Studying the Specificity of hPXR Antagonists Using a Panel of Cell-based Assays

The pregnane X receptor (PXR or SXR; NR1I2) is a member of the nuclear receptor superfamily. It activates the transcription of a large network of genes including cytochrome P450 (CYP) and Mdr1 which play critical roles in chemicals metabolism and transportation. Induction of CYPs contributes to adverse drug-drug interactions. Non-toxic, PXR-specific antagonists will be valuable in attenuating the adverse drug-drug interaction which is the cause of many treatment failures in clinic. However, few hPXR antagonists were reported particularly the specific ones. In this thesis a reporter gene assay was used to study the specificity of hPXR antagonists from a panel of compounds that have been previously indentified as non-toxic hPXR antagonists

Molecular Mechanisms of Nuclear Hormone Receptor Transcriptional Synergy and Autoinduction.

Thyroid hormone (TH) and glucocorticoids (GC) play critical roles in the development and function of the central nervous system (CNS) by binding to their cognate nuclear hormone receptors (NRs), which function as ligand-activated transcription factors. In my dissertation, I studied the TH and GC-mediated regulation of Krüppel like factor 9 (Klf9/Basic Transcription Element Binding Protein 1; Bteb1), a member of the Sp1/KLF family of zinc finger transcription factors that bind GC rich genomic sequences, and plays an important role in neuronal development and plasticity. In prior work Klf9 was found to be a direct TH receptor (TR) target gene. I showed that Klf9 is also directly targeted by the GC receptor (GR), and that TH and GCs cause synergistic induction of Klf9. This synergistic regulation is phylogenetically ancient, and was likely present in the earliest tetrapods and has been evolutionarily conserved from frogs to mammals. I identified a genomic region in the 5’ flanking region of the Klf9 gene (the ‘Klf9 synergy module’) that contains TR/GR binding sites and confers synergistic gene regulation by TH and GC. The synergistic effect of TH and GC on Klf9 can be explained by a TR-dependent increase in the recruitment of the GR and enhanced association of stalled RNA polymerase II at the Klf9 synergy module, and an interaction between the synergy module and the Klf9 promoter by chromosomal looping. I also conducted a genome-wide microarray analysis, the first study to identify transcriptional targets that are coordinately regulated by TH and GC in the brain. Lastly, I demonstrated a role for KLF9 as an accessory transcription factor to support TH-dependent expression of TRβ (autoinduction), a process that is necessary for the progression of amphibian metamorphosis, and normal mammalian brain development. Given the synergistic regulation of the Klf9 gene by TR and GR, and its role in TRβ autoinduction, my findings support that KLF9 is an important intermediate that functions to integrate TH and GC by enhancing the cell sensitivity to hormonal signals. Taken together, my thesis broadens our understanding of the molecular mechanisms of NR cooperativity and autoinduction, with important implications for animal development.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/93977/1/piab_1.pd

Evolučně zachovalé mechanismy regulace genové exprese jadernými receptory.

Transkripční regulace genové exprese eukaryotních organismů se vyvinula během milionů let. Regulační cesta jaderných receptorů představuje evolučně starý, ale zachovalý mechanismus zahrnující asociované akcesorní proteiny, z nichž mnohé tvoří funkční strukturu známou jako Mediátorový komplex, který je účastný v transkripci. Hypotézujeme, že přes universalitu této cesty, v níž vnitřní potenciál NR-Mediátorové cesty zachovalý napříč druhy, přímo překládá regulační signál do biologické odpovědi zapojením nových adaptačních funkcí ve fylogeneticky novějších Metazoidních organizmech a přídavná regulace může být dosažena sekundárními funkcemi základních členů této regulace. Pro podporu této hypotézy jsme studovali schopnost vazby ligandu retinoidního X receptoru ve vločkovci Trichoplax adhaerens a přinesli důkaz podporující koncept, že tato schopnost byla přítomna již na počátku metazoidní evoluce. S ohledem na možné sekundární funkce, majíce inspiraci z našeho předcházejícího výzkumu, jsme identifikovali Mediátorovou podjednotku 28 (MED28) jako jediný známý člen s dokumentovanou duální cytoplasmatickou a jadernou funkcí a mající tedy potenciál přenášet signály z jaderných strukturních stavů do jádra. V důsledku chybění významné zachovalosti sekvence, ale dostupnosti experimentálních přístupů, jsme se...Transcriptional regulation of gene expression in eukaryotes has evolved over millions of years. The regulatory pathways of nuclear receptors represent an evolutionarily ancient, but conserved mechanism with associated accessory proteins, many of them forming a functional nexus known as the Mediator complex involved in transcription. Despite the versatility of the pathway, e.g. through the adoption of new regulatory functions in phylogenetically more recent Metazoa, we hypothesise that the intrinsic potential of the NR-Mediator axis to directly translate a stimulus to a biological response is conserved across species, and additional regulation could also be achieved through secondary functions of its essential members. To support the hypothesis, we assessed the ligand-binding capability of retinoic X receptor in Trichoplax adhaerens and provided evidence to support the concept that this capability was already present at the base of metazoan evolution. With regards to the potential secondary functions, we took inspiration from previous research and identified the Mediator subunit 28 (MED28) as the only known member having documented nuclear and cytoplasmic dual roles, and thus possessing the potential to transmit signals from the cellular structural states to the nucleus. Due to the lack of...BIOCEV, First Faculty of Medicine, Charles UniversityBIOCEV, 1. LF UK1. lékařská fakultaFirst Faculty of Medicin

Regulation of the Pregnane X Receptor Signaling Pathway

Liver-enriched nuclear receptors (NRs) collectively function as metabolic and toxicological `sensors' that mediate liver-specific gene-activation in mammals. NR-mediated gene-environment interaction regulates important steps in the hepatic uptake, metabolism and excretion of glucose, fatty acids, lipoproteins, cholesterol, bile acids, and xenobiotics. While it is well-recognized that ligand-binding is the primary mechanism behind activation of NRs, recent research is revealing that multiple signal transduction pathways modulate NR-function in liver. The interface between specific signal transduction pathways and NRs helps to determine their overall responsiveness to various environmental and physiological stimuli. The pregnane x receptor (PXR, NR1I2) was identified in 1998 as a member of the NR superfamily of ligand-activated transcription factors. PXR is activated by a broad range of lipophilic compounds in a species-specific manner. The primary function ascribed to PXR is the homeostatic control of steroids, bile acids, and xenobiotics. This function is mediated through PXR's ability to coordinately activate gene expression and regulate the subsequent activity of phase I and phase II metabolic enzymes, as well as several membrane transporter proteins. While PXR likely evolved primarily to protect the liver from toxic assault, its activation also represents the molecular basis for an important class of drug-drug, herb-drug, and food-drug interactions. While ligand binding is the primary mode of PXR activation, several signal transduction pathways interface with the PXR protein to determine its overall responsiveness to environmental stimuli. Multiple signaling pathways modulate the activity of PXR, likely through direct alteration of the phosphorylation status of the receptor and its protein cofactors. Therefore, specific combinations of ligand binding and cell signaling pathways affect PXR-mediated gene activation and determine the overall biological response. This dissertation contributes to the molecular understanding of the regulation of PXR by novel agonists, cAMP-dependent protein kinase (PKA) signaling, and phosphorylation. The results presented here were primarily obtained from mouse and tissue culture systems. This dissertation identifies Tian Xian, a traditional Chinese herbal anti-cancer remedy, as a novel PXR activator. This evidence suggests that Tian Xian should be used cautiously by cancer patients taking chemotherapy due to its potential to increase the metabolism of co-administered medications. In addition, data presented here show that activation of PKA signaling modulates PXR activity in a species-specific manner. It is further revealed that PXR exists as phospho-protein in vivo and that the activation of PKA signaling modulates the phospho-threonine status of PXR. Finally, the potential phosphorylation sites within the PXR protein are identified. These phosphorylation sites are characterized, using a phosphomimetic and phospho-deficient site-directed mutagenesis based approach, based on their ability to modulate PXR activity. Taken together, the work presented in this dissertation contributes to understanding the interface between ligands, signal transduction pathways and PXR activity, which is critical for the development of safe and effective therapeutic strategies

Cellular factors that interact with the negative regulatory element of the 5'-long terminal repeat of human immunodeficiency virus type 1

Transcriptional regulation of HlV-1 gene expression has been shown to be regulated by a combination of viral and cellular proteins which bind to regulatory elements in the viral 5' long terminal repeat (5'LTR) Functionally the LTR can be divided into three regulatory regions: the TAR region, extending from nucleotides 1 to 60 relative to the start site of transcription, contains sequences with which the viral trans-activator Tat interacts. The adjacent region from nucleotides -1 to -78 contains the core promoter with elements crucial for both basal and Tat-induced expression. The third region, extending from -79 to -454, contains numerous elements with which a variety of cellular factors may interact, resulting in either positive or negative modulation of LTR-driven transcription. The work contained within this thesis describes the discovery and delineation of two new transcription factor binding sites, designated as site A and site B, within the 5'-LTR of HIV-1. The majority of the work focused on site B itself, involving the characterisation of cellular proteins that specifically interacted with the nucleotide sequences in this site. Site B was found to contain a palindromic sequence TGACC involved in protein-DNA contact separated by a 9 base-pair spacer sequence that was not important for protein binding. This palindrome resembles the consensus binding site for members of the nuclear hormone receptor super-family of transcription factors. Although several members of this super-family of transcription factors were shown to interact in vitro with site B, the predominant protein present in T-lymphocyte nuclear extracts did not correspond to any of those previously characterised. The T-cell protein was shown to have a relative molecular size of 100-110 kD for the monomeric polypeptide and bound to site B as a dimer. Maximal binding to site B required both halves of the palindrome. Functionally site B was shown to act as a repressor element of both basal transcription and of transcription activated by phorbol ester in T- lymphocytes. Site B was also shown to function as a retinoic acid response element (RARE) in a heterologous promoter. The ability to function as either a positive or negative regulatory element is a recognised characteristic of nuclear hormone response elements and in part is a function of the relative abundance of factors able to interact with the site or to form complexes with one another. The overall effect of site B upon LTR-directed transcription may similarly depend upon the complex interaction of multiple factors which themselves depend on the cell type, cell activation state and degree of differentiation

Kidney filtration barrier components nephrin and Neph3 : transcriptional regulation and role in cell adhesion

Glomerular epithelial cells, podocytes, and the specialized cell junctions termed slit diaphragms (SDs) between interdigitating foot processes of the podocytes form the essential components of the filtration barrier in the kidney glomerulus. Nephrin is a transmembrane immunoglobulin superfamily member and a crucial structural and signalling component of the SD. Mutations in the nephrin gene cause congenital nephrotic syndrome of the Finnish type (CNF), leading to disruption of the SD and leakage of plasma proteins into the urine. The Neph-protein family comprises Neph1, Neph2 and Neph3; these are nephrin homologues and also important components of the SD. Interactions between nephrin and Neph1-3 are suggested to play a role in cell adhesion and thus serve as a structural framework of the SD. This thesis investigated the interactions and cell adhesion activities of nephrin and Neph-family members. Neph3 was shown to belong to the nephrin protein complex and to form homodimers. Both Neph1 and Neph3 were demonstrated to participate in the formation of cell-cell contacts by their homophilic interactions. Cell adhesion was also promoted by heterophilic trans-interactions between nephrin and Neph1 or Neph3. These adhesive activities may play an important role in the formation and function of the SD. In nephrin-deficient mice, where SDs are replaced by tight junction-like structures, Neph3 was shown to be up-regulated. This suggests that Neph3 may be involved in molecular mechanisms that are associated with morphological changes in injured podocytes. Nephrin and Neph3 genes, NPHS1 and KIRREL2, respectively, are located on human chromosome 19q13.12 in a head-to-head orientation separated by an approximately 5-kb intergenic region. A similar bidirectional arrangement of nephrin and Neph3 genes is present in mouse and rat chromosomes. The bidirectional and conserved arrangement of nephrin and Neph3 genes together with their similar protein structure and location suggest that nephrin and Neph3 genes may share key features in their regulation. This thesis focused on investigating transcriptional regulatory mechanisms important for nephrin and Neph3 genes. Transcription factors WT1 and NF-κB were shown to function co-operatively in both nephrin and Neph3 gene regulation. Further, DNA methylation participated in silencing both nephrin and Neph3 gene expression. These similar mechanisms in regulating transcription of nephrin and Neph3 genes may produce their similar spatial and temporal expression as well as similar function. In addition, transcription factors GABP and Sp1 were found to regulate nephrin and Neph3 genes, respectively. The results of this thesis widen the current understanding of the role of nephrin and Neph-family members in the formation of the SD; Neph3 belongs to the nephrin protein complex sharing similar binding properties with nephrin, Neph1 and Neph2, and interactions of nephrin, Neph1 and Neph3 promote cell adhesion. Further, nephrin and Neph3 genes were demonstrated to share similar transcriptional regulatory mechanisms. This may improve understanding of the transcriptional regulation of podocytes in general.Munuaisen tehtävänä on suodattaa verestä haitallisia kuona-aineita ja erittää ne virtsan mukana pois elimistöstä. Suodatus tapahtuu munuaiskeräsissä suodatuskalvon läpi joka koostuu endoteelisoluista, tyvikalvosta ja monihaaraisten epiteelisolujen, podosyyttien, välisistä soluliitoksista eli välihiloista. Munuaisen suodatusmekanismin häiriintyminen näkyy valkuaisaineiden erittymisenä virtsaan, valkuaisvirtsaisuutena, joka jatkuessaan saattaa johtaa dialyysihoitoa tai munuaissiirtoa vaativaan munuaisvaurioon. Välihila koostuu useista munuaisen suodatustoiminnassa tärkeässä roolissa olevista proteiineista, joista yksi on nefriini. Mutaatio nefriiniä koodaavassa geenissä aiheuttaa suomalaistyyppistä synnynnäistä nefroosia (CNF), joka johtaa välihilojen puuttumiseen ja voimakkaaseen valkuaisvirtsaisuuteen. Sairautta hoidetaan munuaissiirrolla varhaisessa iässä. Välihilassa ilmentyy ainakin kolme nefriinin kaltaista proteiinia, Neph-proteiinit Neph1, Neph2 ja Neph3. Nefriinin ja Neph-proteiinien välisillä interaktioilla oletetaan olevan tärkeä rooli soluadheesiossa ja siten myös välihilan rakenteessa ja toiminnassa. Väitöskirjatyössä tutkittiin nefriinin ja Neph-proteiinien interaktioita ja roolia soluadheesiossa. Työssä osoitettiin, että Neph3 sitoutuu nefriiniin ja sen muodostamaan proteiinikompleksiin. Lisäksi osoitettiin, että Neph1 ja Neph3 pystyvät yksin indusoimaan soluadheesiota, kun taas nefriinin pitää sitoutua vastakkaisen solun pinnalla olevien Neph1:n tai Neph3:n kanssa, jotta solukontaktit muodostuisivat. Havaitsimme myös, että Neph3:n ilmentymistaso lisääntyy sellaisten hiirten podosyyteissä, joilta puuttuu nefriini. Nefriiniä ja Neph3:a koodaavat geenit, NPHS1 ja KIRREL2, sijaitsevat ihmisen kromosomissa 19 ainoastaan noin 5 kb:n etäisyydellä toisistaan siten, että niiden transkriptio tapahtuu vastakkaisiin suuntiin. Geenien samanlainen järjestäytyminen on säilynyt ainakin hiiren ja rotan kromosomissa. Nefriinin ja Neph3:n geenien järjestäytyminen sekä niiden proteiinien samankaltainen rakenne ja toiminta viittaavat siihen, että nefriinin ja Neph3:n geenejä saatetaan säädellä samalla tavoin. Tässä väitöskirjatyössä selvitettiin nefriinin ja Neph3:n geenien transkriptionaalista säätelyä. Työssä osoitettiin, että transkriptiotekijät WT1 ja NF-κB kooperatiivisesti säätelevät kummankin geenin transkriptiota. Lisäksi havaittiin, että DNA:n metylaatio voi vaimentaa molempien geenien ilmentymistä. Transkriptiotekijän GABP osoitettiin ilmentyvän podosyyteissä ja säätelevän nefriinin geenin transkriptiota ja transkriptiotekijän Sp1 osallistuvan Neph3:n geenin transkriptionaaliseen säätelyyn. Väitöskirjatyössä tehdyt tutkimukset tuovat uutta tärkeää tietoa välihilan rakenteesta ja nefriinin ja Neph3:n geenien ilmentymiseen vaikuttavista säätelymekanismeista. Neph3:lla näytettiin olevan samankaltaisia toiminnallisia ominaisuuksia nefriinin ja muiden Neph-proteiinien kanssa, ja nefriinin ja Neph3:n geenien ilmentymistä näytettiin säädeltävän samanlaisilla mekanismeilla. Nefriinin ja Neph-proteiinien adhesiivisilla ominaisuuksilla saattaa olla tärkeä rooli välihilan rakenteessa ja toiminnassa. Samanlaiset säätelymekanismit nefriinin ja Neph3:n geenien transkriptiossa saattavat vaikuttaa siihen, että näiden geenien koodaamien proteiinien ilmentyminen ja toiminta ovat samankaltaisia. Nefriinin ja Neph3:n geenien säätelyn tarkempi tietämys voi auttaa ymmärtämään paremmin podosyyttien transkriptionaalista säätelyä ja toimintaa yleisellä tasolla. Väitöskirjatyön tulokset saattavat auttaa uusien hoitomuotojen tunnistamisessa

Addressing the roles of the retinoic acid receptors during mammalian development

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Cloning of the Bone Gla Protein gene from the teleost fish Sparus aurata (gilthead seabream). Molecular organization, developmental appearance and evolutionary implications

Tese de Doutoramento, Biologia Molecular, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2001A proteína Bone Gla (BGP, osteocalcina) é uma pequena proteína dependenteda. vitamina K que apresenta resíduos de ácido glutâmico y-carboxilados. A presença destes aminoácidos modificados permite à proteína ligar-se a iões Ca2+ e interagir com os cristais de hidroxiapatite dos tecidos mineralizados.The Bone Gla Protein (BGP, osleocalcin) is a small vilamin K-dependent protein which presenls Ihree y-carboxylated glutamic acid residues. lhe prcsence oí these modified amino acids enables the protein to bind to Ca2+ ions and to interact with hydroxyapatite ciystals of mineralized tissues

Modulation of vitamin D action by local factors in bone

Bone provides rigid mechanical support, protects vital organs and serves as a reservoir of ions, such as calcium and phosphate, that can be mobilized from bone. There are two types of bone: cortical bone, which is composed of densely packed, mineralized collagen laid down in layers and provides rigidity, and trabecular bone which is spongy in appearance, and also consists of mineralized collagen and provides strength and elasticity. There are also two types of bone formation: intramembranous ossification, which primarily takes place in the calvarium and starts within the connective tissue, and endochondral ossification, which takes place in long bones and starts with the formation of cartilage followed by bone formation and mineralization. The mineralized matrix is composed of collagenous and noncollagenous proteins and contains a variety of locally produced growth factors