Correlation of magnetic resonance images with neuropathology of irreversible metronidazole-induced encephalopathy: an autopsy case report.

BACKGROUND: Neurological symptoms and radiographic abnormalities may remain in a small proportion of patients with metronidazole-induced encephalopathy (MIE). Although experimental animal models of MIE have suggested a Wernicke's encephalopathy-like pathology, little is known about the histopathological features of MIE. Here we report the first autopsy case of irreversible MIE. CASE PRESENTATION: A 72-year-old Japanese woman with pancreatic neuroendocrine tumour and metastatic tumours in the liver developed intraabdominal bleeding from a hepatic abscess. She was administered metronidazole for 79 days (1.5 g/day), which caused dysarthria followed by hand tremor and altered mental status. Brain magnetic resonance imaging at the time of onset revealed hyperintensities in the deep white matter of the bilateral parietal lobes and splenium of the corpus callosum on diffusion-weighted imaging (DWI) with reduced apparent diffusion coefficient (ADC) values. Despite the improvement of dysarthria and hand tremor, her cognition remained affected even after the withdrawal of metronidazole. She died of pancreatic neuroendocrine tumour at the age of 74 years. Histopathological examinations of the brain confirmed a combination of severe demyelination and moderate axonal degeneration, which corresponded to the regions showing abnormal signal intensities on DWI with reduced ADC values. There were no pathological findings suggestive of Wernicke's encephalopathy in the brain. CONCLUSION: We have demonstrated the clinical, radiographic and histopathological aspects of irreversible MIE. Hyperintensities on DWI with reduced ADC values in affected regions may indicate a poor clinical prognosis due to irreversible pathological damage.

Identification of a homozygous c.1039C>T (p.R347C) variant in CYP17A1 in a 67-year-old female patient with partial 17α-hydroxylase/17,20-lyase deficiency.

17α-Hydroxylase/17,20-lyase deficiency (17OHD) is caused by pathogenic mutations in CYP17A1. Impaired 17α-hydroxylase and 17,20-lyase activities typically induce hypertension, hypokalemia, sexual infantilism, and amenorrhea. Most patients with 17OHD are diagnosed in adolescence. Here, we report a female (46, XX) patient with 17OHD who was diagnosed at the age of 67 years. Genetic analysis was performed using direct DNA sequencing of polymerase chain reaction (PCR) products and multiplex ligation-dependent probe amplification (MLPA) analysis. Direct DNA sequencing revealed a homozygous c.1039C>T in CYP17A1, corresponding to a p.R347C amino acid change. MLPA probe signals showed that the CYP17A1 mutation was present in the homozygous carrier state. The patient's dehydroepiandrosterone sulfate and androstenedione levels were extremely low, despite elevated adrenocorticotropic hormone (ACTH) and normal cortisol levels. A corticotropin-releasing hormone (CRH) test showed no response of cortisol, despite a normal response of ACTH. Rapid ACTH injection resulted in elevations in the deoxycorticosterone, corticosterone, aldosterone, and 17-hydroxypregnenolone levels, but not in the cortisol level. These results suggested that 17α-hydroxylase/17,20-lyase activities were partially impaired. Computed tomography revealed bilateral adrenal hyperplasia and a hypoplastic uterus. A high basal plasma ACTH level and a discrepancy between ACTH and cortisol responses in a CRH test may provide a definitive diagnostic clue for this disease.

Differential effects of ß-arrestin1 and ß-arrestin2 on somatostatin receptors in murine AtT-20 corticotroph tumor cells.

Autonomous production of adrenocorticotropic hormone (ACTH) from pituitary corticotroph adenomas is the primary cause of Cushing's disease. Somatostatin receptor, a G protein-coupled receptor (GPCR), types 2 (SSTR2) and 5 (SSTR5) mRNA expression is greater than that of other SSTR subtypes in human corticotroph adenomas. Further, the multiligand SOM230 shows potent effects in decreasing ACTH plasma levels and urinary free cortisol levels in patients with Cushing's disease. We previously showed that both Sstr2 and Sstr5 mRNA levels were unaffected by SOM230 treatment, suggesting that both receptors might not be downregulated by the agonist. Intracellular molecules, such as ß-arrestins, modulate ligand activated-receptor responses. In the present study, we determined regulation of ß-arrestin1 and ß-arrestin2 by SOM230 and dexamethasone in murine AtT-20 corticotroph tumor cells. In addition, we examined the effects of ß-arrestin1 and ß-arrestin2 on Sstr mRNA and their protein levels. SOM230 treatment increased ß-arrestin1 mRNA levels and did not alter ß-arrestin2 mRNA levels. SOM230 treatment could induce ß-arrestin1 production in corticotroph tumor cells. Dexamethasone treatment decreased ß-arrestin2 mRNA levels. ß-arrestin2 knockdown increased proopiomelanocortin, and both Sstr2 and Sstr5 mRNA and their protein levels. The ß-arrestin2 knockdown-increased proopiomelanocortin mRNA levels were canceled by SOM230 treatment.

Presence of aberrant adrenocorticotropic hormone precursors in two cases of McCune-Albright syndrome.

McCune-Albright syndrome (MAS) is a rare disorder. MAS is classically defined by the occurrence of fibrous dysplasia, café-au-lait skin macules, and precocious puberty. In addition to precocious puberty, other hyperfunctioning endocrinopathies may occur. We evaluated hypothalamic-pituitary-adrenal function in two cases of typical MAS associated with fibrous dysplasia and growth hormone excess. Pituitary adenoma or hyperplasia was not detected by magnetic resonance imaging. Hormonal data showed normal or low cortisol levels, despite high ACTH levels in the blood. A high ratio of circulating ACTH to cortisol was found in the two cases. Insulin tolerance and CRH tests showed hyper-responses of ACTH and an insufficient increase in cortisol levels. No involvement of 11ß-HSD1 by GH excess was suggested because basal levels of ACTH and cortisol showed no changes, even after therapy for acromegaly by somatostatin analogues. Patients with Cushing's disease cases of pituitary macroadenoma can have high circulating ACTH precursor levels, and elevated ACTH precursors have been observed in ectopic ACTH syndrome. Autonomous cortisol excess was excluded by the level of midnight cortisol and the level of cortisol after a low-dose dexamethasone suppression test in the two cases. Finally, the gel filtration profiles of immunoreactive ACTH contents showed the presence of aberrant ACTH precursors. To the best of our knowledge, there have been no reports of MAS associated with aberrant ACTH precursors. Our findings in these cases emphasize that attention should be to secretion of inactive ACTH precursors in MAS.

Secondary oxalosis induced by xylitol concurrent with lithium-induced nephrogenic diabetes insipidus: a case report.

BACKGROUND: Xylitol is an approved food additive that is widely used as a sweetener in many manufactured products. It is also used in pharmaceuticals. Secondary oxalosis resulting from high dietary oxalate has been reported. However, reported cases of oxalosis following xylitol infusion are rare. CASE PRESENTATION: A 39-year-old man with a 16-year history of organic psychiatric disorder was hospitalized for a laparoscopic cholecystectomy because of cholecystolithiasis. He had been treated with several antipsychotics and mood stabilizers, including lithium. The patient had polyuria (> 4000 mL/day) and his serum sodium levels ranged from 150 to 160 mmol/L. Urine osmolality was 141 mOsm/L, while serum arginine vasopressin level was 6.4 pg/mL. The patient was diagnosed with nephrogenic diabetes insipidus (NDI), and lithium was gradually discontinued. Postoperative urine volumes increased further to a maximum of 10,000 mL/day, and up to 10,000 mL/day of 5% xylitol was administered. The patient's consciousness level declined and serum creatinine increased to 4.74 mg/dL. This was followed by coma and metabolic acidosis. After continuous venous hemodiafiltration, serum sodium improved to the upper 140 mmol/L range and serum creatinine decreased to 1.25 mg/dL at discharge. However, polyuria and polydipsia of approximately 4000 mL/day persisted. Renal biopsy showed oxalate crystals and decreased expression of aquaporin-2 (AQP2) in the renal tubules. Urinary AQP2 was undetected. The patient was discharged on day 82 after admission. CONCLUSIONS: Our patient was diagnosed with lithium-induced NDI and secondary oxalosis induced by excess xylitol infusion. NDI became apparent perioperatively because of fasting, and an overdose of xylitol infusion led to cerebrorenal oxalosis. Our patient received a maximum xylitol dose of 500 g/day and a total dose of 2925 g. Patients receiving lithium therapy must be closely monitored during the perioperative period, and rehydration therapy using xylitol infusion should be avoided in such cases.

Evaluation of growth hormone-releasing peptide-2 for diagnosis of thyrotropin-producing pituitary adenomas.

Thyrotropin (TSH)-producing adenomas are a rare cause of hyperthyroidism and are a type of functional pituitary adenoma. The diagnosis of TSH-producing adenoma is a challenging problem in clinical endocrinology. Since growth hormone-releasing peptide-2 (GHRP-2) fails to induce TSH secretion in normal subjects, the effect of GHRP-2 on TSH levels was therefore examined in patients with TSH-producing adenomas. A total of 5 patients (4 women and 1 man) referred to our departments for further evaluation of pituitary hormones were followed-up using the GHRP-2, TSH-releasing hormone (TRH), octreotide, and bromocriptine tests to examine and evaluate TSH secretory dynamics in TSH-producing adenomas. Of 5 patients, 2 (40%) showed such a significant response, defined as a >50% increase in serum TSH level above baseline in the GHRP-2 test. Additionally, 1 patient showed a 48% increase in serum TSH level. In 1 patient whose adenoma was completely removed, basal serum concentrations of TSH were sufficiently suppressed after the operation, and serum TSH levels failed to increase in response to GHRP-2 administration. In 4 patients (80%), a poor response of serum TSH levels was observed in the TRH test. In 2 out of 5 patients (40%), serum TSH levels were significantly decreased following octreotide administration. No patient demonstrated a significant response to the bromocriptine test. In addition to TRH test, the GHRP-2 test as a potential diagnostic tool for TSH-producing pituitary adenomas.

Regulation of the expression of corticotropin-releasing factor gene by pyroglutamylated RFamide peptide in rat hypothalamic 4B cells.

Pyroglutamylated RFamide peptide (QRFP), an important regulator of metabolism and energy homeostasis, has orexigenic effects. QRFP acts via a specific receptor, Gpr103. Gpr103 mRNA is expressed in the rat hypothalamic paraventricular nucleus (PVN). In the PVN, corticotropin-releasing factor (CRF), which plays a central role in regulating the stress response and is produced in response to stress, stimulates the release of adrenocorticotropic hormone from the anterior pituitary. We hypothesized that QRFP regulates CRF gene expression directly in the hypothalamus, and thus examined the direct effect of QRFP on the promoter activity and mRNA levels of CRF in hypothalamic cells. To examine these pathways, we used hypothalamic 4B cells, a homologous PVN neuronal cell line. Gpr103a and Gpr103b mRNA, and Gpr103 (a and b) proteins were expressed in the hypothalamic cells. The Gpr103 mRNA and protein levels were increased by QRFP. QRFP also stimulated CRF mRNA levels and CRF promoter activity directly in 4B cells following their transfection with the CRF promoter. The protein kinase A (PKA) and protein kinase C (PKC) pathways were involved in the QRFP-induced increases in CRF promoter activity. QRFP stimulated cAMP response element-binding protein (CREB) phosphorylation. CREB phosphorylation was inhibited by a PKC inhibitor. PKC-dependent signaling would be upstream of the CREB phosphorylation. Thus, QRFP-dependent pathways are involved in the regulation of CRF gene expression in the hypothalamus.

Inhibitory effects of trichostatin A on adrenocorticotropic hormone production and proliferation of corticotroph tumor AtT-20 cells.

Cushing's disease is primarily caused by adrenocorticotropic hormone (ACTH)-producing pituitary adenomas. Pituitary tumor-transforming gene 1 (PTTG1) expression, a hallmark of pituitary tumors, stimulates pituitary cell proliferation. Histone deacetylases (HDACs) play an important role in regulating gene transcription and HDAC inhibitors induce cellular differentiation and suppress tumor cell proliferation. HDAC inhibitors also repress PTTG1 mRNA levels. Trichostatin A (TSA) is a potent cell-permeable HDAC inhibitor that blocks cell cycle progression. In the present study, we determined the effect of TSA on ACTH production and cellular proliferation in mouse AtT-20 corticotroph tumor cells. TSA decreased proopiomelanocortin (POMC) mRNA levels in AtT-20 cells and reduced ACTH levels in the culture medium of these cells. The TSA-induced decreases in POMC mRNA levels were not modulated when TSA and dexamethasone were simultaneously administered. Drug treatment also decreased AtT-20 cell proliferation, induced apoptosis, and increased the percentage of cells in G0/G1 phase using flow cytometry. TSA decreased PTTG1 mRNA levels. Furthermore, PTTG1 knockdown inhibited cellular proliferation. Its knockdown also inhibited POMC mRNA and ACTH levels. TSA inhibits ACTH production and corticotroph tumor cell proliferation. TSA may inhibit cellular proliferation, and ACTH synthesis and secretion by decreasing PTTG1 expression.

Mutations in NFKB2 and potential genetic heterogeneity in patients with DAVID syndrome, having variable endocrine and immune deficiencies.

BACKGROUND: DAVID syndrome is a rare condition combining anterior pituitary hormone deficiency with common variable immunodeficiency. NFKB2 mutations have recently been identified in patients with ACTH and variable immunodeficiency. A similar mutation was previously found in Nfkb2 in the immunodeficient Lym1 mouse strain, but the effect of the mutation on endocrine function was not evaluated. METHODS: We ascertained six unrelated DAVID syndrome families. We performed whole exome and traditional Sanger sequencing to search for causal genes. Lym1 mice were examined for endocrine developmental anomalies. RESULTS: Mutations in the NFKB2 gene were identified in three of our families through whole exome sequencing, and in a fourth by direct Sanger sequencing. De novo origin of the mutations could be demonstrated in three of the families. All mutations lie near the C-terminus of the protein-coding region, near signals required for processing of NFΚB2 protein by the alternative pathway. Two of the probands had anatomical pituitary anomalies, and one had growth and thyroid hormone as well as ACTH deficiency; these findings have not been previously reported. Two children of one of the probands carried the mutation and have to date exhibited only an immune phenotype. No mutations were found near the C-terminus of NFKB2 in the remaining two probands; whole exome sequencing has been performed for one of these. Lym1 mice, carrying a similar Nfkb2 C-terminal mutation, showed normal pituitary anatomy and expression of proopiomelanocortin (POMC). CONCLUSIONS: We confirm previous findings that mutations near the C-terminus of NFKB2 cause combined endocrine and immunodeficiencies. De novo status of the mutations was confirmed in all cases for which both parents were available. The mutations are consistent with a dominant gain-of-function effect, generating an unprocessed NFKB2 super-repressor protein. We expand the potential phenotype of such NFKB2 mutations to include additional pituitary hormone deficiencies as well as anatomical pituitary anomalies. The lack of an observable endocrine phenotype in Lym1 mice suggests that the endocrine component of DAVID syndrome is either not due to a direct role of NFKB pathways on pituitary development, or else that human and mouse pituitary development differ in its requirements for NFKB pathway function.

Advances in Molecular Pathophysiology and Targeted Therapy for Cushing's Disease.

Cushing's disease is caused by autonomous secretion of adrenocorticotropic hormone (ACTH) from corticotroph pituitary neuroendocrine tumors. As a result, excess cortisol production leads to the overt manifestation of the clinical features of Cushing's syndrome. Severe complications have been reported in patients with Cushing's disease, including hypertension, menstrual disorders, hyperglycemia, osteoporosis, atherosclerosis, infections, and mental disorders. Cushing's disease presents with a variety of clinical features, ranging from overt to subtle. In this review, we explain recent advances in molecular insights and targeted therapy for Cushing's disease. The pathophysiological characteristics of hormone production and pituitary tumor cells are also explained. Therapies to treat the tumor growth in the pituitary gland and the autonomous hypersecretion of ACTH are discussed. Drugs that target corticotroph pituitary neuroendocrine tumors have been effective, including cabergoline, a dopamine receptor type 2 agonist, and pasireotide, a multi-receptor-targeted somatostatin analog. Some of the drugs that target adrenal hormones have shown potential therapeutic benefits. Advances in potential novel therapies for Cushing's disease are also introduced.

Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells.

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

Biochemical Evaluation by Confirmatory Tests after Unilateral Adrenalectomy for Primary Aldosteronism.

Primary aldosteronism (PA) is the most common cause of endocrine hypertension. Unilateral PA can be cured using unilateral adrenalectomy (Adx). PA surgery outcome (PASO) criteria, which include clinical and biochemical outcomes, have been proposed to evaluate PA cure after Adx. However, clinical outcomes are often inconsistent with biochemical outcomes. In addition, although confirmatory tests are included as endpoints of biochemical outcomes in the PASO criteria, their clinical usefulness has not yet been established. We evaluated clinical parameters and confirmatory test results before and after Adx in 16 patients with PA and assessed the usefulness of the confirmatory tests. The following were the clinical outcomes after Adx: 37.5% complete success, 62.5% partial success, and 0% absent success. The ratio of biochemical complete success was as follows: 69% aldosterone/renin ratio and basal plasma aldosterone concentration, 19% as assessed by the captopril challenge test, 47% as assessed by the saline infusion test, 30% as assessed by the furosemide upright test, and 100% urine aldosterone. Of these, biochemical complete success was judged in four cases by aldosterone/renin ratio and basal plasma aldosterone concentration, one case by captopril challenge test, five cases by saline infusion test, and one case by furosemide upright test. Although clinical outcomes and urine aldosterone levels improved after Adx, confirmatory tests failed to improve in some cases. The current criteria are not considered useful for biochemical evaluation after Adx. To determine whether additional treatment with mineralocorticoid receptor antagonists is required, more accurate biochemical criteria should be established after Adx.

Multiple endocrine neoplasia type 1 with Zollinger-Ellison syndrome: clinicopathological analysis of a Japanese family with focus on menin immunohistochemistry.

Background: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the occurrence of multiple epithelial neuroendocrine tumors (NETs) and non-NETs in various organs. MEN1 encodes a 610-amino acid-long tumor suppressor protein, menin. The optimal treatment for multiple tumors, identification of the most critical tumors for patient prognosis, and menin immunohistochemistry findings remain controversial. Therefore, we aimed to elucidate these issues through a histological analysis of tumors and tumor-like lesions in a Japanese family, comprising a father and his two sons, who had MEN1 with Zollinger-Ellison syndrome (ZES). Patients and methods: All family members had a germline alteration in exon 10, c.1714-1715 del TC of MEN1, and exhibited multiple synchronous and metachronous tumors. The patients had pulmonary NETs, hyperparathyroidism, hypergastrinemia, pituitary adenomas, pancreaticoduodenal NETs, adrenocortical adenoma with myelolipoma, nodular goiter of the thyroid, lipomas, and angiofibroma. Most tumors were resected and histologically examined. We compared their clinical courses and tumor histology, and conducted menin immunohistochemistry (IHC). Results: Two patients died of pulmonary NET G2. One patient who underwent pancreaticoduodenectomy was cured of ZES; however, the two other patients who did not undergo pancreaticoduodenectomy suffered persistent ZES despite treatment with octreotide. Menin IHC revealed varying NET intensities, ranging from positive to negative stains. Conclusion: Pancreaticoduodenectomy is the most effective treatment for ZES. Long-term follow-up is essential for pulmonary NET G2 owing to the risk of distant metastasis and/or multiplicity. Moreover, the variability of menin IHC in MEN1-related tumors may indicate the pattern of tumor formation rather than the diagnostic utility of menin in MEN1.

Diabetic ketoacidosis shortly after COVID-19 vaccination in a non-small-cell lung cancer patient receiving combination of PD-1 and CTLA-4 inhibitors: A case report.

We describe a case of diabetic ketoacidosis (DKA) shortly after the SARS-CoV-2 (COVID-19) vaccination in a 65-year-old woman with non-small-cell lung cancer under a combination treatment of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors. She had no history of diabetic mellitus. A few days after the second shot of COVID-19 vaccination, she developed DKA. We speculate that the immune-related adverse event and immunogenicity of vaccination synergistically induced DKA.

Eosinophil counts can be a predictive marker of immune checkpoint inhibitor-induced secondary adrenal insufficiency: a retrospective cohort study.

Immune checkpoint inhibitors (ICIs) treatment can result in endocrine immune-related adverse events (irAEs), including pituitary dysfunction. Quick diagnosis of secondary adrenal insufficiency (AI) is challenging because no universal definition of ICI-induced secondary AI has been agreed. The aim of this study was to clarify the clinical features of ICI-induced secondary AI that can be used for screening in standard clinical practice. This retrospective study was performed using the medical records of patients who received ICIs at Hirosaki University Hospital between 1 September 2014 and 31 January 2021. Longitudinal clinical data of patients who developed AI were analyzed and compared with the data of thyroid irAEs. Regression analysis showed a significant correlation between ICI-induced secondary AI and absolute or relative eosinophil counts at pre-onset of AI, as well as differences or rate of increase in eosinophil counts at baseline and at pre-onset. Absolute eosinophil counts > 198.36/µL or relative eosinophil counts > 5.6% at pre-onset, and a difference of 65.25/µL or a rate of eosinophil count increase of 1.97 between the baseline and at pre-onset showed the best sensitivity and specificity. This is the first report to demonstrate that eosinophil counts can be a predictor of ICI-induced secondary AI.

Ghrelin stimulates corticotropin-releasing factor and vasopressin gene expression in rat hypothalamic 4B cells.

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) play a central role in regulating the stress response. In response to stress, CRF and AVP neurons in the hypothalamic paraventricular nucleus secrete the peptides to stimulate the release of adrenocorticotropic hormone from the anterior pituitary. Ghrelin, an endogenous ligand of the growth hormone-releasing peptide receptors (GHSR), has been shown to stimulate the release of CRF and AVP by rat hypothalamic explants. However, little is known about the ability of the ghrelin signaling pathways to activate the CRF and AVP genes in the hypothalamus. In the present study, we examined the direct effect of ghrelin on CRF and AVP gene expression in hypothalamic 4B cells, which show the characteristics of the hypothalamic parvocellular paraventricular nucleus neurons. Cells were transfected with CRF or AVP promoter to examine the activity of each promoter. Ghrelin stimulated the promoter activities and mRNA levels for both CRF and AVP. The involvement of a protein kinase pathway was examined using inhibitors. Protein kinase A and phospholipase C pathways were shown to be involved in ghrelin-induced increases in both CRF and AVP promoter activities. GHSR type 1a (GHSR1a) mRNA levels were also increased by ghrelin, and these ghrelin-induced levels were suppressed by a GHSR1a antagonist. Thus, ghrelin-dependent pathways are involved in the regulation of CRF and AVP gene expression in the hypothalamus: ghrelin, an orexigenic hormone, stimulates CRF, an anorexigenic/anxiogenic factor in the hypothalamus, resulting in hypothalamic-pituitary-adrenal axis activation to stimulate the release of glucocorticoids.

Involvement of histone deacetylase 1/2 in adrenocorticotropic hormone synthesis and proliferation of corticotroph tumor AtT-20 cells.

Cushing's disease is mainly caused by autonomous production of adrenocorticotropic hormone (ACTH) from pituitary adenomas. In our previous study, a histone deacetylase (HDAC) inhibitor, trichostatin A, inhibited cell proliferation and ACTH production via decreased pituitary tumor-transforming gene 1 (PTTG1) in AtT-20 mouse corticotroph tumor cells. In the present study, we examined the effects of romidepsin, a potent and selective HDAC1/2 inhibitor, on cell proliferation and ACTH synthesis. To elucidate further potential mechanisms of romidepsin, we examined the effects of HDAC1/2 on proopiomelanocortin (Pomc) and Pttg1 mRNA levels and cell proliferation. Small interfering RNA-mediated knockdown was used to decrease HDAC1 or 2. Romidepsin treatment decreased Pomc and Pttg1 mRNA levels, and cell proliferation. The drug also increased Hdac1 and decreased Hdac2 mRNA levels. Hdac1 knockdown decreased basal Pttg1 mRNA levels and cell proliferation, but not Pomc mRNA levels. Romidepsin treatment decreases ACTH synthesis in corticotroph tumor cells. Romidepsin suppresses cell proliferation via PTTG1. HDAC1 is also involved in the proliferation of corticotroph cells via PTTG1.

Involvement of nuclear factor-kB and Nurr-1 in cytokine-induced transcription of proopiomelanocortin gene in AtT20 corticotroph cells.

OBJECTIVE: The precise mechanism whereby proinflammatory cytokines activate the hypothalamo-pituitary-adrenal axis is still unclear. We examined whether transcription factors nuclear factor (NF)-kappaB and Nurr-1 are involved in the cytokine-induced proopiomelanocortin (POMC) gene expression. METHODS: The mouse corticotropinoma cell line AtT20 was treated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta). Real-time PCR, luciferase assay and Western blotting were conducted to assess the gene expression, promoter activity and protein expression in various conditions. RESULTS: Intrinsic expression of NF-kappaB was confirmed by RT-PCR. An active component of NF-kappaB (p65) was upregulated in the nuclear fraction by both TNF-alpha and IL-1beta treatment in a dose- and time-related manner. These cytokines potently stimulated the promoter activity of NF-kappaB and Nurr-1. We also found rapid upregulation of the Nurr-1 gene and protein after treatment with these cytokines. Cotreatment of the cells with either of the cytokines and corticotropin-releasing hormone resulted in additive effects. Cytokine-induced Nurr-1 transcription and Nurr-1 transcription induced by overexpression of NF-kappaB were both blunted by mutagenesis within the NF-kappaB responsive element, which implies that Nurr-1 upregulation specifically requires NF-kappaB binding to its own DNA-binding site. Proinflammatory cytokines exert positive effects on POMC gene expression, which were inhibited by pretreatment with a specific NF-kappaB inhibitor. CONCLUSION: These results together imply that Nurr-1 expression is a connecting point between neuroendocrine and immune systems in mediating cytokine-induced POMC gene expression.

Cytokines induce NF-κB, Nurr1 and corticotropin-releasing factor gene transcription in hypothalamic 4B cells.

OBJECTIVE: In the hypothalamus, corticotropin-releasing factor (CRF) plays a central role in regulating stress responses. Cytokines are important mediators of the interaction between the neuroendocrine and immune systems, and are implicated in the regulation of CRF expression. Following inflammatory challenges, interleukin (IL)-1 or IL-6 stimulates the hypothalamic-pituitary-adrenal axis. CRF promoter contains multiple nuclear factor (NF)-kappaB and Nurr1 binding sites. In the present study, we determined the ability of the signaling pathways to activate the CRF gene in the hypothalamic paraventricular nucleus following inflammatory challenge. METHODS: Cytokine-induced changes in CRF gene expression were examined in the hypothalamic system. Luciferase assay and Western blotting were performed to assess transcriptional activity and the nuclear translocation of transcriptional factors. RESULTS: IL-1beta, IL-6 and tumor necrosis factor (TNF)-alpha stimulated the nuclear expression levels of NF-kappaB, NF-kappaB-dependent Nurr1 and c-Fos proteins. Direct stimulatory effects of TNF-alpha and IL-1beta, in addition to IL-6, were found on the transcriptional activity of the CRF gene in hypothalamic 4B cells. CONCLUSION: These cytokines are involved in the regulation of CRF gene activity in hypothalamic cells.

Evaluation of plasma, salivary, and urinary cortisol levels for diagnosis of Cushing's syndrome.

As a screening test for Cushing's syndrome, the evaluation of late-night cortisol levels is indispensable. We evaluated the usefulness and accuracy of plasma, urinary, and salivary cortisol levels measured late at night for the diagnosis of Cushing's syndrome. High cortisol levels (> 5 microg/dL) during the night are indicative of Cushing's syndrome, although night plasma cortisol levels are not readily reproducible because of the stressful situation. There was no correlation between plasma and urinary cortisol levels late at night, and late-night urinary cortisol levels provided weak information for the diagnosis of Cushing's syndrome. By contrast, late-night plasma and salivary cortisol levels showed a positive correlation, and salivary cortisol sampling was found to be useful for the diagnosis of Cushing's syndrome, because more than 0.4 microg/dL of late-night salivary cortisol levels gave a sensitivity of 86% and a specificity of 100% in our hospital. This method is also useful for the diagnosis of early or mild stage Cushing's syndrome, so-called subclinical Cushing's syndrome. Inherent differences between assays make it difficult to define optimal diagnostic criteria. However, the relative levels of salivary cortisol ratio, which is presented as a relative level, compared with the mean levels of healthy subjects in each institute, is useful for the screening of Cushing's syndrome as the cut-off level of 1.5 shows both high sensitivity and specificity in subclinical and overt Cushing's syndrome. Late-night salivary cortisol measurement is therefore a primary method of choice in the screening of patients suspected of having Cushing's syndrome.