YM750, an ACAT Inhibitor, Acts on Adrenocortical Cells to Inhibit Aldosterone Secretion Due to Depolarization.

Primary aldosteronism (PA) is considered the most common form of secondary hypertension, which is associated with excessive aldosterone secretion in the adrenal cortex. The cause of excessive aldosterone secretion is the induction of aldosterone synthase gene (CYP11B2) expression by depolarization of adrenocortical cells. In this study, we found that YM750, an Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, acts on adrenocortical cells to suppress CYP11B2 gene expression and aldosterone secretion. YM750 inhibited the induction of CYP11B2 gene expression by KCl stimulation, but not by angiotensin II and forskolin stimulation. Interestingly, YM750 did not inhibit KCl-stimulated depolarization via an increase in intracellular calcium ion concentration. Moreover, ACAT1 expression was relatively abundant in the zona glomerulosa (ZG) including these CYP11B2-positive cells. Thus, YM750 suppresses CYP11B2 gene expression by suppressing intracellular signaling activated by depolarization. In addition, ACAT1 was suggested to play an important role in steroidogenesis in the ZG. YM750 suppresses CYP11B2 gene expression and aldosterone secretion in the adrenal cortex, suggesting that it may be a potential therapeutic agent for PA.

Comparative proteomic analysis to identify the novel target gene of angiotensin II in adrenocortical H295R cells.

Angiotensin II (Ang II) is a well-known peptide that maintains the balance of electrolytes in the higher vertebrates. Ang II stimulation in the adrenal gland induces the synthesis of mineralocorticoids, mainly aldosterone, through the up-regulation of aldosterone synthase (CYP11B2) gene expression. Additionally, it has been reported that Ang II activates multiple signaling pathways such as mitogen-activated protein kinase (MAPK) and Ca2+ signaling. Although Ang II has various effects on the cellular signaling in the adrenal cells, its biological significance, except for the aldosterone synthesis, is still unclear. In this study, we attempted to search the novel target gene(s) of Ang II in the human adrenal H295R cells using a proteomic approach combined with stable isotopic labeling using amino acid in cell culture (SILAC). Interestingly, we found that Ang II stimulation elevated the expression of phosphofructokinase type platelet (PFKP) in both protein and mRNA levels. Moreover, transactivation of PFKP by Ang II was dependent on extracellular-signal-regulated kinase (ERK) 1/2 activation. Finally, we observed that Ang II treatment facilitated glucose uptake in the H295R cells. Taken together, we here identified PFKP as a novel target gene of Ang II, indicating that Ang II not only stimulates steroidogenesis but also affects glucose metabolism.

The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression.

Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+ signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.

Effects of Adipocyte-derived Factors on the Adrenal Cortex.

BACKGROUND AND OBJECTIVE: Obesity is highly complicated by hypertension and hyperglycemia. In particular, it has been proposed that obesity-related hypertension is caused by adipocyte-derived factors that are recognized as undetermined proteins secreted from adipocytes. Adipocyte-derived factors have been known to be related to aldosterone secretion in the adrenal gland. So far, Wnt proteins, CTRP-1, VLDL, LDL, HDL and leptin have been demonstrated to stimulate aldosterone secretion. In contrast, it has not yet been clarified whether adipocyte-derived factors also affect adrenal cortisol secretion. METHODS AND RESULTS: In the present study, we investigated the effect of adipocyte-derived factors on cortisol synthase gene CYP11B1 mRNA expression in vitro study using adrenocortical carcinoma H295R cells and mouse fibroblast 3T3-L1cells. Interestingly, adipocyte-derived factors were demonstrated to have the ability to stimulate CYP11B1 mRNA expression. CONCLUSION: Since CYP11B1 is well known as a limiting enzyme of cortisol synthesis, our study suggests that adipocyte-derived factors may stimulate cortisol secretion, as well as aldosterone secretion. Taken together, adipocyte-derived factors may be the cause of metabolic syndrome due to their stimulating effects on aldosterone/cortisol secretion. Therefore, the innovation of novel drugs against them may possibly be a new approach against metabolic syndrome.

High glucose stimulates expression of aldosterone synthase (CYP11B2) and secretion of aldosterone in human adrenal cells.

Aldosterone synthase is the key rate-limiting enzyme in adrenal aldosterone production, and induction of its gene (CYP11B2) results in the progression of hypertension. As hypertension is a frequent complication among patients with diabetes, we set out to elucidate the link between diabetes mellitus and hypertension. We examined the effects of high glucose on CYP11B2 expression and aldosterone production using human adrenal H295R cells and a stable H295R cell line expressing a CYP11B2 5'-flanking region/luciferase cDNA chimeric construct. d-glucose (d-glu), but not its enantiomer l-glucose, dose dependently induced CYP11B2 transcription and mRNA expression. A high concentration (450 mg·dL-1) of d-glu time dependently induced CYP11B2 transcription and mRNA expression. Moreover, high glucose stimulated secretion of aldosterone into the media. Transient transfection studies using deletion mutants/nerve growth factor-induced clone B (NGFIB) response element 1 (NBRE-1) point mutant of CYP11B2 5'-flanking region revealed that the NBRE-1 element, known to be activated by transcription factors NGFIB and NURR1, was responsible for the high glucose-mediated effect. High glucose also induced the mRNA expression of these transcription factors, especially that of NURR1, but NURR1 knockdown using its siRNA did not affect high glucose-induced CYP11B2 mRNA expression. Taken together, it is speculated that high glucose may induce CYP11B2 transcription via the NBRE-1 element in its 5'-flanking region, resulting in the increase in aldosterone production although high glucose-induced NURR1 is not directly involved in the effect. Additionally, glucose metabolism and calcium channels were found to be involved in the high glucose effect. Our observations suggest one possible explanation for the high incidence of hypertension in patients with diabetes.

A ubiquitin-proteasome inhibitor bortezomib suppresses the expression of CYP11B2, a key enzyme of aldosterone synthesis.

CYP11B2 is a key enzyme involved in the synthesis of the mineralocorticoid aldosterone. CYP11B2 expression in the adrenal glands is controlled by the renin-angiotensin system (RAS), and plays an important role in the maintenance of electrolyte metabolism in higher organisms. Abnormal overexpression of CYP11B2 results in the disruption of mineral balance and can lead to hypertension. Though the molecular mechanism of the regulation of CYP11B2 expression has remained elusive, we hypothesize that compounds that prevent CYP11B2 expression could represent a novel class of antihypertensive drugs. In this study, we established a high-throughput screening system to identify such compounds, and subsequently carried out chemical screening. We found that the ubiquitin-proteasome inhibitor bortezomib could suppress CYP11B2 expression and secretion of aldosterone induced by angiotensin II (Ang II) in adrenocortical H295R cells. Moreover, bortezomib down-regulated the Cyp11b2 mRNA expression facilitated in the adrenal gland of Tsukuba hypertensive mice, resulting in subsequent lowering of their blood pressures. Furthermore, we observed the characteristic alteration of H3K27ac in the adrenal CYP11B2 gene promoter induced by Ang II stimulation, which was found to be disrupted by bortezomib. Taken together, these results suggest the possibility of developing novel antihypertensive drugs that prevent CYP11B2 expression.

Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells.

DNA methylation is closely involved in the regulation of cellular differentiation, including chondrogenic differentiation of mesenchymal stem cells. Recent studies showed that Ten-eleven translocation (TET) family proteins converted 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, 5-formylcytosine and 5carboxylcytosine by oxidation. These reactions constitute potential mechanisms for active demethylation of methylated DNA. However, the relationship between the DNA methylation patterns and the effects of TET family proteins in chondrocyte differentiation is still unclear. In this study, we showed that DNA hydroxylation of 5mC was increased during chondrocytic differentiation of C3H10T1/2 cells and that the expression of Tet1 was particularly enhanced. Moreover, knockdown experiments revealed that the downregulation of Tet1 expression caused decreases in chondrogenesis markers such as type 2 and type 10 collagens. Furthermore, we found that TET proteins had a site preference for hydroxylation of 5mC on the Insulin-like growth factor 1 (Igf1) promoter in chondrocytes. Taken together, we showed that the expression of Tet1 was specifically facilitated in chondrocyte differentiation and Tet1 can regulate chondrocyte marker gene expression presumably through its hydroxylation activity for DNA.

Effects of RXR Agonists on Cell Proliferation/Apoptosis and ACTH Secretion/Pomc Expression.

Various retinoid X receptor (RXR) agonists have recently been developed, and some of them have shown anti-tumor effects both in vivo and in vitro. However, there has been no report showing the effects of RXR agonists on Cushing's disease, which is caused by excessive ACTH secretion in a corticotroph tumor of the pituitary gland. Therefore, we examined the effects of synthetic RXR pan-agonists HX630 and PA024 on the proliferation, apoptosis, ACTH secretion, and pro-opiomelanocortin (Pomc) gene expression of murine pituitary corticotroph tumor AtT20 cells. We demonstrated that both RXR agonists induced apoptosis dose-dependently in AtT20 cells, and inhibited their proliferation at their higher doses. Microarray analysis identified a significant gene network associated with caspase 3 induced by high dose HX630. On the other hand, HX630, but not PA024, inhibited Pomc transcription, Pomc mRNA expression, and ACTH secretion dose-dependently. Furthermore, we provide new evidence that HX630 negatively regulates the Pomc promoter activity at the transcriptional level due to the suppression of the transcription factor Nur77 and Nurr1 mRNA expression and the reduction of Nur77/Nurr1 heterodimer recruiting to the Pomc promoter region. We also demonstrated that the HX630-mediated suppression of the Pomc gene expression was exerted via RXRα. Furthermore, HX630 inhibited tumor growth and decreased Pomc mRNA expression in corticotroph tumor cells in female nude mice in vivo. Thus, these results indicate that RXR agonists, especially HX630, could be a new therapeutic candidate for Cushing's disease.

Retinoic acid receptor-α up-regulates proopiomelanocortin gene expression in AtT20 corticotroph cells.

Cushing's disease is a disorder caused by excessive ACTH secretion from a corticotroph tumor of the pituitary gland. Although its standard therapy is a transsphenoidal surgery, innovation of novel medical treatments for the disease is urgently necessary. Retinoic acid (RA) has been reported to suppress adrenocorticotropic hormone (ACTH) secretion in Cushing's disease. However, the role of RA receptor (RAR) in proopiomelanocortin (Pomc) gene expression remains uncertain. We here examined the involvement of RARα in Pomc regulation using AtT20 corticotroph cells. Surprisingly, a synthetic RARα agonist Am80 increased Pomc mRNA expression, CRH-induced ACTH secretion, and Pomc promoter activity. Small interfering RNA-mediated RARα-knockdown suppressed both basal and Am80-induced Pomc promoter activity. RARα-overexpression dose-dependently increased Pomc promoter activity. Pomc promoter mutation analysis revealed that both Tpit and NeuroD1 binding elements were responsible for the Am80-mediated effect. Am80 increased Tpit expression while RAR antagonist LE540 suppressed the increase. Tpit-overexpression increased Pomc promoter activity. Mammalian two-hybrid assay revealed that Am80 induced NeuroD1-RARα interaction. NeuroD1-overexpression enhanced the Am80-induced Pomc promoter activity, which was suppressed by NeuroD1 truncated mutant-overexpression. RARα thus positively regulates ACTH secretion/Pomc gene expression through interaction with NeuroD1 and Tpit expression increase. The present observation will be useful for the future development of the RA/retinoid-derived therapeutics of the disease.

Angiotensin II receptor blockers differentially affect CYP11B2 expression in human adrenal H295R cells.

We generated a stable H295R cell line expressing aldosterone synthase gene (CYP11B2) promoter/luciferase chimeric reporter construct that is highly sensitive to angiotensin II (AII) and potassium, and defined AII receptor blocker (ARB) effects. In the presence of AII, all ARBs suppressed AII-induced CYP11B2 transcription. However, telmisartan alone increased CYP11B2 transcription in the absence of AII. Telmisartan dose-dependently increased CYP11B2 transcription/mRNA expression and aldosterone secretion. Experiments using CYP11B2 promoter mutants indicated that the Ad5 element was responsible. Among transcription factors involved in the element, telmisartan significantly induced NGFIB/NURR1 expression. KN-93, a CaMK inhibitor, abrogated the telmisartan-mediated increase of CYP11B2 transcription/mRNA expression and NURR1 mRNA expression, but not NGFIB mRNA expression. NURR1 over-expression significantly augmented the telmisartan-mediated CYP11B2 transcription, while high-dose olmesartan did not affect it. Taken together, telmisartan may stimulate CYP11B2 transcription via NGFIB and the CaMK-mediated induction of NURR1 that activates the Ad5 element, independent of AII type 1 receptor.

Schinzel-Giedion syndrome: a further cause of early myoclonic encephalopathy and vacuolating myelinopathy.

Here, we report a male child with Schinzel-Giedion syndrome associated with intramyelinic edema detected on brain magnetic resonance imaging (MRI) and persistent suppression-burst pattern on electroencephalography (EEG) with erratic myoclonus of the extremities and face. Similar to nonketotic hyperglycinemia, Schinzel-Giedion syndrome may be recognized as another causative genetic disease of early myoclonic encephalopathy and vacuolating myelinopathy.

A case of autoimmune hepatitis with Graves' disease treated by propylthiouracil.

A 58-year-old woman was referred to our hospital because of liver dysfunction. Her serum levels of AST (619 IU/l) and ALT (603 IU/l) had increased. Histological findings in the liver biopsy were compatible to autoimmune hepatitis (AIH), and the diagnosis of AIH was confirmed by the diagnostic criteria. She was admitted to a nearby hospital 3 years ago, and diagnosed with Graves' disease. She received methimazole (MMI) at first, which was discontinued due to liver injury in one month, then propylthiouracil (PTU) was administered. One year later, transaminase increased and was decreased by stopping PTU administration. PTU was restarted after her transaminase decreased, but a recurrence of hepatotoxicity was observed, and she was referred to our hospital. Oral prednisolone decreased liver function immediately. In this case, PTU-induced liver injury was suspected as a possible trigger of AIH. While PTU remains a commonly used drug in the treatment of hyperthyroidism, severe liver injury is reported in some cases. If liver injury is observed in patients treated with PTU, rechallenge is not recommended in order to avoid severe hepatotoxicity.

A case with central and peripheral hypomyelination with hypogonadotropic hypogonadism and hypodontia (4H syndrome) plus cataract.

Hypomyelination with hypogonadotropic hypogonadism and hypodontia (4H syndrome) is a rare disease, characterized by both central and peripheral hypomyelination. We describe a 21-year-old male with mildly progressive ataxia, mental retardation, pituitary hypogonadotropic hypogonadism, delayed dentition, and cataract. Brain magnetic resonance imaging showed hypomyelinated white matter, cerebellar atrophy, and a thin corpus callosum. The literature suggests that abnormal findings upon sural nerve biopsy may indicate peripheral hypomyelination, even in the absence of clinically and physiologically evident peripheral neuropathy. A sural nerve biopsy of this patient was normal, and this finding is further discussed. Taken together with previous reports, this case suggests that 4H syndrome can be regarded as a spectrum disorder, the cardinal signs of which may be central hypomyelination, ataxia, hypogonadotropic hypogonadism, and hypodontia.

Respiratory failure was improved by growth hormone substitution in a patient with hypopituitarism.

A 33-year-old man was referred to the Department of Respiratory Medicine in our hospital due to severe hypercapnic respiratory failure in March 2008. His respiratory function test showed severe restrictive pulmonary dysfunction, and respiratory muscle strength assessed by measuring maximal static expiratory and inspiratory mouth pressures was severely impaired. After non-invasive positive pressure ventilation was started, he was referred to the Endocrinology Department as he was diagnosed as hypopituitarism in his childhood. Pituitary MRI demonstrated pituitary stalk agenesis, and hormonal examination showed that he had severe growth hormone (GH) deficiency. GH replacement therapy was started in August 2008 and his arterial blood gas values and respiratory muscle strength were improved in 6 months. The current case demonstrated that GH deficiency could be a cause of severe respiratory failure.

Successful treatment with sumatriptan in a case with cyclic vomiting syndrome combined with 18q- syndrome.

The authors present a 14-year-old girl with 18q- syndrome combined with cyclic vomiting syndrome. Since the age of 5 years, she has been admitted to hospital 30 times. Despite trying many prophylactic treatments, no medication has inhibited the vomiting attacks successfully. Intranasal sumatriptan was effective at halting the vomiting attacks. This is the first case of 18q- syndrome combined with cyclic vomiting syndrome successfully treated with sumatriptan. This report may allow us to consider sumatriptan use in patients suffering from misery attack of cyclic vomiting syndrome combined with chromosomal abnormality of 18q- syndrome.

Genetic analysis of two Japanese patients with non-classical 21-hydroxylase deficiency.

We report two Japanese women with androgen excess symptoms. Analyses of 21-hydroxylase gene demonstrated that a 24-year-old Japanese woman had a homozygous mutation of IVS2-13 A/C>G, while a 25-year-old Japanese woman had a compound heterozygous mutation of I172N and E245del1nt, a novel mutation which would result in completely nonfunctional enzyme due to a frame shift. As IVS2-13 A/C>G and I172N have been classified as mutations leading to severe impairment in enzyme activity, this study not only clarified a novel mutation causing 21-hydroxylase deficiency, but also demonstrated that genotype and phenotype do not correlate well in these cases.

Utility of subtraction ictal SPECT images in detecting focal leading activity and understanding the pathophysiology of spasms in patients with West syndrome.

PURPOSES: The aims of the study were to evaluate the detectability of focal leading activity in three cases of West syndrome having focal abnormal activity on EEG by comparing subtraction ictal images and raw ictal images, and to interpret the results in 16 cases. METHODS: Subtraction images were constructed using iNeurostat (revision 2). RESULTS: In three cases with focal abnormal activity on EEG, subtraction ictal images reflected the EEG findings; in contrast, raw ictal images did not. Diverse degrees of cortical hyperperfusion, ranging from zero to 10 sites, seen in the other 13 cases seemed to reflect spasm pathophysiology and rapid spasm propagation. Subtraction ictal images also allowed the ready detection of hyperperfusion of subcortical structures and of a tight cortico-subcortical relationship in a subset of cases. CONCLUSIONS: We showed the superiority of subtraction ictal images in detecting the focal epileptic region and in showing propagation pathways from the cortex to subcortical structures. A subset of spasms in WS may be focal cortical-onset secondarily generalized seizures. We believe that subtraction analysis is valuable in patients with complex WS who have partial seizures and spasms simultaneously along with focal epileptic EEG activity, as they will likely be candidates for epilepsy surgery.

Direct and indirect modulation of neuropeptide Y gene expression in response to hypoglycemia in rat arcuate nucleus.

Expression of neuropeptide Y (Npy) heteronuclear (hn) RNA, an indicator of gene transcription, was significantly increased in the arcuate nucleus of rats 30min after insulin injection. Npy hnRNA levels were also increased significantly in response to hypoglycemia in rats in which the hypothalamus was deafferentated, although the absolute levels were significantly lower than in sham-operated rats. Direct effects of lowering glucose levels on Npy gene expression were also confirmed in hypothalamic organotypic cultures. Thus, Npy gene transcription in the arcuate nucleus increases rapidly in response to hypoglycemia, and both direct and indirect inputs are involved in the rapid upregulation.

Osteopontin is dispensable for protection against high load systemic fungal infection.

Osteopontin (OPN) is a multi-functional cytokine which is involved in the pathogenesis of autoimmune disease. We previously reported that thrombin-cleaved form of OPN plays a pathogenic role in murine model of rheumatoid arthritis (RA) by using neutralizing antibody (M5) reacting against the cryptic epitope within OPN, exposed by thrombin cleavage of OPN. It has been shown that OPN-deficient mice are susceptible to various infections, demonstrating the protective role of OPN against various infectious diseases. However, it remains to be clarified whether and how OPN is involved in protection against systemic fungal infection. In a murine model of systemic fungal infection, OPN-deficient mice showed the increase in the susceptibility to low load, but not to high load fungal infection, indicating the protective of OPN against mild or severe forms of infections. However, mice treatment with M5 antibody did not alter the susceptibility to both high and low load fungal infection. These experiments suggest that in sharp contrast to the complete abrogation of OPN expression in OPN-deficient mice, the neutralization of OPN by antibody against thrombin-cleaved form of OPN does not interfere with the host defense against high and low load fungal infection. These findings suggest that the neutralizing antibody which is specific for the epitope of thrombin-cleaved OPN may become an attractive therapeutic means for the treatment of RA without interfering host defense system.

Glucocorticoids increase neuropeptide Y and agouti-related peptide gene expression via adenosine monophosphate-activated protein kinase signaling in the arcuate nucleus of rats.

Recent studies suggest that the AMP-activated protein kinase (AMPK) signaling in the hypothalamus is the master regulator of energy balance. We reported in previous studies that glucocorticoids play a permissive role in the regulation of orexigenic neuropeptide Y (Npy) gene expression in the arcuate nucleus. In this study, we examined whether any cross talk occurs between glucocorticoids and AMPK signaling in the hypothalamus to regulate Npy as well as agouti-related peptide (Agrp) gene expression in the arcuate nucleus. In the hypothalamic organotypic cultures, the addition to the medium of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside, increased phosphorylated AMPK (p-AMPK) as well as phosphorylated acetyl-coenzyme A carboxylase (p-ACC) in the explants, accompanied by significant increases in Npy and Agrp gene expression in the arcuate nucleus. The incubation with dexamethasone (DEX) also activated AMPK signaling in the explants, accompanied by significant increases in Npy and Agrp gene expression in the arcuate nucleus. The addition of the AMPK inhibitor compound C to the medium, which blocked increases of p-AMPK and p-ACC by DEX, significantly attenuated Npy and Agrp gene expression stimulated by DEX. Furthermore, p-AMPK and p-ACC levels in the arcuate nucleus were significantly decreased in adrenalectomized rats compared with sham-operated rats, and a replacement of glucocorticoids reversed the AMPK signaling in adrenalectomized rats. Thus, our data demonstrated that glucocorticoids up-regulate the Npy and Agrp gene expression in the arcuate nucleus through AMPK signaling, suggesting that the activation of the hypothalamic APMK signaling by glucocorticoids might be essential to the energy homeostasis.