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Interleukin-4-receptor signal transduction [Elektronische Ressource] : involvement of P62 / vorgelegt von Susanne Bürgis

83 pages
Interleukin-4 ReceptorSignal Transduction:Involvement of P62Den Naturwissenschaftlichen Fakult¨atender Friedrich–Alexander–Universitat Erlangen–Nurnberg¨ ¨zurErlangung des Doktorgradesvorgelegt vonSusanne Bu¨rgisaus Nurnberg¨Als Dissertation genehmigt von denNaturwissenschaftlichen Fakult¨atender Universitat Erlangen–Nurnberg¨ ¨Tag der mu¨ndlichen Pru¨fung: 6. Oktober 2006Vorsitzender derPrufungskommission: Prof. Dr. D.–P. Hader¨ ¨Erstberichterstatter: Prof. Dr. Dr. A. GessnerZweitberichterstatter: Prof. Dr. T. WinklerContents1 Introduction 11.1 The biology of interleukin-4 and its receptor . . . . . . . . . . . . . . . . . 11.1.1 Interleukin-4 and its biological functions . . . . . . . . . . . . . . . 11.1.2 Interleukin-4-receptor complexes . . . . . . . . . . . . . . . . . . . . 21.1.3 Interleukin-4-receptor signaling . . . . . . . . . . . . . . . . . . . . 31.2 The adaptor protein P62 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.3 Link between IL-4R and P62 . . . . . . . . . . . . . . . . . . . . . . . . . . 71.4 Aims of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Materials and Methods 112.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.1.1 Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.1.2 Commercial systems . . . . . . . . . . . . . . . . . . . . . . . . . . 112.1.3 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . .
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Interleukin-4 Receptor Signal Transduction: Involvement of P62
DenNaturwissenschaftlichenFakultaten derFriedrichAlexanderUniversitatErlangenNurnberg zur Erlangung des Doktorgrades
vorgelegt von SusanneBurgis s N berg au urn
Als Dissertation genehmigt von den NaturwissenschaftlichenFakultaten derUniversitatErlangenNurnberg
TagdermundlichenPrufung:
Vorsitzender der Prufungskommission: Erstberichterstatter: Zweitberichterstatter:
6. Oktober 2006
Prof.Dr.D.P.Hader Prof. Dr. Dr. A. Gessner Prof. Dr. T. Winkler
Contents
1 Introduction 1.1 The biology of interleukin-4 and its receptor . . . . . . . . . . . . . . . . . 1.1.1 Interleukin-4 and its biological functions . . . . . . . . . . . . . . . 1.1.2 Interleukin-4-receptor complexes . . . . . . . . . . . . . . . . . . . . 1.1.3 Interleukin-4-receptor signaling . . . . . . . . . . . . . . . . . . . . 1.2 The adaptor protein P62 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Link between IL-4R and P62 . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Aims of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials and Methods 2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2 Commercial systems . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.5 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.6 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.7 Cell lines and bacteria . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.8 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.9 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Genotyping of mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Polymerase chain reaction . . . . . . . . . . . . . . . . . . . . . . . 2.2.3 Sequencing of plasmids and PCR products . . . . . . . . . . . . . . 2.2.4 Isolation of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . 2.2.5 Southern blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.6 Isolation of total RNA . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.7 Reverse transcription RT–PCR . . . . . . . . . . . . . . . . . . . . 2.2.8 Quantitative RT–PCR . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.9 Enzyme linked immuno sorbent assay . . . . . . . . . . . . . . . . . 2.2.10 Generation of a P62 antiserum . . . . . . . . . . . . . . . . . . . . . 2.2.11 Transfection of cells with calcium–phosphate . . . . . . . . . . . . . 2.2.12 Western blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.13 Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.14 Proliferation of cells . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.15 Stimulation of macrophages . . . . . . . . . . . . . . . . . . . . . .
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1 1 1 2 3 5 7 9 11 11 11 11 12 12 13 14 16 17 17 18 18 18 19 19 20 20 20 20 21 21 22 22 22 23 23
iv CONTENTS 2.2.16 Manipulation of mouse embryonic stem cells . . . . . . . . . . . . . 24 2.2.17 Preparation of mouse embryo fibroblasts . . . . . . . . . . . . . . . 24 2.2.18 Class switching of splenic B cells . . . . . . . . . . . . . . . . . . . 25 2.2.19TH2dierentiationofnaıveTcells..................25 2.2.20 Infection withLeishmania major. . . . . . . . 26. . . . . . . . . . . 2.2.21 Oral glucose tolerance of mice . . . . . . . . . . . . . . . . . . . . . 26 2.2.22 OVA induced allergic lung disease . . . . . . . . . . . . . . . . . . . 26 3 Results 27 3.1 Studies in cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.1.1 Phosphorylation of signaling intermediates . . . . . . . . . . . . . . 27 3.1.2 Effect of kinase inhibitors . . . . . . . . . . . . . . . . . . . . . . . 27 3.2 Involvement of P62 in IL-4R signaling . . . . . . . . . . . . . . . . . . . . . 30 3.2.1 Binding of P62 to the IL-4R . . . . . . . . . . . . . . . . . . . . . . 30 3.2.1.1 Colocalization of P62-EGFP and IL-4R-RFP . . . . . . . 30 3.2.1.2 Co-Immunoprecipitation of P62 with the IL-4R . . . . . . 30 3.2.1.3 Trimolecular Co-Immunoprecipitation . . . . . . . . . . . 31 3.2.2 Description of the P62 gene deficient mouse model . . . . . . . . . . 32 3.2.2.1 P62 deficiency on genomic level . . . . . . . . . . . . . . . 32 3.2.2.2 P62 deficiency on transcriptome level . . . . . . . . . . . . 32 3.2.2.3 P62 deficiency on protein level . . . . . . . . . . . . . . . 34 3.2.3 Steady state phenotype of P62 gene-targeted mice . . . . . . . . . . 35 3.2.4 Immunological phenotypes of P62 gene-targeted mice . . . . . . . . 36 3.2.4.1 Oral glucose tolerance . . . . . . . . . . . . . . . . . . . . 36 3.2.4.2 Functionality of macrophages . . . . . . . . . . . . . . . . 37 3.2.4.3 Class switching of splenic B cellsin vitro. . . . . . . . . . 38 3.2.4.4 Differentiation of naıve T cellsin vitro. . . . . . . . . . . 39 3.2.4.5 Infection withLeishmania major. . . . . . . . . . . . . . 41 3.2.4.6 Fibroblast functionality . . . . . . . . . . . . . . . . . . . 42 3.2.4.7 OVA induced allergic asthma . . . . . . . . . . . . . . . . 43 3.3 Generation of a ΔN388 IL-4R mouse model . . . . . . . . . . . . . . . . . 44 3.3.1 Targeting strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.3.2 Homologous recombination . . . . . . . . . . . . . . . . . . . . . . . 45 3.3.3 Blastocyst injection and breeding strategy . . . . . . . . . . . . . . 46 4 Discussion 47 4.1 Mechanism of IL-4 induced proliferation in TF1 cells . . . . . . . . . . . . 47 4.2 Binding of P62 to IL-4R and PKCζ 49. . . . . . . . . .. . . . . . . . . . . . 4.3 Involvement of P62 in IL-4R signal transduction . . . . . . . . . . . . . . . 50 4.4 Generation of a ΔN388 IL-4R knock in mouse model . . . . . . . . . . . . 52 5 Summary 55 A Abbreviations 59 Bibliography 61
Chapter 1
Introduction
1.1 The biology of interleukin-4 and its receptor
1.1.1 Interleukin-4 and its biological functions Murine interleukin-4 (IL-4) is a pleiotropic cytokine of 140 amino acids that is secreted after removal of a signal peptide as a mature protein of 120 amino acids, and has first been described as a growth factor for B cells by the group of William Paul [58]. Similar to other cytokines, it has a compact, globular fold, which is stabilized by three disulphide bonds [18]. The crystal structure has been solved for the human protein, and based on the amino acid sequence identity, the murine protein is expected to have a similar tertiary structure dominated by a four alpha-helix bundle with a left handed twist [153]. IL-4 is mainly sectreted by CD4+T cells that also produce IL-5 and IL-13 (TH2 cells) [128]. In mice, basophils have been shown to secrete IL-4 after infection with the nematode Nippostrongylus brasiliensisin humans upon challenge with allergens [127]; likewise,[93], mast cells have been reported to secrete IL-4 [13, 15]. Additional sources of IL-4 are NK1.1 positive CD4+T cells [20],γ/δ also Recently,T cells [35] and eosinophils [31]. dendritic cells and B cells have been suggested to produce IL-4 [90, 52]. IL-4 is an important cytokine in shaping immune responses and exerts different effects on various hematopoietic cells including lymphocytes (reviewed in [43] and [101]). On B cells, it acts as a growth factor [58] and is responsible for the upregulation of surface molecules such as MHC II [103], the low affinity IgE receptor CD23 [25] and the IL-4R itself [104]. Likewise, IL-4 induces Ig heavy chain class switching of human B cells to IgE and IgG4[39] and is indispensable for class switching of murine B cells to IgE and IgG1 [23, 150]. On T cells, IL-4 acts as a growth factor that induces either proliferation or antiapoptotic effects [60]. IL-4 is also required for the development of TH2 cells from naıve T cells after antigen stimulation. These cells secrete large amounts of IL-4 and other cytokines such as IL-5 or IL-13, thereby initiating a positive feedback loop. Additionally, IL-4 blocks the development of IFN-γsecreting TH1 cells, thus stabilizing a TH2 dominated immune response [59, 128]. In some infectious disease models it is decisive for the outcome of the infection, whether the immune system mounts a TH1- or TH2-dominated immune response. The importance of IL-4 for this decision has been clearly shown in various mouse
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CHAPTER 1. INTRODUCTION
models using either neutralizing anti-IL-4 antibodies [117, 121], soluble IL-4R [113, 44] or IL-4 gene-deficient mice [75]. Apart from lymphocytes, IL-4 also affects other cells of the hematopoietic lineage. It upregulates MHC II expression on monocytes and leads to enhanced antigen presentation by macrophages, while at the same time the production of proinflammatory cytokines is downregulated [10]. IL-4 is also involved in inflammatory processes. It influences the expression of endothelial adhesion molecules like vascular cell adhesion molecule 1 (VCAM-1) [145] or E-selectin [6] and promotes chemokine production, thereby favouring the recruitment of eosinophils to the site of infection. Dysregulation of IL-4 expression results in uncontrolled allergic inflammation in mice and humans [115, 143], so a thorough understanding of IL-4R signal transduction processes is essential for therapeutic applications.
1.1.2 Interleukin-4-receptor complexes IL-4 binds to a high affinity receptor IL-4Rα(Kd20 to 80 pM [87]), that is a member of the type I cytokine receptor family as it contains conserved cysteine residues and an extracellular WSXWS motif, which is required for ligand binding [85]. The receptor is ex-pressed on a variety of hematopoietic (monocytes, macrophages, mast cells, T cells and B cells) and nonhematopoietic cells (fibroblasts, neuroblasts, keratinocytes and hepatocytes) at relatively low frequencies (100 to 5000 copies per cell). Apart from the glycosylated 140 kD transmembrane protein, a 40 kD soluble IL-4R has been identified, that results from an alternatively spliced mRNA [99]. Compared to the transmembrane receptor, the soluble IL-4R binds IL-4 with similar affinity [34] and has been employed in the treatment of human asthma [11] and murine leishmaniasis [44]. Signal tranduction can be initiated through artificial cross-linkage of IL-4Rαchains [80], whereas physiological activity is obtained through heterodimerization of the IL-4Rαchain with either the common gamma chain (γcto yield the type I receptorchain, CD132) complex, or the IL-13Rα The1 chain to yield the type II receptor complex, respectively. majority of cells, including hematopoietic cells, express the type I IL-4R complex in which γc protein is also involved in the signal transduction of receptor Thischain participates. complexes for IL-2, IL-7, IL-9, IL-15 [138] and IL-21 [108]. First, a complex of IL-4 and IL-4Rαis formed and subsequentlyγcchain interacts with this complex [82]; this binding does not increase the affinity for IL-4, but is required for the initiation of signal transduc-tion [119]. Cells lacking theγcchain (mostly nonhematopoietic cells) can signal via the type II IL-4R complex [16, 56]. This receptor can also transduce signals in response to the cytokine IL-13 [92]. Additionally to the IL-13Rα1 chain, an IL-13Rα2 chain exists, that binds IL-13 with even higher affinity but does not contribute to signal transduction; it acts as a decoy receptor for IL-13 [158]. However, most recently the IL-13Rα2 chain was described to transduce IL-13 signals in macrophages, thereby fostering TGF-β-mediated fibrosis [36]. Recently the IL-4Rαchain has been proposed to contribute to the signal transduction of progesterone-induced blocking factor (PIBF), which induces a TH2 dominated cytokine
1.1. THE BIOLOGY OF INTERLEUKIN-4 AND ITS RECEPTOR 3
production through signaling via STAT6. This novel receptor complex consists of the IL-4Rαchain and the GPI-anchored PIBF receptor [77].
1.1.3 Interleukin-4-receptor signaling As a typical member of the hematopoietin receptor family, the IL-4Rαand the associ-ated receptor moleculesγcchain and IL-13Rα1 lack endogenous kinase activity. Thus, for the initiation of signaling cascades, intracellular kinases and adaptor proteins have to be recruited. Janus tyrosine kinases (Jaks) are known to contribute to cytokine signaling [62]: Jak-1, Jak-2 and Jak-3 have been shown to interact with the IL-4R. The IL-4Rα chain itself recruits Jak-1 and in some cell lines also Jak-2 [101]; in doing so, binding is mediated by a conserved motif of the IL-4Rαchain, located in close proximity to the transmembrane region and containing several acidic amino acids (box-1 motif) [27, 95]. Theγcand has been shown to bind Jak-3, whereas thechain also contains a box-1 motif IL-13Rα1 chain is associated with Jak-2 or the tyrosine kinase 2 (Tyk2) [106]. Activation of Jaks or other IL-4R associated kinases leads to tyrosine phosphorylation of the IL-4Rαconserved tyrosine residues of the intracellular receptor Five chain [130]. portion are phosphorylated, namely tyrosines Y475, Y550, Y578, Y606 and Y684 (num-bering according to Mosleyet al. adaptor proteins with Src homology[99]). Subsequently, 2 domains (SH2) or phosphotyrosine-binding domains (PTB) can bind to the receptor. Two major pathways for IL-4R signal transduction have been described [101]: one em-ploys insuline receptor substrates (IRS) and transduces mainly IL-4 induced proliferation signals (IRS-pathway), the other uses signal transducers and activators of transcription (STATs) and mediates gene activation in response to IL-4 (Jak/STAT-pathway).
Figure 1.1:type I and type II. Schematic representation with receptor chains involvedIL-4R and conserved interaction domains for adaptor proteins.
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CHAPTER 1. INTRODUCTION
Signaling via the IRS-pathway is initiated by the recruitment of IRS molecules, that possess a PTB domain, to the phosphorylated consensus motif466PIxxxxNPxYxSxSD480 within the IL-4Rαchain [71] (see figure 1.1). motif is also known as insulin-/IL-4R- This motif (I4R) due to the homology in receptor sequences [155]. Subsequently, IRS molecules are multiply phosphorylated at up to 20 different amino acids by Jaks [140, 160], after which they act as adaptor proteins linking other signal transduction molecules to the re-ceptor [139, 141]. Phosphorylated IRS molecules have been described to bind to the reg-ulatory subunit of the phosphoinositide-3 kinase (PI3-kinase); the activated PI3-kinase phosphorylates membrane lipids that act as second messengers and are crucial for the survival of the cell [37]. Another binding partner of phosphorylated IRS is the adaptor molecule Grb-2, that establishes a link to the Ras/Raf-pathway [26]. Similarly, signaling via the STAT6-pathway requires the recruitment of cytoplasmatic STAT6 molecules to the IL-4Rα three conserved tyrosine residues Y550, Y578chain. The and Y606 of the IL-4Rαact as binding sites for the SH2 domain of STAT6 (see figure 1.1), and bound STAT6 is phosphorylated by Jaks. After tyrosine phosphorylation, STAT6 dis-sociates from the receptor and translocates as a homodimer to the nucleus, where it acts as a transcription factor binding to promotor consensus motifs (GAS-elements) of IL-4 inducible genes [63, 91]. STAT6 regulates genes responsible for allergic reactions, a TH2-dominated immune response and IgE production [110].
Figure 1.2:IL-4R deletion mutants. Schematic representation of C-terminal IL-4Rαdele-tion mutants and their ability to proliferate upon IL-4 stimulation and to activate signaling intermediates.
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