Somatic mutations of the catalytic subunit of the cyclic AMP-dependent protein kinase (PRKACA) gene have recently been identified in about 35% of cortisol-producing adenomas (CPAs), with the affected patients showing overt Cushing's syndrome. Since we recently reported higher prevalence of mutations of the KCNJ5 gene and associations with autonomous cortisol secretion in Japanese aldosterone-producing adenomas than in Western countries, there might be different features of CPAs between Japan and the West. We therefore investigated mutations of the PRKACA gene in Japanese patients with several adrenal tumors secreting cortisol, including overt Cushing's syndrome, subclinical Cushing's syndrome, and aldosterone-producing adenomas (APAs) co-secreting cortisol operated on at Gunma University Hospital. Of the 13 patients with CPA who showed overt Cushing's syndrome, 3 (23%) had recurrent somatic mutations of the PRKACA gene, p.L206R (c.617 T>G), and there were no mutations in subclinical Cushing's syndrome. Among 33 APAs, 24 had somatic mutations of the KCNJ5 gene, either G151R or L168R, 11 (33%) had autonomous cortisol secretion, but there were no mutations of the PRKACA gene. We established a PCR-restriction fragment length polymorphism assay and revealed that the mutated allele was expressed at a similar level to the wild-type allele. These findings demonstrated that 1) the prevalence of Japanese patients with CPA who showed overt Cushing's syndrome and whose somatic mutations in the PRKACA gene was similar to that in Western countries, 2) the mutation might be specific for CPAs causing overt Cushing's syndrome, and 3) the mutant PRKACA allele was expressed appropriately in CPAs.
Obesity arises from impaired energy balance, which is centrally coordinated by leptin through activation of the long form of leptin receptor (Leprb). Obesity causes central leptin resistance. However, whether enhanced peripheral leptin sensitivity could overcome central leptin resistance remains obscure. A peripheral metabolic organ targeted by leptin is the liver, with low Leprb expression. We here show that mice fed a high-fat diet (HFD) and obese patients with hepatosteatosis exhibit increased expression of hepatic helicase with zinc finger 2, a transcriptional coactivator (Helz2), which functions as a transcriptional coregulator of several nuclear receptors, including peroxisome proliferator-activated receptor ? in vitro. To explore the physiological importance of Helz2, we generated Helz2-deficient mice and analyzed their metabolic phenotypes. Helz2-deficient mice showing hyperleptinemia associated with central leptin resistance were protected against HFD-induced obesity and had significantly up-regulated hepatic Leprb expression. Helz2 deficiency and adenovirus-mediated liver-specific exogenous Leprb overexpression in wild-type mice significantly stimulated hepatic AMP-activated protein kinase on HFD, whereas Helz2-deficient db/db mice lacking functional Leprb did not. Fatty acid-? oxidation was increased in Helz2-deficeint hepatocytes, and Helz2-deficient mice revealed increased oxygen consumption and decreased respiratory quotient in calorimetry analyses. The enhanced hepatic AMP-activated protein kinase energy-sensing pathway in Helz2-deficient mice ameliorated hyperlipidemia, hepatosteatosis, and insulin resistance by reducing lipogenic gene expression and stimulating lipid-burning gene expression in the liver. These findings together demonstrate that Helz2 deficiency ameliorates HFD-induced metabolic abnormalities by stimulating endogenous hepatic Leprb expression, despite central leptin resistance. Hepatic HELZ2 might be a novel target molecule for the treatment of obesity with hepatosteatosis.
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 TR?1, 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 TR?1 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.
Thyroid storm (TS) is a life-threatening endocrine emergency. However, the pathogenesis of TS is poorly understood. A 40-year-old man was admitted to a nearby hospital with body weight loss and jaundice. Five days after a contrasted abdominal computerized tomography (CT) scan, he exhibited high fever and disturbance of consciousness. He was diagnosed with TS originating from untreated Graves' disease and was transferred to the intensive care unit (ICU) of our hospital. The patient exhibited impaired consciousness (E4V1M4 in Glasgow coma scale), high fever (39.3 °C), and atrial flutter with a pulse rate 162/min, and was complicated by heart failure, acute hepatic failure, and disseminated intravascular coagulation syndrome (DIC). His circulating level of soluble interleukin-2 receptor (sIL-2R), a serum marker of an activated immune response, was highly elevated (7,416 U/mL, reference range: 135-483). Multiple organ failure (MOF) and DIC were successfully managed by multimodality treatments using inorganized iodide, glucocorticoids, anti-thyroid drugs, beta-blockers, and diuretics as well as an anticoagulant agent and the transfusion of platelet concentrate and fresh frozen plasma. sIL-2R levels gradually decreased during the initial treatment, but were still above the reference range even after thyroidectomy. Mild elevations in serum levels of sIL-2R have previously been correlated with thyroid hormone levels in non-storm Graves' disease. The present study demonstrated, for the first time, that circulating sIL-2R levels could be markedly elevated in TS. The marked increase in sIL-2R levels was speculated to represent an inappropriate generalized immune response that plays an unknown role in the pathogenesis of TS.
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 receptor? (PPAR?). 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 PPAR?, 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 PPAR?-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 PPAR?-mediated gene activation cooperatively with Helz2.
Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, the mechanisms underlying the tissue-selectivity of tumors remain unknown. The product of the Men1 gene, menin, and mixed lineage leukemia (MLL) have been found to cooperatively regulate p27(Kip1)/CDKN1B (p27) and p18(Ink4C)/CDKN2C (p18) genes. However, there are no reports on the tissue distribution of these MEN1-related genes. We investigated the expression of these genes in the endocrine and non-endocrine organs of wild-type, Men1 knockout and MLL knockout mice. Men1 mRNA was expressed at a similar level in endocrine and non-endocrine organs. However, MLL, p27 and p18 mRNAs were predominantly expressed in the endocrine organs. Notably, p27 and MLL mRNAs were expressed in the pituitary gland at levels approximately 12- and 17-fold higher than those in the liver. The heterozygotes of Men1 knockout mice the levels of MLL, p27 and p18 mRNAs did not differ from those in the wild-type mice. In contrast, heterozygotes of MLL knockout mice showed significant reductions in p27 mRNA as well as protein levels in the pituitary and p27 and p18 in the pancreatic islets, but not in the liver. This study demonstrated for the first time the predominant expression MEN1-related genes, particularly MLL and p27, in the endocrine organs, and a tissue-specific haploinsuffiency of MLL, but not menin, may lead to a decrease in levels of p27 and p18 mRNAs in endocrine organs. These findings may provide basic information for understanding the mechanisms of tissue selectivity of the tumorigenesis in patients with MEN1.
Tumors in multiple endocrine neoplasia type 1 (MEN1) are generally benign. Since information on the pathogenesis of MEN1 in malignant cases is limited, we conducted genetic analysis and compared the expression of menin, p27(Kip1)(p27)/CDKN1B and p18(Ink4C)(p18)/CDKN2C with levels in benign cases. We describe the case of a 56 year-old male with an atypical prolactinoma and malignant pancreatic neuroenocrine tumor. At age 50, he had undergone transsphenoidal surgery to remove a prolactinoma. However, the tumor relapsed twice. Histological analysis of the recurrent prolactinoma revealed the presence of prolactin, a high MIB-1 index (32.1 %), p53-positive cells (0.2%), and an unusual association with FSH-positive cells. A few years later, he was also found to have a non-functioning pancreatic tumor with probable metastasis to the extradullar region. The metastatic region tested positive for chromogranin and CD56, and negative for prolactin, with 1.2 % of cells p53-positive. Although genetic analyses of the MEN1, p27, and p18 genes demonstrated no mutation, numbers of menin, p27 and p18 immuno-positive cells were significantly down-regulated in the recurrent prolactinoma, but that of p18 was intact in the metastatic region. Furthermore, MEN1 and p27 mRNA levels of the recurrent prolactinoma were down-regulated, particularly the MEN1 mRNA level, compared to levels in 10 cases of benign prolactinoma, while the p18 mRNA level was similar to that of normal pituitary. The tumor in this case may be a subtype of MEN1 showing more aggressive and malignant features probably induced by low levels of menin and p27.
The liver X receptors (LXR-? and -?) are nuclear oxysterol receptors that play pivotal roles in regulating the expression of genes involved in cholesterol transport and metabolism. Recently, several groups have reported that the LXRs also regulate adrenal steroidogenesis. In the previous report, we demonstrated that LXR-? is dominantly expressed in the pituitary and that LXR-? positively regulates the proopiomelanocortin (POMC) gene promoter at the transcriptional level. In this report, we evaluated the expression levels of LXR-? and -? gene in the human pituitary tumor. Even though LXR-? mRNA levels are not significantly increased in ACTH-secreting adenomas, LXR-?/? expression ratio is significantly higher than other pituitary tumors including normal pituitaries. Furthermore, in At-T20 cells, which express POMC gene, overexpression of LXR-? decreased POMC gene promoter activities. Thus, we concluded that LXR-?/? gene expression ratio is a critical factor to activate POMC gene expression in ACTH-secreting pituitary adenomas.
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder associated mainly with tumors of multiple endocrine organs. Mutations in the MEN1 gene that encodes for the menin protein are the predominant cause for hereditary MEN1 syndrome. Though menin is a tumor suppressor, its molecular mechanism of action has not been defined. Here, we report that menin interacts with AKT1 in vitro and in vivo. Menin downregulates the level of active AKT and its kinase activity. Through interaction with AKT1, menin suppresses both AKT1-induced proliferation and antiapoptosis in nonendocrine and endocrine cells. Confocal microscopy analysis revealed that menin regulates AKT1 in part by reducing the translocation of AKT1 from the cytoplasm to the plasma membrane during growth factor stimulation. Our findings may be generalizable to other cancers, insofar as we found that loss of menin expression was also associated with AKT activation in a mouse model of pancreatic islet adenoma. Together, our results suggest menin as an important novel negative regulator of AKT kinase activity.
We recently identified a novel satiety peptide, nesfatin-1, containing 82 amino acids derived from the precursor peptide, nucleobindin 2 (NUCB2), from a troglitazone (TZ)-induced cDNA library. We examined the molecular mechanism underlying TZ-induced NUCB2 mRNA expression. Although TZ induced the mRNA expression in HTB185 cells, a nuclear run-on assay revealed no significant change in the transcription of the gene. Surprisingly, HTB185 cells possessed no functional peroxisome proliferator-activated receptor-gamma. We therefore examined the effect of TZ on the mRNAs stability. The half-life of NUCB2 mRNA was approximately 6 h, and incubation with TZ increased this to 27 h. Furthermore, this increase was completely inhibited by an ERK inhibitor, PD98059, and phosphorylated ERK1/2 was significantly increased after 30 min incubation with TZ. In addition, we cloned the entire NUCB2 gene and identified four adenylate/uridylate-rich elements (AREs) in the 3 untranslated region (UTR), to which several proteins of HTB185 extracts treated with TZ bound. The reporter assay fused with 3UTR showed that the second and third AREs were crucial. Furthermore, the human NUCB2 gene spanned 55 kb and contained 14 exons and 13 introns. The transcriptional start site formed clusters around 246 bp upstream from the translational start site. We confirmed that a construct containing 5889 bp of the promoter region was very active in neuron-derived cell lines but not stimulated by TZ. These findings demonstrated a novel action of derivatives of thiazolidinediones, oral insulin-sensitizing antidiabetic agents, to stabilize the mRNA of NUCB2 through AREs in the 3UTR by activating the ERK1/2 pathway independently of peroxisome proliferator-activated receptor-gamma.
We have recently reported that an alternative splicing variant of liver X receptor (LXR)-beta acts as an RNA co-activator, which is referred to as LXRBSV. The in vivo role of LXRBSV is yet to be clarified. The LXRBSV gene is expressed in various tissues including the liver and brain. We evaluated the gene expression of LXRBSV in various regions of the brain using real-time quantitative PCR assays in the current study and found that LXRBSV is abundantly expressed in the pituitary. 5-rapid amplification of cDNA ends (5-RACE) revealed that the transcriptional start site (TSS) of LXRBSV is located 40 base pairs (bp) downstream of LXR-beta. We prepared two promoter constructs: -1598/+35 bp and -1598/+75 bp in pGL4 for LXR-beta and LXRBSV, respectively. The latter promoter construct demonstrated significantly higher activity than the former construct in GH3 cells derived from the rat pituitary. On the contrary, the promoter activities of these two constructs were indistinguishable in Hepa1-6 cells derived from mouse hepatocytes. Furthermore, the promoter region specific for LXRBSV itself exerted promoter activity in GH3 cells but not in Hepa1-6 cells. Taken together, we have concluded that LXRBSV is preferentially transcribed and expressed in the pituitary, indicating that LXRBSV plays a role in regulating pituitary gene expression. These data provide clues to elucidating the physiological relevance of LXRBSV.
Sporadic multiple endocrine neoplasia type 1 (MEN1) is defined as the occurrence of tumours in two of three main endocrine tissue types: parathyroid, pituitary and pancreaticoduodenal. A prolactinoma variant or Burin variant of MEN1 was found to occur in three large kindreds, with more prolactinomas and fewer gastrinomas than typical MEN1. MEN1 tumours differ from common tumours by showing features from the MEN1 gene (e.g. larger pituitary tumours). They also show various expressions of tumour multiplicity; however, pituitary tumour in MEN1 is usually solitary. Diagnosis in MEN1 carriers during childhood is not directed at cancers but at benign morbid tumours. Morbid prolactinoma occurred at the age of 5 years in one MEN1 individual; hence, this is the earliest age at which to recommend tumour surveillance in carriers. The MEN1 gene shows biallelic inactivation in 30% of some types of common variety endocrine tumours (e.g. parathyroid adenoma, gastrinoma, insulinoma and bronchial carcinoid), but in only 1-5% of common pituitary tumours. Heterozygous knockout of MEN1 in mice provides a robust model of MEN1 and has been found to support further research on anti-angiogenesis therapy for pituitary tumours. The rarity of MEN1 mutations in some MEN1-like states aids the identification of other mutated genes, such as AIP, HRPT2 and p27(Kip1). We present recent clinical and basic findings about the MEN1 gene, particularly concerning hereditary vs. common variety pituitary tumours.
Stearoyl-CoA desaturase-1 (SCD-1) plays a pivotal role in an increase of triglyceride by an excess of dietary carbohydrate intake. Dietary carbohydrates increase SCD-1 gene expression in liver by sterol response element binding protein (SREBP)-1c-dependent and SREBP-1c -independent pathways. Previous report demonstrated that thyroid hormone (TH) negatively regulates mouse SCD-1 gene promoter before SREBP-1c was revealed. We reported that TH negatively regulates SREBP-1c recently. Therefore, in the current study, we examined whether and how TH regulates human SCD-1 gene expression and evaluated SREBP-1c effect on the negative regulation. Luciferase assays revealed that TH suppresses both mouse and human SCD-1 gene promoter activity. In SREBP-1 knockdown HepG2 cells, TH still suppresses SCD-1 gene promoter activity, and it also exerted the negative regulation under cotransfection of a small amount of SREBP-1c. These data indicated that SREBP-1c does not play the decisive role for the negative regulation by TH. The responsible region for the negative regulation in human SCD-1 gene promoter turned out to be between -124 and -92 bp, referred to as site A. Chromatin immunoprecipitation assays demonstrated that TH receptor-? is recruited to the region upon T(3) administration, although TR-? does not bind directly to site A. In conclusion, TH negatively regulates human SCD-1 gene expression in without direct binding of the TH receptor to the SCD-1 gene promoter.
Adrenal aldosterone-producing adenomas (APA) are rarely associated with the clear co-secretion of cortisol. Somatic mutations of the potassium channel KCNJ5 gene, with the hotspots G151R and L168R, have been recently identified in patients with APA. However, whether APAs that secrete cortisol have these mutations remains unclear. We examined three patients with APAs showing clear autonomous secretion of cortisol who possessed a 1 mg dexamethasone suppression test (DST) with a failure of the serum cortisol level to drop below 3.0 ?g/dL, a morning plasma ACTH level of less than 10 pg/mL, and suppressed accumulation in the intact adrenal on (131)I- adosterol scintigraphy, or postoperative adrenal insufficiency. Laparoscopic adrenectomy revealed all tumors to be golden yellow, and histological examination confirmed them to be adrenocortical adenomas. All these patients required replacement therapy with hydrocortisone after surgery. Sequencing demonstrated that 2 of three cases showed a mutation of the KCNJ5 gene, one with c.451G>A, p.G151R and one with c.503T>G, p.L168R. Furthermore, the mRNA levels of steroidogenic enzymes including CYP11B1, CYP11B2, HSD3B2, CYP17A1, CYP11A1 and KCNJ5 in the 3 cases did not differ from those in 8 pure APAs not showing any of the above conditions for autonomous cortisol secretion. In addition, all 8 pure APAs harbored mutations of the KCNJ5 gene. These findings suggested that at least some aldosterone- and cortisol-co-secreting adrenal tumors have mutations of the KCNJ5 gene, suggesting the origin to be APA, and pure APAs may show a high incidence of KCNJ5 mutations.
Thyrotropin-releasing hormone (TRH) is a major stimulator of thyrotropin-stimulating hormone (TSH) synthesis in the anterior pituitary, though precisely how TRH stimulates the TSH? 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 TSH? 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 TSH? gene, and the NR4A1 gene belonged to the same cluster as and showed a similar expression profile to the TSH? 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 TSH? 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 TSH? 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 TSH? gene, and 2) TRH mediated induction of the TSH? gene, at least in part by sequential stimulation of the NR4A1-TSH? genes through a PKC and ERK1/2 pathway.
Mutations of the KCNJ5 gene have recently been identified in patients with aldosterone-producing adenomas (APA).
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.