Browsing by Subject "Receptors, Cytoplasmic and Nuclear"
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Item Functional Analysis of Liver Receptor Homolog-1 and Farnesoid X Receptor in Enterohepatic Physiology(2008-05-13) Lee, Youn-Koung; Kliewer, StevenLiver receptor homolog-1 (LRH-1), an orphan nuclear receptor, and farnesoid X receptor (FXR), a bile acid receptor, are both highly expressed in liver and intestine, where they regulate bile acid homeostasis. To gain further insight into their biological actions, we investigated their function in vivo using gain-of-function and loss-of-function strategies. For LRH-1, three different experimental approaches were used. First, we generated and analyzed mice deficient for LRH-1 in either hepatocytes or intestinal epithelium. These tissue-specific knockout mice had altered expression of a large number of genes involved in bile acid metabolism. Furthermore, there was a marked change in the composition of the bile acid pool in mice lacking LRH-1 in hepatocytes. In a second experimental approach, a constitutively-active form of LRH-1 (VP16LRH-1) was expressed in the intestine of transgenic mice. The intestines of these mice were profoundly enlarged due to alterations in pathways controlling proliferation and apoptosis. In a third experimental approach, the effect of LRH-1 on early developmental decisions was examined in Xenopus laevis. In animal cap explant assays, LRH-1 induced early molecular markers of endoderm differentiation. Taken together, the Xenopus laevis and mouse studies reveal the diverse roles that LRH-1 plays during development and in adult physiology, including effects on endoderm formation, intestinal proliferation, and bile acid homeostasis. FXR regulates bile acid homeostasis through actions in both liver and intestine. In studies designed to probe for additional actions, we found that FXR has an important role in preventing the overgrowth of bacteria in the small intestine. In summary, these studies reveal the diverse processes regulated by the nuclear receptors LRH-1 and FXR and their profound impact on enterohepatic physiology.Item Identification of Ligands for the Orphan Nuclear Receptor Daf-12 that Govern Dauer Formation and Reproduction in C. Elegans(2006-08-15) Motola, Daniel Lewis; Mangelsdorf, David J.The orphan nuclear hormone receptor, DAF-12, plays a central role in the physiology of free-living nematode, C. elegans. DAF-12 is best known for its role in regulating dauer formation, a non-reproductive larval state entered in harsh environments and marked by developmental arrest, stress resistance, and extended life-span. Genetic screens for genes controlling dauer formation have identified conserved endocrine signaling pathways that converge on DAF-12 to influence the choice between dauer formation and reproductive development. Detailed genetic analysis of these signaling pathways suggests that they promote reproductive development in favorable environments by influencing the production of a ligand for DAF-12 by the cytochrome p450, DAF-9. Despite abundant evidence for hormonal control, the identity of the DAF-12 ligand has remained elusive. Using a cell-based ligand screening assay I initially identified a group of 3-keto-containing sterols that potently activated DAF-12 in a DAF-9-dependent manner. Subsequent analysis using a variety of techniques showed that DAF-9 catalyzes the non-stereo-selective addition of a carboxylic acid to the terminal side-chain methyl groups of 3-keto-sterols, producing 3-keto-cholestenoic acids. In collaboration with the laboratory of Dr. Eric Xu, we demonstrated that 3-keto-cholestenoic acids, referred to as dafachronic acids, directly bind to DAF-12 as bona fide ligands. In collaboration with Dr. Adam Antebi we found that these ligands also potently rescued the phenotypes resulting from mutations in daf-9 or its upstream activating genes. Dafachronic acids are also shown to be endogenous hormones, as they could be detected in DAF-12 activating lipid fractions from wild-type but not daf-9 null worms. Taken together, this work defines 3-keto-cholestenoic acids as the first hormonal ligands for an invertebrate orphan nuclear receptor and the first endogenous steroid hormones in nematodes. In addition, these findings demonstrate that steroid hormone control of reproduction is conserved from worms to humans. Finally, given the existence of DAF-12 homologs in parasitic nematodes, this work raises the possibility of targeting DAF-12 in parasitic nematodes as a means for controlling their growth.Item Nuclear Receptor Controls Nematode Metabolism And Development: Insight Into Man’s Nemesis, the Conqueror Worm(2011-02-01T19:35:41Z) Wang, Zhu; Mangelsdorf, DavidThe nuclear receptor DAF-12 plays a central role in controlling the larval development of C. elegans. Activation of DAF-12 by its ligands called dafachronic acids (DAs) commits the nematode to development into reproductive adult, which will otherwise arrest at a diapause stage called dauer. But the molecular mechanisms remain unclear. Furthermore, whether the DAF-12 signaling pathway is conserved in other nematode species, especially parasitic ones, is also unknown. One aspect of my studies is to investigate the molecular mechanisms by which this DA-DAF-12 signaling pathway regulates the C. elegans development. By measuring a series of metabolic parameters, we demonstrated that DAF-12 activation markedly elevated aerobic utilization of fatty acids. In accordance with this, expression of a network of metabolic genes responsible for energetic catabolism of fatty acids was up-regulated as well. Importantly, inhibition of these metabolic genes abolished the reproductive growth stimulated by DAF-12. These results revealed a DAF-12-controlled metabolic network that coordinates energy metabolism and larval development in C. elegans. The other emphasis of my work is on the role of DAF-12 in parasitic nematodes. Our results showed that, as seen in C. elegans, DAF-12 activation also induced recovery from the infective L3 (iL3), which is the dauer larva of the parasites. Moreover, the metabolic genes controlled by C. elegans DAF-12 were identified in parasitic nematodes. These facts indicate that the DAF-12 signaling pathway is conserved in parasitic nematodes. Importantly, administration of DA dramatically reduced the formation of the pathogenic larvae that are mostly resistant to current anthelmintic drugs, indicating the unique therapeutic potential of DAF-12 ligands to treat nematode parasitic diseases. To understand the pharmacology of targeting DAF-12, we solved the 3-demenstional structure of DAF-12 in a parasitic nematode called Stronglyloides stercoralis that infects human. These results reveal the molecular basis for DAF-12 ligand binding and identify DAF-12 and its downstream metabolic genes as unique therapeutic targets in parasitic nematodes. Based on this, we have discovered several small molecules that activate Stronglyloides stercoralis DAF-12 and these molecules may provide lead compounds for developing novel anthelmintic drugs.Item Nuclear Receptors in Lung Cancer(2007-05-22) Jeong, Yangsik; Mangelsdorf, David J.Lung Cancer is a fatal disease with new diagnoses of more than 150,000 Americans every year. Although it has a relatively well-known etiology (e.g. smoking) and has been widely researched, clinical tools and markers for early diagnosis, prognostic prediction, and therapeutic interventions remain limited. Here, for the first time, I propose a novel translational approach for providing diagnostic, prognostic, mechanistic, and therapeutic information by studying of the expression of the nuclear receptor (NR) superfamily in lung cancer. Using quantitative real-time PCR, mRNA expression levels for the 48 members of the NR superfamily were profiled in 56 lung cell lines. Based on the resulting dataset, further analysis was performed to show the diagnostic and therapeutic potential of the NR profile using both an in vitro cell response assay and an in vivo mouse xenograft model with cognate ligand treatment for selected nuclear receptors. In addition, the NR profiles of 30 microdissected and pair-matched patient tissue samples provided a subset of NRs showing dramatic differences in expression and subgroupings that demonstrate individual variations between the normal and corresponding tumor. Furthermore, I identified several individual NRs as well as a subgroup of NRs with prognostic power. The relevance of NRs to disease pathogenesis was then studied in genetically manipulated human bronchial epithelial cells (HBEC3) and in transgenic K-rasV12 mice, a well-known genetic model for lung adenocarcinoma. In the HBEC3 panel, the induced expression of peroxisome proliferator activating receptor gamma (PPARγ) in the parental HBEC3 introduced by oncogenic K-rasV12 is decreased in a subset of tumorigenic clones derived from the parental cells. It appears to be strongly correlated to the expression of cylooxygenase 2 (COX2), which is shown to be decreased with PPARγ ligand treatment. In the transgenic model, I demonstrated that expression of a subgroup of NRs in wild type mice becomes altered in histologically normal tissues that harbor the K-ras mutation, and become further altered in tumor tissues of the mutant. This observation suggests that NR profiling also provides a valuable tool for understanding disease pathogenesis in lung cancer.Item Steroidogenic Factor 1 and Beta-Catenin: Two Critical Regulators of Endocrine Organ Development(2009-06-18) Reuter, Anne Louise; Repa, Joyce J.The endocrine system comprises a diverse array of organs and hormones that regulate many aspects of development and homeostasis. Steroidogenic hormones - secreted by the adrenal cortex, testis, and ovary - are required for electrolyte balance, maintenance of intermediary metabolism, and reproduction. The nuclear receptor steroidogenic factor 1 (SF-1, officially designated NR5A1) originally was identified as a transcriptional regulator of steroidogenic synthetic enzymes. In addition to expression in the adrenal cortex and somatic cells of the gonads, however, SF-1 is expressed in the ventromedial hypothalamic nucleus (VMH) and pituitary gonadotropes, suggesting a broader role in endocrine physiology. Global knockout of SF-1 in mice confirmed this possibility, as it resulted in complete adrenal and gonadal agenesis and XY sex reversal, causing postnatal death due to adrenal insufficiency. Humans with mutations in SF-1 exhibit a spectrum of phenotypes ranging from 46, XY sex reversal and adrenal insufficiency to patients with normal adrenal function and mild gonadal dysgenesis. Herein is reported a patient with compound heterozygosity for a previously described SF-1 polymorphism and a novel mutation, p.R84C, which impairs DNA binding and transactivation activity. SF-1 interacts with numerous coactivators, including beta-catenin, a central mediator of the canonical Wnt signaling pathway. Upon activation by Wnt, beta-catenin translocates to the nucleus where it enhances the transcription of Wnt target genes, and - as recently appreciated - a subset of SF-1 gene targets. Due to the reported functional synergy between SF-1 and beta-catenin, and as Wnt4 deficiency in mice resulted in adrenal and gonadal defects, we investigated the consequences of beta-catenin disruption in certain SF-1-expressing tissues, specifically the adrenal and pituitary glands, and the VMH. SF-1/Cre-mediated beta-catenin knockout mice died immediately after birth and lacked adrenal glands. In contrast, the VMH and pituitary gland were largely unaffected from a structural viewpoint. Analysis of adrenal development revealed that though the adrenal primordium forms, adrenocortical cell numbers quickly decline due to impaired proliferation. The potential for beta-catenin/SF-1 synergy on a fetal adrenal specific enhancer was investigated. These results implicate beta-catenin - presumably as part of the Wnt signaling pathway - as a required factor in adrenocortical development.Item Studies of Bile Acid-Like Signaling Pathways in Mammals and Nematodes(2010-01-12T18:59:50Z) Zhi, Xiaoyong; Mangelsdorf, David J.Bile acids are not only detergents for lipid solubilization and absorption, but also important signaling molecules. They regulate biological events in mammals by acting on nuclear receptors and membrane-bound receptors. Bile acid homeostasis is maintained in part through a FXR-SHP signaling circuit, in which SHP functions as a transcriptional corepressor. The mechanism whereby SHP represses was one focus of my thesis research. I used a number of biochemical strategies including tandem affinity purification to identify SHP interacting proteins. I also successfully solubilized SHP recombinant protein, which was used to generate crystals that diffracted to 3.2 Angstroms. Bile acid-like molecules function in nematodes to control a variety of life history traits such as dauer and infective L3 formation through the nuclear receptor DAF-12. Although DAF-12 homologues from different nematode species are functionally and structurally conserved, they show differential pharmacological responses to ligands. To that end, I solved the X-ray crystal structure of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain and revealed the molecular basis underlying species specific-ligand binding for DAF-12. Furthermore, DAF-12 was shown to be structurally similar to the bile acid sensor FXR, suggesting bile acid-like signaling pathways have been conserved across evolution. In conclusion, my studies provide new insights into how bile acids are sensed and regulated in mammals and nematodes. [Keywords: bile acids; SHP; FXR; DAF-12; Dafachronic acids; hookworm; protein expression; crystallization]