Report of the 90th Annual Meeting of the Endocrine Society, SAN FRANCISCO – JUNE 15-18, 2008

The progesterone receptor (PR) is a hormone activated transcription factor essential for female reproductive function. PR also activates cell signaling pathways through its interaction with src kinase. In turn, the activities of PR and its coactivators are dependent on phosphorylation by a variety of kinases. Initial studies using the chicken PR (cPR) as a model resulted in the identification of four phosphorylation sites. One of these controls the sensitivity of response to hormone whereas the others are required for optimal transcriptional activity. The activity of cPR is very responsive to alterations in activation of cell signaling pathways. In some cases, activation of a protein kinase or inhibition of a phosphatase is sufficient to activate cPR in the absence of hormone. The phosphorylation of human PR (hPR) is much more complex. It is expressed as two isoforms, the full length PR-B and PR-A, which lacks the first 164 amino acids of PRB. We have identified 14 phosphorylation sites in hPR, most of which are in the aminoterminus with six unique to the PR-B isoform. Initial studies using breast cancer cell lines expressing phosphorylation site mutants show that the requirements for specific phosphorylations are target gene dependent. Activation of specific cell signaling pathways potentiates the activity of hPR and, in some cases, causes the partial antagonist, RU486, to function as an agonist. The activity of hPR also is dependent on cyclin dependent kinases. We find that cyclin A2 is a PR coactivator that is recruited to PR binding sites in target genes. Although PR can be phosphorylated by cyclin A2/Cdk2 in vitro, our data suggest that the kinase is required for phosphorylation of the coactivator, SRC-1, enhancing its interaction with PR. Elimination of cyclin A2 reduces PR dependent induction of a subset of endogenous target genes. Moreover, PR phosphorylation and PR activity vary as a function of the cell cycle with optimal transcriptional activity of an integrated MMTV CAT reporter during S phase, where cyclin A2 expression is highest. Cyclin A2 is overexpressed in breast cancer and may contribute to altered PR activity. In addition, recent studies suggest that administration of progesterone later in life increases risk for breast cancer. These findings support the need for a greater understanding of the regulation of PR function. MAPKs in Beta Cell Function. MH Cobb, Pharmacology, Univ of Texas SW Med Ctr, Dallas, TX Abstract. The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are regulated by glucose and other nutrients, as well as by hormones and growth factors in pancreatic beta cells. These kinases are sensitive to the secretory demand placed on beta cells. ERK1/2 regulate the transcription of numerous genes including the insulin gene, both positively and negatively, promoting transcriptional changes appropriate to the differentiated state, the secretory needs and the stress on beta cells. Nutrients and hormones controlling insulin secretion activate ERK1/2 in a calcium-dependent manner in beta cells, while insulin itself and several growth factors regulate these kinases by calcium-independent mechanisms. We are studying the mechanisms through which ERK1/2 influence transcription. At least five factors that enhance insulin gene transcription show sensitivity to the ERK1/2 pathway inhibitors U0126 and/or PD98059. Direct and indirect findings suggest that at least four of these factors are direct substrates. Three additional factors that inhibit insulin gene transcription also appear to be ERK1/2 substrates as their functions are also inhibited by U0126. The transcription of several other ERK1/2-sensitive genes, including the apoptotic factor CHOP-10 (GADD153/DDIT-3) and c-fos, and the actions of glucose on their transcription are also under study to define the essential regulatory mechanisms. These studies support hypothesis that the genes regulated by ERK1/2 and the mechanisms employed are key to maintaining normal beta cell function. MicroRNAs as Regulators of Cardiac Stress & Thyroid Hormone Responsiveness. E Olson, Molecular Biology, Univ of Texas SW Med Ctr, Dallas, TX Abstract. MicroRNAs act as negative regulators of gene expression by inhibiting the translation and promoting the degradation of target mRNAs. We have identified a signature pattern of microRNAs associated with heart failure in humans and mouse models of heart disease. Gainand loss-of-function studies in mice have revealed key roles for these microRNAs as regulators of growth, contractility, energy metabolism and responsiveness of the heart to stress and thyroid hormone signaling, providing glimpses of new regulatory mechanisms and potential therapeutic targets for heart disease. Comments (prepared by Dr. Nora Saraco) miRNAs are approximately 22 nucleotides in length and inhibit translation by interacting with the 3′ untranslated regions of specific mRNA targets. miRNAs commonly function to modulate or fine-tune cellular phenotypes by repressing expression of proteins that are inappropriate for a particular cell type or by adjusting protein dosage. miRNAs have also been proposed to provide robustness to cellular phenotypes by eliminating extreme fluctuations in gene expression. Numerous miRNAs are upregulated in response to cellular stress. The human genome is predicted to encode as many as a thousand miRNAs. To date, the in vivo functions of only a handful of miRNAs have been determined in mammals. miRNAs are transcribed by RNA polymerase II and can be derived from individual miRNA genes, from introns of protein-coding genes, or from polycistronic transcripts that often encode multiple, closely related miRNAs. Pri-miRNAs, generally several thousand bases long, are processed in the nucleus by the RNase Drosha into 70–100 nucleotide, hairpin-shaped precursors, called pre-miRNAs. Following transport to the cytoplasm, the pre-miRNA is further processed by the RNA endonuclease Dicer to produce a double-stranded miRNA. The fully processed miRNA duplex is then incorporated into a multicomponent protein complex known as RNA-induced silencing complex (RISC). During this process, one strand of the miRNA duplex is selected as a mature miRNA while the other strand, known as miRNA*, is in general rapidly removed and degraded. This selection process is primarily determined by the strength of base pairing at the end of the miRNA:miRNA* duplex. As part of the RISC, miRNAs negatively regulate gene expression through two major mechanisms, translational repression and mRNA cleavage, which depend on the extent of complementarity between the miRNA and its mRNA target and other criteria that are still being defined. Although it was originally thought that perfect base pairing was a requisite for mRNA cleavage, it has now become clear that even imperfect base pairing can lead to a decrease in target mRNA abundance. Cardiac hypertrophy and pathological remodeling The heart responds to chronic and acute injury by hypertrophic growth. Cardiomyocyte hypertrophy is the dominant cellular response to virtually all forms of hemodynamic overload, endocrine disorders, myocardial injury, or inherited mutations in a variety of structural and contractile proteins. Stress-mediated myocardial hypertrophy is a complex phenomenon. In response to pathological stress, cardiac fibroblasts and extracellular matrix proteins accumulate disproportionately and excessively. Myocardial fibrosis, a characteristic of all forms of pathological hypertrophy, leads to mechanical stiffness, which contributes to contractile dysfunction. The upregulation of βMHC, a slow ATPase, and downregulation of αMHC, a fast-contracting ATPase, in response to stress has been implicated in the diminution of cardiac function. βMHC is the predominant myosin isoform expressed in the heart prenatally whereas αMHC is upregulated after birth. Thyroid hormone signaling soon after birth provides the stimulus for the β– to αMHC switch. αMHC represents more than 95% of MHC in the adult rodent heart whereas it constitutes only about 30% of MHC in the adult human heart. Nevertheless, downregulation of αΜΗC and upregulation of βMHC is a common response to cardiac injury irrespective of the species. miRNAs and control of stress-dependent cardiac hypertrophy Cardiac-specific overexpression of miRNA-195 (miR-195), which is consistently upregulated in rodent and human hypertrophic hearts, results in dilated cardiomyopathy and heart failure in mice as early as two weeks of age. Another miRNA consistently induced by cardiac stress, miR-21, appears to function as a regulator of cardiac growth and fetal gene activation in primary cardiomyocytes in vitro. Knockdown of miR-21 can suppress cardiomyocyte growth and fetal gene expression in response to the hypertrophic agonists angiotensin II and phenylephrine. Encoded within intron 27 of the gene encoding αMHC is miR-208. Like αMHC, miR208 is expressed specifically in the heart with trace expression in the lung. miR-208 is processed out of the αMHC pre-mRNA rather than being transcribed as a separate transcript. miR-208 displays a remarkably long half-life of at least 14 days and can thereby exert functions even when αMHC mRNA expression has been downregulated. miR-208, regulates stress-dependent cardiomyocyte growth and gene expression. In the absence of miR-208, the expression of ßMHC is severely blunted in the adult heart in response to pressure overload, activated calcineurin, or hypothyroidism, suggesting that the pathways through which these stimuli induce ßMHC transcription share a common miR-208–sensitive component. In contrast, ßMHC expression was unaltered in the hearts of newborn miR-208–/– mice, demonstrating that miR-208 participates specifically in the mechanism for stress-dependent regulation of ßMHC expression. A clue to the mechanism of action of miR-208 comes from the resemblance of miR208–/– hearts to hyperthyroid hearts, both of which display a block to ßMHC expression, up-regulation of stress-response genes, and protection against pathological hypertrophy and fibrosis. The up-regulation of fast skeletal muscle genes in miR-208–/– hearts also mimics the induction of fast skeletal muscle fibers in the hyperthyroid state. T3 signaling induces αMHC transcription through a positive T3 response element (TRE), whereas a negative TRE in the promoter of the ßMHC gene mediates transcriptional repression in the postnatal heart, and PTU (Propylthiouracil), which causes hypothyroidism (prevents T3 biosynthesis), induces ßMHC. The inability of PTU to induce ßMHC expression in miR-208–/– hearts further implicates miR-208 in the T3 signaling pathway. These results suggest that miR-208 acts, at least in part, by repressing expression of the TR (Thyroid hormone receptor) coregulator THRAP1, which can exert positive and negative effects on transcription. The TR acts through a negative TRE to repress ßMHC expression in the adult heart. Thus, the increase in THRAP1 expression in the absence of miR-208 would be predicted to enhance the repressive activity of the TR toward ßMHC expression, consistent with the blockade to ßMHC expression in miR-208–/– hearts. In contrast, the regulation of αMHC and ßMHC expression during development is independent of T3 signaling (2) and is unaffected by miR-208. It has been proposed that the ßMHC gene may respond to specific TR isoforms. Perhaps THRAP1 acts on specific TR isoforms or selectively on a subset of TR-dependent genes through interactions with promoter-specific factors. Because miRNAs generally act through multiple downstream targets to exert their effects, additional targets are also likely to contribute to the effects of miR-208 on cardiac growth and gene expression. Relatively minor increases in ßMHC composition, as occur during cardiac hypertrophy and heart failure, can reduce myofibrillar ATPase activity and systolic function. Thus, therapeutic manipulation of miR-208 expression or interaction with its mRNA targets could potentially enhance cardiac function by suppressing ßMHC expression. Based on the profound influence of miR-208 on the cardiac stress response, and the regulation of numerous miRNAs in the diseased heart, we anticipate that miRNAs will prove to be key regulators of the functions and responses to disease of the adult heart and possibly other organs. Referentes. van Rooij E, Olson EN. MicroRNAs: powerful new regulators of heart disease and provocative therapeutic targets. J Clin Invest. 2007 Sep;117(9):2369-76. Review. van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN. Control of stressdependent cardiac growth and gene expression by a microRNA. Science. 2007 Apr 27;316(5824):575-9. Gsα Imprinting & Metabolic Control. LS Weinstein, Metabolic Diseases Branch, NIDDK/NIH, Bethesda, MD Abstract. Gsα, the α-subunit of the heterotrimeric G protein Gs, is ubiquitously expressed and mediates the intracellular cAMP response to multiple hormone and other receptors. Activating and inactivating mutations of the Gsα gene GNAS lead to a wide variety of disorders. Heterozygous Gsα loss-of-function mutations lead to characteristic skeletal and neurobehavioral abnormalities known as Albright hereditary osteodystrophy (AHO). Patients with mutations on the maternal allele also develop resistance to various hormones (eg. PTH, TSH) and early-onset obesity (pseudohypoparathyroidism type 1A, PHPIA). Likewise mice with germline disruption of the maternal but not the paternal Gsα allele develop metabolic syndrome, with severe obesity, reduced energy expenditure, insulin resistance, diabetes, and hypertriglyceridemia. These parent-oforigin effects result from tissue-specific genomic imprinting of Gsα leading to suppressed Gsα expression from the paternal allele in a small number of tissues. Genetic studies in PHPIB patients who have PTH resistance without AHO have identified a Gsα imprint control region upstream of the Gsα promoter. Paternal deletion of this region reverses Gsα imprinting and also reverses the metabolic consequences of maternal Gsα mutation, which confirms that the metabolic changes result from severe Gsα deficiency in one or more tissues due to the combined effects of mutation on the maternal allele and imprinting on the paternal allele. The metabolic features of maternal Gsα germline mutation are completely reproduced in mice in which the maternal Gsα allele is disrupted only in the central nervous system, while disruption of the paternal allele in the CNS produces no metabolic phenotype. These findings suggest that Gsα is imprinted in one or more CNS regions involved in metabolic control and that metabolic consequences of maternal Gsα mutations in mice and in PHP1A patients results from dysregulation of metabolic control by the CNS. Melanocortin receptors in the CNS (MC4R, MC3R) negatively regulate energy balance via reduced food intake and increased energy expenditure and are coupled to Gsα. Studies in CNS-specific Gsα knockout mice show that the ability of melanocortins to stimulate energy expenditure are reduced while their anorexic effects are not altered. These findings suggest that the effects of melanocortins on food intake and energy expenditure may involve distinct signaling pathways, neuronal populations, or anatomical sites. Comments. This is a good analysis of the complex subject of Gsα multiple roles and its pathological or experimental disruptions. Just as a remainder it might be good to remember that melanocortins are a group of pituitary peptide hormones that include adrenocorticotropin (ACTH) and the alpha, beta and gamma melanocyte-stimulating hormones (MSH) that derive from the prohormone proopiomelanocortin. Melanocortins act through a multitude of melanocortin receptors designated MC1 through MC5. MC2 is also known as the adrenocorticotrophic hormone receptor since it selectively binds ACTH. The other melanocortin receptors are semi-selective in their ability to bind multiple melanocortins to some degree. As reviewed by Wikberg et al., knowledge of melanocortins and their receptors has increased tremendously over the last few years. The cloning of five melanocortin receptors, and the discovery of two endogenous antagonists for these receptors, agouti and agouti-related peptide, have sparked intense interest in the field. A comprehensive review of the pharmacology, physiology and molecular biology of the melanocortins and their receptors is reviewed. In particular, the roles of the melanocortins in the immune system, behaviour, feeding, the cardiovascular system and melanoma. Moreover, while evidence is discussed suggesting that while many of the actions of the melanocortins are mediated via melanocortin receptors, some appear to be mediated via mechanisms distinct from melanocortin receptors (Wikberg JE, Muceniece R, Mandrika I, Prusis P, Lindblom J, Post C, Skottner A. New aspects on the melanocortins and their receptors. Pharmacol Res. 2000 Nov;42(5):393-420). Cancer & Cell Signaling. J Fagin, Medicine, Mem Sloan-Kettering Cancer Ctr, New York, NY Abstract. Many of the genetic changes associated with human thyroid cancers of follicular origin have been identified. Mutually exclusive mutations of RET, NTRK1, RAS or BRAF (serine/threonine kinase B-RAF) are present in 70% of PTCs, whereas only the latter two are seen in poorly differentiated and anaplastic cancers. As RAS and BRAF are associated with more aggressive disease, it is critical to determine whether these oncoproteins drive the neoplastic process once cancers are fully established. We are exploring these questions using mice with conditional knock-in mutations of HRAS and BRAF, which when targeted to thyroid cells result in endogenous levels of expression levels of the respective oncoproteins. Oncogenic BRAF induces thyroid cancers with high penetrance at an early age, which is associated with downregulation of expression of thyroid-specific gene products, including TPO, NIS and Tg. This is likely a direct effect of BRAF, as doxycycline-induced expression of BRAF in thyroid cells in vivo recapitulates these events within days. The reversibility of these changes is currently under study. By contrast, endogenous expression of HRAS in thyroid cells does not by itself induce cancer development within the first year of life, nor does it alter thyroid function. However, when oncogenic HRAS is expressed in mice with heterozygous loss of Pten, they develop follicular thyroid cancers late in life, associated with loss of function of the remaining Pten allele. Evidence that physiological expression levels of oncogenic BRAF, but not HRAS, result in thyroid dedifferentiation is consistent with data from thyroid cancer patients, and provides a hypothetical framework for reversal of these changes by inhibiting activity of MEK, its immediate downstream effector. Indeed, when tested against a large panel of human thyroid cancer cell lines, MEK inhibitors demonstrate preferential growth-inhibitory activity against cell lines with BRAF mutations. The dominant trend in experimental oncological therapeutics is directed towards the inhibition of oncogenic targets that are intrinsic to the cancer cell, with the notable exception of agents that block tumor angiogenesis. Recent evidence suggests that the tumor microenvironment may play a key role in driving or modulating oncogenic progression. Although this has not yet been established in thyroid cancer, preliminary data indicates that innate immune cells, in particular, may play a significant role in thyroid cancer progression. Nuclear Receptors, Circadian Rhythms & Metabolism. RM Evans, Gene Expression Lab, Salk Inst/Howard Hughes Inst, San Diego, CA Abstract. Nuclear hormone receptors (NRs) are pleotrophic transcription factors that play a fundamental role in body wide physiology by regulating the activity of key genes controlling the synthesis and metabolism of a diverse collection, of lipophilic molecules including, retinoids and steroid hormones as well as hormonal derivatives of common fatty acids, bile acids and cholesterol. In addition NRs and their co-regulators are believed to play increasingly important roles in an elaborate repertoire of sensorcoupled signaling mechanisms that maintain metabolic homeostasis, inflammation and cell growth. The generation of a body wide (anatomic) and circadian (temporal) expression profile of the entire NR family has produced a functional blue print that suggests new cooperative pathways through which receptor cascades control insulin resistance, heart disease and cancer. We suggest that dysregulation of NR regulated gene clusters contributes to metabolic and inflammatory based disorders and suggests novel therapeutic approaches to the treatment of human disease. Comments. Availability of genomic sequence information reveals that humans encode 48 NR family members and mice encode 49 NR family members. In the absence of ligand, NRs are either present in the cytoplasm forming a complex with heat shock proteins and immunophilin chaperones, or in the nucleus constitutively bound to a HRE, forming a repressive complex with a corepressor such as SMRT/NCOR and histone deacetylases (HDAC) complexes. Binding of ligand in the ligand-binding pocket induces conformational changes in the activating AF-2 domain of NRs, which mechanistically facilitates the release of corepressors and HDAC complexes and the recruitment of coactivators and histone acetyl transferase (HAT) complexes. In some cases, the AF-2 peptide is fixed in an active conformation, resulting in constitutive receptor activation. In these cases, the activity of the NR is regulated by nuclear availability of the receptor itself or coactivators, or by signal-induced receptor modifications such as phosphorylation or acetylation. The main NRs discussed discussed in the conference were the following: a) Peroxisome proliferator-activated receptors (PPARs) include 3 members: α, β/δ, and γ, and each of them act as a heterodimer with RXR. As the name suggests, the founding member PPARα was identified as the target of the fibrate-class of anti-hyperlipidemic drug or peroxisome proliferators. Shortly afterwards, PPARα was shown to bind to and function as an endogenous sensor for polyunsaturated fatty acids. a.1) PPARα is highly expressed in liver, heart, muscle and kidney where it regulates fatty acid oxidation and apolipoprotein synthesis. In particular, it induces hepatic peroxisomal fatty acid oxidation during fasting and PPARα deficient mice develop dramatic steatosis upon prolonged fasting or high-fat diet feeding. It is also abundantly present in the vascular wall and in human macrophage foam cells where it is thought to exert anti-inflammatory and anti-atherogenic effects. Though the effects in rodents are still debatable, clinical studies in humans indicated that PPARα agonists are most likely effective not only in correcting dyslipidemia, but also cardiovascular mortality and morbidity. In addition, fibrates suppress satiety and improve insulin resistance in mice, although they are not generally viewed as insulin sensitizers in humans. a.2) PPARγ is the target of the thiazolidinedione (TZD)-class of insulin sensitizers, which commands the largest share of the current oral anti-diabetic drug market. PPARγ is a master regulator of adipogenesis and is most abundantly expressed in adipose tissue. The mechanisms and location of insulin sensitization by TZDs are believed to involve PPARγ activation in adipocytes, an increase in adipocyte fat storage and secretion of insulin sensitizing adipokines such as adiponectin. Consistent with the paradox that PPARγ promotes insulin sensitivity while promoting fat differentiation which could precipitate insulin resistance, a partial loss-of-function Pro12Ala mutation in humans or heterozygosity in mice results in improved insulin sensitivity, while the gain-of-function Pro115Gln mutation in humans results in obesity and insulin resistance. These genetic studies suggest that a PPARγ modulator might exhibit a better insulin sensitizing profile than a full agonist. PPARγ is also abundantly expressed in foam cell macrophages in human aortic atherosclerotic lesions and is activated by proatherogenic lipoprotein, oxidized LDL. PPARγ ligands decrease atherosclerosis in mice, whereas loss-of-PPARγ in bone marrow cells promotes it. The anti-atherogenic activity of PPARγ in macrophages involves anti-inflammatory effects as well as activation of reverse cholesterol transport. a.3) PPARδ is the least studied subtype of PPARs. It is ubiquitously expressed and acts as a sensor for polyunsaturated fatty acids and VLDL lipoprotein particle. Generally, PPARδ activation promotes mitochondrial fatty acid oxidation, energy expenditure and thermogenesis. PPARδ deficient mice are prone to obesity and insulin resistance. b) LXR (liver X receptor) α and β serve as oxysterol (hydroxycholesterol) sensors and are critical for whole body cholesterol homeostasis c) FXR (farnesoid X receptor) serves as a sensor for bile acids, the end products of hepatic cholesterol catabolism, and counteracts LXR in both cholesterol and triglyceride metabolism d) There are three subtypes of ERRs (estrogen receptor related), α, β, and γ, that share a high degree of sequence similarity with estrogen receptors, and can bind to estrogen receptor binding sites in vitro as dimers. However these receptors often act as monomers in physiological settings through consensus half sites found in many genes involved in mitochondrial function. The activity of ERRβ and γ can be modulated by synthetic agonists and antagonists, but it is believed that all of the ERRs are regulated not by ligands, but by ligand-independent binding of inducible coactivators PGC-1α and PGC-1β. PGC-1 expression is abundant in oxidative tissues and under complex regulation by physiological stimuli such as exercise, fasting, and cytokines. e) The xenobiotic receptors PXR and CAR are highly expressed in the liver and intestine. They function as sensors of toxic byproducts derived from endogenous metabolism and of exogenous chemicals. In order to enhance their elimination they act as mediators of drug-induced multi-drug clearance. PXR has a large flexible ligand binding pocket and binds numerous structurally unrelated chemicals, making it a true drug sensor. On the other hand, CAR is constitutively active in the nucleus, and indirectly regulated by nuclear localization elicited by activating drugs through a membrane-juxtaposed kinase signaling pathway. f) Mutations in hepatic nuclear factor (HNF)4α protein cause Maturity Onset Diabetes of the Young (MODY1) in humans. HNF4α is highly expressed in liver, kidney, and intestine as well as in pancreatic islets. Single nucleotide polymorphisms in both liverspecific and pancreatic-specific promoter regions are also associated with adult onset type 2 diabetes. HNF4α is a key regulator of hepatic gene expression and a major activator of HNF1α (MODY3), which in turn activates the expression of a large number of liver-specific genes, including those involved in glucose, cholesterol, and fatty acid metabolism. g) The three members of the NR4A subfamily have recently been shown to act as important regulators of gluconeogenesis in fasting and diabetes. NR4As are transcriptionally induced by the glucagon/cAMP axis in the liver, where they control the expression of many gluconeogenic genes. Elevated expression of NR4A is observed in diabetic animal models and also associated with insulin resistance and diabetes in some human studies. NR4A proteins have also been shown to modulate macrophage inflammatory responses and smooth muscle proliferation, highlighting a potential role for NR4As in cardiovascular disease. h) Small Heterodimer Partner (SHP), along with another NR, DAX-1, is an atypical NR that does not have a DBD and acts by inhibiting other NR pathways. SHP can interact with many NR and control broad aspects of nutrient metabolism. In humans, mutations in the SHP gene are associated with mild obesity, whereas SHP deficiency in mice causes increased energy expenditure, improved pancreatic β cell function and improved glucose homeostasis. In summary, the transcriptional activity of many NRs can be modulated by lipophilic ligands, and in turn NRs control fundamental processes important for metabolic and energy homeostasis. Thus, NRs provide a powerful platform for drug discovery to treat metabolic disease. Indeed, a number of synthetic ligands for endocrine receptors as well as adopted orphan receptors are currently in clinical use or under clinical trial. In addition to the receptors that we described here, there are a number of orphan receptors about which little is known. Based on the therapeutic promise of the adopted orphan receptors, an auspicious future for orphan receptor research is predicted. In addition to typical transcriptional mechanisms via NR binding to elements in promoter regions, NRs can elicit their activity through binding to other transcription factors, signaling molecules, or metabolic enzymes. Indeed, the anti-inflammatory effects of GR, for which synthetic glucocorticoids are most often prescribed, are exerted through interactions with non-NR transcription factors such as NF-κB. Antiinflammatory effects of PPARs and LXRs are also likely mediated by non-DNA binding domain-dependent protein-protein interaction. EDWIN B ASTWOOD AWARD LECTURE: The Glucocorticoid Receptor: One Gene, Many Proteins: New Mechanisms for Tissue Specific Actions of Glucocorticoids. John Cidlowski, NIEHS/NIH, Research Triangle Pk, NC Summary. Abstract Text not Provided Comments. The abstract of an article by Duma, Jewell and Cidlowski is transcribed (J Steroid Biochem Mol Biol. 2006;102:11-21). Glucocorticoids regulate diverse physiological effects in virtually every organ and tissue in the body. Glucocorticoid actions are mediated through the glucocorticoid receptor (GR), a ligand-dependent transcriptional factor that activates or represses gene transcription. Since, the cloning of the human GR in 1985, research efforts have been focused on describing the mechanism of action exerted by one of the GR isoforms, GRalpha. However, recent studies from our lab and others have suggested that multiple isoforms of hGR are generated from one single gene and one mRNA species by the mechanisms of alternative RNA splicing and alternative translation initiation. These isoforms display diverse cytoplasm-to-nucleus trafficking patterns and distinct transcription activities. In addition, this new information predicts that each hGR protein can be subjected to a variety of post-translational modifications, such as phosphorylation, sumoylation and ubiquitination. The nature and degree of posttranslational modification, as well as subcellular localization, may differentially modulate stability and function among the GR isoforms in different tissues providing an additional important mechanism for regulation of GR action. We outline the recent advances made in identifying the processes that generate and modify multiple GR isoforms and the post-translational modifications that contribute to the increasing diversity in the glucocorticoid signaling pathway. CRF, Urocortins & Their Receptors & Binding Protein: Roles in the Integrated Stress Response. Wylie Vale, Salk Inst, La Jolla, CA Abstract. Corticotropin Releasing Factor (CRF), a 41 amino acid peptide initially identified by virtue of its role in the control of the hypothalamic pituitary adrenal axis, is broadly distributed centrally and peripherally and mediates multiple complementary endocrine, autonomic and behavioral responses to stressors. CRF is important in adaptive homeostasis and may be a factor in the development of maladaptive, allostatic states as well. CRF and paralogous peptides signal through CRF1 and CRF2 receptors, related Class B GPCRs that activate adenylate cyclase as well as alternative signaling cascades. The identification of the CRF2 receptor for which CRF had low affinity together with observations of receptor-ligand anatomical mismatch led to the search for new ligands and the recognition of the three mammalian urocortins (1; 2, aka stresscopin-RP; 3, aka stresscopin), all of which have high affinity for the CRF2 receptor. Only urocortin 1 has high affinity for both receptors. A CRF binding and inactivating protein has high affinity for CRF, urocortin 1 and some species of urocortin 2. The four ligands have distinct tissue specific distributions and regulation. Mice null for each receptor display complex endocrine, metabolic, cardiovascular, gastrointestinal and behavioral phenotypes as do mice null for each of the four ligands. Selective peptidic agonists and antagonists for both receptors and small molecule antagonists for the CRF1 receptor have been developed. Understanding the various physiologic and potential pathophysiologic roles of the members of the CRF/urocortin network may lead to therapeutic means of managing stress-related and other disorders. Comments. Indeed, there are 4 related ligands (CRF, urocortins 1, 2, 3) and 2 receptors (CRFR1 and CRFR2). However, specificities, tissue distribution and physiological roles differ. It is of interest to include information on the role of urocortin 3 in stress, published by the speaker ́s group in Endocrinology (Jamieson PM et al. Endocrinology 147: 45784588, 2006). Corticotropin-releasing factor (CRF) is a key effector of the behavioral and neuroendocrine responses to stressors, mediating its effects predominantly via CRF type 1 receptors (CRFR1) within the central nervous system (CNS). The more recently described members of the CRF family of peptides, the urocortins (Ucn 1, 2, and 3), signal preferentially through the CRF type 2 receptor (CRFR2). Within the CNS, both receptors are expressed in regions involved in the neuronal circuitry of stress responses including the amygdala, hippocampus, and the paraventricular nucleus (PVN) of the hypothalamus. CRFR2-selective ligand urocortin 3 is expressed in discrete subcortical brain regions with fibers distributed mainly to hypothalamic and limbic structures. Close anatomical association between major urocortin 3 terminal fields and CRFR2 in hypothalamus, lateral septum, and medial amygdala (MEA) suggest it is well placed to modulate behavioral and hormonal responses to stress. Urocortin 3 was administered intracerebroventricularly to male rats under basal conditions or before a restraint stress, and circulating ACTH, corticosterone, glucose, and insulin were measured. Urocortin 3 activated the hypothalamic-pituitary-adrenal axis under basal conditions and augmented ACTH responses to restraint stress. Elevated blood glucose with lowered insulin to glucose ratios in both groups suggested increased sympathetic activity. Circulating catecholamines were also increased by urocortin 3, providing additional evidence for sympathoadrenomedullary stimulation. Intracerebroventricular urocortin 3 increased vasopressin mRNA expression in the parvocellular division of the hypothalamic paraventricular nucleus, whereas CRF expression was unchanged, providing a possible mechanism by which urocortin 3 mediates its actions. Urocortin 3 mRNA expression was examined after exposure to stress-related paradigms. Restraint increased levels in MEA with a trend to increased expression in the rostral perifornical hypothalamic area, whereas hemorrhage and food deprivation decreased expression in MEA. Adrenalectomy markedly increased expression in the rostral perifornical hypothalamic area, and high-level corticosterone replacement restored this to control levels. The evidence that urocortin 3 has the potential to influence hormonal components of the stress response and the changes in its expression levels after stressors is consistent with a potential function for the endogenous peptide in modulating stress responses. SYMPOSIUM SESSION. TRANSLATION. HOT TOPICS IN MALE REPRODUCTION. Origin & Evolution of Spermatogenesis Genes. JM Graves, Research School of Biological Sciences, Australian Natl Univ, Canberra, ACT, Australia Abstract. Both the X and Y chromosomes have a remarkable enrichment of genes involved in gonadogenesis and gametogenesis. The small human Y chromosome contains the sex determining gene SRY, as well as several genes that are critical for spermatogenesis and are expressed exclusively in the testis. The X chromosome, too, is enriched for genes involved in sex and reproduction. We have taken a comparative approach to ask when, how and why this occurred, making use of the distant relationship to humans of Australias extraordinary mammals. The biased gene content of the X and Y chromosomes can be best understood in terms of the origin and evolution of our sex chromosomes. The Y chromosome is a relic of an ancient autosome pair that differentiated into our X and Y in mammals 180 million years ago. We can see it in its original form as an ordinary autosome in birds, and even platypus, dating the beginnings of our sex chromosomes to about 200 million years ago. The human Y chromosome has degraded so that only 45 of an original 1300 genes survive, mostly because they acquired a critical male-specific role. Using comparisons between distantly related species, we can trace spermatogenesis genes, and even the sex determining gene, back to ancestral genes with functions (often in the brain) in both sexes. Many of these genes on the Y have amplified within palindromic structures, but most of the copies have become inactive we can see this as a race against the degradation that threatens the Y with extinction in a few million years. The X chromosome, too, has evolved and amplified male-advantage genes. We think this is because it is represented as a single copy in males, where it is exposed to selection in males but not females. This accumulation of male-advantage genes is an example of the genomic war of the sexes. Comments prepared by Dr. Esperanza Berensztein. J Graves presented a comparative approach to ask when, how and why the enrichment of genes involved in gonadogenesis and gametogenesis of the X and Y chromosomes occurred, making use of the distant relationship to humans of Australia's extraordinary mammals, monotremes and marsupials. The biased gene content of the X and Y chromosomes can be best understood in terms of the origin and evolution of human sex chromosomes. The human X bears about 1000 genes. Genome sequencing confirms that the “intelligence” genes are in 5-fold excess on the X. Using comparisons between distantly related species, the author can trace spermatogenesis genes, and even the sex determining gene, back to ancestral genes with functions in both sexes. Many of these genes on the Y have amplified within palindromic structures, but most of the copies have become inactive. The X chromosome has evolved and amplified male-advantage genes. X chromosome is represented as a single copy in males, where it is exposed to selection. An ancient region is shared by all mammals and is clearly distinguished by chromosome painting, a technique in which marsupial X chromosome DNA is isolated, tagged with a fluorescent dye, and hybridized to the homologous sequences on the human X. However, a large region (equivalent to the rest of the short arm and including the pseudoautosomal region) is autosomal in marsupials and monotremes, implying that it was added to the placental X between 100 and 180 million years (MY) ago. The small human Y chromosome contains the sex determining gene SRY, as well as several genes critical for spermatogenesis which are expressed exclusively in the testis. The human Y chromosome is composed of a corresponding ancient region shared with marsupials, and an added region, on the Y only in placental mammals. The human Y chromosome has degraded so that only 45 of an original 1300 genes survive, mostly because they acquired a critical male-specific role. The testis is a dangerous place for a chromosome to be for two reasons. Firstly it takes many more cell divisions to make a sperm than an egg, providing additional opportunities for damage. Secondly, the sperm is an oxidative environment and lacks repair enzymes. In addition, the repetitive structure of the human Y chromosome makes deletions very frequent. What causes Y degradation? Many forces are lined up against the heterogametic sex chromosome: a higher mutation rate, and the inefficiency of selection on a nonrecombining chromosome. In mammals, the Y seems to be subject to far more mutation, deletion, and insertion than the rest of the Genome. The average rate of loss of genes from the human Y is calculated from the numbers of genes lost from the human Y (from 1000 to 45) divided by the time over which this loss occurred (?300 MY). This comes to approximately 3.3/MY, and extrapolation of this linear loss leads to a predicted extinction time of about 14 MY. However, it is very unlikely that the rate of loss is uniform over time. Would loss of the Y chromosome and consequent loss of the SRY gene lead to the disappearance of males? If males became extinct, so would humans, because many maternally imprinted genes in the human genome are active only if they are derived from the male parent. The human race therefore must preserve males in order to continue reproducing. One possibility is that the SRY gene that triggers testis determination could be moved or copied onto a safer spot on an autosome. This would create a novel proto-Y and unleash a new round of sex chromosome differentiation. Thus the human race could carry on as if nothing had happened after the Y chromosome—in the long or the short term—becomes extinct. A New Concept for Adult Sertoli Cells: Both Programmable & Proliferative. SJ Meachem, Male Reproductive Endocrinology and Metabolism Gro, Prince Henrys Inst, Clayton, Victoria, Australia Abstract. New data has challenged the convention that the adult Sertoli cell population is terminally differentiated; a finding that has important implications for male fertility. The Sertoli cell has 2 distinct functions, i) formation of the seminiferous cords and ii) provision of nutritional and structural support to developing germ cells. For these to occur successfully, Sertoli cells must undergo numerous maturational changes between fetal and adult life, the main switches occurring around puberty, coincident with the rise in serum gonadotropins, including the loss of proliferative activity and the formation of the blood testis barrier. Follicle stimulating hormone (FSH) plays a key role in promoting Sertoli cell proliferation while thyroid hormone inhibits proliferative activity in early postnatal life. Together these regulate the Sertoli-germ cell complement and sperm output in adulthood. By puberty, the Sertoli cell population is considered to be stable and unmodifiable by hormones. We have challenged this concept by showing that the size of the adult Sertoli cell population is modifiable by hormones and that Sertoli cells can regain proliferative activity in hamster and human models of spermatogenic disruption. Gonadotropin suppression in the adult Djungarian hamster also disturbs blood testis barrier function and the spatial organisation of the inter Sertoli cell tight junction proteins. Administration of FSH stimulates the majority of the Sertoli cell population to re-enter the cell cycle and re-establishes the functional integrity of the blood testis barrier and organisation of tight junction proteins which is associated with the re-initiation of spermatogenesis (2). Likewise, gonadotropin suppression in the human, as induced by androgen-based contraception, results in a proportion of the Sertoli cell population showing proliferative ability while tight junction proteins are disrupted (unpublished data). One must conclude that adult Sertoli cells are not a homogeneous and terminally differentiated population but rather a proliferative and programmable population that is governed by gonadotropins. This new information may be relevant in clinical settings, particularly to some types of infertility and testicular malignancies where Sertoli cells have failed to undergo these important maturational switches. The potential to replenish an adult Sertoli-germ cell compliment to normal in a setting of infertility may now be realised. Programming of Male Reproductive Development: Origin of the Common Male Reproductive Disorders. RM Sharpe, H Scott, G Hutchison, M Jobling, C McKinnell, M Walker, P Saunders, L Smith, M Welsh, MRC Human Reproductive Sciences Unit, Queens Med Res Inst, Edinburgh, Scotland, UK Abstract. Becoming a male is ultimately determined by androgen-induced masculinization. Disorders of this, resulting in hypospadias (abnormal penis development) or cryptorchidism (undescended testes), are common disorders in humans, but their cause is unclear. Together with adult onset disorders (low sperm counts, testis germ cell cancer) they can constitute a testicular dysgenesis syndrome (TDS) in humans, with a proposed common fetal origin that may involve deficiencies in androgen production or action. Masculinization is well studied, but no unifying concept explains male reproductive development and its abnormalities, and encompasses the origin of TDS disorders. This talk will report on our studies in rats using two models that involve perturbation of normal testis development/function (TDS animal model) or blockade of androgen action during selective fetal time windows (flutamide model). Using these models, we show that masculinization of all reproductive tract tissues is programmed by androgen action during a common, fetal time window ( programming window ), which occurs earlier than expected, as it precedes morphological differentiation of the same tissues, a time when androgen action is, surprisingly, unnecessary. Androgen-driven masculinization of females is confined to the same programming window as for normal males. Blocking androgen action only within the programming window induces hypospadias and cryptorchidism and alters penile length in males; these all correlate with anogenital distance (AGD), which provides a non-invasive, lifelong read-out of androgen exposure in the programming window (but not later in gestation). As AGD is measurable in human neonates, it could predict adult onset TDS disorders as well as providing clinically important insights into reproductive tract masculinization and its disorders. We show in our TDS animal model that AGD predicts fetal and adult testis size (and thus sperm production). Though this is consistent with a key role for androgens in regulating fetal Sertoli cell proliferation, it is surprising as the time window for androgen regulation of Sertoli cell proliferation extends beyond the masculinisation programming window (when AGD is determined). Our findings support the view that deficient fetal androgen action is a key feature of TDS; we suggest that delayed onset of fetal testosterone production may also be important in TDS.