These data show that resveratrol inhibits DNA binding of AR, presumably by decreasing its level of acetylation and suggest that acetylation of AR is involved in its accumulation in the nucleus. © 2010 Elsevier Ltd All rights reserved. Source

Mitani T.,Osaka Prefecture University | Yamaji R.,Osaka Prefecture University | Higashimura Y.,Osaka Prefecture University | Harada N.,Osaka Prefecture University | And 2 more authors.
Journal of Steroid Biochemistry and Molecular Biology | Year: 2011

The androgen receptor (AR) acts as a ligand-dependent transcriptional factor controlling development or progression of prostate cancer. Androgen ablation by castration is an effective therapy for prostate cancer, whereas eventually most of the tumors convert from a hormone-sensitive to a hormone-refractory disease state and grow even in a low androgen environment (e.g., 0.1 nM 5α-dihydrotestosterone (DHT)) like the castration-resistant stage. Androgen ablation results in hypoxia, and solid tumors possess hypoxic environments. Hypoxia-inducible factor (HIF)-1, which is composed of HIF-1α and HIF-1β/ARNT subunits, functions as a master transcription factor for hypoxia-inducible genes. Here, we report that hypoxia enhances AR transactivation in the presence of 0.05 and 0.1 nM DHT in LNCaP prostate cancer cells. siRNA-mediated knockdown of HIF-1α inhibited hypoxia-enhanced AR transactivation. Its inhibition by HIF-1α siRNA was canceled by expression of a siRNA-resistant form of HIF-1α. HIF-1α siRNA repressed hypoxia-stimulated expression of the androgen-responsive NKX3.1 gene in the presence of 0.1 nM DHT, but not in the absence of DHT. In hypoxia, HIF-1α siRNA-repressed AR transactivation was restored in mutants in which HIF-1α lacked DNA-binding activity. Furthermore, a dominant negative form of HIF-1α canceled hypoxia-enhanced AR transactivation, and HIF-1β/ARNT siRNAs had no influence on hypoxia-enhanced AR transactivation. These results indicate that hypoxia leads to HIF-1α-mediated AR transactivation independent of HIF-1 activity and that HIF-1β/ARNT is not necessarily required for the transactivation. © 2010 Elsevier Ltd All rights reserved. Source

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