[show abstract][hide abstract] ABSTRACT: The majority of the cases diagnosed as primary aldosteronism (PA) are caused by aldosterone-producing adenoma (APA) or idiopathic hyperaldosteronism (IHA). Histopathologically, both IHA and adjacent adrenal glands of APA demonstrate remodeled subcapsular zone (RSZ) but these zones in two disorders are markedly different in terms of steroidogenesis. 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) expression has been known to be activated synergistically by GATA6 and SF-1, and repressed by DAX1 through abolishing the activation. Nerve growth factor-induced clone B (NGFIB) is also known as one of the transcription factors to bind to and activate 3β-HSD promoter. The results of our immunohistochemical analysis demonstrated the expression levels of 3β-HSD in RSZ of IHA were higher than in RSZ of adjacent adrenals of APA, while those in the zona glomerulosa (ZG) of normal adrenal gland (NA) were in between these two RSZs. The expression levels of GATA6, SF-1 and DAX1 did not prominently differ among these three types of adrenals, especially between in RSZs of IHA and APA cases, indicating the marked difference of 3β-HSD expression was unlikely to be explained by the levels of these three factors. However, the levels of NGFIB expression were significantly higher in RSZ of IHA than in RSZ of adjacent adrenals of APA and the ZG of NA (P<0.05), which may partly account for the expression levels of 3β-HSD among the three groups of adrenals. These results may imply NGFIB plays important roles in the marked differences in steroidogenic functions in the two distinct types of RSZ of PA cases.
[show abstract][hide abstract] ABSTRACT: Although neuroendocrine tumors (NETs) are rare, the number of patients with NET is increasing. However, in Japan, there have been no epidemiological studies on NET since 2005; thus, the prevalence of NET remains unknown.
We reported the epidemiology of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) [pancreatic neuroendocrine tumors (PNETs) and gastrointestinal neuroendocrine tumors (GI-NETs)] in Japan in 2005. Here, we conducted the second nationwide survey on patients with GEP-NETs who received treatment in 2010.
A total of 3,379 patients received treatment for PNETs in 2010, representing a 1.2-fold increase in the number of patients from 2005 to 2010. The prevalence was estimated to be 2.69/100,000, with an annual onset incidence of 1.27/100,000 in 2010. Non-functioning tumor (NF)-PNETs comprised 65.5 % of cases followed by insulinoma (20.9 %) and gastrinoma (8.2 %). Interestingly, the number of patients with NF-PNETs increased ~1.8 fold since 2005. A total of 19.9 % of patients exhibited distant metastasis at initial diagnosis; 4.3 % had complications with multiple endocrine neoplasia type 1 (MEN-1), and only 4.0 % had NF-PNETs associated with MEN-1. Meanwhile, an estimated 8,088 patients received treatment for GI-NETs, representing a ~1.8-fold increase since 2005. The prevalence was estimated to be 6.42/100,000, with an annual onset incidence of 3.51/100,000. The locations of GI-NETs varied: foregut, 26.1 %; midgut, 3.6 %; and hindgut, 70.3 %. Distant metastasis and complications with MEN-1 were observed in 6.0 and 0.42 % at initial diagnosis, respectively. The frequency of carcinoid syndrome in patients with GI-NETs was 3.2 %.
We clarified the epidemiological changes in GEP-NETs from 2005 to 2010 in Japan.
Journal of Gastroenterology 02/2014; · 3.79 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aberrant expression of gonadotropin-releasing hormone receptor (GnRHR) has been reported in human adrenal tissues including aldosterone-producing adenoma (APA). However, the details of its expression and functional role in adrenals are still not clear. In this study, quantitative RT-PCR analysis revealed the mean level of GnRHR mRNA was significantly higher in APAs than in human normal adrenal (NA) (P=0.004). GnRHR protein expression was detected in human NA and neoplastic adrenal tissues. In H295R cells transfected with GnRHR, treatment with GnRH resulted in a concentration-dependent increase in CYP11B2 reporter activity. Chronic activation of GnRHR with GnRH (100 nM), in a cell line with doxycycline-inducible GnRHR (H295R-TR/GnRHR), increased CYP11B2 expression and aldosterone production. These agonistic effects were inhibited by blockers for the calcium (Ca(2+)) signaling pathway, KN93 and calmidazolium. These results suggest GnRH, through heterotopic expression of its receptor, may be a potential regulator of CYP11B2 expression levels in some cases of APA.
Molecular and Cellular Endocrinology 01/2014; · 4.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: Purkinje cell protein 4 (PCP4) is a calmodulin (CaM) binding protein that accelerates calcium association and dissociation with CaM. It has been previously detected in aldosterone-producing adenomas (APA) but details on its expression and function in adrenocortical tissues have remained unknown. Therefore, we performed the immunohistochemical analysis of PCP4 in the following tissues: normal adrenal (NA; n=15), APA (n=15), cortisol producing adenomas (CPA; n=15) and idiopathic hyperaldosteronism cases (IHA; n=5). APA samples (n=45) were also submitted to quantitative RT-PCR (qPCR) of PCP4, CYP11B1, and CYP11B2, as well as DNA sequencing for KCNJ5 mutations. Transient transfection analysis using PCP4 siRNA was also performed in H295R adrenocortical carcinoma cells, following ELISA analysis, and CYP11B2 luciferase assays were also performed after PCP4 vector transfection in order to study the regulation of PCP4 protein expression. In our findings, PCP4 immunoreactivity was predominantly detected in APA and in the zona glomerulosa (ZG) of NA and IHA. In APA, the mRNA levels of PCP4 were significantly correlated with those of CYP11B2 (P<0.0001) and were significantly higher in cases with KCNJ5 mutation than wild-type (P=0.005). Following PCP4 vector transfection, CYP11B2 luciferase reporter activity was significantly higher than controls in the presence of angiotensin-II. Knockdown of PCP4 resulted in a significant decrease in CYP11B2 mRNA levels (P=0.012) and aldosterone production (P=0.011). Our results indicate that PCP4 is a regulator of aldosterone production in normal, hyperplastic and neoplastic human adrenocortical cells.
Journal of Molecular Endocrinology 01/2014; · 3.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neuroendocrine tumors (NET) are highly vascularized, but the process of proliferation and maturation of vascular structures during tumor development and progression has remained unknown. We examined the structural alterations of intratumoral blood vessels in human gastroenteropancreatic NET. Microvessel density was evaluated using the endothelial cell markers vasohibin-1, CD31, and endoglin in 135 cases. Double immunohistochemistry staining was performed to localize endothelium and pericytes on the same vessels using the pericyte marker nestin. The ratio of Ki-67/CD31 was significantly correlated with that of vasohibin-1/CD31 positivity (P < 0.001), indicating that the ratio of vasohibin-1/CD31 also reflects the status of neovascularization in NET. This ratio was higher in NET than in its non-neoplastic counterpart (P = 0.10) and tended to increase according to World Health Organization (WHO) grade, although the differences were not statistically significant (P = 0.32). The ratio of vasohibin-1/nestin-positive vessels, representing the maturation of neovessels, was also significantly higher in NET than in its non-neoplastic counterparts (P = 0.003). Among WHO grades, the ratio increased from grade 1 to grade 2 (P = 0.36) and decreased in neuroendocrine carcinoma (P = 0.34). Our results demonstrated that VASH-1/CD31 can be an ideal immunohistochemical marker for characterizing neovascularization in neuroendocrine tumor. The VASH-1/CD31 content increased with WHO grade, and the vessels covered by pericytes decreased in higher grades. These structural changes in the vessels are considered to play an important role in inducing tumor-cell proliferation.
[show abstract][hide abstract] ABSTRACT: 1.The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate.2.Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41–52 for the CYP11B2 and amino acids 80–90 for the CYP11B1 enzyme.3.The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.
Molecular and Cellular Endocrinology 12/2013; · 4.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: NRF2 (nuclear factor erythroid 2-related factor 2) is an important transcriptional activator involved in the cellular defense mechanisms against electrophilic and oxidative stress. Recent studies have demonstrated that NRF2 protein is up-regulated in several human malignancies and is associated with worse prognosis of these patients. However, pathological and clinical significance of NRF2 has remained largely unknown in the breast cancer patients. Therefore, in this study, we immunolocalized NRF2 in 106 breast cancer cases. NRF2 immunoreactivity was mainly detected in the nucleus of the breast carcinoma cells and was positive in 44% of the cases, while negligible in normal mammary glands. The NRF2 status was significantly associated with histological grade, Ki-67 labeling index, p62 immunoreactivity and NAD(P)H:quinone oxidoreductase 1 (NQO1) immunoreactivity, and adversely associated with progesterone receptor status. In addition, results of multivariate analysis revealed that NRF2 status was an independent adverse prognostic factor for both recurrence and disease-free survival of the patients. Subsequent in vitro studies also demonstrated that NRF2 expression significantly increased cell proliferation activity of MCF7 and SK-BR-3 breast carcinoma cells. These results suggest that nuclear NRF2 protein is accumulated in the breast carcinoma cells and plays important roles in the proliferation and/or progression of breast carcinoma. Nuclear NRF2 immunoreactivity is therefore considered a potent prognostic factor in breast cancer patients.
Endocrine Related Cancer 12/2013; · 5.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: Context:In primary aldosteronism (PA), glomerular hyperfiltration due to excessive aldosterone is considered to underestimate actual renal damage.Objective:To determine prevalence of chronic kidney disease (CKD) in PA and identify the predictors of decreasing estimated glomerular filtration rate (eGFR) after treatment.Design:Twelve-month prospective study.Setting:Patients with PA treated at Tohoku University Hospital.Patients and interventions:All patients were treated according to the results of adrenal venous sampling; 102 patients with aldosterone-producing adenoma (APA) underwent adrenalectomy, and 111 with bilateral hyperaldosteronism (BHA) were treated with mineralocorticoid receptor antagonists.Main outcome measures:Electrolytes, blood pressure (BP), and indicators of renal function were determined at 1 and 12 months after intervention.Results:BP, urinary albumin excretion (UAE) and eGFR, which significantly decreased at 1 month after treatment of PA, did not further decrease at 12 months. Prevalence of CKD, which was 15.7% in APA and 8.1% in BHA at first visit, increased to 37.1% and 28.3% at the end of study, respectively (p < 0.0001). Multivariate regression analysis revealed that higher UAE and lower serum potassium levels were found to be the independent predictors of decreasing eGFR after intervention.Conclusions:This large cohort study shows that prevalence of CKD of PA was increased after treatment, and that higher UAE and lower serum potassium levels at first visit were predictors of decreasing eGFR after treatment of PA. To prevent a large decrease of eGFR after intervention, PA patients should be diagnosed before evolution to severe albuminuria and hypokalemia.
The Journal of clinical endocrinology and metabolism 11/2013; · 6.50 Impact Factor
[show abstract][hide abstract] ABSTRACT: Metallothionein-3 (MT-3) is an intracellular, low molecular weight protein mainly distributed in the central nervous system but also in various peripheral organs and several types of human neoplasms. However, details of MT-3 expression have not been examined in human adrenal cortex and its disorders. The mRNA levels of MT-3 were first evaluated by quantitative RT-PCR (qPCR) in adrenocortical aldosterone-producing adenoma (APA: 11) and cortisol-producing adenoma (CPA: 14). In addition, MT-3 immunohistochemistry was performed in non-pathological adrenal glands (NA: 19), idiopathic hyperaldosteronism (IHA: 10), APA (20), CPA (24), adjacent non-neoplastic adrenal glands of adenoma (AAG: 20), and adrenocortical carcinoma (ACC: 8). H295R cells were also treated with angiotensin-II or forskolin in a time-dependent manner, and the changes of MT-3 mRNA levels were evaluated by qPCR. Results of qPCR analysis demonstrated that MT-3 mRNA levels were significantly higher in APA than CPA (P = 0.0004). MT-3 immunoreactivity was detected in the zona glomerulosa of NA, IHA, and AAG, as well as in APA, CPA, and ACC. When treated with angiotensin-II and forskolin, MT-3 mRNA levels reached a peak by 12 h in H295R cells, with significantly higher levels compared to control non-treated cells (P < 0.01). The presence of MT-3 in the ZG of NA, IHA, and AAG, as well as APA may imply a role in the pathophysiology of aldosterone-producing tissues.
[show abstract][hide abstract] ABSTRACT: Plasma cell mastitis (PCM) is one of the most frequently encountered inflammatory diseases of the nonlactating breast. Histologically, PCM is characterized by infiltration of relatively abundant plasma cells into the mammary ducts. Its pathogenesis has remained unknown. In this study, we immunolocalized intercellular adhesion molecule (ICAM) 1 and 2 and E-selectin, all of which play pivotal roles in the inflammatory process, in 35 cases of PCM. We then compared the results with those of non-PCM and nonpathologic breast tissue. In the ductal epithelium, ICAM-1 immunoreactivity was significantly more pronounced in PCM than in non-PCM (P = .045). Both ICAM-1 (P < .001) and ICAM-2 (P = .001) were significantly more pronounced in PCM than in nonpathologic breast tissue. However, no significant differences in ICAM-2 and E-selectin immunoreactivity were detected between ductal epithelium of PCM and non-PCM. ICAM-1, but not ICAM-2 or E-selectin, demonstrated significantly higher immunoreactivity in endothelial cells of PCM than in nonpathologic breast (P < .001). These results all suggest that ICAM-1 in both ductal epithelium and endothelium plays important roles in the inflammatory process of PCM, possibly through margination, extravasation, and attachment of plasma cells and lymphocytes, which may result in continuous inflammatory cell homing to ductal epithelial cells.
[show abstract][hide abstract] ABSTRACT: Vasohibin 1, an endothelium-derived negative feedback regulator of angiogenesis, is induced by fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor A (VEGF-A). In this study, we retrospectively evaluated immunoreactivity of FGF-2 and VEGF-A as well as microvessel density (MVD) determined by expression of vasohibin 1 and CD34 (MVD-CD34) and correlated the findings with clinical outcomes of 181 patients with hepatocellular carcinoma (HCC). Double immunostaining of an endothelial marker CD34 and vasohibin 1 with Ki-67 was also performed to assess angiogenic activity of endothelial cells in HCC. The ratio of Ki-67-positive endothelial cells in vasohibin 1-positive vessels (22%) was significantly higher than that of CD34-positive vessels (9%, P < .001), suggesting the correlation between vasohibin 1 and neovascularization in endothelial cells of HCC. MVD-CD34 decreased, but the ratio of MVD determined by expression of vasohibin 1 to MVD-CD34 (vasohibin 1/CD34) increased significantly according to histologic grade. Vasohibin 1 was significantly correlated with the status of FGF-2 (P = .007) but not with that of VEGF-A (P = .055). The 10-year overall survival and the 2-year disease-free survival rates of the low vasohibin 1/CD34 group (vasohibin 1/CD34 ≤0.459) were significantly higher than those of the high vasohibin 1/CD34 group (vasohibin 1/CD34 >0.459) (survival, 48% versus 38% and 52% versus 35%; P < .001 and P < .05, respectively). In addition, vasohibin 1/CD34 in HCC patients was an independent marker of poor prognosis, as determined by multivariate analysis (risk ratio, 1.973; 95% confidence interval, 1.049-3.711; P = .035). Vasohibin 1/CD34 could identify the proliferative vessels and could be a useful biomarker for predicting the clinical outcome of HCC patients.
[show abstract][hide abstract] ABSTRACT: DNA methylation patterns are maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not preserved. We demonstrate that stimulatory signals can change the DNA methylation status at a CCAAT/enhancer binding protein (CEBP) binding site and a transcription start site and activate expression of the angiotensinogen gene (AGT). A CEBP binding site in the human AGT promoter was hypomethylated in tissues with high expression of AGT, but not in those with low expression. The transcriptional activity of AGT promoter sequences cloned into a reporter plasmid depended on DNA methylation. In cultured human cells, interleukin 6 stimulation caused DNA demethylation around a CEBP binding site and a transcription start site; demethylation was accompanied by increased CEBP-β recruitment and chromatin accessibility of the AGT promoter. DNA methylation activity decreased in the nucleus. Excess circulating aldosterone upregulated AGT expression and was accompanied by DNA hypomethylation around a CEBP binding site and a transcription start site in human visceral adipose tissue. High salt intake led to upregulation of Agt expression, DNA hypomethylation around 2 CEBP binding sites and a transcription start site, and decreased DNA methylation activity in rat visceral adipose tissue. Taken together, CEBP binding initiates chromatin relaxation and transcription, which are followed by DNA demethylation around a CEBP binding site and a transcription start site in the AGT promoter. Decreased DNA methylation activity in the nucleus may play a role in DNA demethylation. DNA demethylation switches the phenotype of AGT expression from an inactive to an active state.