CONTROL OF HEPATIC AND B-CELL FUNCTION BY CO-ACTIVATORS
Fredric E Wondisford, Chief, Metabolism Division
Johns Hopkins University, Broadway Research Building Suite 117, Baltimore, Md 21205
Grant 3R01DK063349-06S1 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: The overall goal of this proposal is to define the mechanism of cyclic AMP (cAMP) signaling pathways in the liver and pancreatic 2-cell. Elevated intracellular cAMP in the hepatocyte increases hepatic glucose production, while the same signal in the pancreatic 2-cell promotes insulin secretion and proliferation. The cAMP signal activates protein kinase A (PKA), which phosphorylates the nuclear cAMP response element binding protein (CREB). Phosphorylated CREB recruits the nuclear co-activators CREB binding protein (CBP), and the related protein p300. CBP, but not p300, contains an insulin phosphorylation site at serine 436. Insulin phosphorylation at serine 436 is required for efficient dissociation of the CREB-CBP complex and consequent attenuation of CREB-dependent gene transcription. We have generated a knock-in mouse model where CBP is mutated at serine 436 (CBP-S436A), removing this site of insulin phosphorylation. As a result, CREB-dependent gene transcription is increased and resistant to the inhibitory nuclear effects of insulin. These mice show enhanced hepatic glucose production and pancreatic 2-cell hyperplasia associated with a defect in glucose-stimulated insulin secretion. These effects may be mediated by the PPAR3 co-activator 11(PGC-1 1, a cAMP target gene) whose expression is increased in both the hepatocyte and pancreatic 2-cell of these animals. PGC-11 activates a fasting gene program in liver and impairs energy production in the 2-cell. In this proposal, we will attempt to understand if PGC-1 1 mediates the CBP S436A mouse phenotype, since this mutation may affect other signaling pathways (Aim 1). CBP and p300 may have discrete physiological roles based on the presence or absence, respectively, of an insulin phosphorylation site. Aim 2 will establish the relative importance and mechanism of hepatic and 2-cell regulation mediated by these closely related co- activators by studying a mouse model where an artificial insulin phosphorylation site is introduced in p300 (G421S). Finally, CBP remains in the nucleus regardless of its phosphorylation state, while the forkhead protein, FoxO1, is excluded from the nucleus after insulin phosphorylation. FoxO1 has been reported to play a dominant role in activating hepatic gluconeogenesis and inhibiting 2-cell growth based on over-expression mouse models. Aim 3 will compare the effects of insulin phosphorylation mutants of CBP and FoxO1, expressed at allelic levels, on hepatic glucose production and 2-cell regulation and/or growth. These studies are significant because they will establish signaling pathways that normally regulate the hepatocyte and pancreatic 2-cell in vivo and provide a mechanistic understanding of pathophysiological changes found in patients with diabetes mellitus. In this proposal, we will attempt to understand the physiological importance of CBP and p300 in the hepatocyte and pancreatic 2-cell, in the context of other factors reported to mediate activation of genes containing cAMP response elements
Keywords: 3`5`-cyclic ester of AMP; Activation, Gene; Adenosine Cyclic 3`, 5`-Monophosphate; Adenosine Cyclic Monophosphate; Adenosine Cyclic Monophosphate-Dependent Protein Kinases; Adenosine, cyclic 3`, 5`-(hydrogen phosphate); Affect; Amino Acids; Aminoacetic Acid; Animals; Antidiabetic Hormone; Binding; Binding (Molecular Function); Binding Proteins; CBP protein; CBP protein, human (CREB binding protein); CRE; CRE Binding Protein; CREB; CREB Binding Protein; CREB Protein; CREB binding protein, human; CREB-binding protein; CREBBP protein, human; Cell Communication and Signaling; Cell Function; Cell Growth in Number; Cell Multiplication; Cell Nucleus; Cell Process; Cell Proliferation; Cell Signaling; Cell physiology; Cells; Cellular Expansion; Cellular Function; Cellular Growth; Cellular Physiology; Cellular Process; Cellular Proliferation; Cyclic AMP; Cyclic AMP Response Element; Cyclic AMP Response Element-Binding Protein; Cyclic AMP Responsive Element Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cyclic AMP-Responsive DNA-Binding Protein; D-Glucose; Defect; Dephosphorylation; Dextrose; Diabetes Mellitus; Dissociation; E1A-associated p300 protein; EP300; EP300 gene; Fasting; GCG; Gastrointestinal Tract, Pancreas; Gene Activation; Gene Targeting; Gene Transcription; Generalized Growth; Genes; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Glucagon; Glucagon (1-29); Gluconeogenesis; Glucose; Glukagon; Glycine; Goals; Growth; HG-Factor; Hepatic; Hepatic Cells; Hepatic Parenchymal Cell; Hepatocyte; Humulin R; Hyperglycemia; Hyperglycemic-Glycogenolytic Factor; Hyperplasia; Hyperplastic; Hypoglycemia; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin Resistance; Insulin, Regular; Intracellular Communication and Signaling; Knock-in; Knock-in Mouse; L-Serine; Ligand Binding Protein; Liver; Liver Cells; Mammals, Mice; Mediating; Mice; Mice, Mutant Strains; Modeling; Molecular Interaction; Murine; Mus; Mutant Strains Mice; Mutate; Mutation; Novolin R; Nuclear; Nucleus; Orthophosphate[{..}]oxaloacetate carboxy-lyase (phosphorylating); PEPCK; PKA; Pancreas; Pancreatic; Patients; Phenotype; Phospho-CREB Binding Protein; Phosphoenolpyruvate Carboxylase; Phosphorylation; Phosphorylation Site; Physiologic; Physiological; Play; Position; Positioning Attribute; Process; Production; Programs (PT); Programs [Publication Type]; Protein Binding; Protein Dephosphorylation; Protein Kinase A; Protein Phosphorylation; Proteins; RNA Expression; Recruitment Activity; Regulation; Relative; Relative (related person); Reporting; Resistance; Role; Rubinstein-Taybi syndrome protein, human; Serine; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Site; Subcellular Process; Targetings, Gene; Testing; Tissue Growth; Transcription; Transcription, Genetic; Transducers; adenosine 3`5` monophosphate; aminoacid; attenuation; base; biological signal transduction; body system, hepatic; cAMP; cAMP Response Element; cAMP Response Element-Binding Protein; cAMP Responsive Element Binding Protein; cAMP-Dependent Protein Kinases; cell growth; cofactor; diabetes; fasted; fasts; fork head protein; forkhead protein; forkhead transcription factors; gene product; genome mutation; glucose RA; glucose biosynthesis; glucose production; glucose rate of appearance; hepatic gluconeogenesis; homologous recombination; human CREBBP protein; hyperglycemic; hypoglycemic; hypoglycemic episodes; impaired glucose tolerance; in vivo; insulin resistant; insulin secretion; mouse model; mouse mutant; mutant; novel; nuclear protein CBP; ontogeny; organ system, hepatic; p300; p300 E1A-associated coactivator; p300 protein; p300-CBP coactivator; p300/CBP proteins; phospho-CREB-binding protein; phosphoenolpyruvate carboxykinase; programs; protein complex; protein expression; public health relevance; reconstitute; reconstitution; recruit; resistant; social role; transcription factor
Relevance: In this proposal, we will attempt to understand the physiological importance of CBP and p300 in the hepatocyte and pancreatic ¿-cell, in the context of other factors reported to mediate activation of genes containing cAMP response elements
Project start date: 2010-02-04
Project end date: 2010-04-30
Budget start date: 4-FEB-2010
Budget end date: 30-APR-2010
PFA/PA: PA-07-070
3R01DK063349-06S1 (2010): $21000
Sponsored Links Excellgen http://Excellgen.com
Control Of Beta Cell Function By Co-activators
Fredric E Wondisford, Professor And Chief
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637
Grant 3R01DK063349-02S1 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZDK1
Abstract: This grant proposal is in response to RFA DK 02014 entitled "Comprehensive Programs in Beta Cell Biology." My laboratory studies mechanisms of transcriptional regulation by nuclear hormone receptors and has recently become interested in the specific role of co-activators in the beta cell based on the study of nuclear targets for insulin signaling. This proposal is responsive to the RFA in several ways 1) I am a new investigator to the diabetes field interested in transcriptional regulation of the beta-cell by insulin; 2) transgenic and knockout (KO) animal models are proposed to study beta-cell proteins in their physiological context; 3) a study of the role of two co-activators in then-cell, CBP and p300, are proposed; and 4) the role of a beta-cell-specific protein, PDX-1, in insulin-stimulated gene expression and R-cell proliferation will be explored. Regulation of insulin gene transcription in the pancreas involves specific positive and negative cis-acting elements. located in the 5 flanking region. One major regulator of insulin gene expression is the homeobox protein, PDX-1 (also referred to as IPF-1, STF-1, and IDX-1). This protein, first expressed at e8.5 in the mouse, has a major role in pancreatic development and (beta-cell function as demonstrated in both generalized and conditional PDX-1 KO mice; respectively. A second major regulator of both insulin transcription and (beta-cell development is the basic helix-loop-helix (bHLH) protein, NeuroDI43-2, which forms a DNA-binding complex with the ubiquitously expressed E2A proteins. Both PDX-1 and NeuroDI/(3-2 have been shown to interact with the highly related CBP and p300 co-activator proteins. These co-activators have proven to be critical in many aspects of mammalian development, including their function on mitogen-responsive genes; they have also been proposed to be essential for insulin gene transcription based on in vitro studies. It is unclear, however, what role they play in (beta-cell growth and function, and whether their function is constitutive or regulated in the (beta cell. Three Aims are proposed 1) To-define the role of insulin signaling via the AP-1 complex in proliferative responses of the pancreatic (beta cell; 2) To define constitutive and hormonal regulated interaction domains in CBP and p300 important in the pancreatic (beta cell; and 3) To define the role of CBP and p300 in the adult (beta-cell in mice harboring a cell-specific knock out of either CBP or p300. The overall goal of this proposal will be to determine the mechanism of CBP and p300 action in regulating pancreatic beta-cell growth and function
Keywords: cell proliferation, genetic regulatory element, genetic transcription, insulin, pancreatic islet function, protein protein interaction, protein structure function, AP1 protein, DNA binding protein, biological signal transduction, cell differentiation, gene expression, hormone regulation /control mechanism, phosphorylation, gene targeting, genetically modified animal, laboratory mouse
Project start date: 2002-09-30
Project end date: 2006-08-31
3R01DK063349-02S1 (2003): $98224
Grants awarded to Fredric E Wondisford
TSH-B GENE EXPRESSION AND REGULATION BY TH
Fredric E Wondisford, Professor And Chief
Beth Israel Deaconess Medical Center
330 Brookline Avenue, Br 264
boston, Ma 02215
Grant 5R29DK043653-04 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: END
Abstract: Thyrotropin (TSH) is composed of two subunits an alpha subunit, common to pituitary TSH and pituitary and placental gonadotropins, and a unique bet subunit (TSHbeta). Thyroid hormone and its nuclear receptor (c-erbA) bind to and regulate the transcription of a variety of genes including both TSH subunit genes (TSHalpha, TSHbeta) and the rat growth hormone gene (rGH). However, the molecular mechanisms that determine whether a thyroid hormone DNA response element (TRE) negatively (e.g., TSHbeta) or positively (e.g., rGH) regulates gene expression are completely unknown. The proposed studies will determine whether the location, orientation, or a specific DNA sequence defines a TRE as inhibitory or stimulatory. DNA transfection studies in a variety of cell lines will be performed using chimeric constructs containing inhibitory and stimulatory TREs in a variety of locations and orientations. Moreover, since preliminary evidence suggests that an additional nuclear protein(s), other than the thyroid hormone receptor, is involved in the molecular mechanism of thyroid hormone inhibition of human TSHbeta expression (hTSHbeta) an additional goal of these studies will be to define further the cis-acting elements to which this protein(s) binds, and whether it is cell-specific. DNA transfection studies using deletions and mutations of the human TSHbeta TRE in parallel with DNase I footprinting studies of this TRE with nuclear extracts from TSH-secreting and non-secreting cell lines and tissues will be utilized to accomplish this goal. These studies will provide new insight into thyroid hormone action and allow a more detailed model of thyroid hormone action at any TRE to be developed. Since additional nuclear proteins in the thyrotroph may be involved in the molecular mechanism of thyroid hormone inhibition of hTSHbeta expression, the second major goal of these studies will be to define cis-acting elements responsible for thyroid hormone regulation and thyrotroph-specific expression of the hTSHbeta gene. To accomplish this goal, in vitro DNA transfection studies (utilizing chimeric hTSHbeta constructs) and DNA binding assays (DNase I footprinting) will be correlated with in vivo expression of chimeric hTSHbeta genes in transgenic mice. The long range goal of these studies is to determine the interrelationship between cell- specific expression and thyroid hormone regulation of the hTSHbeta gene in the anterior pituitary. Ultimately, a greater understanding of thyroid hormone action in man may be gained
Keywords: gene expression, genetic regulatory element, hormone regulation /control mechanism, thyroid hormone, thyrotropin, transcription factor gene deletion mutation, genetic transcription, growth hormone, hormone receptor, nonhistone nucleoprotein, nucleic acid sequence, receptor binding, reporter gene DNA footprinting, immunocytochemistry, laboratory mouse, neoplastic cell culture for noncancer research, northern blotting, point mutation, polymerase chain reaction, tissue /cell culture, transfection, transgenic animal
Project start date: 1991-06-01
Project end date: 1996-05-31
5R29DK043653-04 (1994): $151709
HUMAN TSH-B GENE EXPRESSION AND REGULATION BY TH
Fredric E Wondisford, Professor And Chief
Beth Israel Deaconess Medical Center
330 Brookline Avenue, Br 264
boston, Ma 02215
Grant 5R29DK043653-03 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: END
Abstract: Thyrotropin (TSH) is composed of two subunits an alpha subunit, common to pituitary TSH and pituitary and placental gonadotropins, and a unique bet subunit (TSHbeta). Thyroid hormone and its nuclear receptor (c-erbA) bind to and regulate the transcription of a variety of genes including both TSH subunit genes (TSHalpha, TSHbeta) and the rat growth hormone gene (rGH). However, the molecular mechanisms that determine whether a thyroid hormone DNA response element (TRE) negatively (e.g., TSHbeta) or positively (e.g., rGH) regulates gene expression are completely unknown. The proposed studies will determine whether the location, orientation, or a specific DNA sequence defines a TRE as inhibitory or stimulatory. DNA transfection studies in a variety of cell lines will be performed using chimeric constructs containing inhibitory and stimulatory TREs in a variety of locations and orientations. Moreover, since preliminary evidence suggests that an additional nuclear protein(s), other than the thyroid hormone receptor, is involved in the molecular mechanism of thyroid hormone inhibition of human TSHbeta expression (hTSHbeta) an additional goal of these studies will be to define further the cis-acting elements to which this protein(s) binds, and whether it is cell-specific. DNA transfection studies using deletions and mutations of the human TSHbeta TRE in parallel with DNase I footprinting studies of this TRE with nuclear extracts from TSH-secreting and non-secreting cell lines and tissues will be utilized to accomplish this goal. These studies will provide new insight into thyroid hormone action and allow a more detailed model of thyroid hormone action at any TRE to be developed. Since additional nuclear proteins in the thyrotroph may be involved in the molecular mechanism of thyroid hormone inhibition of hTSHbeta expression, the second major goal of these studies will be to define cis-acting elements responsible for thyroid hormone regulation and thyrotroph-specific expression of the hTSHbeta gene. To accomplish this goal, in vitro DNA transfection studies (utilizing chimeric hTSHbeta constructs) and DNA binding assays (DNase I footprinting) will be correlated with in vivo expression of chimeric hTSHbeta genes in transgenic mice. The long range goal of these studies is to determine the interrelationship between cell- specific expression and thyroid hormone regulation of the hTSHbeta gene in the anterior pituitary. Ultimately, a greater understanding of thyroid hormone action in man may be gained
Keywords: gene expression, genetic regulatory element, hormone regulation /control mechanism, thyroid hormone, thyrotropin, transcription factor gene deletion mutation, genetic transcription, growth hormone, hormone receptor, nonhistone nucleoprotein, nucleic acid sequence, receptor binding, reporter gene DNA footprinting, immunocytochemistry, laboratory mouse, neoplastic cell culture for noncancer research, northern blotting, point mutation, polymerase chain reaction, tissue /cell culture, transfection, transgenic animal
Project start date: 1991-06-01
Project end date: 1996-05-31
5R29DK043653-03 (1993): $149980
DIABETES RESEARCH AND TRAINING CENTER
Fredric E Wondisford, Chief, Metabolism Division
Johns Hopkins University, 3400 N Charles St, Baltimore, Md 21218
Grant 3P60DK079637-02S2 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: This proposal seeks new funding for a Baltimore Diabetes Research and Training Center (DRTC). The proposed Center will involve investigators from Johns Hopkins University and the University of Maryland. These investigators have primary appointments in numerous departments within and between these Institutions; and while their scientific backgrounds are diverse, they share a common interest in diabetes and endocrinology research and many have established collaborations. The aims of the Center are the following 1) to foster collaborative, multidisciplinary diabetes and endocrinology research in a supportive environment; 2) to enhance the clinical and basic research capabilities of established diabetes investigators; 3) to encourage investigators not involved in diabetes research to become interested in pursuing problems related to diabetes and endocrinology; 4) to develop and implement programs for the training of health care professionals in the diagnosis and management of diabetes; 5) to develop, implement and evaluate programs that deliver cost effective health care for the treatment of persons with diabetes; 6) to speed the translation of advances in basic biomedical and genetic research to the clinical arena where they may be applied to!the diagnosis and treatment of persons with diabetes; and 7) to inform others in both professional and lay settings of the accomplishments, opportunities and advancements in diabetes research and training. This application contains four separate components Administrative, Biomedical Research, and Pilot and Feasibility and Enrichment. The Administrative Component will be responsible for overseeing the operation of the Center as a whole. The Biomedical Research Component will consist of six Core Laboratories to foster collaborative diabetes research. A Pilot and Feasibility Component will foster the participation and interaction of junior investigators with established investigators in research related to diabetes. The Enrichment Program will facilitate the interaction within and between Johns Hopkins University and the University of Maryland. Since these Institutions have complementary research interests, the Enrichment Program as well as the proposed Biomedical Research Cores will strengthen collaborative ties between the Institutions. Taken together, these objectives and components define the new Baltimore Diabetes Research and Training Center (DRTC)
Keywords: Appointment; Baltimore; Basic Research; Basic Science; Biomedical Research; Budgets; Care, Health; Caring; Cities; Clinical; Clinical Faculty; Clinical Nurse Educator; Clinical Research; Clinical Study; Collaborations; Communication; Complications of Diabetes Mellitus; Diabetes Complications; Diabetes Mellitus; Diabetes-Related Complications; Diabetic Complications; Diagnosis; Discipline of Nursing; Education; Educational aspects; Effectiveness; Endocrine Diseases; Endocrine Diseases and Manifestations; Endocrine System Diseases; Endocrinology; Environment; Epidemiology; Faculty; Faculty, Nursing; Feeding behaviors; Fostering; Funding; Genetic; Genetic Research; Goals; Health Care Professional; Health Professional; Health profession; Healthcare; Healthcare professional; Healthcare worker; Human Resources; Humulin R; Individual; Ingestive Behavior; Institution; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin, Regular; Investigators; Laboratories; Manpower; Maryland; Metabolism and Endocrinology; NIH; National Institutes of Health; National Institutes of Health (U.S.); Neuroendocrine; Neuroendocrine System; Neurosecretory Systems; Novolin R; Nurse Educator; Nursing; Nursing Faculty; Nursing Field; Nursing Profession; Nursing Staff Developer; Nursing Staff Development Specialist; Nutritionist; Obesity; Operation; Operative Procedures; Operative Surgical Procedures; Participant; Patients; Persons; Physicians; Play; Populations at Risk; Productivity; Program Description; Programs (PT); Programs [Publication Type]; Public Health Schools; R01 Mechanism; R01 Program; RPG; Recruitment Activity; Research; Research Activity; Research Grants; Research Personnel; Research Project Grants; Research Projects; Research Projects, R-Series; Research Resources; Research Training; Researchers; Resources; Role; Schools; Schools, Medical; Schools, Public Health; Scientist; Speed; Speed (motion); Surgical; Surgical Interventions; Surgical Procedure; Talents; Training Activity; Training Programs; Translations; Underserved Population; United States; United States National Institutes of Health; Universities; adipocyte biology; adiposity; base; corpulence; corpulency; corpulentia; cost effective; diabetes; diabetes management; diabetic; endocrine disorder; feeding-related behaviors; interest; medical schools; member; multidisciplinary; nutrient intake activity; obese; obese people; obese person; obese population; outreach; personnel; programs; recruit; response; social role; surgery; translational study; under served population; underserved people
Project start date: 2009-09-25
Project end date: 2011-08-31
Budget start date: 25-SEP-2009
Budget end date: 31-AUG-2011
PFA/PA: RFA-DK-06-014
3P60DK079637-02S2 (2009): $277045
MECHANISM OF TRH SIGNALING PATHWAYS MEDIATED BY PIT-1
Fredric E Wondisford, Professor And Chief
Beth Israel Deaconess Medical Center
330 Brookline Avenue, Br 264
boston, Ma 02215
Grant 5R01DK050564-04 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: END
Abstract: This proposal addresses the molecular mechanism of thyrotropin-releasing hormone TRH, acting through its cell membrane receptor, modulates the phosphorylation state of a pituitary-specific transcription factor, Pit-I. Pit-I, in turn, regulates anterior pituitary expression of prolactin (Pr1), growth hormone (GH), and thyrotropin-beta subunit (TSH-beta) genes by binding to specific DNA response elements. [Four] Specific aims are proposed to explore further the role of Pit-1 in TRH action (1) TRH- responsive regions of the thyrotropin beta-subunit gene (TSH-beta) will be mapped in vivo before and after TRH stimulation. (2) The importance of individual phosphorylation sites on Pit-1 for TRH action will be determined. [Purified protein kinases and TRH-treated rat pituitary somatotroph (GH3 cells) cellular extract will be used to phosphorylate recombinant wild type or mutant Pit-1 protein preparations in vitro and phosphorylation sites confirmed in vivo in GH3 cells.] The phosphorylation pattern and DNA-binding properties of these Pit-I proteins will then be determined. Transient transfection of GH3 cells with wild type of mutant Pit-1 and its isoforms will also be utilized to study TRH-stimulated expression of the TSH-beta subunit and prolactin (Pr1) genes. [(3) The role of CREB-binding protein (CBP) in mediating TRH-stimulated gene expression of Pr1 and TSH-beta subunit genes via Pit-1 will next be explored. CBP both binds to Pit-1 and enhances TRH stimulated gene expression. The location and phosphorylation dependence of this interaction will be characterized.] (4) Finally, the ability of human Pit- 1 mutations to disrupt TRH action in vitro will be evaluated. [The effect of human Pit-1 mutations on DNA-binding, CBP interaction, and TRH- stimulated expression of Pr1 and TSH-beta subunit genes will be determined. This proposal will provide strong evidence that TRH stimulates gene expression through a phosphorylation-dependent interaction between Pit-1 and CBP.] Moreover, the study of human mutations of the pit-I gene and their effects on TRH signaling will yield new insights into the molecular mechanisms of normal anterior pituitary development, and regulation in man
Keywords: biological signal transduction, gene expression, hormone regulation /control mechanism, molecular genetics, thyrotropin releasing hormone, transcription factor DNA binding protein, binding protein, cAMP response element binding protein, gene mutation, hormone biosynthesis, molecular site, phosphorylation, prolactin, protein isoform, protein kinase C, protein structure /function, thyrotropin cell line, laboratory mouse, luciferin monooxygenase, pituitary gland, transfection, transgenic animal
Project start date: 1997-09-15
Project end date: 2001-05-31
5R01DK050564-04 (2000): $223912
5R01DK050564-03 (1999): $230954
5R01DK050564-02 (1998): $224228
1R01DK050564-01A2 (1997): $217695
Control Of Beta Cell Function By Co-activators
Fredric E Wondisford, Professor And Chief
Johns Hopkins University W400 Wyman Park Building Baltimore, Md 212182680
Grant 7R01DK063349-04 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZDK1
Abstract: This grant proposal is in response to RFA DK 02014 entitled "Comprehensive Programs in Beta Cell Biology." My laboratory studies mechanisms of transcriptional regulation by nuclear hormone receptors and has recently become interested in the specific role of co-activators in the beta cell based on the study of nuclear targets for insulin signaling. This proposal is responsive to the RFA in several ways 1) I am a new investigator to the diabetes field interested in transcriptional regulation of the beta-cell by insulin; 2) transgenic and knockout (KO) animal models are proposed to study beta-cell proteins in their physiological context; 3) a study of the role of two co-activators in then-cell, CBP and p300, are proposed; and 4) the role of a beta-cell-specific protein, PDX-1, in insulin-stimulated gene expression and R-cell proliferation will be explored. Regulation of insulin gene transcription in the pancreas involves specific positive and negative cis-acting elements. located in the 5 flanking region. One major regulator of insulin gene expression is the homeobox protein, PDX-1 (also referred to as IPF-1, STF-1, and IDX-1). This protein, first expressed at e8.5 in the mouse, has a major role in pancreatic development and (beta-cell function as demonstrated in both generalized and conditional PDX-1 KO mice; respectively. A second major regulator of both insulin transcription and (beta-cell development is the basic helix-loop-helix (bHLH) protein, NeuroDI43-2, which forms a DNA-binding complex with the ubiquitously expressed E2A proteins. Both PDX-1 and NeuroDI/(3-2 have been shown to interact with the highly related CBP and p300 co-activator proteins. These co-activators have proven to be critical in many aspects of mammalian development, including their function on mitogen-responsive genes; they have also been proposed to be essential for insulin gene transcription based on in vitro studies. It is unclear, however, what role they play in (beta-cell growth and function, and whether their function is constitutive or regulated in the (beta cell. Three Aims are proposed 1) To-define the role of insulin signaling via the AP-1 complex in proliferative responses of the pancreatic (beta cell; 2) To define constitutive and hormonal regulated interaction domains in CBP and p300 important in the pancreatic (beta cell; and 3) To define the role of CBP and p300 in the adult (beta-cell in mice harboring a cell-specific knock out of either CBP or p300. The overall goal of this proposal will be to determine the mechanism of CBP and p300 action in regulating pancreatic beta-cell growth and function
Keywords: cell proliferation, genetic regulatory element, genetic transcription, insulin, pancreatic islet function, protein protein interaction, protein structure function, AP1 protein, DNA binding protein, biological signal transduction, cell differentiation, gene expression, hormone regulation /control mechanism, phosphorylation, gene targeting, genetically modified animal, laboratory mouse
Project start date: 2002-09-30
Project end date: 2008-08-31
7R01DK063349-04 (2005): $499129
5R01DK063349-03 (2004): $454496
5R01DK063349-02 (2003): $442611
Sponsored Links Excellgen http://Excellgen.com
Fredric E Wondisford, Professor And Chief
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637
Grant 2P60DK020595-269009 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZDK1
Abstract: The Transgenic Core of the DRTC has been in operation since 1991. At the last competitive renewal, the Core was judged to be of excellent merit. Although the Core was though to generally be of high quality, concerns about the extent of the Director s involvement in the DRTC and the adequacy of detailing progress in the Core were noted. In the previous funding period, Elaine Fuchs, Ph.D. had joint responsibility for directing the transgenic cores of both the Cancer and Diabetes Center. This arrangement facilitated the establishment and delivery of high quality and cost-effective service to DRTC investigators. In the proposed funding period, Fredric E. Wondisford, M.D. will serve as Director, and Sally Radovick, M.D. will serve as Co-Director. Both are highly qualified to direct this core and their direct involvement in diabetes and diabetesrelated research should further enhance transgenic services to DRTC investigators. The Technical Director, Ms. Linda Degenstein, is unchanged from the previous funding period. The Core laboratory has also been relocated to a newly renovated pathogen-free barrier facility adjacent to the animal facility. A detailed progress report outlining the growth and effectiveness of the Transgenic Core is include in the proposal.
Keywords: biomedical facility, laboratory mouse, transgenic animal, diabetes mellitus, embryonic stem cell, gene expression, gene targeting
Project start date: 2002-12-01
Project end date: 2007-11-30
TESTING OF PATIENTS WITH SUSPECTED DISORDERS OF TSH SECRETION
Fredric E Wondisford, Professor And Chief
Beth Israel Deaconess Medical Center 330 Brookline Avenue, Br 264 Boston, Ma 02215
Grant 5M01RR001032-240653 from National Center For Research Resources
Abstract: The purpose of this protocol is to 1) help establish the diagnosis of pituitary resistance to thyroid hormone by evaluating a number of physiologic responses to thyroid hormone administration in vivo, and 2) evaluate and confirm the diagnosis of secondary hypothyroidism.
Keywords: diagnosis design /evaluation, endocrine disorder diagnosis, hypothyroidism, thyroid hormone, hormone regulation /control mechanism, pituitary gland, clinical research, human subject
THYROID HORMONE ACTION IN THE PITUITARY
Fredric E Wondisford, Professor And Chief
Beth Israel Deaconess Medical Center 330 Brookline Avenue, Br 264 Boston, Ma 02215
Grant 5R01DK049126-04 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: END
Abstract: The proposed studies address the molecular mechanism of thyroid hormone action in the anterior pituitary. While much has been learned about how thyroid hormone stimulates gene expression, the molecular mechanism underlying thyroid hormone inhibition of gene expression is less well understood. In the thyroid gland, hormone synthesis is regulated by thyroid-stimulating hormone (TSH), which is secreted from the anterior pituitary gland. TSH expression in the pituitary, in turn, is controlled by thyroid hormones which feedback and inhibit the expression of TSH subunit genes, alpha and beta. Although most patients with thyroid disease have normal regulation of TSH secretion, patients with thyroid hormone resistance syndromes, due to mutations in the beta isoform of the thyroid hormone receptor (TR), have dysregulated TSH secretion which is central to the pathogenesis of the disorder. A through understanding of the mechanisms of thyroid hormone inhibition in normal and diseased states, therefore, is warranted. In this proposal, a comprehensive evaluation of pituitary thyroid hormone inhibition with four specific aims is planned. First, the physiologic significance of negative thyroid hormone response elements (nTREs) defined in vitro will be validated in vivo utilizing human TSH-beta luciferase reporter gene constructs in transgenic animals. Second, the role of TR isoforms on negative and positive thyroid hormone regulation will be explored. Human common glycoprotein alpha, TSH-beta, and TRH luciferase reporter gene constructs will be compared with positive TRE (pTRE) containing constructs for these studies. Isoform specificity for thyroid hormone inhibition and mapping of a TR-beta specific thyroid hormone inhibitory domain will be investigated using wild type and chimeric alpha and beta TRs. Third, the effect of RXR isoforms on the structure and function of TR complexes bound to nTRE of the TSH-beta subunit gene will be examined. Finally, the structure and function of TR- beta in patients with selective pituitary resistance to thyroid hormone action (PRTH) will be determined. Each aim is designed to answer a central question about thyroid hormone inhibition, and will, therefore, provide significant insight into the mechanism of thyroid hormone action in the pituitary. They will validate the location and function of nTREs in vivo ; determine whether specific TR isoforms mediate thyroid hormone inhibition and by what mechanism; examine the role, if any, RXRs play in thyroid hormone inhibition; and characterize mutations of TR-beta in patients with a clinically defined and selective pituitary resistance to thyroid hormone. When completed, these studies will provide unique insights into how thyroid hormone both stimulates and inhibits gene transcription and expand our understanding of thyroid hormone resistance syndromes in man.
Keywords: gene induction /repression, genetic regulatory element, hormone receptor, hormone regulation /control mechanism, pituitary thyroid axis, thyrotropin, triiodothyronine, chimeric protein, gene mutation, pituitary gland, protein isoform, retinoid binding protein, thyroid disorder, thyrotropin releasing hormone, transcription factor, gel mobility shift assay, human genetic material tag, human subject, immunocytochemistry, laboratory mouse, northern blotting, nucleic acid sequence, reporter gene, tissue /cell culture, transgenic animal
Project start date: 1995-08-01
Project end date: 2000-07-31
5R01DK049126-04 (1998): $235235
5R01DK049126-03 (1997): $226186
3R01DK049126-04S2 (2000): $87000
3R01DK049126-04S1 (1998): $10321
MOLECULAR MECHANISMS OF THYROID HORMONE ACTION
Fredric E Wondisford, Chief, Metabolism Division
Johns Hopkins University, W400 Wyman Park Building, Baltimore, Md 21218
Grant 5R01DK049126-14 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: The overall goal of this proposal is to gain a better understanding of the central regulation of the hypothalamic-pituitary-thyroid axis (HPT axis). Thyroid stimulating hormone (TSH) is a heterodimeric protein, which is synthesized and secreted from the anterior pituitary, and is essential for activating thyroid hormone (TH) synthesis in the thyroid gland. In response to TSH stimulation, the thyroid generates two thyroid hormones T4 and T3. T4 is thought to be a prohormone, which is activated by its conversion to a higher affinity ligand known as T3. Systemically derived or locally produced T3 then binds to thyroid hormone receptors (TRs) in the thyrotropin-releasing hormone (TRH) neuron of the hypothalamus or pituitary thyrotroph and inhibits gene expression. Negative control of the HPT axis by TH bound to TRs is generally accepted to be the dominant influence on the axis. In this model, TRH and TSH production are maximal when TH is absent. Our recent studies in KO mice challenge this model. We demonstrate that both TRH and TR-beta are necessary for activation of TSH subunit gene expression and TSH production, even in the hypothyroid state. However, the function of TR-alpha in this process and the mechanism of action of unliganded TR-beta are still unknown. In this proposal, therefore, we will define the role and mechanism of TRH and unliganded TRs in stimulating TSH subunit gene expression in the hypothyroid state. When TH is present, the TR-beta isoforms (principally the TR-beta 2 isoform) mediate TH negative feedback at the level of the hypothalamus and pituitary based on the findings of modestly elevated TSH and TH levels in TR-beta KO mice. In contrast, TR-alpha KO mice actually display slightly reduced TH levels associated with slightly elevated TSH levels. These findings suggest that TR-alpha has no role in negative TH regulation of the HPT axis in the euthyroid state. However, it must play some role in negative regulation because supraphysiologic T3 administration to TR-beta KO mice suppresses TSH synthesis, and most impressively KO of both TR-alpha and TR-beta isoforms markedly elevates both TSH and TH levels in mice. What explains this TR isoform difference? In this proposal, we will test the hypothesis that the lower affinity of TR-alphaiDor T3 explains why it has a limited role in regulating the central axis in the euthyroid state
Keywords: 3, 5, 3`, 5`-Tetraiodothyronine; 5-Oxo-L-prolyl-L-histidyl-L-prolinamide; Adenohypophysis; Affinity; Amino Acids; Anterior Lobe of Pituitary; Anterior Lobe of the Pituitary Gland; Anterior Pituitary Gland; Anterior pituitary; Antithyroid Agents; Antithyroid Drugs; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Cell Line; Cell Lines, Strains; CellLine; Chemotherapy-Hormones/Steroids; DNA Binding; DNA Binding Interaction; Data; Diet; ERBA Beta Protein; ERBA1 Gene Products; ERBA2 Gene Products; ERBA2 Protein; Endocrine Gland Secretion; Feedback; Gene Expression; Genes; Genetic Alteration; Genetic Change; Genetic defect; Goals; Gray; Gray unit of radiation dose; Head and Neck, Thyroid; Hormone Receptor; Hormones; Hypophysis; Hypophysis Cerebri; Hypothalamic structure; Hypothalamus; Hypothyroidism; I- element; Iodine; Isoforms; Knock-in; Knock-in Mouse; L-3, 5, 3`, 5`-Tetraiodothyronine; L-Thyroxine; Levothyroxine; Ligand Binding; Ligands; Mammals, Mice; Mediating; Mice; Modeling; Molecular; Molecular Interaction; Murine; Mus; Mutation; NR1A1 Gene Products; NR1A2 Gene Products; Nerve Cells; Nerve Unit; Nervous System, Pituitary; Neural Cell; Neurocyte; Neurons; O-(4-Hydroxy-3, 5-diiodophenyl) 3, 5-diiodo-L-tyrosine; O-(4-Hydroxy-3, 5-diiodophenyl)-3, 5-diiodotyrosine; Pars Anterior Pituitary Gland; Pituitary; Pituitary Gland; Pituitary Gland, Anterior; Play; Precipitation; Principal Investigator; Process; Production; Programs (PT); Programs [Publication Type]; Protein Isoforms; Proteins; Protirelin; Proto-Oncogene Proteins c-erbA; Protyreline; Pyr-His-ProNH2; Receptor Protein; Recombinant TSH; Recombinant Thyroid-Stimulating Hormone; Regulation; Role; T4 Thyroid Hormone; THR Gene; THRA Gene Products; THRB Gene Products; THRB Protein; TR beta; TRH; Testing; Therapeutic Hormone; Therapeutic Levothyroxine; Thyreotropin; Thyroid; Thyroid Antagonists; Thyroid Gland; Thyroid Gland Hormone; Thyroid Hormone Receptor; Thyroid Hormone Receptor Beta; Thyroid Hormone Receptor Gene; Thyroid Hormone Receptor beta 2; Thyroid Hormones; Thyroid Stimulating Hormone; Thyroid hormone receptor alpha; Thyroid-Releasing Hormone; Thyroid-Stimulating Hormone; Thyrotropin; Thyrotropin-Releasing Hormone; Thyroxine; Tyrosine, O-(4-hydroxy-3, 5-diiodophenyl)-3, 5-diiodo-; aminoacid; base; c-erb A Protein; c-erbA Proteins; c-erbA alpha; c-erbA alpha Protein; c-erbA beta; c-erbA-1 Protein; cofactor; cultured cell line; erbA Proto-Oncogene Products; gene product; genome mutation; hormonal regulation; hormone regulation; hypothalamic; in vivo; neuronal; pituitary thyroid axis; programs; prohormone; receptor; response; social role; thyroid inhibitor; thyroxin
Project start date: 1995-08-01
Project end date: 2011-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
5R01DK049126-14 (2010): $294359
5R01DK049126-13 (2009): $297332
2R01DK049126-11A1 (2007): $303400
Sponsored Links Excellgen http://Excellgen.com
CONTROL OF HEPATIC AND B-CELL FUNCTION BY CO-ACTIVATORS
Fredric E Wondisford, Chief, Metabolism Division
Johns Hopkins University, W400 Wyman Park Building, Baltimore, Md 21218
Grant 5R01DK063349-07 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: The overall goal of this proposal is to define the mechanism of cyclic AMP (cAMP) signaling pathways in the liver and pancreatic 2-cell. Elevated intracellular cAMP in the hepatocyte increases hepatic glucose production, while the same signal in the pancreatic 2-cell promotes insulin secretion and proliferation. The cAMP signal activates protein kinase A (PKA), which phosphorylates the nuclear cAMP response element binding protein (CREB). Phosphorylated CREB recruits the nuclear co-activators CREB binding protein (CBP), and the related protein p300. CBP, but not p300, contains an insulin phosphorylation site at serine 436. Insulin phosphorylation at serine 436 is required for efficient dissociation of the CREB-CBP complex and consequent attenuation of CREB-dependent gene transcription. We have generated a knock-in mouse model where CBP is mutated at serine 436 (CBP-S436A), removing this site of insulin phosphorylation. As a result, CREB-dependent gene transcription is increased and resistant to the inhibitory nuclear effects of insulin. These mice show enhanced hepatic glucose production and pancreatic 2-cell hyperplasia associated with a defect in glucose-stimulated insulin secretion. These effects may be mediated by the PPAR3 co-activator 11(PGC-1 1, a cAMP target gene) whose expression is increased in both the hepatocyte and pancreatic 2-cell of these animals. PGC-11 activates a fasting gene program in liver and impairs energy production in the 2-cell. In this proposal, we will attempt to understand if PGC-1 1 mediates the CBP S436A mouse phenotype, since this mutation may affect other signaling pathways (Aim 1). CBP and p300 may have discrete physiological roles based on the presence or absence, respectively, of an insulin phosphorylation site. Aim 2 will establish the relative importance and mechanism of hepatic and 2-cell regulation mediated by these closely related co- activators by studying a mouse model where an artificial insulin phosphorylation site is introduced in p300 (G421S). Finally, CBP remains in the nucleus regardless of its phosphorylation state, while the forkhead protein, FoxO1, is excluded from the nucleus after insulin phosphorylation. FoxO1 has been reported to play a dominant role in activating hepatic gluconeogenesis and inhibiting 2-cell growth based on over-expression mouse models. Aim 3 will compare the effects of insulin phosphorylation mutants of CBP and FoxO1, expressed at allelic levels, on hepatic glucose production and 2-cell regulation and/or growth. These studies are significant because they will establish signaling pathways that normally regulate the hepatocyte and pancreatic 2-cell in vivo and provide a mechanistic understanding of pathophysiological changes found in patients with diabetes mellitus. In this proposal, we will attempt to understand the physiological importance of CBP and p300 in the hepatocyte and pancreatic 2-cell, in the context of other factors reported to mediate activation of genes containing cAMP response elements
Keywords: 3`5`-cyclic ester of AMP; Adenosine Cyclic 3`, 5`-Monophosphate; Adenosine Cyclic Monophosphate; Adenosine Cyclic Monophosphate-Dependent Protein Kinases; Adenosine, cyclic 3`, 5`-(hydrogen phosphate); Affect; Amino Acids; Aminoacetic Acid; Animals; Antidiabetic Hormone; Binding; Binding (Molecular Function); Binding Proteins; CBP protein; CBP protein, human (CREB binding protein); CRE; CRE Binding Protein; CREB; CREB Binding Protein; CREB Protein; CREB binding protein, human; CREB-binding protein; CREBBP protein, human; Cell Communication and Signaling; Cell Function; Cell Growth in Number; Cell Multiplication; Cell Nucleus; Cell Process; Cell Proliferation; Cell Signaling; Cell physiology; Cells; Cellular Expansion; Cellular Function; Cellular Growth; Cellular Physiology; Cellular Process; Cellular Proliferation; Cyclic AMP; Cyclic AMP Response Element; Cyclic AMP Response Element-Binding Protein; Cyclic AMP Responsive Element Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cyclic AMP-Responsive DNA-Binding Protein; D-Glucose; Defect; Dephosphorylation; Dextrose; Diabetes Mellitus; Dissociation; E1A-associated p300 protein; EP300; EP300 gene; Fasting; GCG; Gastrointestinal Tract, Pancreas; Gene Activation; Gene Targeting; Gene Transcription; Generalized Growth; Genes; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Glucagon; Glucagon (1-29); Gluconeogenesis; Glucose; Glukagon; Glycine; Goals; Growth; HG-Factor; Hepatic; Hepatic Cells; Hepatic Parenchymal Cell; Hepatocyte; Humulin R; Hyperglycemia; Hyperglycemic-Glycogenolytic Factor; Hyperplasia; Hyperplastic; Hypoglycemia; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin Resistance; Insulin, Regular; Intracellular Communication and Signaling; Knock-in; Knock-in Mouse; L-Serine; Ligand Binding Protein; Liver; Liver Cells; Mammals, Mice; Mediating; Mice; Mice, Mutant Strains; Modeling; Molecular Interaction; Murine; Mus; Mutant Strains Mice; Mutate; Mutation; Novolin R; Nuclear; Nucleus; Orthophosphate[{..}]oxaloacetate carboxy-lyase (phosphorylating); PEPCK; PKA; Pancreas; Pancreatic; Patients; Phenotype; Phospho-CREB Binding Protein; Phosphoenolpyruvate Carboxylase; Phosphorylation; Phosphorylation Site; Physiologic; Physiological; Play; Position; Positioning Attribute; Process; Production; Programs (PT); Programs [Publication Type]; Protein Binding; Protein Dephosphorylation; Protein Kinase A; Protein Phosphorylation; Proteins; RNA Expression; Recruitment Activity; Regulation; Relative; Relative (related person); Reporting; Resistance; Role; Rubinstein-Taybi syndrome protein, human; Serine; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Site; Subcellular Process; Targetings, Gene; Testing; Tissue Growth; Transcription; Transcription, Genetic; Transducers; adenosine 3`5` monophosphate; aminoacid; attenuation; base; biological signal transduction; body system, hepatic; cAMP; cAMP Response Element; cAMP Response Element-Binding Protein; cAMP Responsive Element Binding Protein; cAMP-Dependent Protein Kinases; cell growth; cofactor; diabetes; fasted; fasts; fork head protein; forkhead protein; forkhead transcription factors; gene product; genome mutation; glucose RA; glucose biosynthesis; glucose production; glucose rate of appearance; hepatic gluconeogenesis; homologous recombination; human CREBBP protein; hyperglycemic; hypoglycemic; hypoglycemic episodes; impaired glucose tolerance; in vivo; insulin resistant; insulin secretion; mouse model; mouse mutant; mutant; novel; nuclear protein CBP; ontogeny; organ system, hepatic; p300; p300 E1A-associated coactivator; p300 protein; p300-CBP coactivator; p300/CBP proteins; phospho-CREB-binding protein; phosphoenolpyruvate carboxykinase; programs; protein complex; protein expression; public health relevance; reconstitute; reconstitution; recruit; resistant; social role; transcription factor
Relevance: In this proposal, we will attempt to understand the physiological importance of CBP and p300 in the hepatocyte and pancreatic ¿-cell, in the context of other factors reported to mediate activation of genes containing cAMP response elements
Project start date: 2002-09-30
Project end date: 2012-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
5R01DK063349-07 (2010): $360554
5R01DK063349-06 (2009): $390474
Clinical/Moleular Studies Of Thyroid Hormone Resistance
Fredric E Wondisford, Professor And Chief
Medicineuniversity Of Chicago
5801 S Ellis Ave
chicago, Il 60637
Grant 1K24DK059449-01 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: ZDK1
Abstract: Taken directly ) The syndrome of resistance to thyroid hormone is characterized by elevated thyroid hormone levels and inappropriate TSH secretion due in almost all cases to point mutations in the TR-b locus. Studies of these naturally occurring TR mutations in vitro have yielded useful insights into the syndrome of RTH. Unfortunately, the generalizability of these studies to the patient´s disorder is not always clear and because of their artificial nature may not always be secure. Confounding these issues is the clinical heterogeneity of RTH in families with the same mutation and the of at least distinct varieties of RTH in man generalized RTH (GRTH) and central RTH (CRTH). The goal of these studies will be to define the clinical differences between these forms of RTH and provide a molecular explanation for these differences. This proposal will allow the Principal Investigator to devote additional time to the clinical study of patients with RTH and the mentoring of new investigators entering the field of thyroid research. The candidate has recently accepted the position of Chief of Endocrinology and Professor of Medicine at the University of Chicago. He has independent support to study thyroid hormone action (R01 DK49126 and R01 DK53036) in animals and man and TRH signaling pathways in the pituitary (R01 DK50564). He directs a CRC protocol to evaluate and categorize patients with RTH. He actively mentors three BC endocrinologists in thyroid research, each having NIH K08 support for their projects. With his previous research and mentoring success, in an environment rich with academic opportunities, this award will allow him to increase his mentoring activities and to enhance his own commitment to patient-oriented research
Keywords: hormone sensitivity /resistance, molecular pathology, sign /symptom, syndrome, thyroid hormone point mutation, secretion, thyrotropin human subject, patient oriented research
Project start date: 2001-07-01
Project end date: 2006-05-31
1K24DK059449-01 (2001): $101218
7K24DK059449-05 (2005): $116335
5K24DK059449-04 (2004): $101218
5K24DK059449-03 (2003): $101218
MOLECULAR MECHANISMS OF THYROID HORMONE ACTION
Fredric E Wondisford, Chief, Metabolism Division
Johns Hopkins University, 3400 N Charles St, Baltimore, Md 21218
Grant 5R01DK053036-11 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: Resistance to thyroid hormone (RTH) is a human syndrome characterized by abnormal regulation of the hypothalamic-pituitary-thyroid axis (HPT axis) leading to inappropriate TSH secretion and elevated serum thyroid hormone levels. A central role for thyroid hormone receptor-beta (TR-beta) gene in thyroid hormone negative regulation of the HPT axis was established by finding TR-13 gene mutations in patients with RTH and by deleting the TR-beta isoforms in mice. Despite years of study, however, the mechanism of thyroid hormone inhibition of the HPT axis remains unclear. In normal thyroid hormone action, TR-a interacts with co-repressor molecules in the absence of ligand (T3) and co-activator molecules in the presence of T3 to stimulate gene expression. In contrast, the mechanism of negative regulation by thyroid hormone is less clear. Understanding how the same receptor and associated cofactors both stimulates and inhibits gene expression would enhance our understanding of transcriptional signaling processes and may provide the basis for discovery of novel signaling pathways. To accomplish this goal three specific aims are proposed A.1 To elucidate the mechanism of T3 inhibition by using a mouse thyrotroph (TSH-secreting) cell line (TaT1.1). Using a variety of methods, a subclone of the TaT1 cell line (TaT1.1) will be used to understand the nature of the protein complexes bound to the TSH-beta nTRE and their functional significance. A.2 To determine the direct role of co-activators in TR-beta mediated T3 inhibition in vivo. A point mutation was introduced into helix 12 (E457A) of the mouse TR-beta locus (E457A knock-in). This mutation specifically interferes with co-activator binding without affecting co-repressor or ligand (T3) binding. The effect of this mutation on both positive and negative T3 regulation will be determined in vivo. A.3 To determine the direct role of co-repressors in TR-beta mediated T3 inhibition in vivo. A point mutation (I280M) will be introduced into mouse TR-beta locus (I280M knock-in). This mutation specifically interferes with co-repressor binding without affecting either co-activator or ligand binding. The effect of this mutation on both positive and negative T3 regulation will be determined in vivo. Study of other animal models, where co-repressor function has been altered, is also planned in this aim
Keywords: 9-cis-Retinoic Acid Receptor; Affect; Animal Model; Animal Models and Related Studies; Binding; Binding (Molecular Function); Blood Serum; CD3; CD3 Antigens; CD3 Complex; CD3 molecule; Cell Line; Cell Lines, Strains; CellLine; Cells; Complex; DNA Alteration; DNA mutation; Diamond; ERBA Beta Protein; ERBA2; ERBA2 Gene Products; ERBA2 Protein; Gene Alteration; Gene Expression; Gene Mutation; Gene Transcription; Generalized Thyroid Hormone Resistance; Genes, TR-beta; Genes, erbA beta; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Genetic mutation; Goals; Human; Human, General; Hypothalamic structure; Hypothalamus; Isoforms; Knock-in; Knock-in Mouse; Ligand Binding; Ligands; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Methods; Mice; Modeling; Molecular; Molecular Interaction; Molecules, Repressor; Murine; Mus; Mutation; NR1A2 Gene Products; Nature; Nuclear; OKT3 antigen; Patients; Point Mutation; Principal Investigator; Programs (PT); Programs [Publication Type]; Protein Isoforms; RNA Expression; RXR; RXR Protein; Receptor Protein; Recruitment Activity; Refetoff Syndrome; Refetoff-DeWind-DeGroot Syndrome; Regulation; Repressor Molecules; Resistance; Retinoic Acid Receptor RXR; Retinoid X Receptors; Role; Sequence Alteration; Serum; Signal Pathway; Syndrome; T3 Antigens; T3 Complex; T3 molecule; THR1; THRB; THRB Gene Products; THRB Protein; THRB gene; THRB1; THRB2; TR beta; TSH, beta Chain; TSH, beta Subunit; TSH-beta; TSHB Gene Product; Testing; Thyroid Gland Hormone; Thyroid Hormone Receptor Beta; Thyroid Hormone Receptor Beta Gene; Thyroid Hormone Resistance; Thyroid Hormone Resistance Syndrome; Thyroid Hormones; Thyroid Stimulating Hormone, beta Subunit; Thyrotropin, beta Chain; Thyrotropin, beta Polypeptide Chain; Thyrotropin, beta Subunit; Thyrotropin-beta; Transcription; Transcription, Genetic; base; beta Subunit Thyrotropin; c-erbA beta; cofactor; computerized data processing; cultured cell line; data processing; genome mutation; hormone response element; hypothalamic; in vivo; model organism; monomer; novel; pituitary thyroid axis; programs; protein complex; receptor; recruit; resistant; signal processing; social role
Project start date: 1999-02-15
Project end date: 2011-01-31
Budget start date: 1-FEB-2009
Budget end date: 31-JAN-2011
5R01DK053036-11 (2009): $342881
5R01DK053036-09 (2007): $349434
5R01DK053036-08 (2006): $358773
2R01DK053036-06A1 (2005): $343125
Sponsored Links Excellgen http://Excellgen.com
MOLECULAR MECHANISMS OF THYROID HORMONE RESISTANCE
Fredric E Wondisford, Professor And Chief
Beth Israel Deaconess Medical Center
330 Brookline Avenue, Br 264
boston, Ma 02215
Grant 5R01DK053036-02 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: END
Abstract: Patients with the syndrome of resistance to thyroid hormone (RTH) exhibit inappropriate section of TSH and elevated thyroid hormone levels due to mutations occurring in the beta gene of the thyroid hormone receptor (TR). The TR-beta gene encodes two isoforms and it is unclear whether both isoforms are equally important in negative regulation of TSH secretion by T/3 or how mutant TR-beta isoforms function to cause RTH. In vitro studies suggest that the TR-beta isoforms differ in their ability to mediate negative regulation of TRH and TSH subunit gene transcription by T/3 and vary in their function as dominant negative inhibitors of gene expression in RTH. Physiological studies in vivo are lacking, however, to confirm the significance of these findings. This proposal will test whether differences in TR-beta isoform regulation of TSH subunit gene expression by T/3 are relevant in vivo and are responsible for distinct syndromes of resistance to thyroid hormone (RTH). Three specific aims are proposed to elucidate the function of TR- beta isoforms in normal and T/3 resistance states. In the first aim, the specific role of the TR-beta2 isoform in the regulation of TRH and TSH subunit gene expression in vivo will be determined through targeted disruption of the TR-beta2 isoform in mice. In the second aim, the function of nuclear hormone receptor co-repressor protein (NCoR) on pituitary thyroid function will be determined in vivo by targeted over- expression of an NCoR inhibitor in transgenic mice. Finally, in third aim, the relative roles of the TR-beta isoforms in mediating distinct syndromes of resistance to thyroid hormone (RTH) will be assessed by expression of mutant TRs, in the context of either a TR-beta1 or TR- beta2 isoform, in the hypothalamus or pituitary or transgenic animals. The overall goal of this proposal is to clarify the function of TR-beta isoforms in vivo by utilizing physiologically relevant model systems. Determination of TR isoform-specific regulation of gene expression in vivo would yield novel insights into thyroid hormone action in man
Keywords: hormone receptor, hormone sensitivity /resistance, receptor expression, thyroid hormone, thyrotropin, thyrotropin releasing hormone gene expression, genetic promoter element, genetic regulatory element, hormone regulation /control mechanism, hypothalamus, pituitary gland, protein isoform gene targeting, histochemistry /cytochemistry, in situ hybridization, laboratory mouse, transfection, transgenic animal
Project start date: 1999-02-15
Project end date: 2001-01-31
5R01DK053036-02 (2000): $262625
1R01DK053036-01A2 (1999): $254974
5R01DK053036-05 (2003): $246852
7R01DK053036-03 (2001): $232682
DIABETES RESEARCH AND TRAINING CENTER
Fredric E Wondisford, Chief, Metabolism Division
Johns Hopkins University, W400 Wyman Park Building, Baltimore, Md 21218
Grant 3P60DK079637-03S1 from National Institute Of Diabetes And Digestive And Kidney Diseases
Abstract: More than 80 genes in the human genome code for voltage-gated ion channels and their relatives in the 6TM ion-channel family. Among their many roles these channels mediate pain and other sensory modalities, perform long-distance signaling in the brain, time the heartbeat and control lymphocyte proliferation. They are of great interest as a rich set of potential targets for drugs and therapies, and are also of intrinsic interest as proteins having a uniquely high sensitivity to membrane potential changes. Single-particle electron cryomicroscopy (cryo-EM) is a method for observing the three-dimensional structure of macromolecular complexes. Although the resolution is inferior to X-ray crystallography, cryo-EM technology is making steady progress in this area; meanwhile it has the great advantage of providing "solution structures" of proteins without the necessity of forming crystals. We have developed methods for cryo-EM single-particle imaging of membrane proteins reconstituted into liposomes, and have recently obtained the first closed-state structure of a eukaryotic 6TM channel, the large-conductance Ca2+activated potassium (BK) channel. The structure has relatively low resolution, limited by the small number of particle images we have been able to acquire. In this application we propose first to greatly increase the data-collection efficiency in order to reach resolutions better than 1 nm. We will then image the BK channel in its various conformational states. In parallel, we will apply the methods to Kv1.2, one of the best-studied voltage-gated potassium channels. By creating membrane potentials in the vesicles, we will be able to trap this channel in its closed as well as open states for structur determination. Voltage-gated ion channels act as molecular switches, controlling the electrical currents in the brain, heart and many other organs. Because there are many (more than 80) varieties, defects in these ion channels give rise to a spectrum of disorders ranging from epilepsy, migraine and muscular paralysis to hypertension and irregular heart rhythm. To understand how they work, we propose to observe the molecular structure of two of these channels, in their various functional states, using novel electron- microscopy techniques
Project start date: 2010-05-10
Project end date: 2011-04-30
Budget start date: 10-MAY-2010
Budget end date: 30-APR-2011
PFA/PA: RFA-DK-06-014
3P60DK079637-03S1 (2010): $88235
Insulin Regulation Of The Gonadotroph
Fredric E Wondisford, Professor And Chief
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637
Grant 1U54HD041859-01A10004 from National Institute Of Child Health And Human Development IRG: ZHD1
Abstract: In the anterior pituitary, the gonadotroph synthesizes luteinizing hormone (LH) and follicle stimulating hormone (FSH), which are critical for normal reproductive function. Recent evidence indicates that the gonadotroph develops from a pituitary progenitor cell by acquisition of GATA2 expression. Subsequently, gonadotrophs synthesize and secrete LH and FSH in response to gonadotropin-releasing hormone (GnRH). GnRH bound to its receptor activates several intracellular signaling pathways shared with insulin and insulin like growth factor 1 ([GF-1). Importantly, GnRH signaling activates an activating protein 1 (AP-1) element in the 5 flanking region of the GnRH receptor (GnRHr) gene, leading to increased GnRHr density on gonadotrophs. Both GATA factors and AP-1 recruit CREB (cAMP response element binding protein) binding protein (CBP) to the transcription complex, where it functions as a potent co-activator. My laboratory has recently identified a phosphorylation site on CBP which is a potential target for both GnRH and insulin signaling pathways. Since insulin signaling is clearly essential for the response of the reproductive axis to nutritional stimuli, its role and interaction with GnRH signaling in the gonadotroph will be explored. Therefore, the goals of this project will be to determine the importance of the pituitary gonadotroph in insulin regulation of the reproductive axis, and the molecular mechanism whereby insulin regulates gonadotroph-specific gene expression. Three specific aims are proposed. In aim 1, the specific role of insulin signaling in the gonadotroph will be determined using a conditional knock out (cKO) of the insulin receptor in the [gonadotroph.] In aim 2, the molecular mechanism(s) of CBP interaction(s) with gonadotroph-speciflc transcription factors, such as AP-1 and Egr-1, will be explored. Finally in Aim 3, the importance of CBP in the gonadotroph will be definitively established through a cKO of this co-activator. These studies will lead to unique insights into mechanisms responsible for insulin and GnRH regulation of gonadotropin secretion in the pituitary.
Keywords: gonadotropin releasing factor, hormone biosynthesis, hormone regulation /control mechanism, insulin, pituitary gland, binding protein, biological signal transduction, cAMP response element binding protein, cooperative study, follicle stimulating hormone, gene expression, luteinizing hormone, protein protein interaction, gene targeting, laboratory mouse, transfection
Project start date: 2003-04-28
Project end date: 2008-03-31
MOLECULAR MECHANISMS OF THYROID HORMONE ACTION
Fredric E Wondisford, Professor And Chief
University Of Chicago 5801 S Ellis Ave Chicago, Il 60637
Grant 5R01DK049126-08 from National Institute Of Diabetes And Digestive And Kidney Diseases IRG: END
Abstract: Adapted from s ) The syndrome of resistance to thyroid hormone is characterized by elevated thyroid hormone levels and inappropriate TSH secretion due in almost all cases to point mutations in the TR-b locus. Studies of these naturally occurring TR mutations in vitro have yielded useful insights into the syndrome of RTH. Unfortunately, the generalizability of these studies to the patient s disorder is not always clear and because of their artificial nature may not always be secure. Confounding these issues are the clinical heterogeneity of RTH in families with the same mutation and the suggestion that at least two distinct varieties of RTH are found in man generalized RTH (GRTH) and central RTH (CRTH). Recently, a TR knockout model in mice has been described having some features in common with RTH but also displaying some significant differences. This autosomal recessive model is in fact pathophysiologically distinct from the vast majority of patients with RTH where a mutant TR expressed from one TR-beta allele dominantly interferes with gene expression by the remaining normal TRs. A few in vivo models of mutant TR-beta overexpression causing RTH have also been described but unfortunately the level and tissue distribution of mutant TR-beta expression may not reproduce that found in RTH patients. Given these limitations, the investigator s laboratory has begun to develop mouse RTH models by introducing point mutations into the TR-beta locus by homologous recombination. This proposal has three aims directed at understanding whether the ligand-independent activity of the TR in vivo is important in the genesis of RTH and whether the selective form, central RTH (CRTH), is a discrete clinical entity. By providing a genetically homogenous background for study and the ability to extensively analyze gene expression in different tissues, these models (Aim 1 and 2) complement ongoing studies in humans with the RTH syndrome (Aim 3).
Keywords: disease /disorder model, hormone sensitivity /resistance, model design /development, thyroid hormone, autosomal recessive trait, hormone receptor, pathologic process, point mutation, gene targeting, laboratory mouse, transgenic animal
Project start date: 1995-08-01
Project end date: 2005-11-30
5R01DK049126-08 (2004): $226972
5R01DK049126-07 (2003): $227176
5R01DK049126-06 (2002): $227374
Sponsored Links Excellgen http://Excellgen.com
5R01DK049126-09 (2005): $226763
2R01DK049126-05A1 (2001): $227566
Fredric E Wondisford
Johns Hopkins University
Project start date: 2008-04-01
Project end date: 2013-01-31
DIABETES RESEARCH AND TRAINING CENTER
Fredric E Wondisford, Chief, Metabolism Division
Johns Hopkins University, 3400 N Charles St, Baltimore, Md 21218
Grant 5P60DK079637-03 from National Institute Of Diabetes And Digestive And Kidney Diseases
Keywords: No Project Terms available
Project start date: 2008-04-01
Project end date: 2013-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: RFA-DK-06-014
5P60DK079637-03 (2010): $1612685
5P60DK020595-27 (2004): $1887403
TESTING OF PATIENTS WITH SUSPECTED DISORDERS OF TSH SECRETION
Fredric E Wondisford, Professor And Chief
Beth Israel Deaconess Medical Center 330 Brookline Avenue, Br 264 Boston, Ma 02215
Grant 5M01RR001032-280653 from National Center For Research Resources
Keywords: diagnosis design /evaluation, endocrine disorder diagnosis, hypothyroidism, thyroid hormone, hormone regulation /control mechanism, pituitary gland, clinical research, human subject
Sponsored Links Excellgen http://Excellgen.com