N-SREBP2 Provides a Mechanism for Dynamic Control of Cellular Cholesterol Homeostasis.

Cholesterol is required to maintain the functional integrity of cellular membrane systems and signalling pathways, but its supply must be closely and dynamically regulated because excess cholesterol is toxic. Sterol regulatory element-binding protein 2 (SREBP2) and the ER-resident protein HMG-CoA reductase (HMGCR) are key regulators of cholesterol biosynthesis. Here, we assessed the mechanistic aspects of their regulation in hepatic cells. Unexpectedly, we found that the transcriptionally active fragment of SREBP2 (N-SREBP2) was produced constitutively. Moreover, in the absence of an exogenous cholesterol supply, nuclear N-SREBP2 became resistant to proteasome-mediated degradation. This resistance was paired with increased occupancy at the HMGCR promoter and HMGCR expression. Inhibiting nuclear N-SREBP2 degradation did not increase HMGCR RNA levels; this increase required cholesterol depletion. Our findings, combined with previous physiological and biophysical investigations, suggest a new model of SREBP2-mediated regulation of cholesterol biosynthesis in the organ that handles large and rapid fluctuations in the dietary supply of this key lipid. Specifically, in the nucleus, cholesterol and the ubiquitin-proteasome system provide a short-loop system that modulates the rate of cholesterol biosynthesis via regulation of nuclear N-SREBP2 turnover and HMGCR expression. Our findings have important implications for maintaining cellular cholesterol homeostasis and lowering blood cholesterol via the SREBP2-HMGCR axis.

Somatic mutations of CADM1 in aldosterone-producing adenomas and gap junction-dependent regulation of aldosterone production.

Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production.

An immunomodulatory role for the Mycobacterium tuberculosis Acr protein in the formation of the tuberculous granuloma.

The tuberculous granuloma is a compact aggregate of dormant bacteria encapsulated by host macrophages. It is commonly regarded as a product of the host defense designed to isolate infectious mycobacteria. This work demonstrates that exposure of macrophages to the Mtb heat-shock protein Acr leads to overproduction of the chemokine CXCL16, allowing the mycobacterium to exploit the innate immune response. This induction of chemokine expression is hypothesized to occur through activation of ADAM proteases, providing an immunomodulatory role for Mtb Acr in the formation of the granuloma.

Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation.

Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics.

DLK1/PREF1 marks a novel cell population in the human adrenal cortex.

The adrenal cortex governs fundamental metabolic processes though synthesis of glucocorticoid, mineralocorticoids and androgens. Studies in rodents have demonstrated that the cortex undergoes a self-renewal process and that capsular/subcapsular stem/progenitor cell pools differentiate towards functional steroidogenic cells supporting the dynamic centripetal streaming of adrenocortical cells throughout life. We previously demonstrated that the Notch atypical ligand Delta-like homologue 1 (DLK1)/preadipocyte factor 1 (PREF1) is expressed in subcapsular Sf1 and Shh-positive, CYP11B1-negative and CYP11B2-partially positive cortical progenitor cells in rat adrenals, and that secreted DLK1 can modulate GLI1 expression in H295R cells. Here we show that the human adrenal cortex remodels with age to generate clusters of relatively undifferentiated cells expressing DLK1. These clusters (named DLK1-expressing cell clusters or DCCs) increased with age in size and were found to be different entities to aldosterone-producing cell clusters, another well-characterized and age-dependent cluster structure. DLK1 was markedly overexpressed in adrenocortical carcinomas but not in aldosterone-producing adenomas. Thus, this data identifies a novel cell population in the human adrenal cortex and might suggest a yet-to be identified role of DLK1 in the pathogenesis of adrenocortical carcinoma in humans.

Oncometabolite induced primary cilia loss in pheochromocytoma.

Primary cilia are sensory organelles involved in regulation of cellular signaling. Cilia loss is frequently observed in tumors; yet, the responsible mechanisms and consequences for tumorigenesis remain unclear. We demonstrate that cilia structure and function is disrupted in human pheochromocytomas - endocrine tumors of the adrenal medulla. This is concomitant with transcriptional changes within cilia-mediated signaling pathways that are associated with tumorigenesis generally and pheochromocytomas specifically. Importantly, cilia loss was most dramatic in patients with germline mutations in the pseudohypoxia-linked genes SDHx and VHL. Using a pheochromocytoma cell line derived from rat, we show that hypoxia and oncometabolite-induced pseudohypoxia are key drivers of cilia loss and identify that this is dependent on activation of an Aurora-A/HDAC6 cilia resorption pathway. We also show cilia loss drives dramatic transcriptional changes associated with proliferation and tumorigenesis. Our data provide evidence for primary cilia dysfunction contributing to pathogenesis of pheochromocytoma by a hypoxic/pseudohypoxic mechanism and implicates oncometabolites as ciliary regulators. This is important as pheochromocytomas can cause mortality by mechanisms including catecholamine production and malignant transformation, while hypoxia is a general feature of solid tumors. Moreover, pseudohypoxia-induced cilia resorption can be pharmacologically inhibited, suggesting potential for therapeutic intervention.

Glial Cells Missing 1 Regulates Equine Chorionic Gonadotrophin Beta Subunit via Binding to the Proximal Promoter.

Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGß-subunit (LHB), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates LHB promoter activity. Here, studies of the LHB proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equine chorionic girdle (ChG) and surrounding tissues. Luciferase assays demonstrated endogenous activity of the LHB promoter in BeWo choriocarcinoma cells with greatest activity by a proximal 335 bp promoter fragment. Transactivation studies in COS7 cells using an equine GCM1 expression vector showed GCM1 could transactivate the proximal 335 bp LHB promoter. Chromatin immunoprecipitation using primary ChG trophoblast cells showed GCM1 to preferentially bind to the most proximal GCM1-binding site over site 2. Mutation of site 1 but not site 2 resulted in a loss of endogenous promoter activity in BeWo cells and failure of GCM1 to transactivate the promoter in COS-7 cells. Together, these data show that GCM1 binds to site 1 in the LHB promoter but also requires the upstream segment of the LHB promoter between -119 bp and -335 bp of the translation start codon for activity. GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, were found to be differentially expressed in the ChG between days 27 and 34 and are excellent candidates for this role. In conclusion, GCM1 was demonstrated to drive the LHB promoter, through direct binding to a predicted GCM1-binding site, with requirement for another factor(s) to bind the proximal promoter to exert this function. Based on these findings, we hypothesize that ETV7 and HOXA13 act in concert with GCM1 to initiate LHB transcription between days 30 and 31, with ETV1 partnering with GCM1 to maintain transcription.

Modeling Congenital Adrenal Hyperplasia and Testing Interventions for Adrenal Insufficiency Using Donor-Specific Reprogrammed Cells.

Adrenal insufficiency is managed by hormone replacement therapy, which is far from optimal; the ability to generate functional steroidogenic cells would offer a unique opportunity for a curative approach to restoring the complex feedback regulation of the hypothalamic-pituitary-adrenal axis. Here, we generated human induced steroidogenic cells (hiSCs) from fibroblasts, blood-, and urine-derived cells through forced expression of steroidogenic factor-1 and activation of the PKA and LHRH pathways. hiSCs had ultrastructural features resembling steroid-secreting cells, expressed steroidogenic enzymes, and secreted steroid hormones in response to stimuli. hiSCs were viable when transplanted into the mouse kidney capsule and intra-adrenal. Importantly, the hypocortisolism of hiSCs derived from patients with adrenal insufficiency due to congenital adrenal hyperplasia was rescued by expressing the wild-type version of the defective disease-causing enzymes. Our study provides an effective tool with many potential applications for studying adrenal pathobiology in a personalized manner and opens venues for the development of precision therapies.

Kisspeptin Is a Novel Regulator of Human Fetal Adrenocortical Development and Function: A Finding With Important Implications for the Human Fetoplacental Unit.

Context: The human fetal adrenal (HFA) is an integral component of the fetoplacental unit and important for the maintenance of pregnancy. Low kisspeptin levels during pregnancy are associated with miscarriage, and kisspeptin and its receptor are expressed in the HFA. However, the role of kisspeptin in fetal adrenal function remains unknown. Objective: To determine the role of kisspeptin in the developing HFA. Design: Experiments using H295R and primary HFA cells as in vitro models of the fetal adrenal. Association of plasma kisspeptin levels with HFA size in a longitudinal clinical study. Setting: Academic research center and tertiary fetal medicine unit. Participants: Thirty-three healthy pregnant women were recruited at their 12-week routine antenatal ultrasound scan. Main Outcome Measures: The spatiotemporal expression of Kiss1R in the HFA. The production of dehydroepiandrosterone sulfate (DHEAS) from HFA cells after kisspeptin treatment, alone or in combination with adrenocorticotropic hormone or corticotropin-releasing hormone. Fetal adrenal volume (FAV) and kisspeptin levels at four antenatal visits (∼20, 28, 34, and 38 weeks' gestation). Results: Expression of Kiss1R was present in the HFA from 8 weeks after conception to term and was shown in the inner fetal zone. Kisspeptin significantly increased DHEAS production in H295R and second-trimester HFA cells. Serial measurements of kisspeptin confirmed a correlation with FAV growth in the second trimester, independent of sex or estimated fetal weight. Conclusions: Kisspeptin plays a key role in the regulation of the HFA and thus the fetoplacental unit, particularly in the second trimester of pregnancy.

A model for small heat shock protein inhibition of polyglutamine aggregation.

Polyglutamine (polyQ) repeat expansions that lead to the formation of amyloid aggregates are linked to several devastating neurodegenerative disorders. While molecular chaperones, including the small heat shock proteins (sHsp), play an important role in protection against protein misfolding, the aberrant protein folding that accompanies these polyQ diseases overwhelms the chaperone network. By generating a model structure to explain the observed suppression of spinocerebellar ataxia 3 (SCA3) by the sHsp αB-crystallin, we have identified key vulnerabilities that provide a possible mechanism to explain this heat shock response. A docking study involving a small bioactive peptide should also aid in the development of new drug targets for the prevention of polyQ-based aggregation.

Dlk1 up-regulates Gli1 expression in male rat adrenal capsule cells through the activation of ß1 integrin and ERK1/2.

The development and maintenance of the zones of the adrenal cortex and their steroidal output are extremely important in the control of gluconeogenesis, the stress response, and blood volume. Sonic Hedgehog (Shh) is expressed in the adrenal cortex and signals to capsular cells, which can respond by migrating into the cortex and converting into a steroidogenic phenotype. Delta-like homologue 1 (Dlk1), a member of the Notch/Delta/Serrate family of epidermal growth factor-like repeat-containing proteins, has a well-established role in inhibiting adipocyte differentiation. We demonstrate that Shh and Dlk1 are coexpressed in the outer undifferentiated zone of the male rat adrenal and that Dlk1 signals to the adrenal capsule, activating glioma-associated oncogene homolog 1 transcription in a ß1 integrin- and Erk1/2-dependent fashion. Moreover, Shh and Dlk1 expression inversely correlates with the size of the zona glomerulosa in rats after manipulation of the renin-angiotensin system, suggesting a role in the homeostatic maintenance of the gland.

FGF signalling through Fgfr2 isoform IIIb regulates adrenal cortex development.

Developmental signalling pathways are implicated in the formation and maintenance of the adrenal gland, but their roles are currently not well defined. In recent years it has emerged that Sonic hedgehog (Shh) and Wnt/ß catenin signalling are crucial for the growth and development of the adrenal cortex. Here we demonstrate that Fibroblast growth factor receptor (Fgfr) 2 isoforms IIIb and IIIc are expressed mainly in the adrenal subcapsule during embryogenesis and that specific deletion of the Fgfr2 IIIb isoform impairs adrenal development, causing reduced adrenal growth and impaired expression of SF1 and steroidogenic enzymes. The hypoplastic adrenals also have thicker, disorganised capsules which retain Gli1 expression but no longer express Dlk1. Fgfr2 ligands were detected in both the capsule and the cortex, suggesting the importance of signalling between the capsule and the cortex in adrenal development.

Etiologic diagnosis of nonsyndromic genetic hearing loss in adult vs pediatric populations.

OBJECTIVES: Determine the diagnostic yield of a shared genetic testing algorithm in adult and pediatric populations with sensorineural hearing loss (SNHL) and recommend effective testing strategies to evaluate for genetic causes of deafness in patients presenting with idiopathic sensorineural hearing loss. STUDY DESIGN: Hospital-based cohort study. SETTING: University of Miami outpatient otology clinics between 2001 and 2010. SUBJECTS: Two hundred twenty-one adult and 163 pediatric patients with nonsyndromic sensorineural hearing loss. METHODS: Peripheral blood samples were screened for mutations in GJB2 and GJB6 and mitochondrial DNA mutations 1555A>G, 7444G>A, and 3243A>G. Audiometric data and family history were also collected. RESULTS: GJB2/GJB6-related deafness was diagnosed in 23 of 163 pediatric patients (14%) compared with only 3 of 221 adults (1%). All adults had a family history of hearing loss, and 2 patients noted deafness onset at birth. Nineteen GJB2 mutations were identified with 35delG the most common mutation. The 35delG homozygous state was the most common pathogenic genotype (54%). Mitochondrial DNA (mtDNA) mutations were found in 6 adult probands (3%). No mtDNA mutations were found in pediatric patients. CONCLUSION: Testing for common GJB2/GJB6 mutations in pediatric patients has considerable value in establishing an etiologic diagnosis for SNHL. Similar testing in adults is of very low yield except perhaps in cases of early-onset SNHL or strong family history. Mitochondrial DNA testing should be considered in adults with idiopathic SNHL.

Expression of guanylyl cyclase-B (GC-B/NPR2) receptors in normal human fetal pituitaries and human pituitary adenomas implicates a role for C-type natriuretic peptide.

C-type natriuretic peptide (CNP/Nppc) is expressed at high levels in the anterior pituitary of rats and mice and activates guanylyl cyclase B receptors (GC-B/Npr2) to regulate hormone secretion. Mutations in NPR2/Npr2 can cause achondroplasia, GH deficiency, and female infertility, yet the normal expression profile within the anterior pituitary remains to be established in humans. The current study examined the expression profile and transcriptional regulation of NPR2 and GC-B protein in normal human fetal pituitaries, normal adult pituitaries, and human pituitary adenomas using RT-PCR and immunohistochemistry. Transcriptional regulation of human NPR2 promoter constructs was characterized in anterior pituitary cell lines of gonadotroph, somatolactotroph, and corticotroph origin. NPR2 was detected in all human fetal and adult pituitary samples regardless of age or sex, as well as in all adenoma samples examined regardless of tumor origin. GC-B immunoreactivity was variable in normal pituitary, gonadotrophinomas, and somatotrophinomas. Maximal transcriptional regulation of the NPR2 promoter mapped to a region within -214 bp upstream of the start site in all anterior pituitary cell lines examined. Electrophoretic mobility shift assays revealed that this region contains Sp1/Sp3 response elements. These data are the first to show NPR2 expression in normal human fetal and adult pituitaries and adenomatous pituitary tissue and suggest a role for these receptors in both pituitary development and oncogenesis, introducing a new target to manipulate these processes in pituitary adenomas.

Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency.

Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.

MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans.

An interesting variant of familial glucocorticoid deficiency (FGD), an autosomal recessive form of adrenal failure, exists in a genetically isolated Irish population. In addition to hypocortisolemia, affected children show signs of growth failure, increased chromosomal breakage, and NK cell deficiency. Targeted exome sequencing in 8 patients identified a variant (c.71-1insG) in minichromosome maintenance-deficient 4 (MCM4) that was predicted to result in a severely truncated protein (p.Pro24ArgfsX4). Western blotting of patient samples revealed that the major 96-kDa isoform present in unaffected human controls was absent, while the presence of the minor 85-kDa isoform was preserved. Interestingly, histological studies with Mcm4-depleted mice showed grossly abnormal adrenal morphology that was characterized by non-steroidogenic GATA4- and Gli1-positive cells within the steroidogenic cortex, which reduced the number of steroidogenic cells in the zona fasciculata of the adrenal cortex. Since MCM4 is one part of a MCM2-7 complex recently confirmed as the replicative helicase essential for normal DNA replication and genome stability in all eukaryotes, it is possible that our patients may have an increased risk of neoplastic change. In summary, we have identified what we believe to be the first human mutation in MCM4 and have shown that it is associated with adrenal insufficiency, short stature, and NK cell deficiency.

A mechanism of action for small heat shock proteins.

Molecular dynamics simulations of a fitted multimeric structure of Mycobacterium tuberculosis α-crystallin (Mtb Acr) identify solvent exclusion from the ß(4)-ß(8) hydrophobic groove as a critical factor driving subunit assembly. Dehydration is also implicated as a determinant factor governing the chaperone activity of the dimer upon its dissociation from the oligomer. Two exposed hydrogen bonds, responsible for stabilizing the ß(8)-ß(9) fold are identified as key mechanistic elements in this process. Based on the overproduction of the chemokine CXCL16, observed after macrophage exposure to Mtb Acr, the proteases ADAM10 and ADAM17 are mooted as possible targets of this chaperone activity.

Tyrosine kinase inhibition: Ligand binding and conformational change in c-Kit and c-Abl.

The conformational flexibility exhibited by protein kinases poses an enormous challenge to the design of cancer therapeutics. Additionally the high degree of structural conservation within the kinase superfamily often leads to inhibitors that exhibit little selectivity and substantial cross reactivity. This work investigates the conformational changes that accompany the binding of Gleevec, or imatinib mesylate, to the tyrosine kinases c-Kit and c-Abl. Our analysis is that this fit is driven, at least in part, by the need to exclude water from solvent-exposed backbone hydrogen bonds. Both experimental and molecular modeling studies of the active state inhibitor of the tyrosine kinase c-Abl indicate that solvent exclusion also plays a role in this system.

Effects of ACTH and expression of the melanocortin-2 receptor in the neonatal mouse testis.

ACTH has been shown to stimulate androgen production by the fetal/neonatal mouse testis through the melanocortin type 2 receptor (MC2R). This study was designed to localize the expression of MC2R in the neonatal mouse testis and characterize the effects of ACTH on testicular androgen production. Using immunohistochemistry, MC2R was localized to the fetal-type Leydig cell population of the neonatal testis. ACTH caused a time-dependent increase in cyclic AMP (cAMP) and testosterone production by isolated cells with an increase in cAMP apparent in < 3 min. There was no additive effect of maximally stimulating doses of ACTH and human chorionic gonadotropin (hCG). Androgen production in response to ACTH and hCG was reduced by UO126 and dexamethasone, which are the inhibitors of ERK1/2 and phospholipase A2 respectively. Expression of mRNA encoding StAR was increased fourfold by both ACTH and hCG, although expression of mRNA encoding for steroidogenic enzymes was not markedly affected. The potency of N-terminal fragments of ACTH to stimulate androgen production was similar to that seen previously in the adrenal. Data indicate that both LH and ACTH, acting through their respective receptors, stimulate steroidogenesis by fetal-type Leydig cells via arachidonic acid, protein kinase A, and ERK1/2 activation of StAR.

Interaction of the melanocortin 2 receptor with nucleoporin 50: evidence for a novel pathway between a G-protein-coupled receptor and the nucleus.

The adrenocorticotropin (ACTH) receptor (melanocortin 2 receptor, or MC2R) is the smallest G-protein-coupled receptor that, when activated by the peptide hormone ACTH, stimulates cAMP production and adrenal steroidogenesis. Receptor expression is dependent on a specific membrane trafficking process involving an accessory protein (melanocortin 2 receptor accessory protein, or MRAP) and other unidentified components. In an attempt to discover novel receptor interacting proteins, the C-terminal tail of the MC2R was used to screen a mouse adrenal Y6 cell cDNA library using the bacterial two-hybrid system. This identified the nucleoporin Nup 50 (Npap60) as the major full-length interacting protein. Interaction was confirmed by a GST pulldown assay and by coimmunoprecipitation in human H295R cells (which express both proteins endogenously). Deletion analysis identified the region between residues 143 and 466 in Nup50 as being required for interaction with the MC2R. Stimulation of H295R cells with ACTH (10(-6) M) was followed by a gradual translocation of the Nup50-MC2R complex from the membrane to the nucleus after 30 min. This time course is most consistent with MC2R internalization dynamics and may suggest a novel role for Nup50.