Browsing by Subject "Receptors, Cytoplasmic and Nuclear"
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Item DAF-12: A Novel Drug Target in Parasitic Nematodes(2014-07-24) Schaffer, Nathaniel Elliot; Phillips, Margaret A.; Mangelsdorf, David J.; Kliewer, Steven A.; Brown, Michael S.; Ranganathan, RamaThe nuclear receptor DAF-12, first identified in C. elegans, controls nematode species' entry into and exit from metabolically hypoactive resting states. In all of the parasitic species studied thus far, that resting state is the infectious third larval (iL3) stage, and activation of the receptor in iL3 worms outside of the host induces a premature and lethal molt (Wang et al. 2009; Motola et al. 2006). In contrast, the relatively uncontrolled soybean parasite Heterodera glycines, which causes USD 1 billion of crop damage annually in the U.S. (Wrather and Koenning 2006; Davis and Tylka 2005), does not go through an iL3 stage but rather arrests as the preinfectious second juvenile (piJ2) within the egg (Davis and Tylka 2005). However, just as iL3 worms only molt to L4 when they are inside their host (Wang et al. 2009), H. glycines nematodes only hatch as infectious J2s when they are near the plant roots required to support their development (Davis and Tylka 2005). This work demonstrates the importance of the DAF-12 ortholog in H. glycines for regulating hatching and introduces the the first well-defined molecular target for controlling the spread of this important pathogen. Additionally, an ortholog with significant sequence and likely functional similarity is identified in the related parasite Globodera pallida, which causes an identical disease in other major agricultural crops including potato and tomato plants and exhibits the same arrest phenotype inside the egg as H. glycines. Finally, a DAF-12 ortholog is cloned from Onchocerca gutturosa, one of several species in the Onchocerca genus that causes the disease onchocerciasis in both humans and livestock. Importantly, since onchocerciasis continues to elude the eradication efforts of several well-funded international programs, both an existing agonist and an avenue to identify endogenous agonists for the receptor are identified. These studies offer direction for the development of novel therapeutics to control these socially and economically important pathogens.Item Exploration of Nuclear Receptor Activity and siRNA-Derived Phenotypes as Therapeutics in Non-Small Cell Lung Cancer(2015-04-10) Carstens, Ryan Murray; Kittler, Ralf; Minna, John D.; Mangelsdorf, David J.; Kliewer, Steven A.; White, Michael A.Nuclear hormone receptors are master regulators of diverse cellular functions implicated in tumor pathogenesis and as oncogenic drivers of many human cancers. To better understand what role these important receptors might be playing in lung cancer, three interconnected studies were initiated to assess nuclear receptor function, expression, and drugability within the lung cancer context. First, a "technology development" project was undertaken to produce and troubleshoot a CLIA-certifiable, high-throughput biomarker platform capable of mRNA expression signature assessment from FFPE specimens. The platform was used to assess NR/CoReg expression levels across a 500+ sample FFPE dataset. Categorical NR/CoReg downregulation upon tumor progression as well as survival benefits for patients retaining a non-pathological NR/CoReg expression pattern were discovered. Second, a panel of 110 NR ligands was screened across a 100-member cell line panel representative of all clinically-relevant facets of lung and breast cancers to pharmacologically interrogate these receptors as novel lung cancer targets. Following completion of this screening effort, three classes of ligands targeting the estrogen, glucocorticoid, and vitamin D receptors (ER, GR, and VDR respectively) that exert anti-proliferative phenotypes on specific subsets of the lung cancer cell lines were identified. Of particular note, several of these agents are routinely used in current clinical practice (particularly dexamethasone) and represent excellent candidates for rapid clinical translation of these findings. Finally, an RNAi-based systematic functional interrogation of NR/CoReg function was undertaken in a 100+ member cell line panel representative of all clinically-relevant facets of lung and breast cancers. A reproducible classification of lung and breast cancers was defined based on their holistic functional states as represented by the RNAi dataset. Each of these "clades" of cancer cell lines was demonstrated to be specifically targetable by unique siRNA reagents capable of inducing growth attenuation or amplification in only that clade of cell lines. Further investigation into the mechanisms of action of these siRNA reagents unexpectedly revealed that the phenotypes were largely mediated by miRNA-like seed sequence based effects rather than target-directed siRNA total complementarity silencing. Following this discovery, efforts were undertaken and subsequently completed to identify the "true" targets of these clade-specific siRNAs.Item Functional Analysis of Liver Receptor Homolog-1 and Farnesoid X Receptor in Enterohepatic Physiology(2008-05-13) Lee, Youn-Kyoung; Kliewer, Steven A.Liver 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 Identification of Oncogenic KRAS-Associated Vulnerabilities in Non-Small Cell Lung Cancer(2016-05-26) Kim, Ji Mi; Lum, Lawrence; White, Michael A.; Fontoura, Beatriz; Cobb, Melanie H.Activating mutations in KRAS are frequently involved in the pathogenesis of non-small cell lung cancer (NSCLC), the disease responsible for the most cancer-related deaths in the US. Despite intensive efforts to develop drugs that directly interfere with KRAS activity over the past decade, no effective inhibitor has been developed. As an alternative, synthetic-lethal therapeutic opportunities are being pursued using large-scale, RNAi-based, functional genomics platforms. We first addressed two major challenges associated with RNAi-based primary synthetic-lethal screens; a prevalent miRNA-like behavior of siRNA and cell line-dependent phenotypic diversity within intra-lineage KRAS-driven cancer. In consideration of these, we performed a whole-genome synthetic-lethal siRNA screen, powered by 106 NSCLC lines and integrated with gene set enrichment analysis. This identified components of nuclear transport machinery as selectively essential for KRAS mutant NSCLC lines. We found that pharmacological inhibition of a key nuclear export receptor, XPO1 (a.k.a. CRM1), was sufficient to induce robust and selective apoptosis in KRAS mutant NSCLC cells in vitro and to cause significant impairment of KRAS mutant tumor growth in vivo. Mechanistically, XPO1-depedent nuclear export machinery was required to maintain NFκB-mediated survival signaling. We discovered that a subset of KRAS mutant NSCLC lines bypassed the addiction to XPO1-dependent nuclear export via YAP1 activation as a consequence of previously unappreciated co-occurring loss-of-function mutations in FSTL5 and mutations in Hippo pathway. The intrinsic resistance was reversed by coadministration of YAP1/TEAD inhibitor. Thus, our study suggests that XPO1 can be exploited for a promising therapeutic opportunity for KRAS mutant lung cancer and provides strategies for genomics-guided application of clinically available XPO1 inhibitors.Item An In Vivo Functional Genomics Screen Identifies New Regulators of Tumorigenesis in Non-Small Cell Lung Cancer(2015-11-24) Hight, Suzie K.; Shay, Jerry W.; Minna, John D.; Roth, Michael G.; White, Michael A.Cancer cells are characterized by the aberrant regulation of signaling pathways that govern responses to growth stimuli, resulting in dysregulated cellular proliferation. The accumulated genomic alterations that cause this malignant growth phenotype can also result in acquired vulnerabilities in the tumor. Loss-of-function screening using pooled short hairpin RNAs (shRNAs) is a powerful method for identifying new therapeutic targets in cancer. We have tested the use of parallel in vitro and in vivo screens using a mini-library of shRNAs to identify previously unknown acquired vulnerabilities in lung cancer. Using a mini-library of 1062 lentiviral shRNAs targeting nuclear hormone receptors and their coregulators (120 genes total), we screened the lung adenocarcinoma cell line NCI-H1819 for dependency on these genes during in vitro and in vivo growth. We identified six genes required for survival in vitro (BRCA1, CCND1, MED1, PHB, HNRNPU, and PELP1), and three genes that were required for tumor survival in vivo, but not in vitro: FOXA1, HDAC1, and NCOR2. None of these genes were mutated by full exome sequencing of H1819, however FOXA1 was found to be co-amplified with NKX2-1 on chromosome 14q. Here we report that NSCLC cell lines and tumor samples have significantly higher FOXA1 expression compared to normal lung epithelial cells and tissues, and that 14q-amplified NSCLC cell lines are preferentially dependent on FOXA1 for clonogenicity and in vivo growth. Integrative transcriptomic and cistromic analyses identified a subset of direct FOXA1 targets as positive regulators of key growth signaling pathways, and negative regulators of several growth inhibitory mechanisms. This regulatory function appears to be partially independent of NKX2-1, and combing de novo motif discovery with expression analyses has identified several other putative FOXA1 coregulators. Our findings provide new insight into the functional consequences of 14q amplification in lung adenocarcinomas and suggest exploration of new transcriptional networks for potential therapeutic vulnerabilities.Item Liver Receptor Homolog-1 Regulates Kisspeptin Expression in the Arcuate Nucleus to Promote Reproductive Axis Function(2014-04-08) Atkin, Stan Dean; Elmquist, Joel; Mangelsdorf, David J.; Kliewer, Steven A.; Takahashi, Joseph; Mendelson, Carole R.The timing of ovulation in mammals is set by a complex hypothalamic pituitary neuroendocrine axis. Kisspeptin neurons in the arcuate nucleus (Arc) are thought to secrete kisspeptin (Kiss1) to stimulate the release of follicle stimulating hormone (FSH) from the pituitary. However, mechanisms that drive Arc KISS1 output and maintain adequate FSH secretion required for folliculogenesis are not well understood. Here, we report that the nuclear receptor, liver receptor homolog-1 (LRH-1), is expressed in kisspeptin neurons of the Arc. Kiss1 is found to be a direct target gene of LRH-1, whereby LRH-1 sets a basal tone of kiss1 expression in the Arc. Deletion of Lrh-1 from kisspeptin neurons causes decreased Arc Kiss1 expression. This leads to reduced plasma FSH, prolongation of the estrous cycle, and decreased follicle maturation and ovulation. These defects ultimately compromise fertility. Overexpression of Lrh-1 in a kisspeptin neuron-specific transgenic mouse model increases Arc Kiss1 expression and plasma FSH. Chromatin immunoprecipitation and luciferase-based promoter assays demonstrate that LRH-1 binds to a putative LRH-1 response element in the Kiss1 promoter to stimulate activity. In conclusion, LRH-1 is expressed in the Arc to set the basal tone of Kiss1 output needed to promote FSH secretion for folliculogenesis prior to ovulation.Item Natural Products as Selective Chemotherapeutic Agents and as Chemical Probes to Understand Biological Processes(2013-09-26) Carrasco, Yazmin Paulina; Tambar, Uttam; MacMillan, John; Roth, Michael G.; Ready, Joseph M.Aware of the important role that terrestrial microbial natural products play in the discovery of therapeutics and the decrease in rate of discovery of new natural products in the pharmaceutical industry, there is an immediate need to explore novel sources of microbial natural products with biological relevance. Marine bacteria are an excellent source of bioactive metabolites. With this idea in mind our laboratory has developed new techniques to isolate >600 species of marine actinomycetes and has created a natural product fraction library from these bacterial strains. My research efforts have focused on the isolation of natural products that exhibit selective activity against a panel of tumor derived cell lines that include lung, colon, melanoma, pancreatic cancer and glioblastoma. In parallel to structure elucidation efforts of our active metabolite, the determination of its mode of action was ongoing in the laboratory of Dr. White. Through the use of a new screening tool, called FUSION (Functional Signature Ontology) we were able to determine that our active compound had similar activity to inhibitors of the TBK1 signaling pathway. Additionally, a novel polyketide was isolated from the marine-derived bacteria Salinispora arenicola, believed to be a key intermediate in the biosynthetic pathway of saliniketal, an inhibitor of ornithine decarboxylase induction. In addition, we have utilized the natural product leptomycin B (LMB), as a chemical tool to understand its inhibition of CRM1, a protein involved in export of cargo from the nucleus to the cytoplasm and a possible chemotherapeutic target. This work was done in collaboration with Dr. Chook. We observed that LMB irreversibly binds to CRM1 due to the hydrolysis of its lactone moiety causing stabilization of the protein-LMB complex. In contrast, compounds that lack the capability of being hydrolyzed by CRM1 are reversible inhibitors of CRM1. These research findings probe the question of designing molecules capable of reversibly inhibiting CRM1 and this perhaps will lead to reduced toxicity. This work illustrates the importance that natural products have not only as therapeutic agents for the treatment of diseases such as cancer but also as important chemical tools to understand complex biological processes.Item Nuclear Export Receptor CRM1 Recognizes Nuclear Export Signals with Diverse Conformations(2017-04-13) Fung, Ho Yee Joyce; Nam, Yunsun; Chook, Yuh Min; Grishin, Nick V.; Yu, Hongtao; Zhang, XuewuThe Chromosome Region of Maintenance 1 or CRM1 protein facilitates export of hundreds of proteins and RNA molecules from eukaryotic cell nuclei. CRM1 recognizes its protein cargoes by their 8-15 residues-long nuclear export signals or NESs, which bind to a hydrophobic groove in CRM1. NESs are highly variable in sequence and structure. Their sequences are described by multiple sequence patterns of four variably-spaced hydrophobic residues, and three previous structures showed CRM1-bound NESs adopting either helix-strand or mostly extended conformations while the CRM1 groove remains unchanged. The plasticity of CRM1-NES interaction and the repertoire of NES conformations were unclear. Many NES sequences also seem incompatible with the asymmetric and seemingly structurally invariant NES-bound CRM1 groove. I developed a general strategy to crystallize CRM1 bound to NES peptides in order to study how diverse sequences bind CRM1. In the first study, I solved crystal structures of CRM1 bound to NESs with unusual sequences, which bound the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). Comparison of minus and plus NESs identified structural and sequence determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome. In the second study, I solved eight additional structures of diverse NESs, which show peptide conformations ranging from mostly loop-like to all-helical NESs, occupying the CRM1 groove to different extents. Comparison of >13 structures show a total of 5-6 different NES conformations where the only conserved structural element is one turn of helix, which has dihedral angles that proceed from helical to β-strand. All NESs also participate in hydrogen bonds with the human CRM1 Lys568 side chain, which functions as a specificity filter that prevents binding of non-NES peptides. The large conformational range of NES backbones explains the lack of a fixed pattern for its 3-5 hydrophobic anchor residues, which in turn explains the large array of peptide sequences that can function as NESs. We now have comprehensive structural knowledge for NESs of most known patterns. The structural information obtained is now the foundation for a new peptide docking/modeling approach to improve the accuracy of NES prediction.Item Nuclear Export Signal Recognition by CRM1 Carrying the Oncogenic E571K Mutation and Structure-Based NES Prediction(2020-08-01T05:00:00.000Z) Baumhardt, Jordan Matthew; Erzberger, Jan; Chook, Yuh Min; Grishin, Nick; Lin, Milo; Fontoura, BeatrizNuclear-cytoplasmic trafficking is an essential cellular process in eukaryotes that maintains and regulates the spatial distribution of key cellular processes in the nucleus and the cytoplasm. CRM1 is the major nuclear export mediator, which facilitates nuclear export of hundreds of protein and RNA molecules. CRM1 is essential to the survival of eukaryotic cells and is overexpressed or mutated in a variety of cancers. This involvement of CRM1 in cancer cell survival has led to the development of novel small molecule inhibitors such as XpovioTM or Selinexor, which has been approved for treatment of advanced and relapsed multiple myeloma and is also in many clinical trials for a variety of cancers. In addition, the point mutant CRM1(E571K) is found in a variety of tumors and is highly prevalent in some B-cell lymphomas. A deep understanding of how CRM1 recognizes the diverse NESs in hundreds to thousands of cargos is needed to understand the role of CRM1 in cancer pathogenesis and the mechanism of action of its inhibitors. My work has focused on expanding our foundational knowledge of CRM1-NES interactions in the presence and absence of the oncogenic E571K mutation. Previous studies measured the affinities of CRM1 interactions with 22 different nuclear export signals (NESs) from protein cargos using a low-throughput differential photobleaching assay. However, hundreds of very diverse CRM1 cargos and their NESs have yet to be characterized, limiting our understanding of the biological roles of CRM1 in disease. I developed a high-throughput, quantitative fluorescence polarization assay to measure the affinity of >100 NES peptides for WT CRM1 and for the oncogenic CRM1(E571K) to understand how the mutation affects NES binding. The large body of CRM1-NES affinity data was also used to develop a new structure-based NES prediction method. The CRM1 oncogenic mutation E571K is highly prevalent in some subtypes of B-cell lymphomas and can drive tumors in mouse B-cell models, but the mechanism of tumorigenesis is unclear. Using structural and biophysical approaches, I studied the recognition of 27 diverse NESs by CRM1(E571K) and showed that while most cargos are unaffected by the mutation, a small subset of highly charged NESs have greater than 10-fold affinity changes for the cancer mutant. To study whether these affinity changes cause nuclear export defects in cells, I used CRISPR/Cas to generate generated HEK 293 cells with either monoallelic CRM1WT/E571K or biallelic CRM1E571K/E571K. HEK 293 cells with CRM1WT/E571K or CRM1E571K/E571K had decreased proliferation and cells with homozygous CRM1E571K/E571K had obvious cell cycle defects. The eIF4E-Transporter which binds 10-fold weaker to CRM1(E571K) is mislocalized in HEK 293 cells with CRM1(E571K) and in chronic lymphocytic leukemia patient cells burdened with the CRM1 mutation. Additionally, I solved crystal structures of CRM1 bound to covalent KPT inhibitors, which showed that the mutation site, position 571 of CRM1, is located far from the bound drugs and thus unlikely to substantially affect their ability to inhibit CRM1. In order to understand the effects of the E571K on CRM1 activity and further illuminate possible cancer mechanisms, it is crucial to identify many more NESs across the entire human proteome. Therefore, NES prediction is of great interest, but existing sequence-based approaches give high false positive rates. My work also shows that may NES-like peptides are not accessible in the full-length cargos to to CRM1, in ways that many existing NES predictors cannot identify. To improve NES prediction, our collaborators in the Grishin Lab utilized CRM1-NES crystal structures as templates to develop a structure-based NES predictor and incorporated full-length protein context considerations to accurately identify novel NESs that are likely to be functional. I contributed to this work by expanding the CRM1-NES structural dataset and I helped to interpret modeling outliers to iteratively improve the CRM1-bound NES models. I also assisted the Grishin lab in using this structure-based predictor to map somatic mutations found in cancer patients that may disrupt nuclear export of the NES-containing proteins. Overall, I have added >100 new CRM1-NES binding affinity measurements and 16 CRM1 (WT or E571K) crystal structures to the field. These results have shown how the oncogenic E571K mutation binds very differently to small subset of highly charged cargos, and provided a robust dataset to develop a new structure-based NES prediction tool. Future work will build on this foundation to clearly illustrate how nuclear export defects lead to oncogenesis, and potentially give key insights into improving use of CRM1-targeted therapeutics.Item Nuclear Hormone Receptor-Mediated Changes of Cholesterol, Triglyceride, and Bile Acid Physiology in Response to Alterations in Cholesterol Absorption and Bile Acid Pool Size in Mice(2013-06-25) Jones, Ryan Dale; Horton, Jay D.; Souza, Rhonda; Kittler, Ralf; Repa, Joyce J.The regulation of lipid metabolism is an interwoven series of pathways and events acting in concert to control the nutritional and metabolic needs of the body. When alterations in lipid balance occur they can result in disease, thus an understanding of the pathophysiology, as well as the molecular mediators of these alterations are vital to the advancement of treatment for these diseases. Many of these mediators are members of a class of proteins called nuclear hormone receptors, which respond to lipophilic compounds to regulate gene transcription. I have undertaken three separate, but related projects relating to lipid metabolism with a particular focus on nuclear hormone receptor function. First, I sought to understand how a drug that blocks cholesterol absorption can ameliorate the onset of hepatic steatosis. What I found was that in the early stages of disease onset, Ezetimibe prevents hepatic steatosis independently from the nuclear hormone receptor liver X receptor, and actually paradoxically stimulates hepatic lipogenesis despite decreased liver TG levels. Second, I determined the molecular and physiological changes that occur with bile acid pool size restoration in mice that have a bile acid deficiency. This project highlighted the importance of bile acids in cholesterol absorption, the function of the bile acid-responsive nuclear hormone receptor farnesoid X receptor, and whole body lipid homeostasis. Additionally, we established a rationale for feeding low physiological amounts of bile acids to experimental animals, especially when those animals have a defect in bile acid production. Lastly, I studied the tissue distribution and nuclear hormone receptor-mediated regulation of a class of enzymes called carboxylesterases. This family of enzymes, which participate in neutral lipid hydrolysis, often came up on microarray analysis of metabolic studies, yet little was known about their regulation or function with regards to lipids. Therefore, I classified the tissue distribution of each of the carboxylestase family members in mice, and tested their gene expression upon treatment with synthetic agonists for the lipid-sensing NHRs. Thus, the information provided in this dissertation provides a valuable resource as the molecular mechanisms of disease and the role of nuclear hormone receptors begin to become clearer.Item Nuclear Receptor Controls Nematode Metabolism And Development: Insight Into Man’s Nemesis, the Conqueror Worm(2011-02-01) Wang, Zhu; Mangelsdorf, David J.The 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.; Minna, John D.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(2010-01-12) 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.Item [UT Southwestern Medical Center News](2006-08-25) Despres, Cliff