FADD Regulates Early T Cell Development By Modulating Notch Signaling

Fas-associated death domain protein(FADD)is a 28 KD adaptor molecular known to transmit apoptotic signal induced by death receptors(DRs),such as Fas.Apart from transmitting apoptotic signal,FADD has been found involved in other physiological processes,e.g.,embryogenesis,cell survival and proliferation,cell cycle procession.Although there is a better understanding about FADD-mediated apoptotic signaling pathway,relatively little is known about the nature of FADD-dependent non-apoptotic mechanism.One of the major studies on non-apoptotic FADD focused on T lymphocytes using FADD or its mutant transgenic mice.These mice are suggested to fall into three modes according to the different construction strategies:FADD domain negative mice(FADD-DN),FADD-/-chimeras in a background devoid of immune system(FADD-/-?RAG-1-/-),and T cell-specific FADD knockout mice.Based on studies on these mice,a principal conclusion that FADD is required for T cell proliferation can be drawn.However,the results gained was controversial among these models,just as FADD deficiency induced a severe thymic development arrest in Lck-cre FADD mice and FADD-/-?RAG-1-/-mice but not in CD4-cre FADD mice or Lck-cre/CD4-cre FADD-EGFP mice(1).Moreover,the detailed mechanism of how FADD regulates T cell development is still remained unraveled.However,there are obvious differences between protein sequence of FADD-EGFP and Wt FADD.Considering that the function of a protein is dependent on its structure,we think that the introduction of EGFP at the C-terminus of FADD will essentially change the conformation of wild type FADD,which may be responsible for above-mentioned questions.To address these questions and also to investigate the essential roles of FADD in T lineage,we produce two Cre/loxP-mediated T cell-specific FADD knockout mice:CD4-Cre-depended T cell-specific FADD knockout mice(CD4-FADD)and Lck-Cre-depended T cell-specific FADD knockout mice(Lck-FADD).It is crucial to generated both CD4-FADD mice and Lck-FADD mice although the latter has been produced and reported in another study.In this study,CD4-FADD mice and Lck-FADD mice are equally important to explore the essential function of FADD in T cell development and this combination will making our data more impersonal and more convictive.Thymocytes that 'pass' ?-selection enter the last proliferative burst they will encounter inthe thymus.They initiate CD4 and CD8 expression(becoming 'double positive",DP)andTCRa gene rearrangement,resulting in the surface expression of TCR?? complexes,During early stage of T cell lineage development,the differentiation,proliferation and survival of thymocytes was regulated by several signaling pathways.Pre-TCR and Notch signaling are both required for progression through the ?-selection checkpoint.It has been stated that Notchl provides important metabolic functions in DN3 cells through the phosphoinositol-3 kinase(PI3K)-AKT pathway at ?-selection and T cells do not survive when Notch signals are withdrawn prior to ?-selection.In this article,we generated and compared two Cre/loxP-mediated T cell-specific FADD knockout mice in which FADD was deleted in different stages of thymocytes.Our observation that FADD deficiency caused a reduction in overall thymocyte numbers and a partial block at the DN-to-DP transition only in Lck-FADD mice demonstrates that FADD plays a crucial role in ?-selection,an important checkpoint occurring from the DN3 stage to DN4 stage of thymocyte development.While the percentage of DN3b cells and the expression of iTCR ?,CD25 and CD69 in DN3 cells were not changed in Lck-FADD mice,it showed that the pre-TCR signal may remain intact and the development arrest is likely due to other factors.When focusing on the mechanism inducing the decrease of DN4 cells,a mild proliferation arrest and a severe apoptosis was observed at the DN4 stage which may account for the violent cellular decrease(and can not be rescued by introduction of TCR ?).In addition,Notch signaling is positive regulated on DN4 and DP thymocytes in T cell-specific FADD-deletion mice,which express higher levels of a subset of Notch target genes,including Hesl,Deltex1 and CD25.Abnormal phosphorylation of Akt and decreased glucose transport may result in the increased apoptosis in DN4 thymocytes from Lck-FADD mice.Moreover,a transcriptional repressor of Notchl,NKAP is down-regulated coupled with the loss of FADD in thymocytes and is found to associate with FADD.These data suggest that as a death receptor,FADD is also required for cell survival in ?-selection as a regulator of Notch1 expression.