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Hirotaka Shibata

Publications and source records attributed to Hirotaka Shibata.

At least 19 recordsLinked to original sources
PubMed2006

Coactivation of the N-terminal transactivation of mineralocorticoid receptor by Ubc9.

Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme.

Aldosterone↗
PubMed2006

Primary malignant hepatic pheochromocytoma with negative adrenal scintigraphy.

A 60-year-old male patient with hypertension was referred to our hospital because of insufficient blood pressure control (190/98 mmHg) and to rule out secondary hypertension. A computed tomography scan revealed no adrenal tumor but a large liver mass (5 x 5 cm), and magnetic resonance imaging showed a high signal intensity lesion on the T2-weighted image. Twenty-four hour urinary excretion of catecholamine metabolites was markedly increased, although a 123I-metaiodobenzyl guanidine (MIBG) scintigram failed to show accumulation in the hepatic mass, and no difference was noted between the catecholamine concentration in the tumor-drainage vein and that obtained from the vein draining from the non-tumor area. Liver biopsy did show features compatible with pheochromocytoma (i.e., chromogranin A-positive cells). Transcatheter arterial embolization of the liver tumor was conducted and resulted in a marked (50%) decrease in the 24-h urine normetanephrine excretion. Several metastatic foci were noted in the spinal bone and transcatheter arterial embolization (TAE) was also conducted with successful results. Thus, we experienced a case of primary malignant hepatic pheochromocytoma with negative 123I-MIBG scanning.

3-Iodobenzylguanidine↗
PubMed2006

A case of estrogen-secreting adrenocortical carcinoma with subclinical Cushing's syndrome.

A 25-year-old man was found to have a large right adrenal mass detected by abdominal echography and computed tomography, and presented with a mild gynecomastia. Endocrine study showed increased serum concentrations and urinary excretion of estrogens and dehydroepiandorosterone sulfate (DHEA-S). The patient had no Cushingoid features but autonomous cortisol secretion, compatible with the diagnosis of subclinical Cushing's syndrome. Surgical removal of the adrenal tumor led to normalization of serum and urinary excretion of estrogens and DHEA-S. Histopathological examination revealed a high-grade adrenocortical carcinoma (ACC). The disorganized expression of all the steroidogenic enzymes in individual tumor cells was demonstrated by immunohistochemical analysis, and the abundant expression of both aromatase mRNA and insulin-like growth factor (IGF)-II mRNA was shown by RT-PCR. These data suggest the excessive secretion of estrogen as well as the ineffective steroidogenesis by the adrenal tumor. This is a very rare case of estrogen-secreting ACC associated with subclinical Cushing's syndrome.

Adrenal Cortex Neoplasms↗
PubMed2005

New genetic causes of pheochromocytoma: current concepts and the clinical relevance.

Pheochromocytoma and paraganglioma are tumors of the autonomous nervous system mainly occurring in the adrenal medulla, but also in the extraadrenal paraganglias of the abdomen, thorax, neck and skull basis. The etiology comprises germline mutations of now 6 genes. About 10 years known are the RET gene susceptible for multiple endocrine neoplasia type 2, the VHL gene for von Hippel-Lindau Disease, and the NF 1 gene for neurofibromatosis Recklinghausen (neuro- fibromatosis type 1). Since 2000 the genes for succinatedehydrogenase subunits SDHB, SDHC, and SDHD have been identified for paraganglioma syndromes type 4, type 3, and type 1 respectively. Investigations of series of pheochromocytoma patients identified germline mutations in one of the genes SDHB, SDHD, VHL and RET in 24% to 50% of the patients. Multifocal tumors, young age and positive family history, known features associated with inheritence, have not been present in all patients. Therefore, analyses of blood DNA for mutations in these genes are recommended. Positive tests provide the patients and their relatives with essential platforms for clinical care. Experiences in this field of medicine have shown that optimal management of patients with pheochromocytoma-associated syndromes is a high challenge. National registries may be instrumental in order to provide with adequate facilities.

Adrenal Gland Neoplasms↗
PubMed2004

Ubc9 and Protein Inhibitor of Activated STAT 1 Activate Chicken Ovalbumin Upstream Promoter-Transcription Factor I-mediated Human CYP11B2 Gene Transcription.

Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.

Animals↗
PubMed2004

Proteasome-mediated mineralocorticoid receptor degradation attenuates transcriptional response to aldosterone.

The ubiquitin-proteasome pathway regulates the turnover of many nuclear hormone receptors, such as the estrogen receptor. For estrogen receptor, proteasome inhibition decreases ligand-mediated transcription. We provide evidence that the mineralocorticoid receptor (MR) is degraded by the ubiquitin-proteasome pathway in a ligand-dependent manner and that proteasomal inhibition results in increased accumulation of the MR with enhancement of transcriptional response to aldosterone. Examination of the primary sequence of human and rat MR has identified two candidate PEST degradation motifs. Mutation of lysine 715 and/or 367 within this PEST element failed to prevent degradation of MR protein or transcriptional activity mediated by aldosterone, indicating that other lysine residues are targeted by proteasomal degradation of MR. These findings demonstrate a coupling between MR up-regulation and transcriptional hyperactivity.

Aldosterone↗
PubMed2004

FHL2, UBC9, and PIAS1 are novel estrogen receptor alpha-interacting proteins.

Estrogen plays important roles in the pathophysiology of atherosclerosis and cardiovascular diseases mediated by estrogen receptor alpha (ERalpha). To elucidate the molecular mechanisms, we screened ERalpha-interacting proteins from a human heart cDNA library using a yeast two-hybrid system, and identified the four and a half of LIM-only protein 2 (FHL2). FHL2 interacted with ERalpha in the presence of 17beta-estradiol, but not of tamoxifen or raloxifene in yeast. FHL2 mainly interacted with N-terminal A/B domain of ERalpha but not C-terminal ligand-binding domain. However, overexpression of full-length FHL2 did not affect ERalpha-dependent transcriptional activities of a reporter containing 3 copies of estrogen response element in COS-1 cells. Since tissue distribution of FHL2 was highly restricted to the heart, the function of FHL2 may be observed in a cell type- or promoter-specific manner. We have also detected strong interactions of ERalpha with Ubc9 and PIAS1 in yeast. Ubc9 and PIAS1, small ubiquitin-related modifier-1 (SUMO-1) conjugating enzyme and ligase, respectively, markedly interacted with ERalpha in a 17beta-estradiol-dependent manner. These proteins mainly interacted with the DNA-binding and ligand-binding domains of ERalpha. Overexpression of Ubc9 or PIAS1 potentiated ERalpha-mediated transcriptional activities in COS-1 cells in a dose-dependent manner, indicating that both Ubc9 and PIAS1 function as coactivators of ERalpha. In addition, the SUMOylation-defective mutant, Ubc9 (C93S) continued to enhance ERalpha-dependent transcriptional activities. These findings suggest that coactivator abilities and SUMOylation capacities of Ubc9 and PIAS1 are separable and distinct. The present studies indicate that ERalpha exhibit tissue-specific functions utilizing multiple tissue-restricted receptor-interacting proteins.

Animals↗
PubMed2004

COUP-TF and transcriptional co-regulators in adrenal steroidogenesis.

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) and steroidogenic factor-1 (SF-1) play key roles in the transcriptional regulation of steroidogenic P450 genes. Transfection studies showed that SF-1 activated bovine CYP17 promoter activity, whereas COUP-TFs repressed it from the CRS2 element in a mutually exclusive manner in mouse adrenocortical Y-1 cells. COUP-TFI and SF-1 competitively bind to the Ad5 element of the human CYP11B2 gene promoter. Unexpectedly, overexpression of COUP-TFI increased the CYP11B2 promoter activity, whereas overexpression of SF-1 repressed it in human adrenocortical H295R cells. In cortisol-producing adrenal cortical adenomas, down-regulation of nuclear receptors, including COUP-TFs was found. We therefore screened for COUP-TFI-interacting proteins using a yeast two-hybrid system and have identified Ubc9 and PIAS1, SUMO-1 conjugating enzyme and ligase, respectively. Coexpression of Ubc9 and PIAS1 synergistically enhanced COUP-TFI-mediated trans-repression of CYP17 gene as well as transactivation of CYP11B2 gene. The SUMOylation-defective mutants of these proteins continued to function as co-regulators of COUP-TFI. These findings indicate that Ubc9 and PIAS1 can function as transcriptional co-regulators of COUP-TFI to modulate adrenal cortical steroidogenesis in a SUMOylation-independent manner.

Adrenal Cortex↗
PubMed2003

Regulation of differential COUP-TF-coregulator interactions in adrenal cortical steroidogenesis.

Hyperfunctioning adrenocortical adenomas produce excessive amounts of various corticosteroids due to dysregulated expression of steroidogenic enzymes. Since no genetic mutations in steroidogenic enzyme genes have been identified as yet, the dysregulated expression at the transcription level may be crucial. Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) and steroidogenic factor-1 (SF-1) play key roles in the transcriptional regulation of steroidogenic P450 genes. Transfection studies showed that SF-1 activated and COUP-TFs repressed the transcription of bovine CYP17 gene promoter from the CRS2 element in a mutually exclusive manner in Y-1 cells. The results indicate that COUP-TFs negatively regulate the transcriptional activity of SF-1, a steroidogenic cell-specific activator of various steroidogenic P450 genes. Expression of both COUP-TFI and COUP-TFII was significantly decreased in the cortisol-producing adenomas, in which CYP17 was drastically overexpressed, indicating that decreased expression of COUP-TFs play a key role in overexpression of CYP17 in this type of tumors. We then screened for COUP-TFI-interacting proteins from a cortisol-producing adenoma cDNA library using a yeast two-hybrid system and identified a novel RING finger-containing protein which can function as a coregulator for COUP-TFI. Notably, COUP-TFI activated rather than repressed several target genes including the human CYP11B2 gene promoter, the results of which were opposite to those of the CYP17 promoter. The bifunctional activities of COUP-TFI may be derived from the promoter context and our newly identified COUP-TFI coregulator.

Adrenal Cortex↗
PubMed2003

Nuclear receptors and co-regulators in adrenal tumors.

We have reported that excessive steroid hormone production in adrenal cortical tumors results from the disordered expression and activity of specific steroidogenic enzymes. Since no genetic mutations in these steroidogenic enzymes have as yet been identified, disordered expression at the transcription level may be crucial for excessive hormone production in adrenocortical tumors. Nuclear receptors SF-1 and COUP-TF/DAX-1 have been shown to activate and repress, respectively, the transcription of CYP17 gene in a mutually exclusive manner in Y-1 cells. Interestingly, the expression of COUP-TF and DAX-1 is significantly decreased in the cortisol-producing adenomas, in which CYP17 is overexpressed. Conversely, DAX-1 is highly expressed in deoxycorticosterone-producing adenomas, where CYP17 expression is almost absent. These expression profiles indicate the possibility that COUP-TF and DAX-1 play important roles in the transcriptional repression of CYP17 in adrenal tumors. To clarify the mechanisms of COUP-TF-mediated repression, we therefore screened for COUP-TF-interacting proteins using a yeast two-hybrid system from a cortisol-producing adenoma cDNA library. We then cloned a novel COUP-TF-interacting protein-1 (CIP-1), which interacts with COUP-TFI, COUP-TFII, and SF-1. Functionally, CIP-1 can act as a transcriptional co-repressor for COUP-TF repression activity. CIP-1 expression profiles parallel those of COUP-TFI in steroidogenic tissues, strongly suggesting that, together, COUP-TFI and CIP-1 play an important role in steroidogenesis.

Adenoma↗
PubMed2003

Relation between blood pressure and rhinitis in a Japanese adolescent population.

Recent studies have reported an association between systolic blood pressure (BP) and rhinitis. The prevalence of allergic rhinitis is 20% to 30% in Japan. The present cross-sectional study was performed to assess the relation between BP and allergic rhinitis in a sample of 2,292 male adolescent high school students who attended the annual school health examination. Of the 2,292 students (mean age, 16 years), 26.6% were considered to have allergic rhinitis based on their responses to a questionnaire, 25.1% were diagnosed with allergic rhinitis by an otolaryngologist, and 12.6% were consistently diagnosed with allergic rhinitis by both questionnaire and an otolaryngologist. There was no difference in systolic BP between subjects with and without allergic rhinitis. Diastolic BP, however, was higher in subjects without allergic rhinitis than in those with allergic rhinitis as diagnosed by questionnaire alone or by both questionnaire and an otolaryngologist. When the subjects were divided into three BP categories (normal, high normal, and hypertension) according to the criteria in the JSH 2000, the rates of allergic rhinitis were similar in the three BP categories. These findings do not confirm a relation between allergic rhinitis and systolic BP.

Adolescent↗
PubMed2002

Expression and regulation of nuclear receptor coactivators in glucocorticoid action.

Nuclear receptor coactivators are involved in receptor-mediated transcriptional activation of target genes in a hormone-sensitive manner, and the mechanism of their transactivation has been studied in recent years. The glucocorticoid receptor (GR) interacts with several coactivators, including steroid receptor coactivator-1 (SRC-1) family and CREB-binding protein (CBP). Since coactivators function as transcription amplifiers, subtle changes in expression levels of coactivators in certain cells would markedly intensify receptor-mediated transcriptional activity. The regulation of coactivators by glucocorticoid action, however, has not yet been clarified. In this study, we have shown that one of the coactivators interacting with GR, SRC-1, is downregulated by dexamethasone (DEX) both in vivo and in vitro. In experiments on Sprague-Dawley rats in vivo, the downregulation of SRC-1 was observed in heart, stomach, kidney, liver, and cerebrum, and in experiments on two types of kidney-derived cells in vitro, similar downregulation of SRC-1 was demonstrated in both types of cells. DEX-mediated downregulation of SRC-1 mRNA recovered in 4-8 h, while the downregulation of SRC-1 protein lasted for 12 h and its levels returned to the basal level, 24 h after DEX treatment. Other coactivators examined in this study showed no remarkable changes in expression by DEX treatment, implying that ligand-mediated downregulation of SRC-1 has a pivotal role in the physiology of glucocorticoid action.

Animals↗