Isao Kurihara

Effect of KCNJ5 Mutations on Gene Expression in Aldosterone-Producing Adenomas and Adrenocortical Cells

CONTEXT Primary aldosteronism is a heterogeneous disease that includes both sporadic and familial forms. A point mutation in the KCNJ5 gene is responsible for familial hyperaldosteronism type III. Somatic mutations in KCNJ5 also occur in sporadic aldosterone producing adenomas (APA). OBJECTIVE The objective of the study was to define the effect of the KCNJ5 mutations on gene expression and aldosterone production using APA tissue and human adrenocortical cells. METHODS A microarray analysis was used to compare the transcriptome profiles of female-derived APA samples with and without KCNJ5 mutations and HAC15 adrenal cells overexpressing either mutated or wild-type KCNJ5. Real-time PCR validated a set of differentially expressed genes. Immunohistochemical staining localized the KCNJ5 expression in normal adrenals and APA. RESULTS We report a 38% (18 of 47) prevalence of KCNJ5 mutations in APA. KCNJ5 immunostaining was highest in the zona glomerulosa of NA and heterogeneous in APA tissue, and KCNJ5 mRNA was 4-fold higher in APA compared with normal adrenals (P < 0.05). APA with and without KCNJ5 mutations displayed slightly different gene expression patterns, notably the aldosterone synthase gene (CYP11B2) was more highly expressed in APA with KCNJ5 mutations. Overexpression of KCNJ5 mutations in HAC15 increased aldosterone production and altered expression of 36 genes by greater than 2.5-fold (P < 0.05). Real-time PCR confirmed increases in CYP11B2 and its transcriptional regulator, NR4A2. CONCLUSIONS KCNJ5 mutations are prevalent in APA, and our data suggest that these mutations increase expression of CYP11B2 and NR4A2, thus increasing aldosterone production.

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.

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.

FHL2, UBC9, and PIAS1 are Novel Estrogen Receptor α‐Interacting Proteins

Estrogen plays important roles in the pathophysiology of atherosclerosis and cardiovascular diseases mediated by estrogen receptor α (ERα). To elucidate the molecular mechanisms, we screened ERα‐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 ERα in the presence of 17β‐estradiol, but not of tamoxifen or raloxifene in yeast. FHL2 mainly interacted with N‐terminal A/B domain of ERα but not C‐terminal ligand‐binding domain. However, overexpression of full‐length FHL2 did not affect ERα‐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 ERα with Ubc9 and PIAS1 in yeast. Ubc9 and PIAS1, small ubiquitin‐related modifier‐1 (SUMO‐1) conjugating enzyme and ligase, respectively, markedly interacted with ERα in a 17β‐estradiol‐dependent manner. These proteins mainly interacted with the DNA‐binding and ligand‐binding domains of ERα. Overexpression of Ubc9 or PIAS1 potentiated ERα‐mediated transcriptional activities in COS‐1 cells in a dose‐dependent manner, indicating that both Ubc9 and PIAS1 function as coactivators of ERα. In addition, the SUMOylation‐defective mutant, Ubc9 (C93S) continued to enhance ERα‐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 ERα exhibit tissue‐specific functions utilizing multiple tissue‐restricted receptor‐interacting proteins.

Quenching the Thirst in Dialysis Patients

In a double-blind cross-over trial, 22 stable end-stage renal failure patients on maintenance haemodialysis were subjected to conventional dialysis with dialysate containing 137 mEq/l sodium and constant ultrafiltration (UF) and to a different dialysis therapy, in which, by linear sodium modelling, the dialysate sodium was reduced from 137 to 128 mEq/l. A computerized UF program was used to gradually reduce the UF to a minimum towards the end of the session. Severity of thirst, interdialytic weight gain and intradialytic complications were less with low sodium dialysate. It allowed adequate UF with absolute hemodynamic stability. The reduced incidence of complication with low sodium dialysate therapy was probably because they required less UF.

Angiography with Nonionic X-Ray Contrast Media in Severe Chronic Renal Failure: Renal Function and Contrast Retention

The effects of contrast media on renal function and the cortical retention of contrast media after abdominal angiography were investigated. Sixteen nondiabetic patients with predialytic chronic renal failure received either the nonionic dimeric contrast medium iodixanol or the monomeric contrast medium iohexol in a double-blind randomized study. All patients were well hydrated before, during and after angiography. Mean 99mTc-DTPA clearance was 14.0 ml/min/1.73 m2 in the iodixanol group, and 9.3 ml/min/1.73 m2 in the iohexol group at baseline. No statistically significant changes were seen after angiography. Serum creatinine increased significantly 48 and 72 h after angiography in both groups, and then normalized. Creatinine clearance was reduced only in the iohexol group, at 72-96 h. The urinary excretion of renal enzymes and of total protein did not change significantly. No patients developed oliguria or required dialysis during the 7-day observation period. Increased attenuation in the renal cortex, measured by computed tomography and probably reflecting intracellular retention of contrast medium, peaked at 24 h, and was observed in both groups during the follow-up period. Thus, although transient and minor changes in glomerular filtration rate were noted, both iodixanol and iohexol were safe for use in angiography in nondiabetic patients with severe chronic failure when the patients were well hydrated.

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.

Clinicopathological study of SDHB mutation-related pheochromocytoma and sympathetic paraganglioma

Pheochromocytoma (PCC) and paraganglioma (PGL) are genetically and phenotypically heterogeneous catecholamine-producing neoplasms. They can occur sporadically or as a part of hereditary disease. Approximately 30% of PCC/PGL are believed to be caused by germline mutations (Welander et al. 2011). Of these, succinate dehydrogenase subunit B (SDHB) gene mutation is considered a high-risk factor for malignancy. Loss of heterozygosity at the SDHB locus (1p36)wasobserved inall tumorswithSDHBmutation, and Gimenez-Roqueplo et al. (2003) strongly suggested that SDHB is a tumor suppressorgene. Subsequently, loss of SDHB protein immunoreactivity in SDHB-mutated PCC/PGL (SDHB–PCC/PGL) was reported with 100% sensitivity and 84% specificity (van Nederveen et al. 2009). Thus, SDHB immunohistochemistry can be used to screen SDHB– PCC/PGL using paraffin-embedded pathological materials. SDHB mutation is the only established factor that indicates future metastasis. Therefore, it is important to analyze the histological characteristics of SDHB–PCC/PGL. It is generally accepted that it is difficult to distinguish histological differences between benign and malignant PCC/PGL. The current consensus is that a long-term follow-up is required after the surgery to screen for recurrence or metastasis in all PCC/PGL patients, regardless whether hereditary or sporadic in origin. Kimura et al. (2014) proposed a histological grading system called the Grading of Adrenal PCC and PGL (GAPP) classification for predicting metastasis. GAPP is composed of six factors: histological pattern, cellularity, presence or absence of comedo-type necrosis, vascular or capsular invasion, Ki67labeling index (%), and elevated catecholamine type. Each factor was assigned a point and the number of points was summated. Tumor scores of 0–2, 3–6, and 7–10 were classified into well differentiated (WD), moderately

NF-YC Functions as a Corepressor of Agonist-bound Mineralocorticoid Receptor

The role of aldosterone has been implicated in the metabolic syndrome and cardiovascular diseases. The biological actions of aldosterone are mediated through mineralocorticoid receptor (MR). Nuclear receptor-mediated gene expression is regulated by dynamic and coordinated recruitment of coactivators and corepressors. To identify new coregulators of the MR, full-length MR was used as bait in yeast two-hybrid screening. We isolated NF-YC, one of the subunits of heterotrimeric transcription factor NF-Y. Specific interaction between MR and NF-YC was confirmed by yeast two-hybrid, mammalian two-hybrid, coimmunoprecipitation assays, and fluorescence subcellular imaging. Transient transfection experiments in COS-7 cells demonstrated that NF-YC repressed MR transactivation in a hormone-sensitive manner. Moreover, reduction of NF-YC protein levels by small interfering RNA potentiated hormonal activation of endogenous target genes in stably MR-expressing cells, indicating that NF-YC functions as an agonist-dependent MR corepressor. The corepressor function of NF-YC is selective for MR, because overexpression of NF-YC did not affect transcriptional activity mediated by androgen, progesterone, or glucocorticoid receptors. Chromatin immunoprecipitation experiments showed that endogenous MR and steroid receptor coactivator-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner, and endogenous NF-YC was sequentially recruited to the same element. Immunohistochemistry showed that endogenous MR and NF-YC were colocalized within the mouse kidney. Although aldosterone induces interaction of the N and C termini of MR, NF-YC inhibited the N/C interaction. These findings indicate that NF-YC functions as a new corepressor of agonist-bound MR via alteration of aldosterone-induced MR conformation.

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.