Takakazu Mitani

Hypoxia enhances transcriptional activity of androgen receptor through hypoxia-inducible factor-1α in a low androgen environment

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., 0.1nM 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 0.1nM 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 0.1nM 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.

Coordinated Action of Hypoxia-inducible Factor-1α and β-Catenin in Androgen Receptor Signaling

Background: The mechanism that regulates androgen receptor (AR) function in castration-resistant prostate cancer (CRPC) remains unclear. Results: Hypoxia-inducible factor (HIF)-1α enhances β-catenin-mediated AR transactivation and promotes nuclear translocation of β-catenin. Conclusion: Coordinated action of HIF-1α and β-catenin contributes to AR function in CRPC. Significance: The novel functions of HIF-1α in AR signaling are important for understanding the development of CRPC. 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-1α 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.

Castration influences intestinal microflora and induces abdominal obesity in high-fat diet-fed mice.

Late-onset hypogonadism (i.e. androgen deficiency) raises the risk for abdominal obesity in men. The mechanism for this obesity is unclear. Here, we demonstrated that hypogonadism after castration caused abdominal obesity in high-fat diet (HFD)-fed, but not in standard diet (SD)-fed, C57BL/6J mice. Furthermore, the phenotype was not induced in mice treated with antibiotics that disrupt the intestinal microflora. In HFD-fed mice, castration increased feed efficiency and decreased fecal weight per food intake. Castration also induced in an increase of visceral fat mass only in the absence of antibiotics in HFD-fed mice, whereas subcutaneous fat mass was increased by castration irrespective of antibiotics. Castration reduced the expression in the mesenteric fat of both adipose triglyceride lipase and hormone-sensitive lipase in HFD-fed mice, which was not observed in the presence of antibiotics. Castration decreased thigh muscle (i.e. quadriceps and hamstrings) mass, elevated fasting blood glucose levels, and increased liver triglyceride levels in a HFD-dependent manner, whereas these changes were not observed in castrated mice treated with antibiotics. The Firmicutes/Bacteroidetes ratio and Lactobacillus species increased in the feces of HFD-fed castrated mice. These results show that androgen (e.g. testosterone) deficiency can alter the intestinal microbiome and induce abdominal obesity in a diet-dependent manner.

Autophagic degradation of the androgen receptor mediated by increased phosphorylation of p62 suppresses apoptosis in hypoxia

Prostate cancer grows under hypoxic conditions. Hypoxia decreases androgen receptor (AR) protein levels. However, the molecular mechanism remains unclear. Here, we report that p62-mediated autophagy degrades AR protein and suppresses apoptosis in prostate cancer LNCaP cells in hypoxia. In LNCaP cells, hypoxia decreased AR at the protein level, but not at the mRNA level. Hypoxia-induced AR degradation was inhibited not only by knockdown of LC3, a key component of the autophagy machinery, but also by knockdown of p62. Depletion of p62 enhanced hypoxia-induced poly(ADP-ribose) polymerase cleavage and caspase-3 cleavage, markers of apoptosis, whereas simultaneous knockdown of p62 and AR suppressed hypoxia-induced apoptosis. Hypoxia increased the formation of a cytosolic p62-AR complex and enhanced sequestration of AR from the nucleus. Formation of this complex was promoted by the increased phosphorylation of serine 403 in the ubiquitin-associated domain of p62 during hypoxia. An antioxidant and an AMP-activated protein kinase (AMPK) inhibitor reduced hypoxia-induced p62 phosphorylation at serine 403 and suppressed hypoxia-induced complex formation between AR and p62. These results demonstrate that hypoxia enhances the complex formation between p62 and AR by promoting phosphorylation of p62 at serine 403, probably through activating AMPK, and that p62-mediated autophagy degrades AR protein for cell survival in hypoxia.

Involvement of three glutamine tracts in human androgen receptor transactivation.

The androgen receptor (AR) possesses a polymorphic polyglutamine tract (polyQ), whose length is inversely correlated with its transcriptional activity. Here, we investigated whether 6 and 5 repetitive glutamine tracts (Q6 and Q5, respectively) in the N-terminal domain of AR also have effects on AR transactivation. In a reporter gene assay using two-tandem repeats of an androgen response element, deletion of glutamine tracts significantly increased AR transactivation in the following order: wild-type<a single deletion of polyQ or Q5<double deletion of polyQ and Q6<double deletion of polyQ and Q5<triple deletion. Deletion of polyQ alone or combined deletion of polyQ and Q5 from an AR mutant lacking the ligand-binding domain, which is constitutively active due to activation function-1, increased AR transactivation. However, the glutamine tracts had no influence on activation function-1 activity, suggesting that the glutamine tracts modulate the binding of AR to DNA. Q5, like polyQ, was found to be involved in the interaction between the NH(2)- and COOH-terminal regions of AR (N-C interaction). These results indicate that the inhibitory effects of polyQ and Q5 on AR transactivation are the due, at least in part, to their negative regulation of N-C interaction.

Kelch-like 20 up-regulates the expression of hypoxia-inducible factor-2α through hypoxia- and von Hippel-Lindau tumor suppressor protein-independent regulatory mechanisms.

Despite their structural similarity, hypoxia-inducible factor (HIF)-1α and HIF-2α have distinct functional properties and exhibit distinct spatiotemporal expression patterns, suggesting that the expressions of the two proteins are regulated by different mechanisms. To clarify the HIF-2α-specific regulatory mechanism, we screened HIF-2α-associated proteins in a yeast two-hybrid system and identified kelch-like 20 (KLHL20). HIF-2α, but not HIF-1α, interacted with KLHL20. siRNA-mediated knockdown of KLHL20 decreased HIF-2α protein, but not HIF-2α mRNA or HIF-1α protein. Depletion of KLHL20 decreased hypoxia-induced HIF activity, and consequently resulted in decreased expression levels of HIF-2α-responsive genes such as VEGF and CITED2. In contrast, overexpression of KLHL20 increased the expression levels and transcriptional activities of the O(2)-sensitive wild-type and O(2)-insensitive mutant forms of HIF-2α. KLHL20 siRNA also inhibited HIF-2 activity in von Hippel-Lindau tumor suppressor protein (pVHL)-deficient 786-O cells. These results indicate that KLHL20 is a novel player that regulates HIF-2α protein expression through mechanisms independent of hypoxia and pVHL.

Enzymatically synthesized glycogen inhibits colitis through decreasing oxidative stress

Abstracts Inflammatory bowel diseases are a group of chronic inflammation conditions of the gastrointestinal tract. Disruption of the mucosal immune response causes accumulation of oxidative stress, resulting in the induction of inflammatory bowel disease. In this study, we investigated the effect of enzymatically synthesized glycogen (ESG), which is produced from starch, on dextran sulfate sodium (DSS)‐ and 2,4,6‐trinitrobenzenesulfonic acid (TNBS)‐induced colitis in C57BL/6 mice. Oral administration of ESG suppressed DSS‐ and TNBS‐induced shortening of large intestine in female mice and significant decreased DSS‐induced oxidative stress and TNBS‐induced pro‐inflammatory cytokine expression in the large intestine. ESG increase in the expression levels of heme oxygenase‐1 (HO‐1) and NF‐E2‐related factor‐2 (Nrf2), a transcription factor for HO‐1 expressed in the large intestine. Furthermore, ESG‐induced HO‐1 and Nrf2 were expressed mainly in intestinal macrophages. ESG is considered to be metabolized to resistant glycogen (RG) during digestion with &agr;‐amylase in vivo. In mouse macrophage RAW264.7 cells, RG, but not ESG decreased 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH)‐induced reactive oxygen species (ROS). Knockdown of Nrf2 inhibited RG‐induced HO‐1 expression and negated the decrease in AAPH‐induced ROS brought about by RG. RG up‐regulated the protein stability of Nrf2 to decrease the formation of Nrf2‐Keap1 complexes. RG‐induced phosphorylation of Nrf2 at Ser40 was suppressed by ERK1/2 and JNK inhibitors. Our data indicate that ESG, digested with &agr;‐amylase to RG, suppresses DSS‐ and TNBS‐induced colitis by increasing the expression of HO‐1 in the large intestine of mice. Furthermore, we demonstrate that RG induces HO‐1 expression by promoting phosphorylation of Nrf2 at Ser40 through activation of the ERK1/2 and JNK cascade in macrophages. Graphical abstract Figure. No Caption available. HighlightsESG prevented DSS‐induced colitis in mice by decreasing oxidative stress.ESG administration induced Nrf2 and HO‐1 expression in colon tissues of mice.ESG metabolite RG induced Nrf2 and HO‐1 expression in RAW264.7 macrophages.Knockdown of Nrf2 and HO‐1 in RAW264.7 cells negated RG‐induced antioxidant effect.

Luteolin suppresses TCDD-induced wasting syndrome in a cultured adipocyte model.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various toxic effects, including wasting syndrome, through activation of an aryl hydrocarbon receptor (AhR). Our previous report demonstrated that certain flavonoids inhibit the activation of AhR and suppress its DNA binding activity. In this study, we searched for an active compound among 13 flavonoids that suppressed TCDD-induced loss of lipid accumulation using 3T3-L1 adipocytes as a cell culture model for wasting syndrome. Two flavonoids, luteolin and epigallocatechin gallate, suppressed TCDD-induced loss of lipid accumulation in this model. We further investigated luteolin to clarify the underlying molecular mechanism and confirmed that luteolin inhibited nuclear translocation of AhR caused by TCDD. Luteolin also inhibited the TCDD-driven decrease in protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Although TCDD alone did not change protein expression of C/EBPβ and C/EBPδ, luteolin and TCDD up-regulated C/EBPδ expression in a dose-dependent manner. On the other hand, TCDD significantly decreased DNA binding of C/EBPβ and C/EBPδ, and luteolin completely canceled TCDD-decreased DNA binding of them. We conclude that luteolin suppresses the TCDD-induced loss of lipid accumulation in 3T3-L1 adipocytes by preventing a decrease in protein expression of PPARγ and C/EBPα, the master regulators of adipocyte differentiation and in DNA binding of C/EBPβ and C/EBPδ. Moreover, luteolin was rapidly incorporated and accumulated in 3T3-L1 adipocytes. Thus, luteolin is an attractive compound for the prevention of TCDD-induced wasting syndrome.

Solventless extraction methods for immature fruits: Evaluation of their antioxidant and cytoprotective activities

In this study, extraction of immature fruits using an environmentally friendly pressurized hot water extraction (PHWE) method was compared with the traditional reflux method. Extracts were tested for their polyphenol content using the Folin-Ciocalteu assay and for their antioxidant activity using the oxygen radical absorbance capacity (ORAC) assay. The highest amount of polyphenol was extracted from grape (stem) using PHWE at 100°C, or reflux extraction. This was followed by reflux extraction of grape (fruit). The results were similar for the ORAC assay. All samples extracted using PHWE at 100°C showed cytoprotective activity against H2O2-induced oxidative stress in Crandell-Reese feline kidney (CRFK) cells. This study demonstrated that beneficial compounds can be extracted from immature fruits without the use of organic solvents. The utilization of beneficial compounds present in immature fruits can also contribute to the reduction in agriculture waste generated.

Role of phosphate groups on antiviral activity of casein phosphopeptide against feline calicivirus as a surrogate for norovirus

BACKGROUND Current research on the gastrointestinal digestion of milk-casein strongly suggests the existence of novel bioactive peptides with antiviral activities that are attributable to their immunostimulatory effects. In the present study, we investigated the antiviral activity of casein peptides rich in phosphate groups, such as casein phosphopeptide (CPP-III). RESULTS We prepared two types of CPP with different phosphorylation levels to clarify the role of the phosphate group. Further phosphorylation of CPP-III was conducted by dry heating with sodium pyrophosphate, whereas dephosphorylation was performed enzymatically using alkaline phosphatase and alkaline treatment. Feline calicivirus (FCV) strain F9, a typical norovirus surrogate, and Crandell Rees feline kidney cells were used as the target virus and host cells, respectively. Antiviral activity was determined based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and quantitative polymerase chain reaction quantification of antiviral cytokine mRNA expression. Higher cell viability was observed in the host cells treated with phosphorylated CPP-III, and a significant up-regulation of type 1 interferon expression was induced compared to that treated with native CPP-III. However, dephosphorylation of CPP-III resulted in a decrease in the anti-FCV effect. CONCLUSION The CPP effect was enhanced by the introduction of additional phosphates and conversely weakened by their elimination. Therefore, CPP-III phosphorylation represents an emerging approach for the production of food-grade antiviral agents.