Yasuyo Nakajima

Expression and Mutations of KCNJ5 mRNA in Japanese Patients with Aldosterone-Producing Adenomas

CONTEXT Mutations of the KCNJ5 gene have recently been identified in patients with aldosterone-producing adenomas (APA). OBJECTIVE Our objective was to investigate the expression and mutations of the KCNJ5 gene in Japanese patients with APA. DESIGN AND PATIENTS We sequenced KCNJ5 cDNA and measured KCNJ5 mRNA levels in 23 patients with APA operated on at Gunma University Hospital. MAIN OUTCOME MEASURES Mutations and mRNA levels of the KCNJ5 gene were examined and compared to those in cortisol-producing adenomas (Cushings syndrome) and pheochromocytomas. RESULTS Of the 23 patients with APA, 15 (65.2%) had two somatic mutations of the KCNJ5 gene: 12 cases of p.G151R (eight with c.451G>A, and four with c.451G>C) and three cases of p.L168R (c.503T>G). Levels of KCNJ5 mRNA were significantly higher in the APA with mutations than those without. Immunohistochemistry also showed a stronger staining of KCNJ5 on the cell membrane in the tumor with a mutation. Furthermore, a PCR-restriction fragment length polymorphism assay with c.503T>G revealed the mutant mRNA to be expressed at a similar level to the wild type. The level of KCNJ5 mRNA in cortisol-producing adenomas was approximately 30% of that in APA, and almost no expression was observed in pheochromocytomas. CONCLUSION We found that: 1) a significant number of patients with APA had somatic mutations of the KCNJ5 gene; 2) KCNJ5 mRNA levels were higher in the APA with KCNJ5 mutations; and 3) the expression of KCNJ5 mRNA was significantly higher in APA than cortisol-producing adenomas and pheochromocytomas.

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.

Subclinical Hypothyroidism and Indices for Metabolic Syndrome in Japanese Women: One-Year Follow-Up Study

CONTEXT Subclinical hypothyroidism (SCH) and metabolic syndrome (MetS) increase with age; however, their relationship remains unclear. OBJECTIVE Our objective was to investigate the relationship between SCH and indices of metabolic syndrome and follow up subjects for 1 year. DESIGN Cross-sectional and longitudinal follow-up studies of cases were collected from Takasaki Hidaka Hospital between 2003 and 2007. PARTICIPANTS Overall, 11 498 participants of health checkups were analyzed. The mean age was 48 ± 9 years. MAIN OUTCOME MEASURES The relationship between SCH and indices of MetS were examined. RESULTS Serum free T4 levels were lower in women than men in most of the age groups, and the prevalence of SCH, 6.3% in women vs 3.4% in men, increased with age, reaching 14.6% in 70-year-old women. Multivariate logistic-regression analyses revealed that waist circumference and the serum triglyceride and low-density lipoprotein-cholesterol levels were significantly higher in subjects with SCH than without among women. Reflecting these findings, the adjusted odds ratio of MetS in patients with SCH was higher than in the euthyroid subjects in women with an odds ratio of 2.7 (95% confidence interval 1.1-5.6; P = .017) but not in men. Furthermore, progression from euthyroid into SCH resulted in a significant increase in the serum triglyceride levels but not low-density lipoprotein-cholesterol in women. CONCLUSION Japanese women exhibited a high prevalence of SCH associated with low free T4 levels. There was a strong association between SCH and several indices of metabolic syndrome in women. SCH may affect serum triglyceride levels and be a risk factor for metabolic syndrome.

Cardiovascular Complications of Patients with Aldosteronism Associated with Autonomous Cortisol Secretion

CONTEXT Primary aldosteronism (PA) is sometimes associated with the autonomous secretion of cortisol. OBJECTIVE Our objective was to investigate the effect of autonomous cortisol secretion on the prevalence of cardiovascular events (CVE) in patients with PA. DESIGN This was a retrospective cross-sectional study of cases collected from Gunma University Hospital between 2002 and 2010. PATIENTS Seventy-six consecutive patients hospitalized for an evaluation of PA were analyzed. MAIN OUTCOME MEASURES Rates of CVE dependent on autonomous cortisol secretion were examined. RESULTS Of the 76 patients with PA, 21 (28%) had a history of CVE, including 14 with stroke, one with myocardial infarction, and six with atrial fibrillation. The multivariate logistic-regression and receiver operating characteristic analyses revealed that PA patients with CVE had significantly higher midnight cortisol levels than those without CVE; the adjusted odds ratio with a cutoff value of 7.4 μg/dl was 7.0 (95% confidence interval, 1.8-30.6; P = 0.006). In addition, results of the 1-mg dexamethasone suppression test with a cutoff value of 3.0 μg/dl differed significantly (odds ratio, 5.0; 95% confidence interval, 1.4-20.7; P = 0.018). Conversely, 67 and 50% of the PA patients with a midnight cortisol level of at least 7.4 μg/dl and 1-mg dexamethasone suppression test of at least 3.0 μg/dl had a history of CVE. Other factors such as age, expected glomerular filtration rate, blood pressure, glucose intolerance, the serum aldosterone concentration, plasma renin activity, and the duration of hypertension had no effect. CONCLUSION The patients with PA associated with autonomous cortisol secretion had high incidence of CVE, and this association may further increase the risk of CVE in patients with PA.

NR4A1 (Nur77) Mediates Thyrotropin-Releasing Hormone-Induced Stimulation of Transcription of the Thyrotropin β Gene: Analysis of TRH Knockout Mice

Thyrotropin-releasing hormone (TRH) is a major stimulator of thyrotropin-stimulating hormone (TSH) synthesis in the anterior pituitary, though precisely how TRH stimulates the TSHβ gene remains unclear. Analysis of TRH-deficient mice differing in thyroid hormone status demonstrated that TRH was critical for the basal activity and responsiveness to thyroid hormone of the TSHβ gene. cDNA microarray and K-means cluster analyses with pituitaries from wild-type mice, TRH-deficient mice and TRH-deficient mice with thyroid hormone replacement revealed that the largest and most consistent decrease in expression in the absence of TRH and on supplementation with thyroid hormone was shown by the TSHβ gene, and the NR4A1 gene belonged to the same cluster as and showed a similar expression profile to the TSHβ gene. Immunohistochemical analysis demonstrated that NR4A1 was expressed not only in ACTH- and FSH- producing cells but also in thyrotrophs and the expression was remarkably reduced in TRH-deficient pituitary. Furthermore, experiments in vitro demonstrated that incubation with TRH in GH4C1 cells increased the endogenous NR4A1 mRNA level by approximately 50-fold within one hour, and this stimulation was inhibited by inhibitors for PKC and ERK1/2. Western blot analysis confirmed that TRH increased NR4A1 expression within 2 h. A series of deletions of the promoter demonstrated that the region between bp -138 and +37 of the TSHβ gene was responsible for the TRH-induced stimulation, and Chip analysis revealed that NR4A1 was recruited to this region. Conversely, knockdown of NR4A1 by siRNA led to a significant reduction in TRH-induced TSHβ promoter activity. Furthermore, TRH stimulated NR4A1 promoter activity through the TRH receptor. These findings demonstrated that 1) TRH is a highly specific regulator of the TSHβ gene, and 2) TRH mediated induction of the TSHβ gene, at least in part by sequential stimulation of the NR4A1-TSHβ genes through a PKC and ERK1/2 pathway.

Resistance to Thyroid Hormone Due to a Novel Thyroid Hormone Receptor Mutant in a Patient with Hypothyroidism Secondary to Lingual Thyroid and Functional Characterization of the Mutant Receptor

BACKGROUND We describe a rare case of congenital hypothyroidism and an extremely high serum thyrotropin (TSH) level caused by a combination of resistance to thyroid hormone (RTH) and a lingual thyroid. As the RTH mutant, R316C, was new, the optimum dose of levothyroxine was unclear. To aid in assessment of the therapy, we characterized the mutant R316C thyroid hormone receptor (TR) and compared it with a common mutant, R316H, using in vitro studies. SUMMARY The patient was a newborn female having severe hypothyroidism with a free thyroxine level of 0.36 ng/dL and a serum TSH level of 177 microU/mL. A scintiscan showed ectopic lingual thyroid tissue without a normal thyroid gland. Supplementation with levothyroxine at a dose of >350 microg/day did not normalize the serum TSH level; however, the patient showed normal growth and intelligence at 14 years of age. Consistent with the results of a computer analysis, the binding of R316C to triiodothyronine (T3) was significantly decreased to 38% that of the wild type. Electrophoretic mobility shift assay demonstrated that like R316H, R316C did not form a homodimer, but formed a heterodimer with RXR. However, a glutathione-S-transferase pull-down assay showed reduced binding of R316C with NCoR in the absence of T3 and impaired release in the presence of T3. In addition, transient transfection experiments demonstrated that unlike R316H, R316C had severe impairment of transcriptional activity on genes both positively and negatively regulated by thyroid hormone. It also had a clear dominant negative effect on genes negatively, but not positively, regulated by thyroid hormone, including the TSH-releasing hormone and TSHbeta genes. CONCLUSION This is the first reported case of a R316C TR mutation. The characteristics of the R316C mutant differed from those of the R316H mutant. Our findings suggest that R316C causes reduced association with and impaired release of NCoR, resulting in RTH predominantly at the pituitary level, and that slightly elevated serum TSH level with high dose of levothyroxine might be optimum for normal growth.

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.

GNAS mutations in adrenal aldosterone-producing adenomas [Rapid Communication]

Mutations in GNAS, which encodes Gsα, have been documented in detail, particularly in human pituitary GH-secreting adenomas. Mutations have also recently been reported in adrenal cortisol-producing adenomas (CPAs), in addition to those in the PRKACA gene. However, mutations have not yet been examined in aldosterone-producing adenomas (APAs). Therefore, we herein investigated mutations in the GNAS gene in APAs. Two of the 15 (13%) CPAs with overt Cushings syndrome and one of the 9 (11%) CPAs with subclinical Cushings syndrome examined had the somatic mutations, p.R201S and p.R201C in the GNAS gene. We identified mutations in the GNAS gene (p.R201C) in 2 out of the 33 (6%) APAs tested, both of which showed autonomous cortisol secretion, while 24 APAs had mutations in the KCNJ5 gene (18 with p.G151R and 6 with p.L168R). These GNAS and KCNJ5 mutations were mutually exclusive in these adenomas. We herein demonstrated for the first time the presence of GNAS mutations in APAs, as well as in some cortisol-secreting adenomas. Our results suggest that these mutations, in addition to mutations in the KCNJ5 gene and other genes such as ATP1A1, ATP2B3 and CACNA1D, may be responsible for the tumorigenesis of APAs and CPAs with subclinical Cushings syndrome.

Changes in visceral and subcutaneous fat mass in patients with pheochromocytoma

CONTEXT Overproduction of catecholamine induces not only hypertension but also glucose intolerance and hyperlipidemia. However, little is known about its effect on visceral and subcutaneous fat. OBJECTIVE Our objective was to investigate changes of metabolic factors including visceral and subcutaneous fat areas in patients with pheochromocytoma (Pheo). DESIGN AND PATIENTS This was a cross-sectional and longitudinal follow-up study of cases collected from Gunma University Hospital between 2002 and 2013. Forty-two patients with Pheo and 23 with non-functioning adrenal adenoma (NFA) were analyzed before and after adrenalectomy. RESULTS Multivariate logistic-regression analysis adjusted by age and gender revealed that glucose intolerance was more common in patients with Pheo than in patients with NFA (21/42, 51% vs. 4/23, 17%, p<0.05). Abdominal visceral fat area (VFA) and subcutaneous fat area (SFA) were significantly lower in patients with Pheo than in those with NFA (80.2±38.7 vs. 124.3±61.8cm(2), p<0.05; 114.6±58.9 vs. 164.3±40.3cm(2), p<0.05, respectively). Significant correlations were observed between fractionated urine noradrenaline level and serum HDL-cholesterol level (r = 0.36, p<0.05), urine normetanephrine level and tumor size (r=0.57, p<0.01), and urine adrenaline level and systolic blood pressure (r=0.35, p<0.05) in Pheo. However, there were no significant correlations between adrenaline and noradrenaline levels and other parameters, including serum LDL-cholesterol and triglyceride levels, and HbA1c. Furthermore, both VFA and SFA, body weight, and BMI were significantly increased, and serum HbA1c as well as HDL-cholesterol levels were decreased after adrenalectomy in Pheo. CONCLUSION These findings suggest for the first time that catecholamines might regulate the serum HDL-cholesterol level and both abdominal visceral and subcutaneous fat mass in men.

Fyn phosphorylates AMPK to inhibit AMPK activity and AMP- dependent activation of autophagy

We previously demonstrated that proto-oncogene Fyn decreased energy expenditure and increased metabolic phenotypes. Also Fyn decreased autophagy-mediated muscle mass by directly inhibiting LKB1 and stimulating STAT3 activities, respectively. AMPK, a downstream target of LKB1, was recently identified as a key molecule controlling autophagy. Here we identified that Fyn phosphorylates the α subunit of AMPK on Y436 and inhibits AMPK enzymatic activity without altering the assembly state of the AMPK heterotrimeric complex. As pro-inflammatory mediators are reported modulators of the autophagy processes, treatment with the pro-inflammatory cytokine TNFα resulted in 1) increased Fyn activity 2) stimulated Fyn-dependent AMPKα tyrosine phosphorylation and 3) decreased AICAR-dependent AMPK activation. Importantly, TNFα induced inhibition of autophagy was not observed when AMPKα was mutated on Y436. 4) These data demonstrate that Fyn plays an important role in relaying the effects of TNFα on autophagy and apoptosis via phosphorylation and inhibition of AMPK.