Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1, encoded by Hsd11b1). Here, we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth, but reduced in vivo tumor progression, which corresponded with increased frequencies of CD8+ tumor-infiltrating lymphocytes (TILs) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of conventional T cell activation in tumor-infiltrating Tregs. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11beta-HSD1 reduced tumor growth to the same degree as gene knockout and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11beta-HSD1 is well tolerated in clinical studies, these data suggest that targeting 11beta-HSD1 may be a beneficial adjunct in cancer therapy.

J Clin Invest | 2023 | M. D. Taves; S. Otsuka; M. A. Taylor; K. M. Donahue; T. J. Meyer; M. C. Cam; J. D. Ashwell | DOI 10.1172/JCI164599 | Mouse | Supernatant

Context: Pathogenic variants in KCNJ5, encoding the GIRK4 (Kir3.4) potassium channel, have been implicated in the pathogenesis of familial hyperaldosteronism type-III (FH-III) and sporadic primary aldosteronism (PA). In addition to aldosterone, glucocorticoids are often found elevated in PA in association with KCNJ5 pathogenic variants, albeit at subclinical levels. However, to date no GIRK4 defects have been linked to Cushing syndrome (CS).
Patient: We present the case of a 10-year-old child who presented with CS at an early age due to bilateral adrenocortical hyperplasia (BAH). The patient was placed on low-dose ketoconazole (KZL), which controlled hypercortisolemia and CS-related signs. Discontinuation of KZL for even 6 weeks led to recurrent CS.
Results: Screening for known genes causing cortisol-producing BAHs (PRKAR1A, PRKACA, PRKACB, PDE11A, PDE8B, ARMC5) failed to identify any gene defects. Whole-exome sequencing showed a novel KCNJ5 pathogenic variant (c.506T>C, p.L169S) inherited from her father. In vitro studies showed that the p.L169S variant affects conductance of the Kir3.4 channel without affecting its expression or membrane localization. Although there were no effects on steroidogenesis in vitro, there were modest changes in protein kinase A activity. In silico analysis of the mutant channel proposed mechanisms for the altered conductance.
Conclusion: We present a pediatric patient with CS due to BAH and a germline defect in KCNJ5. Molecular investigations of this KCNJ5 variant failed to show a definite cause of her CS. However, this KCNJ5 variant differed in its function from KCNJ5 defects leading to PA. We speculate that GIRK4 (Kir3.4) may play a role in early human adrenocortical development and zonation and participate in the pathogenesis of pediatric BAH.

Journal of Clinical Endocrinology & Metabolism | 2021 | C. Tatsi; A. G. Maria; C. Malloy; L. Lin; E. London; N. Settas; C. Flippo; M. Keil; F. Hannah-Shmouni; D. A. Hoffman; C. A. Stratakis | DOI 10.1210/clinem/dgab118 | Human | Cell culture

Chronic stress contributes to numerous human pathologies including cognition impairments and psychiatric disorders. Glucocorticoids are primary stress hormones that activate two closely related nuclear receptors, the glucocorticoid (GR) and mineralocorticoid receptor (MR), that are both highly expressed in the hippocampus. To investigate potential combinatorial actions of hippocampal GR and MR, we developed mice with conditional knockout of both GR and MR in the hippocampus and compared them to their single knockout counterparts. Mice lacking MR alone or both GR and MR in the hippocampus exhibited altered expression of multiple CA2-specific neuronal markers and enhanced cue-dependent learning in a conditioned fear test. Provocatively, in contrast to the single knockouts, mice depleted of both GR and MR showed profound neurodegeneration of the hippo-campus. Neuronal death was increased and neurogenesis was reduced in the dentate gyrus of the double knockout mice. Global gene expression assays of the knockout mice revealed a synergistic increase in the number of dysregulated genes in the hippocampus lacking both GR and MR. This large cohort of genes reliant on both GR and MR for expression was strongly associated with cell death and cell proliferation pathways. GR/MR com-plexes were detected in CA1 and dentate gyrus neurons suggesting receptor heterodimers contribute to the joint actions of GR and MR. These findings reveal an obligate role for MR signaling in regulating the molecular phenotype of CA2 neurons and demonstrate that combinatorial actions of GR and MR are essential for preserving dentate gyrus neurons and maintaining hippocampal health.

Neurobiology of Stress | 2021 | R. H. Oakley; S. D. Whirledge; M. G. Petrillo; N. V. Riddick; X. J. Xu; S. S. Moy; J. A. Cidlowski | ARTN 10036910.1016/j.ynstr.2021.100369 | Mouse | Serum

Adrenocortical cancer (ACC) is a rare and aggressive type of endocrine tumor with high risk of recurrence and metastasis. The overall survival of patients diagnosed with ACC is low and treatment for metastatic stages remain limited to mitotane, which has low efficiency in advanced stages of the disease and is associated with high toxicity. Therefore, identification of new biological targets to improve ACC treatment is crucial. Blockade of the Wnt/beta-catenin pathway decreased adrenal steroidogenesis and increased apoptosis of NCI-H295 human ACC cells, in vitro and in a xenograft mouse model. Aurora kinases play important roles in cell division during the G1M phase and their aberrant expression is correlated with a poor prognosis in different types of tumors. Hence, we hypothesized that inhibition of aurora kinases activity combined with the beta-catenin pathway blockade would improve the impairment of ACC cell growth in vitro. We studied the combinatorial effects of AMG 900, an aurora kinase inhibitor and PNU-74654, a beta-catenin pathway blocker, on proliferation, survival and tumor progression in multiple ACC cell lines: NCI-H295, CU-ACC1 and CU-ACC2. Exposure of ACC cells to the combination of AMG 900 with PNU-74654 decreased cell proliferation and viability compared to either treatment alone. In addition, AMG 900 inhibited cell invasion and clonogenesis compared to PNU-74654, and the combination showed no greater effects. In contrast, PNU-74654 was more effective in decreasing cortisol secretion. These data suggest that inhibition of aurora kinases activity combined with blockade of the beta-catenin pathway may provide a combinatorial approach for targeting ACC tumors.

Molecular and Cellular Endocrinology | 2021 | A. G. Maria; K. S. Borges; R. C. P. Lira; C. H. Thome; A. Berthon; L. Drougat; K. Kiseljak-Vassiliades; M. E. Wierman; F. R. Faucz; V. M. Faca; L. G. Tone; C. A. Stratakis | ARTN 111243
10.1016/j.mce.2021.111243 | Human | Cell culture

Introduction: Sickle cell disease (SCD) is a chronic illness that presents with a wide range of phenotypic variation. Stress may be a contributing factor to differences that are found in this population. Objectives: Our objective is to determine the relationship between hair cortisol content (HCC), a biomarker of stress, and other clinical measures in individuals with SCD. Methods: We collected hair samples and other clinical measures from 73 subjects with SCD (mean age: 39 +/- 12 years, 63% female). Results: HCC was lower among individuals who had greater than 30% hemoglobin S, compared with those who had less than 30% hemoglobin S (W=272.5, P=0.01). Lower HCC was also associated with report of not being on a chronic transfusion program (beta=48.34, SE=14.09, P=0.001) and higher ferritin levels (beta=-0.006, SE=0.002, P=0.02). Furthermore, HCC was significantly correlated with serum cortisol (rs=0.26, P=0.03) and corticosterone (rs=0.29, P=0.01). We also observed a consistent pattern of low steroid values among our population. Conclusion: Our findings suggest that individuals with higher hemoglobin S and ferritin, both markers of severe SCD, may have decreased cortisol levels. This is consistent with the relationship we observed between higher HCC among individuals who are on a chronic blood transfusion program, which typically increases quality of life. Our results suggest that hair cortisol may be an indicator in patients with SCD who could be at risk for developing adrenal insufficiency. We recommend that clinicians treating patients with SCD follow the Endocrine Society guidelines for testing for adrenal insufficiency and treat accordingly.

American Journal of Blood Research | 2021 | B. M. Hollister; M. Zilbermint; C. P. Minniti; A. J. Buscetta; K. E. Abdallah; S. You; S. J. Soldin; J. S. Meyer; C. A. Stratakis; V. L. Bonham | PMID: 34079627 | Human | Hair

Alcohol use disorder (AUD) is an enormous public health problem that poses significant social, medical, and economic burdens. Under AUD, the liver is one of the most adversely affected organs. As current therapies and protective drugs for AUD-mediated liver injury are very limited, the prevention and therapy of alcoholic liver disease are urgently needed. The present study aims to investigate the beneficial effects of tartary buckwheat extract (TBE), the important component of Maopu tartary buckwheat liquor, on both alcoholic-induced acute and chronic liver injuries. We show that the TBE administration, similar to curcumin, significantly reduces the elevated serum aspartate aminotransferase and alanine aminotransferase levels, improves liver index, alleviates the elevated contents of hepatic malondialdehye, and restores the decreased contents of hepatic glutathione both in acute and chronic liver injuries in alcohol-exposed rats. Furthermore, histopathological analyses show that a medium dose of TBE (16.70 ml/kg body weight) alleviates hepatocyte morphology changes in both acute and chronic alcohol exposure models. We also show the protective effects of TBE on the cell death rates of alcohol-exposed primary cultured hepatocytes, HepG2 hepatoma, and Huh 7 hepatoma cells. Furthermore, we demonstrate that TBE exerts hepatoprotection partly through inhibiting the mitochondrial cell death pathway by reducing cytochrome c release, caspase-9 and -3 activities, and the number of TUNEL-positive cells. These effects of TBE were accompanied by enhanced levels of Bcl-2 and Bcl-xL and autophagic cell death pathway by reducing Beclin-1 expression, as well as through promoting its anti-oxidant capacity by suppressing reactive oxygen species production. This study demonstrates, for the first time, the protective effect of TBE against alcohol-induced acute and chronic liver injury in vivo and in vitro. Given the dietary nature of tartary buckwheat, pueraria, lycium barbarum, and hawthorn, the oral intake of TBE or liquor contained TBE, e.g.. Maopu Tartary buckwheat liquor, compared with pure liquor consumption alone, may have the potential to alleviate alcoholic-induced liver injuries.

American Journal of Translational Research | 2020 | Q. Yang; C. L. Luo; X. M. Zhang; Y. C. Liu; Z. F. Wang; P. Cacciamani; J. Shi; Y. C. Cui; C. L. Wang; B. Sinha; B. Peng; G. Q. Tong; G. Das; E. Shah; Y. Gao; W. Li; Y. Y. Tu; D. Y. Qian; K. Shah; M. Akbar; S. H. Zhou; B. J. Song; X. Wang | PMID: 32051738 | Rat | Tissue homogenate

Local and systemic factors that influence renal structure and function in aging are not well understood. The secretory protein C1q/TNF-related protein 1 (CTRP1) regulates systemic metabolism and cardiovascular function. We provide evidence here that CTRP1 also modulates renal physiology in an age- and sex-dependent manner. In mice lacking CTRP1, we observed significantly increased kidney weight and glomerular hypertrophy in aged male but not female or young mice. Although glomerular filtration rate, plasma renin and aldosterone levels, and renal response to water restriction did not differ between genotypes, CTRP1-deficient male mice had elevated blood pressure. Echocardiogram and pulse wave velocity measurements indicated normal heart function and vascular stiffness in CTRP1-deficient animals, and increased blood pressure was not due to greater salt retention. Paradoxically, CTRP1-deficient mice had elevated urinary sodium and potassium excretion, partially resulting from reduced expression of genes involved in renal sodium and potassium reabsorption. Despite renal hypertrophy, markers of inflammation, fibrosis, and oxidative stress were reduced in CTRP1-deficient mice. RNA sequencing revealed alterations and enrichments of genes in metabolic processes in CTRP1-deficient animals. These results highlight novel contributions of CTRP1 to aging-associated changes in renal physiology.

Faseb Journal | 2020 | S. Rodriguez; H. C. Little; P. Daneshpajouhnejad; B. D. Shepard; S. Y. Tan; A. Wolfe; M. U. Cheema; S. Jandu; O. M. Woodward; C. C. Talbot; D. E. Berkowitz; A. Z. Rosenberg; J. L. Pluznick; G. W. Wong | DOI 10.1096/fj.201900558RR | Mouse | Serum

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Nature Chemical Biology | 2020 | E. D. Mock; M. Mustafa; O. Gunduz-Cinar; R. Cinar; G. N. Petrie; V. Kantae; X. Y. Di; D. Ogasawara; Z. V. Varga; J. Paloczi; C. Miliano; G. Donvito; A. C. M. van Esbroeck; A. M. F. van der Gracht; I. Kotsogianni; J. K. Park; A. Martella; T. van der Wel; M. Soethoudt; M. Jiang; T. J. Wendel; A. P. A. Janssen; A. T. Bakker; C. M. Donovan; L. I. Castillo; B. I. Florea; J. Wat; H. van den Hurk; M. Wittwer; U. Grether; A. Holmes; C. A. A. van Boeckel; T. Hankemeier; B. F. Cravatt; M. W. Buczynski; M. N. Hill; P. Pacher; A. H. Lichtman; M. van der Stelt | DOI 10.1038/s41589-020-0528-7 | Mouse | Serum

Inosine triphosphate pyrophosphatase (ITPA) hydrolyzes inosine triphosphate (ITP) and other deaminated purine nucleotides to the corresponding nucleoside monophosphates. In humans, ITPA deficiency causes severe encephalopathy with epileptic seizure, microcephaly, and developmental retardation. In this study, we established neural stem cell-specific Itpa-conditional KO mice (Itpa-cKO mice) to clarify the effects of ITPA deficiency on the neural system. The Itpa-cKO mice showed growth retardation and died within 3 weeks of birth. We did not observe any microcephaly in the Itpa-cKO mice, although the female Itpa-cKO mice did show adrenal hypoplasia. The Itpa-cKO mice showed limb-clasping upon tail suspension and spontaneous and/or audiogenic seizure. Whole-cell patch-clamp recordings from entorhinal cortex neurons in brain slices revealed a depolarized resting membrane potential, increased firing, and frequent spontaneous miniature excitatory postsynaptic current and miniature inhibitory postsynaptic current in the Itpa-cKO mice compared with ITPA-proficient controls. Accumulated ITP or its metabolites, such as cyclic inosine monophosphates, or RNA containing inosines may cause membrane depolarization and hyperexcitability in neurons and induce the phenotype of ITPA-deficient mice, including seizure.

JCI Insight | 2020 | Y. Koga; D. Tsuchimoto; Y. Hayashi; N. Abolhassani; Y. Yoneshima; K. Sakumi; H. Nakanishi; S. Toyokuni; Y. Nakabeppu | DOI 10.1172/jci.insight.140229 | Mouse | Serum

Glucocorticoids are lipid-soluble hormones that signal via the glucocorticoid receptor (GR), a ligand-dependent transcription factor. Circulating glucocorticoids derive from the adrenals, but it is now apparent that paracrine glucocorticoid signaling occurs in multiple tissues. Effective local glucocorticoid concentrations and whether glucocorticoid delivery can be targeted to specific cell subsets are unknown. We use fluorescence detection of chromatin-associated GRs as biosensors of ligand binding and observe signals corresponding to steroid concentrations over physiological ranges in vitro and in vivo. In the thymus, where thymic epithelial cell (TEC)-synthesized glucocorticoids antagonize negative selection, we find that CD4(+)CD8(+)TCR(hi) cells, a small subset responding to self-antigens and undergoing selection, are specific targets of TEC-derived glucocorticoids and are exposed to 3-fold higher levels than other cells. These results demonstrate and quantitate targeted delivery of paracrine glucocorticoids. This approach may be used to assess in situ nuclear receptor signaling in a variety of physiological and pathological contexts.

Cell Reports | 2019 | M. D. Taves; P. R. Mittelstadt; D. M. Presman; G. L. Hager; J. D. Ashwell | Cell Rep 10.1016/j.celrep.2019.02.108 | Mouse | Plasma