PROGRAM IN DEVELOPMENTAL ENDOCRINOLOGY AND GENETICS
The program studies a wide spectrum of subjects that all converge in developmental endocrine and genetic issues. The investigators have equally diverse backgrounds that range from biochemistry to molecular endocrinology and genetics to clinical obesity research.
Greti Aguilera’s Section on Endocrine Physiology focuses on the molecular mechanisms of the hypothalamic stress response. Research during the past year provided novel information on the transcriptional regulation of corticotropin releasing hormone (CRH) and the physiological actions of vasopressin (VP) during chronic stress adaptation. Thus, potentiation of early transcriptional activation and late repression of the CRH gene by minor increases in cAMP explain the rapid regulation of CRH expression by the non–cAMP–dependent regulators norepinephrine and glutamate, which are the major neurotransmitters released in the paraventricular nucleus during stress. Contrary to the conventional view, phosphorylation of the transcription factor CREB is not sufficient for activation of CRH transcription but requires recruitment of additional factors. New findings demonstrate novel roles for the prominent increases in VP activity during chronic stress. In addition to minor effects in modulating pituitary ACTH secretion, VP mediated mitogenic responses in the pituitary and antiapoptotic actions in the brain.
Jeffrey Baron’s Section on Growth and Development has continued to investigate the cellular and molecular mechanisms governing longitudinal bone growth in childhood. Bone growth, which occurs at the growth plate, requires chondrocytes to undergo a two-step differentiation program—from stem-like cells in the resting zone into the rapidly dividing chondrocytes of the proliferative zone and then into the terminally differentiated chondrocytes of the hypertrophic zone. The section developed a novel technique that allows quantitative assessment of gene expression within individual zones of the growth plate, which, when combined with functional studies, provided evidence that a bone-morphogenetic signaling gradient across the growth plate functions as a key mechanism for chondrocyte differentiation. Analogous studies provided insight into the roles of fibroblast growth factor and insulin-like growth factor signaling in the spatial and temporal regulation of growth plate chondrogenesis.
Carolyn Bondy’s Section on Women’s Health primarily studied Turner syndrome and its cardiovascular effects in late age. Using high-resolution magnetic resonance angiography (MRA), the section demonstrated cardiovascular anomalies in about 50 percent of study subjects, in contrast to the previously accepted prevalence of 20 to 30 percent based on echocardiographic studies. MRA revealed a high prevalence of major venous malformations, including partial anomalous pulmonary venous return and persistent left superior vena cava affecting over 20 percent of the study population. The most common anomaly was a distinctive aortic deformation, termed elongated transverse arch of the aorta, which seems to predict aortic complications and hence mandates vigilant surveillance of aorta dimensions in affected patients.
Kevin Catt’s Section on Hormonal Regulation investigates the molecular mechanisms of activation, signaling, and function of G protein–coupled receptors, particularly those for angiotensin II (AT1R and AT2R) and gonadotropin-releasing hormone (GnRHR) and their interactions with the epidermal growth factor receptor (EFGR). Studies in hepatic C9 cells identified specific and shared signaling pathways and interactions between the AT1R and the EGFR and their dependence on caveolin. In addition, the section identified the invariant chain (Ii/CD74) as a novel interacting protein of the AT1R and as a negative regulator of its expression. In cultured GnRH neurons, pulsatile neurosecretion was dependent on the neurons’ endogenous G protein–activated inwardly rectifying K+ (GIRK) channels, activation of which prevents the firing of action potentials and inhibits episodic GnRH release. Bioluminescence resonance energy transfer (BRET) analysis performed in GnRH neurons revealed heterodimerization of the GnRHR and GPR54, the receptor that mediates kisspeptin-induced activation of GnRH secretion. The laboratory also investigates the mechanisms by which GnRH activates several G proteins in the GnRH neuron. Studies on GFP-expressing GnRH neurons in rat brain slices are investigating the regulatory roles of endogenous estrogen receptor subtypes in the control of GnRH secretion in the adult animal.
Janice Chou’s Section on Cellular Differentiation studies glycogen storage disease type I (GSD-I), which is caused by deficiencies in the glucose-6-phosphatase-alpha (G6Pase-alpha)-glucose-6-phosphate transporter (G6PT) complex. Deficiencies in G6Pase-alpha cause GSD-Ia while deficiencies in G6PT cause GSD-Ib. Both manifest symptoms of disturbed glucose homeostasis; GSD-Ib also presents with myeloid dysfunctions. Chou’s section showed that an increase in cellular cholesterol efflux and antioxidant capacity in the sera of GSD-Ia patients may protect against premature atherosclerosis. Using G6PT–/– mice, the group showed that myeloid dysfunctions in GSD-Ib are intrinsically linked to G6PT deficiency in bone marrow and neutrophils and that an adenoviral vector–mediated gene transfer improved metabolic and myeloid functions. The group recently characterized a G6Pase-alpha isoform, G6Pase-beta, that, like G6PT, is ubiquitously expressed. The section showed that G6Pase-beta–null mice manifest myeloid dysfunctions mimicking GSD-Ib and that G6Pase-beta–deficient neutrophils undergo ER stress and enhanced rate of apoptosis, thereby demonstrating that a functional G6Pase-beta-G6PT complex is critical for normal neutrophil function and defining a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology.
Maria Dufau’s Section on Molecular Endocrinology investigates the molecular basis of hormonal regulation of gonadal function. The section focuses on modes of transcriptional repression and derepression of the human luteinizing hormone (LHR) and prolactin receptor (PRLR) and the functions of novel short PRLR inhibitory forms, identified in this laboratory, in physiological regulation and cancer. The laboratory also investigates mechanisms involved in the progress of spermatogenesis and Leydig cell function. Recent studies revealed cell-specific contributions of phosphatases (PP2A, PP1) in trichostatin A–induced activation of LHR transcription. Release of a phosphatase by changes in chromatin structure favors phosphorylation of Sp1 by PI3K/PKCz, causing the release of the p107 inhibitor and the transcriptional activation of the LHR. Other studies demonstrated conformational determinants required for the inhibitory action of the PRLR short form on prolactin-induced signaling through the long form. Studies on the gonadotropin-regulated testicular helicase (GRTH/Ddx25), a protein discovered by this laboratory that is essential for completion of spermatogenesis, demonstrated the helicase’s multifunctionality in nuclear transport, storage/degradation of messages, and translation. The section found a mis-sense mutation (Arg242H) of GRTH in infertile patients; its expression revealed lack of the phosphorylated form, which could be relevant to germ cell development and/or function.
David Klein’s Section on Neuroendocrinology is interested in biological rhythms and, to that end, focuses on the pineal gland. The section works to identify highly expressed genes in the gland, the genes’ physiological role, and the mechanisms involved in regulation of gene expression. Of central interest are the melatonin pathway and the trans-synaptic system that switches melatonin synthesis on and off. At the heart of the pathway is arylalkylamine N-acetyltransferase, which controls the large daily change in the production and circulating levels of melatonin. The section identified a critical residue in a floppy loop of the enzyme that confers instability, thereby promoting movement of the enzyme and enhancing substrate/product flux. The section also helped develop an inhibitor of the enzyme with potential as a drug and biochemical tool. The section discovered an important, previously ignored signaling system involving activation of dopamine type 4 receptors, thus advancing our understanding of the trans-synaptic regulation of the pineal gland; the system appears to inhibit actions of norepinephrine, and regulation of the receptor requires input from both cyclic AMP and thyroid hormone. On a higher level of organization involving circadian organization, the section found that the two closely related genes IA-2 and IA-2B (Ptprn and Ptprn2) are essential for circadian rhythms in cardiovascular physiology, temperature, and activity and determined that the genes are localized in the suprachiasmatic nucleus, which is the location of the central clock, and that the electrophysiology of the suprachiasmatic nucleus is dramatically altered in mice lacking these genes.
Anil Mukherjee’s Section on Developmental Genetics conducts both laboratory and clinical investigations into the molecular mechanisms of heritable childhood neurodegenerative diseases and inflammatory/autoimmune disorders. The section’s investigations focus primarily on two genes: palmitoyl-protein thioesterase-1, mutation of which causes infantile Batten disease (IBD), and uteroglobin (UG), an anti-inflammatory protein. UG-knockout (UG-KO) mice develop IgA-nephropathy and allergic airway inflammation and are susceptible to tumorigenesis. During the past year, the section showed that (1) increased cPLA2-catalyzed production of lysophosphatidylcholine in the brain mediates microglial recruitment and activation in PPT1-KO mice, a model for IBD; (2) ER and oxidative stresses are common manifestations of both neurodegenerative and non-neurodegenerative storage disorders; and (3) UG-KO mice are highly susceptible to developing pulmonary fibrosis. The laboratory is also continuing a Bench-to-Bedside clinical trial to determine if a combined regimen of Cystagon™ and N-acetylcysteine (Mucomyst®) is beneficial for patients with IBD.
The Section on Genetic Disorders of Drug Metabolism, headed by Ida Owens, studies the regulation of UDP-glucuronosyltransferase (UGT) isozymes, focusing on understanding how these endoplasmic reticulum–bound enzymes detoxify innumerable endogenous chemical toxins, as well as those encountered in our diet and environment, by linkage to glucuronic acid. The group’s recent findings include evidence that (1) endogenous reactive oxygen species–related oxidants behave as second messengers to sustain phosphokinases C (PKC)–dependent signaling and UGT phosphorylation to support the constitutive glucuronidation process, (2) UGTs require regulated phosphorylation by distinct PKCs, and (3) their transient downregulation disrupts glucuronidation, leading to marked increases in uptake and efficacy of the widely used immunosuppressant mycophenolic acid. In addition, the section discovered that the critically important family 2B isozymes, UGT2B7 and UGT2B15, which respectively detoxify endogenous genotoxic catechol-estrogens and dihydrotestosterone, require tyrosine phosphorylation, with UGT2B15 also requiring serine/threonine phoshorylation. Importantly, UGT2B7 activity in normal and tumor breast tissue was associated with tyrosine phosphorylation and an active tyrosine kinase, indicating that phospho-tyrosine(s) in UGT2B7 supports metabolism of genotoxic estrogen metabolites in breast tissue.
Forbes Porter’s Section on Molecular Dysmorphology studies a group of human and mouse malformation syndromes attributable to inborn errors of cholesterol synthesis, the most common of which is Smith-Lemli-Opitz syndrome (SLOS). The group studies both basic science and clinical aspects of SLOS, with the goal of developing and testing therapeutic interventions for the condition. A clinical trial evaluating the safety and efficacy of simvastatin therapy in SLOS has completed enrollment. This past year, the group received a Bench-to-Bedside award to investigate the role of impaired glycosphingolipid transport in SLOS. The section continues to study other inborn errors of cholesterol synthesis and recently described the biochemical and phenotypic consequences of disrupting sterol Δ14-reductase. The section initiated a clinical study of patients with Niemann-Pick disease, type C (NPC), a neurodegenerative disorder caused by impaired intracellular transport of cholesterol and glycosphingolipids. The new protocol is designed to investigate biochemical markers and clinical aspects of NPC for potential use as outcome measures in a future clinical trial.
Constantine Stratakis’s Section on Endocrinology and Genetics studies the genetic and molecular mechanisms leading to disorders that affect the adrenal cortex, with emphasis on those that are developmental, hereditary, and associated with adrenal hypoplasia or hyperplasia, multiple tumors, and abnormalities in other endocrine glands (especially the pituitary gland and, to a lesser extent, the thyroid gland). The section studied congenital adrenal hypoplasia caused by triple A syndrome and multiple endocrine deficiencies; familial hyperaldosteronism; adrenocortical and thyroid cancer; pituitary tumors; Carney complex (CNC)—an autosomal dominant disease; multiple endocrine neoplasia (MEN) syndromes affecting the pituitary, thyroid, and adrenal glands. The regulatory subunit type 1-a of protein kinase A (PKA) (the PRKAR1A gene) is mutated in most CNC patients; phosphodiesterase-11A (PDE11A) mutations were found in patients with isolated adrenal hyperplasia and Cushing’s syndrome. A significant part of the section’s work focuses on cyclic AMP/PKA-stimulated signaling pathways, PKA effects on tumor suppression and/or development, the cell cycle, and chromosomal stability. These projects are facilitated by prkar1a and pde11a gene mouse models with the respective genes knocked out. Genome-wide searches for other genes responsible for CNC and related diseases of the adrenal and pituitary glands are ongoing.
Charles Strott’s Section on Steroid Regulation investigates the molecular mechanisms and biologic implications of the modification of substances by sulfonation. The group clones human and animal sulfotransferase genes to examine gene products, tissue expression, transcriptional regulation, and biological significance and studies the human SULT2B1 gene, which encodes two isozymes that sulfonate steroids/sterols in order. The SULT2B1a isoform produces pregnenolone sulfate, an important neurosteroid that plays a role in learning and memory. The SULT2B1b isoform is selectively expressed in skin, produces the multifunctional molecule cholesterol sulfate, and, by virtue of the latter’s ability to bind tightly to the orphan nuclear receptor RORalpha, plays an important role in epidermal development (barrier formation), an action reminiscent of a typical hormone. Rather than producing an active molecule, SULT2B1b detoxifies cytotoxic oxysterol metabolites of cholesterol such as 7-ketocholesterol, a compound involved in the development of macular degeneration and atherosclerosis.
Jack Yanovski’s Unit on Growth and Obesity studies metabolic and behavioral factors involved in body weight regulation and body composition during childhood. During the past year, the unit showed that children with function-altering polymorphisms in the melanocortin 3 receptor (MC3R) gene exhibit greater energy intake during laboratory meal studies but no differences in energy expenditure compared with children with wild-type MC3R alleles. The unit is now investigating the effects of these mutations in a murine model. The laboratory also investigated the role played by BDNF in pediatric overweight, characterizing a severely obese child with a chromosomal abnormality that affects BDNF gene expression; the unit found that children with the WAGR syndrome who have deletions of one copy of the BDNF gene are significantly heavier than those retaining both copies of BDNF, suggesting that obesity is associated with BDNF haploinsufficiency. Ongoing studies are attempting to identify genetic abnormalities that predispose children to binge eating.
