[Show abstract][Hide abstract]ABSTRACT: Germline mutations in genes encoding succinate dehydrogenase subunits are associated with the development of familial pheochromocytomas and paragangliomas [hereditary paraganglioma/pheochromocytoma syndrome (HPPS)]. In particular, a mutation in succinate dehydrogenase subunit B (SDHB) is highly associated with abdominal paraganglioma and subsequent distant metastasis (malignant paraganglioma), indicating the importance of SDHB genetic testing. The discovery of HPPS suggests an association among genetic mitochondrial defects, tumor development, and catecholamine oversecretion. To investigate this association, we transfected pheochromocytoma cells (PC12) with SDHB-specific siRNA. SDHB silencing virtually abolished complex II activity, demonstrating the utility of this in vitro model for investigating the pseudo-hypoxic drive hypothesis. Lack of complex II activity resulting from RNA interference of SDHB increased tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamine biosynthesis) activity and catecholamine secretion. Reduced apoptosis was observed accompanied by Bcl-2 accumulation in PC12 cells, consistent with the phenotypes of paragangliomas with SDHB mutations. In addition, SDHB silencing increased reactive oxygen species (ROS) production and nuclear HIF1α stabilization under normoxic conditions. Furthermore, phenotypes induced by complex II activity knockdown were abolished by pretreatment with N-acetyl cysteine (an ROS scavenger) and by prior HIF1α knockdown, indicating an ROS- and HIF1α-dependent mechanism. Our results indicate that increased ROS may act as signal transduction messengers that induce HIF1α stabilization and may be necessary for the pseudo-hypoxic states observed in our experimental model. To our knowledge, this is the first study demonstrating that pseudo-hypoxic states resulting from SDHB knockdown are associated with increased TH activity and catecholamine oversecretion.
[Show abstract][Hide abstract]ABSTRACT: Germline mutations in the succinate dehydrogenase complex subunit B (SDHB) gene (SDHB) cause susceptibility to paragangliomas and pheochromocytomas; however, it is exceedingly rare in childhood and especially in sporadic cases. We report the first Japanese pediatric case of paraganglioma with a de novo mutation in the SDHB gene. A 6-year-old girl with convulsions and hypertension was found to have a paravertebral abdominal tumor. Urinary and blood examinations revealed markedly elevated levels of norepinephrine. Following treatment for hypertension, the tumor was removed completely and histological findings were consistent with paraganglioma. Immunohistochemistry studies demonstrated the absence of SDHB protein expression, indicating an underlying SDH mutation with high probability. Germline mutation analysis of the SDHB gene revealed a heterozygous splice site mutation in intron 4 (C.423 + 1G > A). Subsequently, a second somatic genetic change was confirmed by multiplex ligation-dependent probe amplification (MLPA) analysis, showing that deletion of the wild-type allele resulted in loss of function of SDHB. No germline mutations in SDHB were detected in her parents.
Genetic testing should be considered for pediatric patients with paragangliomas, even in the absence of familial history, as closer lifelong screening to detect the development of malignancy will be required for patients with SDHB mutations. What is Known • Most sporadic cases of paraganglioma with SDHB mutations occur between adolescence and adulthood. • Screening methods for carriers of SDHB mutations assessing recurrence and detecting developing metastases are yet to be standardized. What is New • The current case of an extra-adrenal paraganglioma with a de novo SDHB mutation had an onset at 6 years. • We suggest much closer periodical observation for these high-risk children.
Full-text Article · Aug 2015 · European Journal of Pediatrics
[Show abstract][Hide abstract]ABSTRACT: Chromogranin A (CHGA) is a major protein in the secretory granules of chromaffin cells. CHGA also gives rise to cardiovascular/metabolism regulatory peptides, such as catestatin (CST) and pancreastatin (PST). While CST is a potent inhibitor of catecholamine secretion, PST is a potent physiological inhibitor of glucose-induced insulin secretion. Recently, several SNPs were identified in the CST and PST domains of CHGA locus in different populations. Among the discovered SNPs, CST variant allele Ser-364 was associated with blood pressure alteration and PST variant allele Ser-297 was associated with significantly higher plasma glucose level. In this study, we examined whether these CST and PST variant alleles exist and influence cardiovascular and metabolic phenotypes in Japanese population. Our study comprised of 343 Japanese subjects aged 45-85 years (143 men and 200 women, mean age 66 ± 8 years). We determined the genotypes of CST and PST by PCR-direct sequencing method and carried out genotype-phenotype association analysis. In 343 participants, the minor allele frequency of CST variant Ser-364 was 6.10%. On the other hand, we did not detect the PST variant Ser-297 in this entire study population. The presence of Ser-364 allele was associated with increased in baPWV (an index of systemic arterial stiffness) that suggests an initiation and/or progression atherogenesis and hypertension. The Ser-364 allele was also associated with elevated systolic blood pressure and pulse pressure, consistent with increased baPWV. In conclusion, the CST Ser-364 allele may increase the risk for cardiovascular diseases in Japanese population.
[Show abstract][Hide abstract]ABSTRACT: Transcriptional regulation of metabolic genes in the liver is the key to maintaining systemic energy homeostasis during starvation. The membrane-bound transcription factor cAMP-responsive element-binding protein 3-like 3 (CREB3L3) has been reported to be activated during fasting and to regulate triglyceride metabolism. Here, we show that CREB3L3 confers a wide spectrum of metabolic responses to starvation in vivo. Adenoviral and transgenic overexpression of nuclear CREB3L3 induced systemic lipolysis, hepatic ketogenesis, and insulin sensitivity with increased energy expenditure, leading to marked reduction in body weight, plasma lipid levels, and glucose levels. CREB3L3 overexpression activated gene expression levels and plasma levels of antidiabetic hormones, including fibroblast growth factor 21 and IGF-binding protein 2. Amelioration of diabetes by hepatic activation of CREB3L3 was also observed in several types of diabetic obese mice. Nuclear CREB3L3 mutually activates the peroxisome proliferator-activated receptor α promoter in an autoloop fashion and is crucial for the ligand transactivation of peroxisome proliferator-activated receptor α by interacting with its transcriptional regulator, PGC-1α. CREB3L3 directly and indirectly controls fibroblast growth factor 21 expression and its plasma level, which contributes at least partially to the catabolic effects of CREB3L3 on systemic energy homeostasis in the entire body. Therefore, CREB3L3 is a therapeutic target for obesity and diabetes.
[Show abstract][Hide abstract]ABSTRACT: Measuring the levels of the plasma free metanephrines (PFMs) represents a recently developed and promising test for the diagnosis of pheochromocytoma in the United States and Europe. As this test has not yet been evaluated in Japan, it is necessary to evaluate the diagnostic efficacy of measuring the levels of PFMs compared with the standard measurement of the urinary excretion of metanephrines (uMNs) whose reliability is well established to detect of pheochromocytoma. A total of 101 Japanese subjects clinically suspected of having pheochromocytoma in were included in this study. Subsequently, we prospectively measured the PFMs levels in all patients, compared with those of biochemical markers of the catecholamine secretion and metabolisms in the plasma and urine. All subjects with adrenal tumors underwent tumor excision. Data were available for 84 of the 101 patients, 47 of whom had histopathologically proven pheochromocytoma and 37 were finally diagnosed with non-pheochromocytoma. The results of comparisons in the accuracy of measurement for diagnosis of pheochromocytoma between PFMs and the urinary excretion of metanephrines (uMNs) were 0.980 VS 0.951 for AUC of receiver operatorating characteristic (ROC) curve, 0.957 VS 0.894 for sensitivity, and 0.973 VS 0.946 for specificity, respectively. Although the differences were small, the results of our study definitely demonstrated that measurement of PFMs was not inferior to standard urinary metanephrines (uMNs) measurement, which is established to be the most reliable biochemical method to detect pheochromocytoma. This study clearly shows measuring the PFMs levels to be a reliable and efficient method for diagnosing pheochromocytoma in Japanese patients, as demonstrated in previous reports.
[Show abstract][Hide abstract]ABSTRACT: Dear Editor,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) was observed in all tumors with SDHB mutation, and Gimenez-Roqueplo et al. (2003) strongly suggested that SDHB is a tumor suppressor gene. 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 GAPP (Grading of Adrenal Pheochromocytoma and Paraganglioma) classification for predicting metastasis. GAPP is composed of six factors: histological pattern, cellularity, presence or absence of comedo-type necrosis, vascular or capsular invasion, Ki67 labeling index (%), and elevated catecholamine type. Each factor was assigned a point and the number of points was summated.
Full-text Article · Mar 2014 · Endocrine Related Cancer
[Show abstract][Hide abstract]ABSTRACT: It is insufficient to distinguish benign tumors from malignant pheochromocytoma using histological analyses of resected tissue alone. We experienced an 18-year-old woman who complained of severe headaches in whom hypertension was revealed. She was suspected of having a malignant tumor based on her clinical characteristics, despite showing no evidence of metastatic lesions. The patient was diagnosed with an aggressive form of hereditary pheochromocytoma-paraganglioma syndrome (HPPS) based on immunohistochemical analyses and genetic testing. The present case indicates that conducting genetic testing, including SDHB mutation analyses, is required to determine the prognosis in patients highly suspected of having HPPS.
[Show abstract][Hide abstract]ABSTRACT: Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors, and recently, it has been shown to be an active agent for the treatment of malignant pheochromocytomas. Previously, we demonstrated that sunitinib directly inhibited mTORC1 signaling in rat pheochromocytoma PC12 cells. Although autophagy is a highly regulated cellular process, its relevance to cancer seems to be complicated. It is of note that inhibition of mTORC1 is a prerequisite for autophagy induction. Indeed, direct mTORC1 inhibition initiates ULK1/2 autophosphorylation and subsequent Atg13 and FIP200 phosphorylation, inducing autophagy. Here, we demonstrated that sunitinib significantly increased the levels of LC3-II, concomitant with a decrease of p62 in PC12 cells. Following sunitinib treatment, immunofluorescent imaging revealed a marked increased punctate LC3-II distribution. Furthermore, Atg13 knockdown significantly reduced its protein level, which in turn abolished sunitinib-induced autophagy. Moreover, inhibition of autophagy by siRNAs targeting Atg13 or by pharmacological inhibition with ammonium chloride, enhanced both sunitinib-induced apoptosis and anti-proliferation. Thus, sunitinib-induced autophagy is dependent on the suppression of mTORC1 signaling and the formation of ULK1/2-Atg13-FIP200 complexes. Inhibition of autophagy may be a promising therapeutic option for improving the anti-tumor effect of sunitinib.
Article · Dec 2012 · Journal of Pharmacological Sciences
[Show abstract][Hide abstract]ABSTRACT: Unlabelled:
Nonalcoholic steatohepatitis (NASH) is associated with obesity and type 2 diabetes, and an increased risk for liver cirrhosis and cancer. ELOVL family member 6, elongation of very long chain fatty acids (Elovl6), is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids (FAs). We have shown previously that Elovl6 is a major target for sterol regulatory element binding proteins in the liver and that it plays a critical role in the development of obesity-induced insulin resistance by modifying FA composition. To further investigate the role of Elovl6 in the development of NASH and its underlying mechanism, we used three independent mouse models with loss or gain of function of Elovl6, and human liver samples isolated from patients with NASH. Our results demonstrate that (1) Elovl6 is a critical modulator for atherogenic high-fat diet-induced inflammation, oxidative stress, and fibrosis in the liver; (2) Elovl6 expression is positively correlated with severity of hepatosteatosis and liver injury in NASH patients; and (3) deletion of Elovl6 reduces palmitate-induced activation of the NLR family pyrin domain-containing 3 inflammasome; this could be at least one of the underlying mechanisms by which Elovl6 modulates the progress of NASH.
Hepatic long-chain fatty acid composition is a novel determinant in NASH development, and Elovl6 could be a potential therapeutic target for the prevention and treatment of NASH.
[Show abstract][Hide abstract]ABSTRACT: Sunitinib is an oral, small molecule multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that primarily targets vascular endothelial growth factor receptors (VEGFRs). Although sunitinib is an active agent for the treatment of malignant pheochromocytomas, it is unclear whether sunitinib acts through only antiangiogenic mechanisms or also directly targets tumor cells. We previously showed that sunitinib directly induced apoptosis of PC-12 cells. To further confirm these direct effects, we examined the effects of sunitinib on tyrosine hydroxylase (TH) (the rate-limiting enzyme in catecholamine biosynthesis) activity and catecholamine secretion in PC-12 cells and the underlying mechanisms. Sunitinib inhibited TH activity in a dose-dependent manner, and decreased TH protein levels. Consistent with this finding, sunitinib decreased TH phosphorylation at Ser(31) and Ser(40) and significantly decreased catecholamine secretion. VEGFR-2 knockdown attenuated these effects, including inhibition of TH activity and catecholamine secretion, suggesting that they were mediated by VEGFR-2. Sunitinib significantly decreased phospholipase C (PLC)-γ phosphorylation and subsequent protein kinase C (PKC) activity. Because Ser(40) phosphorylation significantly affects TH activity and is known to be regulated by PKC, sunitinib may inhibit Ser(40) phosphorylation via the VEGFR-2/PLC-γ/PKC pathway. Additionally, sunitinib markedly decreased the activity of extracellular signal-regulated kinase (ERK), but not c-Jun NH(2)-terminal kinase or p38 mitogen-activated protein kinase. Therefore, sunitinib may reduce TH Ser(31) phosphorylation through inhibition of the VEGFR-2/PLC-γ/PKC/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/ERK pathway. Sunitinib also significantly reduced inositol 1,4,5-trisphosphate production. However, because PC-12 cells do not precisely reflect the pathogenesis of malignant cells, we confirmed the key findings in a human neuroblastoma cell line, SK-N-SH. In conclusion, sunitinib directly inhibits catecholamine synthesis and secretion in pheochromocytoma PC-12 cells.
Full-text Article · Aug 2012 · AJP Endocrinology and Metabolism
[Show abstract][Hide abstract]ABSTRACT: Recently, TMEM127 was shown to be a new pheochromocytoma susceptibility gene; this is consistent with its function as a tumour suppressor gene (Journal of Clinical Endocrinology and Metabolism, 2009, 94, 2817). Most pheochromocytomas arise from the adrenal medulla, and in approximately half of the cases, the tumours are bilateral (Journal of Clinical Endocrinology and Metabolism, 2009, 94, 2817; Journal of the American Medical Association, 2004, 292, 943; Human Mutation, 2010, 31, 41; Science, 2009, 325, 1139). The aim of the present study was to determine whether TMEM127 mutations are involved in the pathogenesis of pheochromocytomas/paragangliomas in Japanese subjects.
For this study, 74 unrelated patients with pheochromocytoma/paraganglioma who tested negative for mutations and deletions in RET, VHL, SDHB and SDHD were recruited through a multi-institutional collaborative effort in Japan. The TMEM127 gene sequence was determined in their germline DNA, and tumour DNA was analysed for the loss of heterozygosity. In addition, their TMEM127 gene sequences were compared with sequences from 114 normal healthy, ethnically matched controls.
Among the 74 eligible patients, two unrelated patients (2·7%) with bilateral adrenal pheochromocytoma were found to have an identical germline TMEM127 mutation (c.116_119delTGTC, p.Ile41ArgfsX39) associated with 2q deletion loss of heterozygosity, which was also previously described in a Brazilian case (Journal of the American Medical Association, 2004, 292, 943). We also determined that none of the 114 normal healthy controls had this deletion mutation.
This is the first report showing that TMEM127 mutation plays a pathological role in pheochromocytoma in an Asian population. Although our surveillance is limited, the prevalence and the phenotype of this gene mutation appear to be similar to those reported in previous studies.
[Show abstract][Hide abstract]ABSTRACT: A 29-year-old female patient presented with shock and dyspnea due to heart failure and pulmonary edema. Echocardiography indicated excessive contraction limited to the left ventricular apex and akinesis of the basal and middle ventricle, which were confirmed by emergency left ventriculography. The finding was diagnostic of inverted Takotsubo cardiomyopathy. An abdominal computed tomography scan showed a tumor in the left adrenal gland with a central low-density area, and the plasma and urinary catecholamines were strikingly elevated. Taken together, these findings suggested the presence of a hemorrhagic pheochromocytoma. A myocardial biopsy in the very acute stage on the day of admission revealed neutrophilic infiltration and contraction-band necrosis, which was indistinguishable from the previously reported pathology in the acute phase of idiopathic Takotsubo cardiomyopathy without pheochromocytoma. The diagnosis of pheochromocytoma in this case was confirmed 7 weeks later by surgical removal of the left adrenal gland with massive hemorrhage at the center of the pheochromocytoma. The marked similarity of the endomyocardial pathology between this case and cases with idiopathic Takotsubo cardiomyopathy strongly points to catecholamine excess as a common causality for Takotsubo cardiomyopathy with or without pheochromocytoma.
[Show abstract][Hide abstract]ABSTRACT: F-box and WD repeat domain-containing 7α (Fbw7α) is the substrate recognition component of a ubiquitin ligase that controls
the degradation of factors involved in cellular growth, including c-Myc, cyclin E, and c-Jun. In addition, Fbw7α degrades
the nuclear form of sterol regulatory element-binding protein (SREBP)-1a, a global regulator of lipid synthesis, particularly
during mitosis in cultured cells. This study investigated the in vivo role of Fbw7α in hepatic lipid metabolism. siRNA knockdown of Fbw7α in mice caused marked hepatosteatosis with the accumulation
of triglycerides. However, inhibition of Fbw7α did not change the level of nuclear SREBP-1 protein or the expression of genes
involved in fatty acid synthesis and oxidation. In vivo experiments on the gain and loss of Fbw7α function indicated that Fbw7α regulated the expression of peroxisome proliferator-activated
receptor (PPAR) γ2 and its target genes involved in fatty acid uptake and triglyceride synthesis. These genes included fatty
acid transporter Cd36, diacylglycerol acyltransferase 1 (Dgat1), and fat-specific protein 27 (Cidec). The regulation of PPARγ2 by Fbw7α was mediated, at least in part, by the direct degradation of the Krüppel-like factor
5 (KLF5) protein, upstream of PPARγ2 expression. Hepatic Fbw7α contributes to normal fatty acid and triglyceride metabolism,
functions that represent novel aspects of this cell growth regulator.
Article · Nov 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: F-box and WD repeat domain-containing 7α (Fbw7α) is the substrate recognition component of a ubiquitin ligase that controls the degradation of factors involved in cellular growth, including c-Myc, cyclin E, and c-Jun. In addition, Fbw7α degrades the nuclear form of sterol regulatory element-binding protein (SREBP)-1a, a global regulator of lipid synthesis, particularly during mitosis in cultured cells. This study investigated the in vivo role of Fbw7α in hepatic lipid metabolism. siRNA knockdown of Fbw7α in mice caused marked hepatosteatosis with the accumulation of triglycerides. However, inhibition of Fbw7α did not change the level of nuclear SREBP-1 protein or the expression of genes involved in fatty acid synthesis and oxidation. In vivo experiments on the gain and loss of Fbw7α function indicated that Fbw7α regulated the expression of peroxisome proliferator-activated receptor (PPAR) γ2 and its target genes involved in fatty acid uptake and triglyceride synthesis. These genes included fatty acid transporter Cd36, diacylglycerol acyltransferase 1 (Dgat1), and fat-specific protein 27 (Cidec). The regulation of PPARγ2 by Fbw7α was mediated, at least in part, by the direct degradation of the Krüppel-like factor 5 (KLF5) protein, upstream of PPARγ2 expression. Hepatic Fbw7α contributes to normal fatty acid and triglyceride metabolism, functions that represent novel aspects of this cell growth regulator.
Article · Sep 2011 · Journal of Biological Chemistry