title: Analysis of the physiological function of TNF Receptor I Associated Death Domain Protein (TRADD) and Familial Cylindromatosis Protein (CYLD) by using conditional gene targeting in mice
creator: Ermolaeva, Maria A.
subject: ddc-570
subject: 570 Life sciences
description: The aim of the project was to apply the conditional gene targeting approach based on the usage of Cre/LoxP system of site-specific DNA recombination to investigate physiological function of two putative mediators of inflammation – TNF Receptor 1 Associated Death Domain Protein (TRADD) and Familial Cylindromatosis Protein (CYLD), in a mouse model.  TRADD is an adaptor molecule postulated to be essential for signal transduction through TNF Receptor 1 (TNFR1). The in vivo physiological role of TRADD has not been determined so far. CYLD is a tumor suppressor that has been described as a negative regulator of TNFR1-mediated signaling and signaling by Toll like Receptors (TLRs). TLRs are essential components of mammalian innate immunity belonging to a group of sensors that directly recognize bacterial and viral products as well as markers of tissue stress. Upon activation TLRs induce intracellular signaling events leading to production of cytokines, chemokines and other mediators that promote protective responses. Tumor Necrosis Factor (TNF) is a pleiotropic cytokine produced by a variety of cells upon TLR stimulation. It plays a key role in the amplification of the initial immune response. The majority of effects that are induced by TNF are dependant on TNFR1. TLR signaling and TNF signaling through TNFR1 share common mechanism of negative regulation that is based on the the removal of K63-linked polyubiquitin chains from specific key components of receptor-associated complexes. Two de-ubiquitinating enzymes – A20 and CYLD are currently known to be responsible for this process. The physiological function of A20 is well characterized by gene knockout studies while the precise role of CYLD remains enigmatic.  We successfully generated mice carrying “floxed” (modified by the insertion of LoxP sites and extra DNA fragments at specific locations) alleles of TRADD and CYLD by using homologous recombination in embryonic stem cells. In case of TRADD the deletion of the LoxP-flanked sequence would generate a null TRADD allele; in case of CYLD the last exon of the gene would be replaced by a mutated copy resulting in the expression of C-terminally truncated form of CYLD that is lacking catalytic activity. We then generated TRADD knockout and CYLD complete mutant mice by crossing the homozygous “floxed” animals to a ubiquitous Cre-Deleter strain.  By analyzing TRADD knockout mice we could observe that TNFR1-mediated apoptosis was completely blocked in these mice while TNF-induced pro-inflammatory and anti-bacterial responses were dramatically reduced but still present. We obtained similar results by evaluating the response of TRADD deficient primary cells to TNF. To our surprise we discovered that TRADD knockout mice had impaired immediate responses to stimulation of Toll like receptors 3 and 4. Consistent with this observation TRADD deficient primary cells demonstrated reduced cytokine production as well as impaired activation of NF-kB and MAP kinases upon stimulation with poly(I:C). On the basis of co-expression experiments performed in HEK293T cells we propose that TRADD is recruited to TLR adaptor TRIF via Receptor-Interacting Protein 1 (RIP1) and acts as a mediator of TRIF-dependant TLR signaling. To our surprise CYLD homozygous mutant mice did not survive until the age of weaning. By carefully following the pups we observed that the mutants died within minutes after birth showing signs of cyanosis and respiratory distress. Mutant pups were smaller then control littermates and demonstrated altered morphology of the tail. We then produced mutant mouse embryonic fibroblasts (MEFs) and analyzed the response of these cells to cytokines. Consistent with the role of CYLD as a negative regulator of pro-inflammatory signaling, mutant cells showed elevated activation of NF-kB and JNK cascades upon stimulation with TNF and IL-1beta
date: 2008
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/8784/1/Ermolaeva_thesis_corrected.pdf
identifier: DOI:10.11588/heidok.00008784
identifier: urn:nbn:de:bsz:16-opus-87840
identifier:   Ermolaeva, Maria A.  (2008) Analysis of the physiological function of TNF Receptor I Associated Death Domain Protein (TRADD) and Familial Cylindromatosis Protein (CYLD) by using conditional gene targeting in mice.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/8784/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng