Pituitary Tumors: Molecular Insights, Diagnosis, and Targeted Therapy.

The anterior pituitary gland comprises a heterogeneous population of pituitary cells [...].

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.

Vasopressin Expressed in Hypothalamic CRF Neurons Causes Impaired Water Diuresis in Secondary Adrenal Insufficiency.

Patients with secondary adrenal insufficiency can present with impaired free water excretion and hyponatremia, which is due to the enhanced secretion of vasopressin (AVP) despite increased total body water. AVP is produced in magnocellular neurons in the paraventricular nucleus of the hypothalamus (PVH) and supraoptic nucleus and in parvocellular corticotropin-releasing factor (CRF) neurons in the PVH. This study aimed to elucidate whether magnocellular AVP neurons or parvocellular CRF neurons coexpressing AVP are responsible for the pathogenesis of hyponatremia in secondary adrenal insufficiency. The number of CRF neurons expressing copeptin, an AVP gene product, was significantly higher in adrenalectomized AVP-floxed mice (AVPfl/fl) than in sham-operated controls. Adrenalectomized AVPfl/fl mice supplemented with aldosterone showed impaired water diuresis under ad libitum access to water or after acute water loading. They became hyponatremic after acute water loading, and it was revealed under such conditions that aquaporin-2 (AQP2) protein levels were increased in the kidney. Furthermore, translocation of AQP2 to the apical membrane was markedly enhanced in renal collecting duct epithelial cells. Remarkably, all these abnormalities observed in the mouse model for secondary adrenal insufficiency were ameliorated in CRF-AVP-/- mice that lacked AVP in CRF neurons. Our study demonstrates that CRF neurons in the PVH are responsible for the pathogenesis of impaired water excretion in secondary adrenal insufficiency.

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.

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.

Molecular Mechanisms Underlying Stress Response and Resilience.

A variety of stressors induce various physiological responses by modulating sympathetic, neuroendocrine, and behavioral systems [...].

Growth differentiation factor-15 modulates adrenocorticotropic hormone synthesis in murine AtT-20 corticotroph cells.

Growth differentiation factor-15 (GDF15) is a stress-responsive cytokine that plays important roles in regulation of inflammatory responses, cell growth, and cell differentiation. However, the nature of these roles remains unclear. Here, we aimed to examine the regulatory effects of dexamethasone on Gdf15 expression in murine AtT-20 corticotroph cells. Human Gdf15 promoter-driven luciferase reporter constructs were transfected into corticotroph cells to analyze their promoter activity. The effects of time and concentration of dexamethasone on Gdf15 and proopiomelanocortin (Pomc) mRNA levels were assessed using quantitative real-time polymerase chain reaction. Dexamethasone induced Gdf15 transcription and mRNA levels as well as GDF15 production in transfected cells, whereas reduced the Pomc mRNA levels. GDF15 modulated adrenocorticotropic hormone (ACTH) synthesis, and the dexamethasone-mediated reduction in Pomc mRNA levels were partially relieved upon Gdf15 knockdown. We concluded that GDF15 modulated ACTH production in pituitary corticotrophs in an autocrine manner by suppressing Pomc expression and subsequently mediating the negative feedback effect of glucocorticoids, thereby contributing to pituitary stress response and homeostasis.

Effects of tubastatin A on adrenocorticotropic hormone synthesis and proliferation of AtT-20 corticotroph tumor cells.

Cushing's disease is an endocrine disorder characterized by hypercortisolism, mainly caused by autonomous production of ACTH from pituitary adenomas. Autonomous ACTH secretion results in excess cortisol production from the adrenal glands, and corticotroph adenoma cells disrupt the normal cortisol feedback mechanism. Pan-histone deacetylase (HDAC) inhibitors inhibit cell proliferation and ACTH production in AtT-20 corticotroph tumor cells. A selective HDAC6 inhibitor has been known to exert antitumor effects and reduce adverse effects related to the inhibition of other HDACs. The current study demonstrated that the potent and selective HDAC6 inhibitor tubastatin A has inhibitory effects on proopiomelanocortin (Pomc) and pituitary tumor-transforming gene 1 (Pttg1) mRNA expression, involved in cell proliferation. The phosphorylated Akt/Akt protein levels were increased after treatment with tubastatin A. Therefore, the proliferation of corticotroph cells may be regulated through the Akt-Pttg1 pathway. Dexamethasone treatment also decreased the Pomc mRNA level. Combined tubastatin A and dexamethasone treatment showed additive effects on the Pomc mRNA level. Thus, tubastatin A may have applications in the treatment of Cushing's disease.

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.

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.

Hypothalamic Regulation of Corticotropin-Releasing Factor under Stress and Stress Resilience.

This review addresses the molecular mechanisms of corticotropin-releasing factor (CRF) regulation in the hypothalamus under stress and stress resilience. CRF in the hypothalamus plays a central role in regulating the stress response. CRF stimulates adrenocorticotropic hormone (ACTH) release from the anterior pituitary. ACTH stimulates glucocorticoid secretion from the adrenal glands. Glucocorticoids are essential for stress coping, stress resilience, and homeostasis. The activated hypothalamic-pituitary-adrenal axis is suppressed by the negative feedback from glucocorticoids. Glucocorticoid-dependent repression of cAMP-stimulated Crf promoter activity is mediated by both the negative glucocorticoid response element and the serum response element. Conversely, the inducible cAMP-early repressor can suppress the stress response via inhibition of the cAMP-dependent Crf gene, as can the suppressor of cytokine signaling-3 in the hypothalamus. CRF receptor type 1 is mainly involved in a stress response, depression, anorexia, and seizure, while CRF receptor type 2 mediates "stress coping" mechanisms such as anxiolysis in the brain. Differential effects of FK506-binding immunophilins, FKBP4 and FKBP5, contribute to the efficiency of glucocorticoids under stress resilience. Together, a variety of factors contribute to stress resilience. All these factors would have the differential roles under stress resilience.

Differential Effects of Fkbp4 and Fkbp5 on Regulation of the Proopiomelanocortin Gene in Murine AtT-20 Corticotroph Cells.

The hypothalamic-pituitary-adrenal axis is stimulated in response to stress. When activated, it is suppressed by the negative feedback effect of glucocorticoids. Glucocorticoids directly inhibit proopiomelanocortin (Pomc) gene expression in the pituitary. Glucocorticoid signaling is mediated via glucocorticoid receptors, 11ß-hydroxysteroid dehydrogenases, and the FK506-binding immunophilins, Fkbp4 and Fkbp5. Fkbp4 and Fkbp5 differentially regulate dynein interaction and nuclear translocation of the glucocorticoid receptor, resulting in modulation of the glucocorticoid action. Here, we explored the regulation of Fkbp4 and Fkbp5 genes and their proteins with dexamethasone, a major synthetic glucocorticoid drug, in murine AtT-20 corticotroph cells. To elucidate further roles of Fkbp4 and Fkbp5, we examined their effects on Pomc mRNA levels in corticotroph cells. Dexamethasone decreased Pomc mRNA levels as well as Fkpb4 mRNA levels in mouse corticotroph cells. Dexamethasone tended to decrease Fkbp4 protein levels, while it increased Fkpb5 mRNA and its protein levels. The dexamethasone-induced decreases in Pomc mRNA levels were partially canceled by Fkbp4 knockdown. Alternatively, Pomc mRNA levels were further decreased by Fkbp5 knockdown. Thus, Fkbp4 contributes to the negative feedback of glucocorticoids, and Fkbp5 reduces the efficiency of the glucocorticoid effect on Pomc gene expression in pituitary corticotroph cells.

Renal Injuries in Primary Aldosteronism: Quantitative Histopathological Analysis of 19 Patients With Primary Adosteronism.

[Figure: see text].

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.

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.

Association between higher urinary normetanephrine and insulin resistance in a Japanese population.

Since activation of the sympathetic nervous system is associated with both impaired insulin secretion and insulin resistance, or namely with diabetes, evaluation of such activation in ordinary clinical settings may be important. Therefore, we evaluated the relationships between urinary concentrations of the catecholamine metabolites, urinary normetanephrine (U-NM) and urinary metanephrine (U-M), and glucose metabolism in a general population. From 1,148 participants in the 2016 population-based Iwaki study of Japanese, enrolled were 733 individuals (gender (M/F): 320/413; age: 52.1±15.1), who were not on medication affecting serum catecholamines, not diabetic, and had complete data-set and blood glucose levels appropriate for the evaluation of insulin secretion and resistance, using homeostasis model assessment (HOMA-ß and HOMA-R, respectively). Univariate linear regression analyses revealed significant correlations between both U-NM and U-M, and HOMA-ß, but adjustment for multiple factors correlated with HOMA indices abolished these (ß = -0.031, p = 0.499, and ß = -0.055, p = 0.135, respectively). However, the correlation between U-NM and HOMA-R observed using univariate linear regression analysis (ß = 0.132, p<0.001) remained significant even after these adjustments (ß = 0.107, p = 0.007), whereas U-M did not correlate with HOMA-R. Furthermore, use of the optimal cut-off value of U-NM for the prediction of insulin resistance (HOMA-R >1.6) determined by ROC analysis (0.2577 mg/gCr) showed that individuals at risk had an odds ratio of 2.65 (confidence interval: 1.42-4.97) after adjustment for the same factors used above. Higher U-NM concentrations within the physiologic range are a significant risk factor for increased insulin resistance in a general Japanese population.

Ubiquitin-specific protease 8 inhibitor suppresses adrenocorticotropic hormone production and corticotroph tumor cell proliferation.

Cushing's disease is primarily caused by autonomic hypersecretion of adrenocorticotropic hormone (ACTH) from a pituitary adenoma. In Cushing's disease, mutations in the ubiquitin-specific protease 8 (USP8) have been detected. These mutations are associated with hyperactivation of USP8 that prevent epidermal growth factor receptor (EGFR) degradation. This leads to increased EGFR stability and results in the maintenance of EGFR signaling in Cushing's disease. USP8 inhibitors can suppress the growth of various tumors. In this study, the effects of a potent USP8 inhibitor, DUBs-IN-2, on ACTH production and cell proliferation were examined in mouse corticotroph tumor (AtT-20) cells. Proopiomelanocortin (Pomc) mRNA levels and ACTH levels were decreased in AtT-20 cells by DUBs-IN-2. Further, cell proliferation was inhibited, and apoptosis was induced by DUBs-IN-2. Transcript levels of pituitary tumor-transforming gene 1 (Pttg1), a pituitary tumor growth marker, were increased; and transcript levels of stress response growth arrest and DNA damage-inducible 45 (Gadd45ß) and Cdk5 and ABL enzyme substrate 1 (Cables1) mRNA levels were increased in response to the drug. Gadd45ß or Cables1 knockdown partially inhibited the DUBs-IN-2-induced decrease in cell proliferation, but not Pomc mRNA levels. Both GADD45ß and CABLES1 may be responsible, at least in part, for the USP8-induced suppression of corticotroph tumor cell proliferation. USP-8 may be a new treatment target in Cushing's disease.

Two Cases of Hypopituitarism Caused by Intrasellar Aneurysm.

Giant internal carotid artery aneurysms sometimes extend into the sellar region, which rarely but occasionally results in hypopituitarism due to the compression of the normal pituitary gland or hypothalamus. Hyponatremia is a known complication of hypopituitarism. We herein report two cases of hypopituitarism caused by intrasellar aneurysm of different origins, resulting in hyponatremia. Untreated hypopituitarism may lead to lethargy, coma, cardiac arrhythmia, and death. Therefore, we must be alert for the occurrence of giant intrasellar aneurysm, as it causes hypopituitarism. The prompt diagnosis and treatment of hypopituitarism are necessary to prevent this fatal outcome.

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.

Progression of Hypopituitarism and Hypothyroidism after Treatment with Pembrolizumab in a Patient with Adrenal Metastasis from Non-small-cell Lung Cancer.

Pembrolizumab, or anti-programmed death receptor 1 antibody, is an immune checkpoint inhibitor that can cause immune-related adverse events. We herein report for the first time the progression of hypopituitarism and hypothyroidism after treatment with pembrolizumab in a patient with adrenal metastasis of non-small-cell lung cancer. Severe primary hypothyroidism occurred three weeks after the first administration of pembrolizumab. Four months after the discontinuation of pembrolizumab, isolated adrenocorticotropic hormone (ACTH) deficiency was noted. Corticotropin-releasing hormone and rapid ACTH tests performed repeatedly showed that the patient's pituitary and adrenal function had been gradually deteriorating. It is important to diagnose adrenal insufficiency without delay in order to prevent adrenal crisis.