Biomedical subjects

Taijiro Okabe

Publications and source records attributed to Taijiro Okabe.

16 recordsLinked to original sources

PubMed2005

Modulation of androgen receptor transactivation by FoxH1. A newly identified androgen receptor corepressor.

Androgen signaling plays key roles in the development and progression of prostate cancer, and numerous ongoing studies focus on the regulation of androgen receptor (AR) transactivity to develop novel therapies for the treatment of androgen-independent prostate cancer. FoxH1, a member of the Forkhead-box (FOX) gene family of transcription factors, takes part in mediating transforming growth factor-beta/activin signaling through its interaction with the Smad2.Smad4 complex. Using a series of experiments, we found that FoxH1 repressed both ligand-dependent and -independent transactivation of the AR on androgen-induced promoters. This action of FoxH1 was independent of its transactivation capacity and activin A but relieved by Smad2.Smad4. In addition, the repression of the AR by FoxH1 did not require deacetylase activity. A protein-protein interaction was identified between the AR and FoxH1 independently of dihydrotestosterone. Furthermore, a confocal microscopic analysis of LNCaP cells revealed that the interaction between the AR and FoxH1 occurred in the nucleus and that FoxH1 specifically blocked the foci formation of dihydrotestosterone-activated AR, which has been shown to be correlated with the AR transactivation potential. Taken together, our results indicate that FoxH1 functions as a new corepressor of the AR. Our observations not only strengthen the role of FoxH1 in AR-mediated transactivation but also suggest that therapeutic interventions based on AR-coregulator interactions could be designed to block both androgen-dependent and -independent growth of prostate cancer.

Activins↗

PubMed2005

Impaired nuclear translocation, nuclear matrix targeting, and intranuclear mobility of mutant androgen receptors carrying amino acid substitutions in the deoxyribonucleic acid-binding domain derived from androgen insensitivity syndrome patients.

CONTEXT: Recent imaging studies revealed that androgen receptor (AR) is ligand-dependently translocated from the cytoplasm into the nucleus and forms intranuclear fine foci. In this study, we examined whether intracellular dynamics of mutant ARs detected in two androgen insensitivity syndrome (AIS) patients was impaired. OBJECTIVE: ARs with mutations in the DNA-binding domain were functionally characterized and compared with the wild-type AR. PATIENTS: In a complete AIS patient (subject 1), cysteine residue 579 in the first zinc finger motif of AR was substituted for phenylalanine (AR-C579F). Another mutation (AR-F582Y) was found in a partial AIS patient (subject 2). RESULTS: AR-F582Y retained less than 10% of the transactivation activity of the wild-type AR, whereas no ligand-dependent transactivation was detected for AR-C579F. Image analyses of the receptors fused to green fluorescent protein showed that the wild-type AR was ligand-dependently translocated into the nucleus in which it formed fine subnuclear foci. Surprisingly, after the addition of dihydrotestosterone, the two mutant ARs initially formed large cytoplasmic dots, many of which were found to be close to mitochondria by electron microscopy. Subsequently, a part of the ligand-bound mutant ARs gradually entered the nucleus to form a smaller number of larger dots, compared with the wild-type AR. Fluorescence recovery after photobleaching analysis revealed that the intranuclear mobility of the mutant ARs decreased, compared with that of the wild-type AR. CONCLUSIONS: These results suggest that the abnormal translocation, localization, and mobility of the mutant ARs may be the cause of AIS in these subjects.

Active Transport, Cell Nucleus↗

PubMed2005

Dehydroepiandrosterone negatively regulates the p38 mitogen-activated protein kinase pathway by a novel mitogen-activated protein kinase phosphatase.

Dehydroepiandrosterone-sulfate, the sulfated form of dehydroepiandrosterone, is the most abundant steroid in young adults, but gradually declines with aging. In humans, the clinical application of dehydroepiandrosterone targeting some collagen diseases, such as systemic lupus erythematosus, as an adjunctive treatment has been applied in clinical trial. Here, we report that dehydroepiandrosterone may negatively regulate the mitogen-activated protein kinase pathway in humans via a novel dual specificity protein phosphatase, DDSP (dehydroepiandrosterone-enhanced dual specificity protein phosphatase). DDSP is highly homologous to LCPTP/HePTP, a tissue-specific protein tyrosine phosphatase (PTP) which negatively regulates both ERK and p38-mitogen-activated protein kinase, and is transcribed from the PTPN7 locus by alternative splicing. Although previous reports have shown that the mRNA expression of the LCPTP/HePTP gene was inducible by extracellular signals such as T-cell antigen receptor stimulation, reverse transcribed (RT)-PCR experiments using specific sets of primers suggested that the expression of LCPTP/HePTP was constitutive while the actual inducible sequence was that of DDSP. Furthermore DDSP was widely distributed among different types of human tissues and specifically interacted with p38-mitogen-activated protein kinase. This inducible negative regulation of the p38-mitogen-activated protein kinase-dependent pathway may help to clarify the broad range of dehydroepiandrosterone actions, thereby aiding the development of new preventive or adjunctive applications for human diseases.

Amino Acid Sequence↗

PubMed2005

Androgen receptor null male mice develop late-onset obesity caused by decreased energy expenditure and lipolytic activity but show normal insulin sensitivity with high adiponectin secretion.

Androgen receptor (AR) null male mice (AR(L-/Y)) revealed late-onset obesity, which was confirmed by computed tomography-based body composition analysis. AR(L-/Y) mice were euphagic compared with the wild-type male (AR(X/Y)) controls, but they were also less dynamic and consumed less oxygen. Transcript profiling indicated that AR(L-/Y) mice had lower transcripts for the thermogenetic uncoupling protein 1, which was subsequently found to be ligand-dependently activated by AR. We also found enhanced secretion of adiponectin, which is insulin sensitizing, from adipose tissue and a relatively lower expression of peroxisome proliferator-activated receptor-gamma in white adipose tissue in comparison to AR(X/Y) mice. Both factors might explain why the overall insulin sensitivity of AR(L-/Y) mice remained intact, despite their apparent obesity. The results revealed that AR plays important roles in male metabolism by affecting the energy balance, and it is negative to both adiposity and insulin sensitivity.

Adiponectin↗

PubMed2004

Activation of peroxisome proliferator-activated receptor-gamma and retinoid X receptor inhibits aromatase transcription via nuclear factor-kappaB.

Our previous studies demonstrated that a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, troglitazone (TGZ),and/or a retinoid X receptor (RXR) ligand, LG100268 (LG), decreased the aromatase activity in both cultured human ovarian granulosa cells and human granulosa-like tumor KGN cells. In the present study, we further found that a combined treatment of TGZ+LG decreased aromatase promoter II (ArPII) activity in both ovarian KGN cells and fibroblast NIH-3T3 cells in a PPARgamma-dependent manner. Furthermore, the inhibition of both aromatase activity and the transcription of ArPII by TGZ+LG was completely eliminated when nuclear factor-kappaB (NF-kappaB) signaling was blocked by specific inhibitors, suggesting NF-kappaB, which is endogenously expressed in both fibroblast and granulosa cells, might be a mediator of this inhibition. Interestingly, activation of NF-kappaB by either forced expression of the p65 subunit or NF-kappaB-inducing kinase up-regulated ArPII activity. Positive regulation of aromatase by endogenous NF-kappaB was also suggested by the fact that NF-kappaB-specific inhibitors suppress basal activity of the aromatase gene. A concomitant formation of high-order complex between NF-kappaB p65 and ArPII was also observed by chromatin immunoprecipitation assay. Although activation of PPARgamma and RXR affected endogenous expression levels of neither inhibitory kappaBalpha nor p65, it impaired the interaction between NF-kappaB and ArPII and the p65 based transcription as well. Altogether, these results indicate that activation of a nuclear receptor system, constituted by PPARgamma and RXR, down-regulates aromatase expression through the suppression of NF-kappaB-dependent aromatase activation and thus provide a new insight in the mechanism of regulation of the aromatase gene.

Aromatase↗

PubMed2004

SF-1/Ad4BP transforms primary long-term cultured bone marrow cells into ACTH-responsive steroidogenic cells.

Bone marrow stem cells develop into haematopoietic and mesenchymal lineages, but have not been known to participate in steroidogenic cell production. Steroidogenic factor 1 (SF-1), also designated adrenal 4 binding protein (Ad4BP), is an essential orphan nuclear receptor for steroidogenesis as well as for adrenal and gonadal gland development. In the present study, we revealed that the adenovirus-mediated forced expression of SF-1 can transform cultured primary long-term cultured bone marrow cells into steroidogenic cells, showing the de novo synthesis of multiple steroid hormones in response to adrenocorticotropic hormone (ACTH). This finding may provide an initial step in innovative autograft cell transfer therapy for steroid hormone deficiencies.

Adenoviridae↗

PubMed2004

Adrenopause.

Dehydroepiandrosterone(DHEA) and DHEA-S are steroids that are abundantly produced by the adrenal gland. Plasma concentrations of DHEA and DHEA-S increase during adrenarche but decrease steadily after puberty. Although DHEA and DHEA-S have few intrinsic androgenic actions, they have recently attracted widespread attention due to their beneficial anti-aging effects. We clarified the beneficial effects of DHEA as an anti-aging steroid with regard to its stimulation of the immune system and its anti-diabetes, anti-atherosclerosis, anti-dementia (neurosteroid), anti-obesity and anti-osteoporosis effects. There are two possible biochemical and molecular mechanisms: direct action via the DHEA receptor on the target gene; and indirect action. We identified a high affinity of DHEA binding in human T-lymphocytes by searching for the target genes that are induced in activated T-lymphocytes in the presence of DHEA, determined the gene sequence and named DHEA-induced dual p38-specific phosphatase (DDSP). DDSP transgenic mice have been created to identify the anti-aging effects of DDSP. The conversion of DHEA to estrone by cytochrome P450 aromatase in primary cultured human osteoblasts was clarified. We are currently undertaking an open trial of DHEA replacement therapy.

Adrenal Glands↗

PubMed2003

A benzimidazole fungicide, benomyl, and its metabolite, carbendazim, induce aromatase activity in a human ovarian granulose-like tumor cell line (KGN).

Endocrine disruptor chemicals are known to cause a range of abnormalities in sexual differentiation and reproduction. One mechanism underlying such effects may be via alteration of aromatase activity, which is responsible for estrogen production. A good screening system for identifying endocrine disruptors has long been desired. We have recently established a human ovarian granulosa-like tumor cell line, KGN, which possesses a relatively high level of aromatase expression and is considered a useful mammalian model for investigating the in vitro effects of various chemicals on aromatase activity. In this study we screened 55 different candidate chemicals for endocrine disruptors by assaying aromatase activity. Only benomyl, known as both a benzimidazole fungicide and a microtubule-interfering agent, was found to induce aromatase activity in association with increased levels of aromatase mRNA in KGN cells. The effect of benomyl was presumed to be mediated by its metabolite carbendazim, because it produced an effect equivalent to that of benomyl. The mechanism underlying the benomyl-induced increase in aromatase activity appears independent of the cAMP-protein kinase A pathway. Treatment with taxol, another class of microtubule-interfering agents, also caused induction of aromatase in KGN cells. Both benomyl and taxol changed KGN cell morphology, including the development of cell roundness and a disorganized network of microtubules. These results indicate that benomyl is a potential endocrine disruptor that provides a novel estrogenicity and operates through a microtubule-interfering mechanism.

Aromatase↗

PubMed2003

Functional characterization of a new human Ad4BP/SF-1 variation, G146A.

Ad4BP/SF-1 plays key roles at all levels of the hypothalamic-pituitary-steroidogenic organ axis and its functional disruption causes endocrine disorders of these organs. However, only three human subjects with Ad4BP/SF-1 mutations have been reported to date, suggesting limited clinical significance as a cause of inborn adrenal or sexual abnormalities. We report the first functional characterization of a new variation found in the hinge region of human Ad4BP/SF-1, G146A. Resulting from a single nucleotide shift (GGG-->GCG), G146A bears slightly diminished transactivation activity evidenced by both adrenal specific cyp11A promoter and ovary specific cyp19 promoter II. The variation does not affect protein expression or stability, exhibiting no dominant negative effect. G146A has a normal interaction pattern with standard co-regulators and subnuclear distribution pattern, and can be considered as a nonsynonymous single nucleotide polymorphism, since it occurs in normals and patients with adrenal diseases. In normal Japanese the allele C frequency is 8%, while in a preliminary population of patients with adrenal diseases it is elevated to 30%; suggesting the G146A variation might be of clinical importance.

Adrenal Gland Diseases↗

PubMed2003

Protein kinase A potentiates adrenal 4 binding protein/steroidogenic factor 1 transactivation by reintegrating the subcellular dynamic interactions of the nuclear receptor with its cofactors, general control nonderepressed-5/transformation/transcription domain-associated protein, and suppressor, dosage-sensitive sex reversal-1: a laser confocal imaging study in living KGN cells.

The mechanism through which protein kinase A (PKA) potentiates the transactivation ability of adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) is currently unclear. In the present study, we investigated the mechanism by applying laser confocal microscopy and fluorescence recovery after photobleaching technique. In KGN cells, forskolin (a PKA stimulator) could reorganize wild-type Ad4BP/SF-1, but not mutant Ad4BP/SF-1 (G35E), from a diffuse distribution pattern to foci formation in the nucleus. The subcellular distributions of GCN5 (general control nonderepressed) and TRRAP (transformation/transcription domain-associated protein), both of which were recently proved to be working in the same complex as the third class of nuclear receptor coactivators, were unexpectedly diffuse inside and outside the nucleus, respectively, when they were separately transfected. However TRRAP was translocated into the nucleus in the presence of GCN5, and together with GCN5 colocalized with Ad4BP/SF-1 in the same foci when PKA was activated. A luciferase assay also indicated that these two cofactors enhanced Ad4BP/SF-1 transactivation.Dosage-sensitive sex reversal (DAX-1) interacts with and thus inhibits Ad4BP/SF-1 transactivation. The coexistence of the two proteins dramatically altered their respective subnuclear distributions. They colocalized extensively, suggestive of binding, and Ad4BP/SF-1 was sharply immobilized when DAX-1 was coexpressed, whereas PKA could maintain mobility, as evidenced by Fluorescence Recovery After Photobleaching showing that Ad4BP/SF-1 mobility recovered after forskolin treatment.Therefore, the PKA signal pathway may modify the interaction between Ad4BP/SF-1 and its activators and repressor (GCN5 and TRRAP are integrated, whereas DAX-1 is disassociated), and thus stimulate the Ad4BP/SF-1 transactivation.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Activation function-1 domain of androgen receptor contributes to the interaction between two distinct subnuclear compartments.

The nucleus contains different sets of functional compartments often called "speckles". The splicing factor compartment (SFC) has been speculated to consist of SFs and transcription factors, which thus make transcription-splicing coupling possible at the periphery of SFC. Androgen receptor (AR), as well as glucocorticoid receptor (GR), is unique since most, if not all, of its activities are mediated via the constitutive activity of the activation function-1 (AF-1) function. Transcriptionally active AR produces 250-400 subnuclear fine speckles11 shared with GR or estrogen receptor (ER), which colocalize with chiefly activation function-2 (AF-2)-interacting p160 family- or CBP-related speckles. We herein report the isolation of ANT-1 (AR N-terminal domain (NTD) transactivating protein-1) enhancing autonomous AF-1 transactivation function of AR or GR, but not of estrogen receptor alpha (ERalpha). The ANT-1 was identical to a binding protein of human splicing factor U5 snRNP (U5 snRNP-associated protein). ANT-1 was compartmentalized into 15-20 coarse SFC speckles which were spatially distinct from but surrounded by the AR compartments. Our results suggest that ANT-1 may play a key role in the molecular interaction between two spatially distinct subnuclear compartments in a receptor-specific fashion, and thereby induce the strong autonomous transactivation functions either of AR- or GR-AF-1.

Binding Sites↗

PubMed2003

Aromatase in bone: roles of Vitamin D3 and androgens.

We have mainly focused on the regulatory mechanism of cytochrome P450 aromatize in bone cells. Our previous study demonstrated a strong positive correlation of serum dehydroepiandrosterone sulfate (DHEA-S) and estrone (E1) with BMD in postmenopausal women but no correlation between serum estradiol (E2) and BMD in the same group. In addition, administration of DHEA to ovariectomized rat significantly increased BMD. These in vivo findings strongly suggested that circulating adrenal androgen may be converted to estrogen in osteoblast and may contribute to BMD maintenance. Actually, in cultured human osteoblast cells, DHEA was found to convert to androstenedione by 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity and then androstenedione to estrone through the apparent aromatase activity. The aromatase activity in cultured human osteoblast cells was significantly increased by dexamethasone (DEX). Interestingly, DEX and 1alpha,25-dihydroxyvitamin D3 (VD3) synergistically enhanced aromatase activity as well as P450arom mRNA expression. A little stronger induction of aromatase activity by DEX and VD3 was observed in cultured human fibroblasts. The increase of the aromatase activity by DEX and VD3 was accompanied with the increase of luciferase activity of fibroblast cells transfected with Exon 1b-promoter-luciferase construct, but not of osteoblasts transfected with the same construct, suggesting a different regulatory mechanism of aromatase by DEX and 1alpha,25-dihydroxyvitamin D3 (VD3) between these two cells despite the same promotor usage. In human bone cells, intracrine mechanism through aromatase activity, together with a positive regulation of aromatase activity by glucocorticoid and VD3, may contribute to the local production of estrogens, thus leading to protective effect against osteoporosis especially after menopause. The effect of sex steroids (estrogen versus testosterone) in bone remodeling was also briefly reviewed based on several recent findings in this field.

Androgens↗

PubMed2003

Activin signaling through type IB activin receptor stimulates aromatase activity in the ovarian granulosa cell-like human granulosa (KGN) cells.

In addition to a stimulatory effect on FSH production by the pituitary gland, activin is thought to have a paracrine or autocrine role in follicular development in the ovary, where it is produced. Recently, we established a human ovarian granulosa tumor cell line, KGN, which possesses in vivo characteristics of granulosa cells, namely the expression of functional FSH receptors and cytochrome P-450 aromatase. Here, we have demonstrated the activin signaling pathway and its role in KGN cells. A series of transient transfection experiments revealed that activin type IB receptor (ActRIB) is an essential component of the activin signaling pathway in KGN cells. Smad2 was found to act downstream of ActRIB as an intracellular signal transmitter. Smad7, but not Smad6, was an inhibitory Smad in the pathway. Finally, we show that FSH receptor expression and cytochrome P-450 (P-450) aromatase activity was up-regulated by activin stimulation through ActRIB in KGN cells. These results show that we have clarified the signaling mechanisms and the roles of activin in the human granulosa cell line, KGN. Activin signaling mediated by ActRIB-Smad2 system in the ovary may thus be essential for the regulation of follicular differentiation.

Activin Receptors, Type I↗

PubMed2003

[Trials of regeneration and gene therapies in endocrine organs, especially in adrenal glands].

Recent progress in trials of regeneration and gene therapy in endocrine organs, especially in adrenal glands has been reviewed. Gene therapies using adenovirus have been most frequently tested in vivo and in vitro, aiming at improvement of steroidogenesis and suppression of adrenal tumor growth. Although the effects were temporal, promising results have been obtained. Interestingly, adrenocortical tissue was shown to be formed by transplantation of adrenocortical cells and to replace the adrenal functions of adrenalectomized animals. Engineered ES cells stably expressing Ad4BP/SF-1 were shown to be directed toward steroidogenic lineage, suggesting a future possibility of regeneration of adrenal cells.

Adenoviridae↗

PubMed2002

Activation function-1 domain of androgen receptor contributes to the interaction between subnuclear splicing factor compartment and nuclear receptor compartment. Identification of the p102 U5 small nuclear ribonucleoprotein particle-binding protein as a coactivator for the receptor.

In the androgen receptor (AR), most of its transactivation activity is mediated via the activation function-1 (AF-1). By employing yeast two-hybrid assay, we isolated a cDNA sequence encoding a protein binding to AR-AF-1. This protein, named ANT-1 (AR N-terminal domain transactivating protein-1), enhanced the ligand-independent autonomous AF-1 transactivation function of AR or glucocorticoid receptor but did not enhance that of estrogen receptor alpha. In contrast, the ANT-1 did not enhance any ligand-dependent AF-2 activities. Furthermore, the ligand-independent interaction between AR-AF-1 and ANT-1 was confirmed in vivo and in vitro. The ANT-1 sequence was identical to that of a protein that binds to U5 small nuclear ribonucleoprotein particle, a human homologue of yeast splicing factor Prp6p, involved in spliceosome. ANT-1 was compartmentalized into 20-40 coarse splicing factor compartment speckles against the background of the diffuse reticular distribution. AR colocalized with ANT-1 only in the diffusely distributed area, whereas the ANT-1 speckles were spatially distinct from but surrounded by the AR compartments. The active gene transcription has been shown to couple simultaneously with pre-mRNA processing at the periphery of the splicing factor compartment. The molecular interaction between two spatially distinct subnuclear compartments mediated by ANT-1 may therefore recruit AR into the transcription-splicing-coupling machinery.

Animals↗

PubMed2002

Mechanism of action of anti-aging DHEA-S and the replacement of DHEA-S.

The plasma ACTH and cortisol levels do not change during aging. On the other hand, the plasma dehydroepiandrosterone sulfate (DHEA-S) changes remarkably during aging. Before puberty, the plasma DHEA-S level both in males and females is very low, however, it rapidly increases at puberty, and thereafter significantly decreases both linearly and age-dependently. Cytochrome P450c17 has two enzyme activities, 17-alpha-hydroxylase and 17,20-lyase. Cortisol is synthesized by 17-alpha-hydroxylase, and DHEA is synthesized by 17,20-lyase. The mechanism of dissociation of cortisol and DHEA synthesis in aging depends on another regulator of 17,20-lyase of cytochrome P450c17 such as cytochrome P450 reductase. We demonstrated significant decrease in cytochrome P450 reductase activity in bovine aged adrenal glands. We clarified the beneficial effects of DHEA as an anti-aging steroid based on both in vitro and in vivo experiments, such as the stimulatory effect of immune system, anti-diabetes mellitus, anti-atherosclerosis, anti-dementia (neurosteroid), anti-obesity and anti-osteoporosis. It is very important to identify the mechanism of action of DHEA. We clarified the conversion of DHEA to estrone by cytochrome P450 aromatase in primary cultured human osteoblasts. We indentified high affinity of DHEA binding with K(d)=6.6 nM in antigen and DHEA stimulated human T lymphocytes. We searched for the target genes that are specifically induced in activated T lymphocytes in the presence of DHEA by subtractive hybridization screening for differentially expressed transcripts. The double blind, randomized human replacement therapies utilizing DHEA are also reviewed.

Aging↗