Transducin ß-like 1, X-linked and nuclear receptor co-repressor cooperatively augment the ligand-independent stimulation of TRH and TSHß gene promoters by thyroid hormone receptors.

Mutations in TBL1X, a component of the nuclear receptor co-repressor (N-CoR) and silencing mediator of retinoic acid and thyroid hormone receptor co-repressor complexes, have recently been implicated in isolated central hypothyroidism (CeH). However, the mechanisms by which TBL1X mutations affect negative feedback regulation in the hypothalamus-pituitary-thyroid axis remain unclear. N-CoR was previously reported to paradoxically enhance the ligand-independent stimulation of TRH and TSHß gene promoters by thyroid hormone receptors (TR) in cell culture systems. We herein investigated whether TBL1X affects the unliganded TR-mediated stimulation of the promoter activities of genes negatively regulated by T3 in cooperation with N-CoR. In a hypothalamic neuronal cell line, the unliganded TR-mediated stimulation of the TRH gene promoter was significantly enhanced by co-transfected TBL1X, and the co-transfection of TBL1X with N-CoR further enhanced promoter activity. In contrast, the knockdown of endogenous Tbl1x using short interfering RNA significantly attenuated the N-CoR-mediated enhancement of promoter activity in the presence of unliganded TR. The co-transfection of N365Y or Y458C, TBL1X mutants identified in CeH patients, showed impaired co-activation with N-CoR for the ligand-independent stimulation of the TRH promoter by TR. In the absence of T3, similar or impaired enhancement of the TSHß gene promoter by the wild type or TBL1X mutants, respectively, was observed in the presence of co-transfected TR and N-CoR in CV-1 cells. These results suggest that TBL1X is needed for the full activation of TRH and TSHß gene promoters by unliganded TR. Mutations in TBL1X may cause CeH due to the impaired up-regulation of TRH and/or TSHß gene transcription despite low T3 levels.

Pituitary NR4A1 is negatively regulated by thyroid hormone without direct binding of thyroid hormone receptors on the gene.

We previously reported that TRH stimulated pituitary TSHß gene expression via an immediate increase in NR4A1 in thyrotrophs. We demonstrated that NR4A1 mRNA levels are regulated by thyroid hormone. Pituitary NR4A1 mRNA levels were decreased in mice injected with L-T4. NR4A1 promoter activity was increased by the overexpression of TRßs, and these increases were decreased by T3, and the -27∼+152 bp region was responsible for these changes in vitro. An EMSA showed the lack of TRßs-isoforms binding, and a ChIP assay demonstrated the recruitment of TRßs and NCoR in the -147∼+148 bp region in the absence of T3, whereas T3 induced their release. Experiments on the overexpression and knockdown of NCoR, and using the mutant TRs supported the involvement of NCoR in the TR-induced stimulation. These results demonstrate that thyroid hormone down-regulated basal NR4A1 mRNA levels in the pituitary, and the direct binding of TR was not required.

Coordinated regulation of transcription and alternative splicing by the thyroid hormone receptor and its associating coregulators.

Emerging evidence has indicated that the transcription and processing of precursor mRNA (pre-mRNA) are functionally coupled to modulate gene expression. In collaboration with coregulators, several steroid hormone receptors have previously been shown to directly affect alternative pre-mRNA splicing coupled to hormone-induced gene transcription; however, the roles of the thyroid hormone receptor (TR) and its coregulators in alternative splicing coordinated with transcription remain unknown. In the present study, we constructed a luciferase reporter and CD44 alternative splicing (AS) minigene driven by a minimal promoter carrying 2 copies of the palindromic thyroid hormone-response element. We then examined whether TR could modulate pre-mRNA processing coupled to triiodothyronine (T3)-induced gene transcription using luciferase reporter and splicing minigene assays in HeLa cells. In the presence of cotransfected TRß1, T3 increased luciferase activities along with the inclusion of the CD44 variable exons 4 and 5 in a dose- and time-dependent manner. In contrast, cotransfected TRß1 did not affect the exon-inclusion of the CD44 minigene driven by the cytomegalovirus promoter. T3-induced two-exon inclusion was significantly increased by the cotransfection of the TR-associated protein, 150-kDa, a subunit of the TRAP/Mediator complex that has recently been shown to function as a splicing factor. In contrast, T3-induced two-exon inclusion was significantly decreased by cotransfection of the polypyrimidine tract-binding protein-associated splicing factor, which was previously shown to function as a corepressor of TR. These results demonstrated that liganded TR in cooperation with its associating cofactors could modulate alternative pre-mRNA splicing coupled to gene transcription.

Formation of ethyl ferulate by rice koji enzyme in sake and mirin mash conditions.

Formation mechanism of ethyl ferulate (EF) in sake and mirin mash conditions was investigated to understand EF level control in the manufacturing process. Rice koji formed EF from ferulic acid (FA) and ethanol and decomposed EF to FA. This did not occur in sake yeast and chemical esterification was rare. Esterification of FA and hydrolysis of EF by rice koji might be due to feruloyl esterase(s). The rice koji enzyme showed normal Michaelis-Menten kinetics for FA in ethyl esterification and for EF in hydrolysis, but not for ethanol in the esterification reaction. Substrate specificity of the rice koji enzyme for hydroxycinnamic acids suggested that the main enzyme involved might be similar to type A feruloyl esterase. We studied the rice koji enzyme properties, short-term digestion of steamed rice grains with exogenous ethanol and small scale mirin making with pH adjustment. Our results suggested differences in the esterification and hydrolysis properties of the enzyme, in particular, different pH dependencies and different behaviors under high ethanol conditions; these factors might cause the differing EF levels in sake and mirin mashes.

THRAP3 interacts with HELZ2 and plays a novel role in adipocyte differentiation.

Using yeast two-hybrid screen, we previously isolated HELZ2 (helicase with zinc finger 2, transcriptional coactivator) that functions as a coregulator of peroxisome proliferator-activated receptorγ (PPARγ). To further delineate its molecular function, we here identified thyroid hormone receptor-associated protein3 (THRAP3), a putative component of the Mediator complex, as a protein stably associating with HELZ2 using immunoprecipitation coupled with mass spectrometry analyses. In immunoprecipitation assays, Thrap3 could associate with endogenous Helz2 as well as Pparg in differentiated 3T3-L1 cells. HELZ2 interacts with the serine/arginine-rich domain and Bcl2 associated transcription factor1-homologous region in THRAP3, whereas THRAP3 directly binds 2 helicase motifs in HELZ2. HELZ2 and THRAP3 synergistically augment transcriptional activation mediated by PPARγ, whereas knockdown of endogenous THRAP3 abolished the enhancement by HELZ2 in reporter assays. Thrap3, similar to Helz2, is evenly expressed in the process of adipogenic differentiation in 3T3-L1 cells. Knockdown of Thrap3 in 3T3-L1 preadipocytes using short-interfering RNA did not influence the expression of Krox20, Klf5, Cebpb, or Cebpd during early stages of adipocyte differentiation, but significantly attenuated the expression of Pparg, Cebpa, and Fabp4/aP2 and accumulation of lipid droplets. Pharmacologic activation of Pparg by troglitazone could not fully restore the differentiation of Thrap3-knockdown adipocytes. In chromatin immunoprecipitation assays, endogenous Helz2 and Thrap3 could be co-recruited, in a ligand-dependent manner, to the PPARγ-response elements in Fabp4/aP2 and Adipoq gene enhancers in differentiated 3T3-L1 cells. These findings collectively suggest that Thrap3 could play indispensable roles in terminal differentiation of adipocytes by enhancing PPARγ-mediated gene activation cooperatively with Helz2.

Isolation of a novel leptin receptor gene promoter preferentially functioning in neuronal cells.

Leptin exerts its metabolic effects by binding to the leptin receptor (Ob-R). In humans, the promoter of the Ob-R gene-related protein (Ob-RGRP) gene and the B219/Ob-R promoter have been speculated to spatially and temporarily regulate Ob-R gene transcription; however, the promoter function of the Ob-R gene has not been directly analyzed. Using 5' rapid amplification of the cDNA end, we isolated novel ob-r transcripts starting from the 3' portions of the B219/ob-r 5'-untranslated region in the adult mouse brain. The proximal promoter containing these start sites showed robust activity in neuron-derived, but not non-neuronal cell lines. The promoter activity was comparable with that of the ob-rgrp promoter in a neuronal cell, but significantly weaker in non-neuronal cells. Deletion analyses identifying two enhancer elements were located in this promoter. The identification of a novel ob-r gene promoter might provide a useful tool to study neuron specific expression and hormonal regulation of the ob-r gene.

Roles of proteasomal 19S regulatory particles in promoter loading of thyroid hormone receptor.

19S regulatory particles (19SRP) of 26S proteasome participate in multiple steps of gene transcription in yeast. We previously showed that Tat-binding protein-1 (TBP-1), an ATPase of 19SRP, interacts with thyroid hormone receptor (TR) and enhances TR-mediated transcription synergistically with steroid receptor coactivator-1 (SRC-1). To further elucidate the roles of ATPases and a non-ATPase component of 19SRP in gene regulation by TR, we investigated whether knockdown (KO) of TBP-1, TRIP1 or Rpn10 using small interfering RNA affects TR-mediated transactivation in HeLa cells. KO of individual subunits attenuated TR-mediated transactivation through the thyroid hormone response element (TRE) in the absence or presence of cotransfected SRC-1 without altering TR and SRC-1 protein levels. KO of TBP-1 disrupted ligand-induced loading of TR, SRC-1, and RNA polymerase II in chromatin immunoprecipitation assays. Collectively, both ATPase and non-ATPase components of 19SRP play critical roles in TR-mediated transactivation by coordinating the proper loading of liganded TR to TRE.

Tat-binding protein-1 (TBP-1), an ATPase of 19S regulatory particles of the 26S proteasome, enhances androgen receptor function in cooperation with TBP-1-interacting protein/Hop2.

The 26S proteasome, which degrades ubiquitinated proteins, appears to contribute to the cyclical loading of androgen receptor (AR) to androgen response elements of target gene promoters; however, the mechanism whereby the 26S proteasome modulates AR recruitment remains unknown. Using yeast two-hybrid screening, we previously identified Tat-binding protein-1 (TBP-1), an adenosine triphosphatase of 19S regulatory particles of the 26S proteasome, as a transcriptional coactivator of thyroid hormone receptor. Independently, TBP-1-interacting protein (TBPIP) was also identified as a coactivator of several nuclear receptors, including AR. Here, we investigated whether TBP-1 could interact with and modulate transcriptional activation by AR cooperatively with TBPIP. TBP-1 mRNA was ubiquitously expressed in human tissues, including the testis and prostate, as well as in LNCaP cells. TBP-1 directly bound TBPIP through the amino-terminal domain possessing the leucine zipper structure. AR is physically associated with TBP-1 and TBPIP in vitro and in LNCaP cells. TBP-1 similarly and additively augmented AR-mediated transcription upon coexpression with TBPIP, and the ATPase domain, as well as leucine zipper structure in TBP-1, was essential for transcriptional enhancement. Overexpression of TBP-1 did not alter AR protein and mRNA levels. In the chromatin immunoprecipitation assay, TBP-1 was transiently recruited to the proximal androgen response element of the prostate-specific antigen gene promoter in a ligand-dependent manner in LNCaP cells. These findings suggest that a component of 19S regulatory particles directly binds AR and might participate in AR-mediated transcriptional activation in cooperation with TBPIP.

Lung cancer producing granulocyte colony-stimulating factor and rapid spreading to peritoneal cavity.

A-73-year-old man, a heavy smoker, was admitted to our hospital for pain in the right lower quadrant of the abdomen. The patient had no complaint of respiratory symptoms. But he had noted a skin lesion on his right hip. Physical examination revealed mild tenderness in the right lower quadrant of the abdomen and a soft swelling on the right hip. Laboratory analysis revealed remarkable leukocytosis (38,600 mul: 86% neutrophils) and evaluated C-reactive protein (8.6 mg/dl). Chest radiograph revealed a mass shadow in the right upper field of the lung. A computed tomography of the chest on admission revealed a heterogeneous mass with mediastinal lymphadenopathy. Abdominal computed tomography revealed multiple metastases in the adrenal glands, gallbladder, intestine, and peritoneum. Fluorodeoxyglucose positron emission tomography demonstrated increased uptake in the peritoneal cavity and primary site of lung. There was no evidence of systemic infection. The histologic finding of poorly differentiated carcinoma was confirmed by a needle biopsy of the skin lesion on his right hip. His white blood cell count was elevated, 38,600 to 121,000 mul within 10 days. Suspecting a granulocyte colony-stimulating factor (G-CSF)-producing tumor, we measured serum G-CSF and subsequently found it to be elevated to 372 pg/ml. His condition was rapidly deteriorated, and he died of multiple organ failure 14 days after admission. The poorly differentiated carcinoma found at autopsy revealed positive immunoreactivity for G-CSF. There was evidence of multiple metastases in the adrenal glands, gallbladder, intestine, pancreas, liver, skin, and peritoneum. The final diagnosis obtained at autopsy demonstrated diffuse metastases spreading to peritoneal cavity resulting from G-CSF-producing lung cancer.

Acute pancreatitis in a patient treated with micafungin.

CASE SUMMARY: A 73-year-old man (height, 158.2 cm; weight, 49.8 kg) presented with upper abdominal tenderness after 3 weeks of treatment with 150 mg/d of micafungin (3 mg/kg . d) (Mycamine, Astellas Pharma US Inc., Deerfield, Illinois) intravenously for pulmonary aspergillosis accompanied by [DOSAGE ERROR CORRECTED] pulmonary Mycobacterium avium complex (MAC) infection. Pulmonary aspergillosis was noninvasively diagnosed by a fungus lump in a cavity in the right upper lung field with a high value of 1,3-beta-D-glucan and a positive result for aspergillosis antigen. The patient had a medical history of gastrectomy due to gastric cancer and idiopathic thrombocytopenic purpura (ITP). He had been prescribed 800 mg/d of clarithromycin, 400 mg/dL of rifampicin, and 750 mg/d of ethambutol hydrochloride for pulmonary MAC infection for 2 years and 5 mg/d of prednisolone for ITP for 7 years. No traditional or homeopathic medicine had been received/administered. Laboratory tests at the onset of abdominal pain revealed a white blood cell count of 4300/microL with 51% neutrophils. There was no eosinophilia. Platelet count was 15,100/muL, with normal coagulation. Immunoglobulin G and immunoglobulin M were 1720 and 154 mg/dL, respectively. The patient had no history of allergy, biliary tract disease, hyperlipidemia, or hypercalcemia. He did not report alcohol use. The laboratory findings, magnetic resonance imaging, and upper abdominal tenderness were consistent with acute pancreatitis. After cessation of all drugs, his symptoms improved with bowel rest and parenteral nutrition. His laboratory measurements normalized thereafter. All drugs, except micafungin, were readministered for pulmonary MAC infection and ITP, and itraconazole was administered for pulmonary aspergillosis after the recovery from pancreatitis. During 16 months of follow-up, the pancreatitis did not recur. DISCUSSION: We performed a literature search of all available English-language articles published on MEDLINE between January 1966 and January 2007 using the key terms micafungin (text and indexed terms) and pancreatitis (text and indexed terms). Based on the search of MEDLINE, there have been no reports of acute pancreatitis associated with micafungin. The Naranjo adverse drug reaction (ADR) probability scale was used to assess the probability of micafungin-associated acute pancreatitis. A score of 6 was obtained, indicating a probable ADR from micafungin treatment. CONCLUSION: We report a case of acute pancreatitis probably associated with micafungin use in an elderly patient.

Loss of consciousness and hypokalemia in an elderly man with a mutation of the thiazide-sensitive Na-Cl cotransporter gene.

An 80-year-old man was referred to our department for evaluation of repetitive loss of consciousness and faintness with hypokalemia. He had relatively low blood pressure, hypomagnesemia, hypocalciuria and chondrocalcinosis in the knee, clinically suggesting Gitelman's syndrome. A renal clearance study could not be carried out due to the patient's age and complications of the heart. Sequence analysis of the gene of thiazide-sensitive Na-Cl cotransporter (TSC) showed a heterozygous missense mutation from C to T at 1712 base pairs from the translation start site, with resultant changes in codon 569 from alanine to valine (A569V). Treatment with oral administration of potassium chloride improved all the symptoms. Although Gitelman's syndrome has been considered to be autosomal recessive, cases of only heterozygous mutation detected have recently been reported. Therefore, the mutation found in this patient may be responsible for Gitelman's syndrome.

Isolation and characterization of a transcriptional cofactor and its novel isoform that bind the deoxyribonucleic acid-binding domain of peroxisome proliferator-activated receptor-gamma.

Using the DNA-binding domain (DBD) and hinge region of human peroxisome proliferator-activated receptor (PPAR)-gamma as bait in yeast two-hybrid screen, we isolated partial cDNA identical with that of the C terminal of KIAA1769. KIAA1769 encodes a 2080-amino acid protein (molecular mass, 231 kDa) that was recently identified to interact with PPARalpha and termed PPARalpha-interacting cofactor 285 (here referred to as PPARgamma-DBD-interacting protein 1 (PDIP1)-alpha). PDIP1 mRNA was expressed in 3T3-L1 adipocytes and THP-1 macrophages. We also identified the expression of the N terminal extended form of PDIP1alpha (referred to as PDIP1beta) consisting of 2649 amino acids (295 kDa) in human cultured cell lines by RT-PCR, and 5' rapid amplification of cDNA ends. Ribonuclease protection assay revealed that PDIP1beta mRNA was expressed more abundantly than PDIP1alpha mRNA. The C-terminal region of PDIP1 directly binds DBD of PPARgamma, and multiple LXXLL motifs in PDIP1 were not required for the interaction. PDIP1alpha and -beta similarly enhanced PPARgamma-mediated transactivation in transfection assays and short interfering RNA targeting PDIP1 mRNA significantly reduced transactivation by PPARgamma. No potent intrinsic activation domain was identified in either PDIP1 isoforms in mammalian one-hybrid assays, and mutation of all LXXLL motifs did not affect enhancement of PPARgamma-mediated transactivation. PDIP1alpha and -beta similarly augmented transactivation by PPARalpha, PPARdelta, thyroid hormone receptor (TR)-alpha1, TRbeta1, and retinoid X receptor-alpha. PDIP1alpha also enhanced estrogen receptoralpha- and androgen receptor-mediated transactivation, whereas PDIP1beta did not. PDIP1alpha showed receptor-specific synergism with activation function-2-interacting coactivators in PPARgamma- and TRbeta1-mediated transactivation. Together, PDIP1 might function as a transcriptional cofactor for a broad range of nuclear receptors, possibly in collaboration with specific activation function-2 interacting coactivators.

The histone-binding code of nuclear receptor co-repressors matches the substrate specificity of histone deacetylase 3.

Ligands for nuclear receptors facilitate the exchange of co-repressors for coactivators, leading to chromatin modifications that favour the activation of gene transcription. Here, we show that the repressed state of an endogenous retinoic acid-regulated gene is quickly re-established after ligand removal. As expected, repression is characterized by recruitment of N-CoR/SMRT-HDAC3 (histone deacetylase 3) co-repressor complexes, leading to local histone hypoacetylation. The achievement of the repressed state involves the ordered deacetylation of lysines in H4 tails. This order is determined by the inherent substrate specificity of HDAC3, and unexpectedly predicts the binding preference of N-CoR/SMRT for submaximally acetylated H4 tails. The match between the specificity of acetyl-histone deacetylation by HDAC3 and the histone-binding preference of N-CoR/SMRT allows the co-repressor complex to stabilize and propagate repression of nuclear hormone receptor gene targets.

Corepressors selectively control the transcriptional activity of PPARgamma in adipocytes.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is the master regulator of adipogenesis as well as the target of thiazolidinedione (TZD) antidiabetic drugs. Many PPARgamma target genes are induced during adipogenesis, but others, such as glycerol kinase (GyK), are expressed at low levels in adipocytes and dramatically up-regulated by TZDs. Here, we have explored the mechanism whereby an exogenous PPARgamma ligand is selectively required for adipocyte gene expression. The GyK gene contains a functional PPARgamma-response element to which endogenous PPARgamma is recruited in adipocytes. However, unlike the classic PPARgamma-target gene aP2, which is constitutively associated with coactivators, the GyK gene is targeted by nuclear receptor corepressors in adipocytes. TZDs trigger the dismissal of corepressor histone deacetylase (HDAC) complexes and the recruitment of coactivators to the GyK gene. TZDs also induce PPARgamma-Coactivator 1alpha (PGC-1alpha), whose recruitment to the GyK gene is sufficient to release the corepressors. Thus, selective modulation of adipocyte PPARgamma target genes by TZDs involves the dissociation of corepressors by direct and indirect mechanisms.

The nuclear receptor corepressor deacetylase activating domain is essential for repression by thyroid hormone receptor.

Nuclear receptor corepressor (N-CoR) mediates repression by thyroid hormone receptor (TR) as well as other nuclear hormone receptors and transcription factors. N-CoR contains several repression domains that repress transcription when fused to a heterologous DNA binding domain, but their relative importance in the full-length N-CoR molecule is unknown. Here we addressed this important issue by depleting N-CoR in human cells and replacing it with mutant and wild-type murine N-CoR. Although the N-terminal RD binds transducin beta-like protein 1 (TBL1), TBLR1, and mSin3, deletion of this region did not affect the ability of N-CoR to mediate repression by TR. By contrast, deletion of the deacetylase activating domain (DAD) that binds and activates histone deacetylase 3 dramatically hampered N-CoR's function as a TR corepressor. Introduction of a single amino acid mutation in the DAD similarly disabled the corepressor function of N-CoR. Thus, the DAD domain of N-CoR is singularly essential for repression by TR.

The N-CoR/histone deacetylase 3 complex is required for repression by thyroid hormone receptor.

Nuclear receptor corepressors (N-CoR) and silencing mediator for retinoid and thyroid receptors (SMRT) have both been implicated in thyroid hormone receptor (TR)-mediated repression. Here we show that endogenous N-CoR, TBL1, and histone deacetylase 3 (HDAC3), but not HDAC1, -2, or -4, are recruited to a stably integrated reporter gene repressed by unliganded TR as well as the orphan receptor RevErb. Unliganded TR also recruits this complex to a transiently transfected reporter, and transcriptional repression is associated with local histone deacetylation that is reversed by the presence of thyroid hormone. Knockdown of N-CoR using small interfering RNAs markedly reduces repression by the TR ligand binding domain in human 293T cells, whereas knockdown of SMRT has little effect. RevErb repression appears to involve both corepressors in this system. Knockdown of HDAC3 markedly reduces repression by both TR and RevErb, while knockdown of HDAC1 or 2 has more modest, partly nonspecific effects. Thus, HDAC3 is critical for repression by multiple nuclear receptors and the N-CoR HDAC3 complex plays a unique and necessary role in TR-mediated gene repression in human 293T cells.

A SANT motif in the SMRT corepressor interprets the histone code and promotes histone deacetylation.

Nuclear receptor corepressors SMRT (silencing mediator of retinoid and thyroid receptors) and N-CoR (nuclear receptor corepressor) recruit histone deacetylase (HDAC) activity to targeted regions of chromatin. These corepressors contain a closely spaced pair of SANT motifs whose sequence and organization is highly conserved. The N-terminal SANT is a critical component of a deacetylase activation domain (DAD) that binds and activates HDAC3. Here, we show that the second SANT motif functions as part of a histone interaction domain (HID). The HID enhances repression by increasing the affinity of the DAD-HDAC3 enzyme for histone substrate. The two SANT motifs synergistically promote histone deacetylation and repression through unique functions. The HID contribution to repression is magnified by its ability to inhibit histone acetyltransferase enzyme activity. Remarkably, the SANT-containing HID preferentially binds to unacetylated histone tails. This implies that the SMRT HID participates in interpreting the histone code in a feed-forward mechanism that promotes and maintains histone deacetylation at genomic sites of SMRT recruitment.