Heterozygous mutations of GATA3, which encodes a dual zinc-finger transcription factor, cause hypoparathyroidism with sensorineural deafness and renal dysplasia. Here, we have investigated the role of GATA3 in parathyroid function by challenging Gata3+/– mice with a diet low in calcium and vitamin D so as to expose any defects in parathyroid function. This led to a higher mortality among Gata3+/– mice compared with Gata3+/+ mice. Compared with their wild-type littermates, Gata3+/– mice had lower plasma concentrations of calcium and parathyroid hormone (PTH) and smaller parathyroid glands with a reduced Ki-67 proliferation rate. At E11.5, Gata3+/– embryos had smaller parathyroid-thymus primordia with fewer cells expressing the parathyroid-specific gene glial cells missing 2 (Gcm2), the homolog of human GCMB. In contrast, E11.5 Gata3–/– embryos had no Gcm2 expression and by E12.5 had gross defects in the third and fourth pharyngeal pouches, including absent parathyroid-thymus primordia. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays showed that GATA3 binds specifically to a functional double-GATA motif within the GCMB promoter. Thus, GATA3 is critical for the differentiation and survival of parathyroid progenitor cells and, with GCM2/B, forms part of a transcriptional cascade in parathyroid development and function.
Irina V. Grigorieva, Samantha Mirczuk, Katherine U. Gaynor, M. Andrew Nesbit, Elena F. Grigorieva, Qiaozhi Wei, Asif Ali, Rebecca J. Fairclough, Joanna M. Stacey, Michael J. Stechman, Radu Mihai, Dorota Kurek, William D. Fraser, Tertius Hough, Brian G. Condie, Nancy Manley, Frank Grosveld, Rajesh V. Thakker
Hypothyroidism in humans is characterized by severe neurological consequences that are often irreversible, highlighting the critical role of thyroid hormone (TH) in the brain. Despite this, not much is known about the signaling pathways that control TH action in the brain. What is known is that the prohormone thyroxine (T4) is converted to the active hormone triiodothyronine (T3) by type 2 deiodinase (D2) and that this occurs in astrocytes, while TH receptors and type 3 deiodinase (D3), which inactivates T3, are found in adjacent neurons. Here, we modeled TH action in the brain using an in vitro coculture system of D2-expressing H4 human glioma cells and D3-expressing SK-N-AS human neuroblastoma cells. We found that glial cell D2 activity resulted in increased T3 production, which acted in a paracrine fashion to induce T3-responsive genes, including ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), in the cocultured neurons. D3 activity in the neurons modulated these effects. Furthermore, this paracrine pathway was regulated by signals such as hypoxia, hedgehog signaling, and LPS-induced inflammation, as evidenced both in the in vitro coculture system and in in vivo rat models of brain ischemia and mouse models of inflammation. This study therefore presents what we believe to be the first direct evidence for a paracrine loop linking glial D2 activity to TH receptors in neurons, thereby identifying deiodinases as potential control points for the regulation of TH signaling in the brain during health and disease.
Beatriz C.G. Freitas, Balázs Gereben, Melany Castillo, Imre Kalló, Anikó Zeöld, Péter Egri, Zsolt Liposits, Ann Marie Zavacki, Rui M.B. Maciel, Sungro Jo, Praful Singru, Edith Sanchez, Ronald M. Lechan, Antonio C. Bianco
Obese patients have chronic, low-grade inflammation that predisposes to type 2 diabetes and results, in part, from dysregulated visceral white adipose tissue (WAT) functions. The specific signaling pathways underlying WAT dysregulation, however, remain unclear. Here we report that the PPARγ signaling pathway operates differently in the visceral WAT of lean and obese mice. PPARγ in visceral, but not subcutaneous, WAT from obese mice displayed increased sensitivity to activation by its agonist rosiglitazone. This increased sensitivity correlated with increased expression of the gene encoding the ubiquitin hydrolase/ligase ubiquitin carboxyterminal esterase L1 (UCH-L1) and with increased degradation of the PPARγ heterodimerization partner retinoid X receptor α (RXRα), but not RXRβ, in visceral WAT from obese humans and mice. Interestingly, increased UCH-L1 expression and RXRα proteasomal degradation was induced in vitro by conditions mimicking hypoxia, a condition that occurs in obese visceral WAT. Finally, PPARγ-RXRβ heterodimers, but not PPARγ-RXRα complexes, were able to efficiently dismiss the transcriptional corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) upon agonist binding. Increasing the RXRα/RXRβ ratio resulted in increased PPARγ responsiveness following agonist stimulation. Thus, the selective proteasomal degradation of RXRα initiated by UCH-L1 upregulation modulates the relative affinity of PPARγ heterodimers for SMRT and their responsiveness to PPARγ agonists, ultimately activating the PPARγ-controlled gene network in visceral WAT of obese animals and humans.
Bruno Lefebvre, Yacir Benomar, Aurore Guédin, Audrey Langlois, Nathalie Hennuyer, Julie Dumont, Emmanuel Bouchaert, Catherine Dacquet, Luc Pénicaud, Louis Casteilla, Francois Pattou, Alain Ktorza, Bart Staels, Philippe Lefebvre
Studies in rodents have shown that male sexual function can be disrupted by fetal or neonatal administration of compounds that alter endocrine homeostasis, such as the synthetic nonsteroidal estrogen diethylstilbestrol (DES). Although the molecular basis for this effect remains unknown, estrogen receptors likely play a critical role in mediating DES-induced infertility. Recently, we showed that the orphan nuclear receptor small heterodimer partner (Nr0b2), which is both a target gene and a transcriptional repressor of estrogen receptors, controls testicular function by regulating germ cell entry into meiosis and testosterone synthesis. We therefore hypothesized that some of the harmful effects of DES on testes could be mediated through Nr0b2. Here, we present data demonstrating that Nr0b2 deficiency protected mice against the negative effects of DES on testis development and function. During postnatal development, Nr0b2-null mice were resistant to DES-mediated inhibition of germ cell differentiation, which may be the result of interference by Nr0b2 with retinoid signals that control meiosis. Adult Nr0b2-null male mice were also protected against the effects of DES; however, we suggest that this phenomenon was due to the removal of the repressive effects of Nr0b2 on steroidogenesis. Together, these data demonstrate that Nr0b2 plays a critical role in the pathophysiological changes induced by DES in the mouse testis.
David H. Volle, Mélanie Decourteix, Erwan Garo, Judy McNeilly, Patrick Fenichel, Johan Auwerx, Alan S. McNeilly, Kristina Schoonjans, Mohamed Benahmed
Congenital hyperinsulinism is a condition of dysregulated insulin secretion often caused by inactivating mutations of the ATP-sensitive K+ (KATP) channel in the pancreatic β cell. Though most disease-causing mutations of the 2 genes encoding KATP subunits, ABCC8 (SUR1) and KCNJ11 (Kir6.2), are recessively inherited, some cases of dominantly inherited inactivating mutations have been reported. To better understand the differences between dominantly and recessively inherited inactivating KATP mutations, we have identified and characterized 16 families with 14 different dominantly inherited KATP mutations, including a total of 33 affected individuals. The 16 probands presented with hypoglycemia at ages from birth to 3.3 years, and 15 of 16 were well controlled on diazoxide, a KATP channel agonist. Of 29 adults with mutations, 14 were asymptomatic. In contrast to a previous report of increased diabetes risk in dominant KATP hyperinsulinism, only 4 of 29 adults had diabetes. Unlike recessive mutations, dominantly inherited KATP mutant subunits trafficked normally to the plasma membrane when expressed in COSm6 cells. Dominant mutations also resulted in different channel-gating defects, as dominant ABCC8 mutations diminished channel responses to magnesium adenosine diphosphate or diazoxide, while dominant KCNJ11 mutations impaired channel opening, even in the absence of nucleotides. These data highlight distinctive features of dominant KATP hyperinsulinism relative to the more common and more severe recessive form, including retention of normal subunit trafficking, impaired channel activity, and a milder hypoglycemia phenotype that may escape detection in infancy and is often responsive to diazoxide medical therapy, without the need for surgical pancreatectomy.
Sara E. Pinney, Courtney MacMullen, Susan Becker, Yu-Wen Lin, Cheryl Hanna, Paul Thornton, Arupa Ganguly, Show-Ling Shyng, Charles A. Stanley
Thyroid hormonogenesis requires secretion of thyroglobulin, a protein comprising Cys-rich regions I, II, and III (referred to collectively as region I-II-II) followed by a cholinesterase-like (ChEL) domain. Secretion of mature thyroglobulin requires extensive folding and glycosylation in the ER. Multiple reports have linked mutations in the ChEL domain to congenital hypothyroidism in humans and rodents; these mutations block thyroglobulin from exiting the ER and induce ER stress. We report that, in a cell-based system, mutations in the ChEL domain impaired folding of thyroglobulin region I-II-III. Truncated thyroglobulin devoid of the ChEL domain was incompetent for cellular export; however, a recombinant ChEL protein (“secretory ChEL”) was secreted efficiently. Coexpression of secretory ChEL with truncated thyroglobulin increased intracellular folding, promoted oxidative maturation, and facilitated secretion of region I-II-III, indicating that the ChEL domain may function as an intramolecular chaperone. Additionally, we found that the I-II-III peptide was cosecreted and physically associated with secretory ChEL. A functional ChEL domain engineered to be retained intracellularly triggered oxidative maturation of I-II-III but coretained I-II-III, indicating that the ChEL domain may also function as a molecular escort. These insights into the role of the ChEL domain may represent potential therapeutic targets in the treatment of congenital hypothyroidism.
Jaemin Lee, Bruno Di Jeso, Peter Arvan
The hormone estradiol affects the auditory system both by itself and by its interaction with neuroprotective factors. In this study, we examined the role of estrogen receptors (ERs) in response to auditory trauma. We found a ligand-dependent protective role for ERβ in the auditory system by investigating mice deficient in ERα (ERKO mice), ERβ (BERKO mice), and aromatase (ARKO mice). Basal auditory brainstem response (ABR) thresholds were similar in all animals. An acoustic trauma causing a temporary hearing loss raised ABR thresholds in male and female BERKO and ARKO mice compared with WT and ERKO mice. The ERα-selective agonist, propyl(1H) pyrazole-1,3,5-triyl-trisphenol (PPT), partially protected ARKO mice from trauma, while the ERβ-selective agonist, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN), protected WT and ARKO mice. Immunohistochemistry and western blotting confirmed the expression of ERβ in cochlea of WT males and females. Levels of brain-derived neurotrophic factor (BDNF), a neuroprotective peptide that can be induced by estrogen, was lower in BERKO and ARKO mice compared with WT. DPN treatment increased BDNF expression in ARKO mice. These data indicate ERβ-mediated neuroprotection involving BDNF in the auditory system of males and females.
Inna Meltser, Yeasmin Tahera, Evan Simpson, Malou Hultcrantz, Konstantina Charitidi, Jan-Åke Gustafsson, Barbara Canlon
Thyroid hormone is a critical determinant of cellular metabolism and differentiation. Precise tissue-specific regulation of the active ligand 3,5,3′-triiodothyronine (T3) is achieved by the sequential removal of iodine groups from the thyroid hormone molecule, with type 3 deiodinase (D3) comprising the major inactivating pathway that terminates the action of T3 and prevents activation of the prohormone thyroxine. Using cells endogenously expressing D3, we found that hypoxia induced expression of the D3 gene DIO3 by a hypoxia-inducible factor–dependent (HIF-dependent) pathway. D3 activity and mRNA were increased both by hypoxia and by hypoxia mimetics that increase HIF-1. Using ChIP, we found that HIF-1α interacted specifically with the DIO3 promoter, indicating that DIO3 may be a direct transcriptional target of HIF-1. Endogenous D3 activity decreased T3-dependent oxygen consumption in both neuronal and hepatocyte cell lines, suggesting that hypoxia-induced D3 may reduce metabolic rate in hypoxic tissues. Using a rat model of cardiac failure due to RV hypertrophy, we found that HIF-1α and D3 proteins were induced specifically in the hypertrophic myocardium of the RV, creating an anatomically specific reduction in local T3 content and action. These results suggest a mechanism of metabolic regulation during hypoxic-ischemic injury in which HIF-1 reduces local thyroid hormone signaling through induction of D3.
Warner S. Simonides, Michelle A. Mulcahey, Everaldo M. Redout, Alice Muller, Marian J. Zuidwijk, Theo J. Visser, Frank W.J.S. Wassen, Alessandra Crescenzi, Wagner S. da-Silva, John Harney, Felix B. Engel, Maria-Jesús Obregon, P. Reed Larsen, Antonio C. Bianco, Stephen A. Huang
The function of the adult thyroid is regulated by thyroid-stimulating hormone (TSH), which acts through a G protein–coupled receptor. Overactivation of the TSH receptor results in hyperthyroidism and goiter. The Gs-mediated stimulation of adenylyl cyclase–dependent cAMP formation has been regarded as the principal intracellular signaling mechanism mediating the action of TSH. Here we show that the Gq/G11-mediated signaling pathway plays an unexpected and essential role in the regulation of thyroid function. Mice lacking the α subunits of Gq and G11 specifically in thyroid epithelial cells showed severely reduced iodine organification and thyroid hormone secretion in response to TSH, and many developed hypothyroidism within months after birth. In addition, thyrocyte-specific Gαq/Gα11-deficient mice lacked the normal proliferative thyroid response to TSH or goitrogenic diet, indicating an essential role of this pathway in the adaptive growth of the thyroid gland. Our data suggest that Gq/G11 and their downstream effectors are promising targets to interfere with increased thyroid function and growth.
Jukka Kero, Kashan Ahmed, Nina Wettschureck, Sorin Tunaru, Tim Wintermantel, Erich Greiner, Günther Schütz, Stefan Offermanns
Estrogen drives both transcriptional activation and proteolysis of estrogen receptor α (ERα; encoded by ESR1). Here we observed variable and overlapping ESR1 mRNA levels in 200 ERα-negative and 50 ERα-positive primary breast cancers examined, which suggests important posttranscriptional ERα regulation. Our results indicate that Src cooperates with estrogen to activate ERα proteolysis. Inducible Src stimulated ligand-activated ERα transcriptional activity and reduced ERα t1/2. Src and ERα levels were inversely correlated in primary breast cancers. ERα-negative primary breast cancers and cell lines showed increased Src levels and/or activity compared with ERα-positive cancers and cells. ERα t1/2 was reduced in ERα-negative cell lines. In both ERα-positive and -negative cell lines, both proteasome and Src inhibitors increased ERα levels. Src inhibition impaired ligand-activated ERα ubiquitylation and increased ERα levels. Src siRNA impaired ligand-activated ERα loss in BT-20 cells. Pretreatment with Src increased ERα ubiquitylation and degradation in vitro. These findings provide what we believe to be a novel link between Src activation and ERα proteolysis and support a model whereby crosstalk between liganded ERα and Src drives ERα transcriptional activity and targets ERα for ubiquitin-dependent proteolysis. Oncogenic Src activation may promote not only proliferation, but also estrogen-activated ERα loss in a subset of ERα-negative breast cancers, altering prognosis and response to therapy.
Isabel Chu, Angel Arnaout, Sophie Loiseau, Jun Sun, Arun Seth, Chris McMahon, Kathy Chun, Bryan Hennessy, Gordon B. Mills, Zafar Nawaz, Joyce M. Slingerland