The protective action of ursodeoxycholic acid (UDCA) in cholestatic liver diseases may be mediated by choleresis, detoxification, and cytoprotection against oxidative stress. Nrf2, one transcription factor, serves as a cellular stress sensor and is a key regulator for hepatic induction of detoxifying enzymes, antioxidative stress genes, and numerous Mrp family members. We aimed to investigate whether UDCA induces hepatic Mrp expression along with that of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. The protein level, subcellular localization, and mRNA level of Mrp family members were assessed in livers of Keap1 gene-knockdown (Keap1-kd) mice and those of UDCA-fed wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. Nuclear levels of Nrf2 in livers of Keap1-kd mice markedly increased, resulting in constitutive activation of Nrf2. Keap1-kd mice have high-level expression of hepatic Mrp2, Mrp3, and Mrp4 relative to WT mice. UDCA potently increased nuclear Nrf2 expression level in livers of WT mice, and the treatment showed maximal hepatic induction of Mrp2, Mrp3, and Mrp4 in association with enhanced membranous localizations in an Nrf2-dependent manner. UDCA similarly increased nuclear Nrf2 expression level in rat hepatocytes. Chromatin immunoprecipitation assays using mouse hepatocytes revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of Mrp2, Mrp3, and Mrp4. These findings demonstrate an important role of Nrf2 in the induction of Mrp family members in livers and suggest that a therapeutic mechanism of UDCA action is, via Nrf2 activation, a stimulation of detoxification and antioxidative stress systems, along with Mrp-mediated efflux transport.

To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants, which accumulates excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared with the wild type. DNA sequencing of the VPE gene in glup3 mutants revealed either amino acid substitutions or the appearance of a stop codon within the coding region. Microscopic observations showed that alpha-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild-type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSVs.

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The androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily of ligand-dependent transactivation factors. Androgens such as testosterone and 5-a-dihydrotestosterone (DHT) act as agonists of AR. AR mediates various biological effects such as the development of male reproductive tissues, sexual development, and spermatogenesis. [1][2][3][4] Since androgen declining with age contributes to age-related bone and muscle loss and increase in fat mass, 5) the anabolic effect of androgen is attractive for the maintenance of health. Furthermore, it is known that AR is involved in androgen-dependent prostate cancer growth. Thus, antagonists of AR (flutamide, bicalutamide, and nilutamide) are used in anti-androgen therapy for prostate cancer. 8,9) AR mediates the expression of androgen-regulated genes, as represented by prostate specific antigen (PSA) [10][11][12] and FK506-binding protein 51 (FKBP51). [13][14][15] In the absence of a ligand, AR is localized in the cytoplasm, where it forms complexes with chaperones. Upon ligand binding, AR translocates into the nucleus. Following nuclear translocation, AR binds to androgen responsive elements (ARE) in the promoter regions of its target genes as a homodimer. Generally, the transcriptional activity of nuclear receptors is modulated by their interaction with cofactors such as coactivators and corepressors. [16][17][18][19] The type of ligand that binds to the receptor determines which type of cofactor is chosen. In the case of agonists, AR interacts with coactivators dominantly over corepressors, and vice versa in the case of antagonists.Unlike other nuclear receptors, AR AF2 demonstrates weak transcriptional activity. However, ligand-dependent interaction between NTD and LBD/AF2 (which is termed as the N/C interaction) endows AR with synergistic transactivation potential. [20][21][22][23] Thus, the N/C interaction is important for the ligand-dependent transactivation potential of AR.Synthetic AR ligands are useful for treatment of prostate cancer and age-related diseases such as osteopenia and sarcopenia. Previously, we prepared novel synthetic steroids.24) In our preliminary screening for novel AR ligands, a steroid compound, (17a,20E)-17,20-[(1-methoxyethylidene) bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), behaved apparently as a partial agonist of AR in an ARE-luciferase reporter assay (unpublished data). We were interested in this partial agonistic nature of YK11. In this report, we will show that YK11 blocks the N/C-interaction required for the full-agonistic function of wild-type AR, inducing selective AR-target genes owing to the constitutive transactivation potential of AF-1 subdomain in endogenous AR-expressing MDA-MB 453 cells. MATERIALS AND METHODS ChemicalsThe compounds tested in our preliminary screening for novel AR ligands were prepared by previously

Morphological changes induced by clofibrate in type-1 predominant soleus, type-2 predominant tensor fasciae latae, and type-1 and -2 mixed biceps femoris muscles and diaphragm in rats were investigated. Administration of the agent at 500 or 750 mg/kg/day by oral gavage for 14 or 28 days caused lesions in the soleus muscle and diaphragm, bur no changes in the tensor fasciae latae and biceps femoris muscles. In soleus muscle, vacuolation of muscle fibers was observed in all animals treated with clofibrate, and degeneration of muscle fibers and infiltration of leukocytes were noted at 750 mg/kg/day. In diaphragm, vacuolation of muscle fibers was also observed in all animals treated with clofibrate, and these lesions were located in type-1 skeletal muscles densely stained with NADH-TR. The vacuoles seen in soleus muscle and diaphragm were positive for oil red O staining. In addition, increase of lipid droplets and mitochondrial hypertrophy was seen in soleus muscle, ultrastructurally. These data suggest that sensitivity to clofibrate-induced muscle toxicity differs among muscles, with type-1 fibers being susceptible.