Satoru Sakihara

Pituitary adenylate cyclase-activating polypeptide stimulates corticotropin-releasing factor, vasopressin and interleukin-6 gene transcription in hypothalamic 4B cells

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are the two major regulatory peptides in the hypothalamic-pituitary-adrenal axis. CRF, produced in the hypothalamic paraventricular nucleus (PVN) in response to stress, is secreted into the pituitary portal circulation, resulting in the release of adrenocorticotropic hormone from the anterior pituitary. AVP is synthesized in the PVN and supraoptic nucleus by various stressors. Hypothalamic 4B cells coexpress CRF and AVP. In 4B cells transfected with either a CRF or an AVP promoter-luciferase construct, forskolin increased the transcriptional activity of CRF or AVP. In the present study, we tried to determine whether pituitary adenylate cyclase-activating polypeptide (PACAP) regulates both CRF and AVP genes in the hypothalamic cells, because receptors for PACAP were expressed in the hypothalamic cells. PACAP stimulated activity of both CRF and AVP promoter via protein kinase A pathway. PACAP stimulated interleukin (IL)-6 promoter activity and the levels of IL-6 mRNA and protein. IL-6 stimulated activity of both CRF and AVP promoter in a dose-dependent manner. Finally, we found that the stimulatory effects of PACAP on both activities were significantly inhibited by treatment with anti-IL-6 monoclonal antibody. These data suggest that PACAP is involved in regulating the synthesis of IL-6 mRNA and IL-6 protein, and that the increase in endogenous IL-6 also contributes to stimulate the expression of both CRF and AVP genes. Taken together, these findings indicate that PACAP stimulates the transcription of CRF, AVP, and IL-6 genes in hypothalamic 4B cells.

Corticotropin-releasing factor (CRF) is involved in the acute anorexic effect of α-melanocyte-stimulating hormone: A study using CRF-deficient mice

Alpha-melanocyte-stimulating hormone (alpha-MSH) and its receptors are critical and indispensable for maintaining appropriate feeding behavior and energy homeostasis in both mice and humans. Corticotropin-releasing factor (CRF) is a candidate for mediating the anorexic effect of alpha-MSH. In the present study, we examined whether CRF and its receptors are involved in the anorexic effect of alpha-MSH, using CRF-deficient (CRFKO) mice and a CRF receptor antagonist. Intracerebroventricular administration of NDP-MSH, a synthetic alpha-MSH analogue, suppressed food intake in wild-type (WT) mice. This effect was abolished by pretreatment with a non-selective CRF receptor antagonist, astressin, suggesting that the effect of alpha-MSH-induced anorexia was mediated by a CRF receptor. In CRFKO mice, administration with NDP-MSH did not affect food intake at an early phase (0-4h). In addition, CRF mRNA levels in the hypothalamus were significantly increased in NDP-MSH-treated mice. Therefore, our findings, using CRFKO, strongly support evidence that CRF is involved in the acute anorexic effect of alpha-MSH. On the other hand, NDP-MSH administered to CRFKO mice led to suppressed food intake at the late phase (4-12h), similar to the effect in WT mice. Further, NDP-MSH similarly reduced food intake during the late phase in all types of mice, including WT, CRFKO, and CRFKO with corticosterone replacement. The results would suggest that alpha-MSH-induced suppression of food intake at late phase was independent of glucocorticoids and CRF.

Differential regulation of corticotropin-releasing factor receptor type 1 (CRF1 receptor) mRNA via protein kinase A and mitogen-activated protein kinase pathways in rat anterior pituitary cells.

Corticotropin-releasing factor (CRF) receptor type 1 (CRF(1) receptor) mRNA levels are down-regulated by CRF via the cyclic AMP-protein kinase A (PKA) pathway. In this study, we focused on the involvement of both the mitogen-activated protein (MAP) kinase pathway and PKA in this regulation. Real-time PCR (RT-PCR) revealed that a MAP kinase, extracellular signal-regulated kinases 1/2, pathway was also involved in the down-regulation of CRF(1) receptor mRNA levels by CRF in the rat anterior pituitary (AP). Down-regulation of CRF(1) receptor mRNA levels was caused by a post-transcriptional system such as mRNA degradation, as incubation with CRF significantly decreased the half-life of CRF(1) receptor mRNA. Furthermore, pre-treatment with a PKA inhibitor completely blocked CRF-induced CRF(1) receptor mRNA destabilization, while pre-treatment with an extracellular signal-regulated kinases 1/2 inhibitor had no inhibitory effect. These results suggested that in the rat AP, down-regulation of CRF(1) receptor mRNA levels is caused by mRNA degradation via PKA, but not by the MAP kinase pathway.

A Multihormonal Pituitary Adenoma with Growth Hormone and Adrenocorticotropic Hormone Production, Causing Acromegaly and Cushing Disease

Pituitary adenoma with growth hormone (GH) and corticotropin (ACTH) production causing apparent acromegaly and Cushing disease is extremely rare. A 45-year-old woman had a pituitary macroadenoma and severe insulin resistance. Physical examination showed a fully developed acromegaly associated with mild Cushingoid features. Serum GH, insulin-like growth factor-I, ACTH, and cortisol levels were all elevated. Hormonal loading tests resulted in GH levels increasing paradoxically in response to thyrotropin-releasing hormone (TRH), but not corticotropin-releasing hormone (CRH). A similar unexpected increase in ACTH and cortisol levels occurred in response to TRH and GH-releasing hormone. After trans-sphenoidal resection of the pituitary macroadenoma immunohistochemistry revealed the presence of either diffuse but faintly GH-positive cells or sparse but distinct ACTH-stained cells. A marked amelioration of insulin resistance was observed postoperatively. The elevated ACTH and cortisol levels should therefore be investigated by CRH and dexamethasone suppression tests for the coexistence of Cushing disease to exclude the possibility of underlying ACTH-producing tumors.

A case of multiple endocrine neoplasia type II accompanied by thyroid medullary carcinoma and pheochromocytomas expressing corticotropin-releasing factor and urocortins.

A 38-year-old woman with RET gene mutation presented with tumors in her thyroid and bilateral adrenal glands. I-metaiodobenzylguanidine scintigraphy revealed accumulation of the radioisotope in both adrenal glands. Both plasma adrenaline and noradrenaline levels were elevated. The circadian rhythms for plasma adrenocorticotropic hormone (ACTH) and cortisol levels were disturbed. Plasma ACTH and cortisol levels failed to be suppressed by an overnight dexamethasone test, suggesting autonomic secretion of ACTH and cortisol, although the patient had no typical Cushingoid features, hypertension, or impaired glucose tolerance. Pathological examination showed that these tumors were pheochromocytoma and thyroid medullary carcinoma, respectively, both of which highly expressed corticotropin-releasing factor, urocortin1, and urocortin3. Together with the endocrinological and pathological observations, the patient was diagnosed as multiple endocrine neoplasia type II with corticotropin-releasing factor- and urocortin-producing tumors that stimulated ACTH and glucocorticoid secretion.

Regulation and role of p21-activated kinase 3 by corticotropin-releasing factor in mouse pituitary.

Corticotropin-releasing factor (CRF) is a major regulatory peptide in the hypothalamic-pituitary-adrenal (HPA) axis under stress conditions. In response to stress, CRF, produced in the hypothalamic paraventricular nucleus, releases adrenocorticotropic hormone (ACTH) from the anterior pituitary (AP). ACTH in turn stimulates the release of glucocorticoid from the adrenal glands. Glucocorticoid then inhibits hypothalamic production of CRF and pituitary production of ACTH. Mice lacking a functional gene for CRF (CRF KO) showed severe impairment of the HPA axis, indicating that CRF is required for its regulation. We applied oligonucleotide microarray analysis to the AP of CRF KO to identify gene expression induced by CRF. Twenty-four genes showed less than 60% expression in CRF KO compared with normal mice. Real-time PCR analysis revealed that p21-activated kinase 3 (Pak3), prohormone convertase type 1 (PC1), and CRF-binding protein (BP) mRNA expression levels were increased by CRF in AP cells. Both Pak3 and PC1 were also increased by dexamethasone in AP cells, while CRF-BP mRNA levels were reduced. Therefore, both Pak3 and PC1 mRNA levels would be regulated by both CRF and glucocorticoids. Pak3 knockdown inhibited CRF-induced cell viability in AtT-20 cells, suggesting the important role of Pak3 in the proliferation of corticotrophs.

Involvement of regulatory elements on corticotropin-releasing factor gene promoter in hypothalamic 4B cells.

Introduction: Corticotropin-releasing factor (CRF) plays a central role in controlling the hypothalamic-pituitary-adrenal (HPA) axis during stressful periods. CRF is synthesized and secreted in the hypothalamic paraventricular nucleus (PVN) in response to stress, and stimulates ACTH in the pituitary corticotrophs. ACTH stimulates the release of glucocorticoids from the adrenal glands, and glucocorticoids sequentially inhibit hypothalamic PVN production of CRF and pituitary production of ACTH. The effects of glucocorticoids on CRF gene regulation, however, are possibly tissue-specific since glucocorticoids stimulate CRF gene expression in the placenta and the bed nucleus of the stria terminalis, while they inhibit it in the hypothalamus. Methods and results: In a hypothalamic cell line, 4B, we found that forskolin-stimulated CRF gene transcription was mediated by a functional cAMP-response element (CRE), which included −220 to −233 bp on the CRF 5′-promoter region. Protein kinase A, protein kinase C, and p38 mitogen-activated protein kinase pathways contributed to forskolin-induced transcriptional activity of CRF in hypothalamic 4B cells. Glucocorticoid-dependent repression of cAMP-stimulated transcriptional activity of CRF was localized to promoter sequences between −278 and −233 bp, which included a glucocorticoid regulatory element and a serum response element. Conclusion: Taken together, these findings indicate that the regulatory elements, including CRE, negative glucocorticoid regulatory element, and a serum response element on the promoter, contribute to the regulation of CRF gene transcription in hypothalamic 4B cells.

Pheochromocytoma with histologic transformation to composite type, complicated by watery diarrhea, hypokalemia, and achlorhydria syndrome.

OBJECTIVE To describe the rare occurrence of histologic transformation of a pheochromocytoma to a composite type of tumor during a long-term follow-up, which was complicated by watery diarrhea, hypokalemia, and achlorhydria syndrome. METHODS We report the case of a 12-year-old girl who presented with headache, hypertension, and elevated catecholamine levels in the blood and urine. A tumor was found in the right adrenal gland and resected. When she was 15 years of age, multiple metastatic nodules were found in the lung and liver. Intensive chemotherapy was ineffective, and she underwent follow-up with conservative therapy. At 25 years of age, she complained of diarrhea. Laboratory studies revealed hypokalemia and an increase in the level of serum vasoactive intestinal polypeptide (VIP). A year later, she died of extensive metastatic disease. The primary and recurrent tumors at autopsy were histologically examined. RESULTS The primary tumor was pure pheochromocytoma, and the tumors at autopsy were a composite type of pheochromocytoma and ganglioneuroma. Only a few VIP-positive cells were found in the primary tumor, whereas both pheochromocytoma and ganglioneuroma cells of composite tumors were frequently positive for VIP. CONCLUSION Our case showed histologic transformation from pheochromocytoma to a composite type of tumor during a 14-year clinical course, which was associated with additional hormone production and a change in symptoms. Careful attention should be paid to the alteration of endocrine symptoms and hormone levels during prolonged follow-up of pheochromocytoma in young patients.

A Case of Adrenocortical Oncocytoma Occurring with Aldosteronoma

A 48-yr-old woman was referred to our hospital for evaluation of a left adrenal mass. An abdominal computed tomography scan demonstrated a 5.4 3.7-cm tumor in the left adrenal gland (Fig. 1, A and B). She had untreated hypertension on admission and no Cushingoid features. When in a seated position, the patient’s urinary aldosterone excretion rate was 19.4 g/d, plasma aldosterone concentration (PAC) was 20.8 ng/dl, and plasma renin activity (PRA) was 0.8 ng/ml h mid morning. Her plasma aldosterone to renin ratio was 26.0. She was diagnosed with normokalemic (serum potassium, 3.8 mmol/ liter) primary aldosteronism after captopril challenge (PAC, 15.2 ng/dl; PRA, 0.4 ng/ml h at 2 h) and furosemide-plus-upright tests (PAC, 65.4 ng/dl; PRA, 0.3 ng/ ml h at 2 h) (1). 18F-Fluorodeoxyglucose (FDG) positron emission tomography demonstrated a marked uptake of FDG in the left adrenal mass (Fig. 1, C and D). The mass was therefore clinically diagnosed as an aldosterone-producing adrenocortical carcinoma (2). After left adrenalectomy, the patient’s hypertension improved with normalizationofaldosterone to renin ratio.Lightmicroscopic examination revealed the cells within the main tumor body to have abundant eosinophilic cytoplasm and three positive scores in Weiss criteria, namely, nuclear atypia, architecture, and an eosinophilic cytoplasm (Fig. 2A). The eosinophilic tumor cells were positive for steroidogenic factor-1, but negative for steroidogenic enzymes [3 -hydroxysteroid dehydrogenase (3BHSD), P450c21, P450c17, and dehydroepiandrosterone sulfotransferase]. This main tumor body was therefore diagnosed as a true nonfunctioning adrenocortical tumor, an “oncocytoma” (3). A wellcircumscribed localized mass of clear cortical cells demonstrating marked P450 side-chain cleavage enzyme, 3BHSD and P450c21, and weak P450c17 immunoreactivity was detected around one margin of the oncocytoma (Fig. 2, B–E).Theattachedzonaglomerulosawashyperplasticbut negative for 3BHSD, evidence pointing toward a nonfunctioning albeit hyperplastic response in the neighboring normal glomerulosa. This part was therefore histopathologically diagnosed as an aldosteronoma. Although it could be one tumor with heterogeneity, this is the first

Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.