In Vivo Localization of Fas-Associated Death Domain Protein in the Nucleus and Cytoplasm of Normal Thyroid and Liver Cells

FADD (Fas-associated death domain) is the main death receptor adaptor molecule that transmits apoptotic signal. Recently, FADD protein was shown to be expressed both in the cytoplasm and nucleus of in vitro cell lines. In contrast to the cytoplasmic FADD, the nuclear FADD was shown to protect cells from apoptosis. However, in vivo subcellular localization of FADD was still unknown. Here, we demonstrated that FADD protein was expressed in both cytoplasmic and nuclear compartment in ex vivo thyroid cells demonstrating that nuclear sublocalization of FADD protein was a relevant phenomenon occurring in vivo. Moreover, we showed that in the nucleus of untransformed thyroid cells FADD localized mainly on euchromatin. We confirmed the nuclear localization of FADD in ex vivo liver and showed that in this organ FADD and MBD4 interact together. These results demonstrate that FADD is physiologically expressed in the nucleus of cells in at least two mouse organs. This particular localization opens new possible role of FADD in vivo either asan inhibitor of cell death, or as a transcription factor, or as a molecular link between apoptosis and genome surveillance

N-terminal and C-terminal domains of calmodulin mediate FADD and TRADD interaction

FADD (Fas–associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins are important regulators of cell fate in mammalian cells. They are both involved in death receptors mediated signaling pathways and have been linked to the Toll-like receptor family and innate immunity. Here we identify and characterize by database search analysis, mutagenesis and calmodulin (CaM) pull-down assays a calcium-dependent CaM binding site in the α-helices 1–2 of TRADD death domain. We also show that oxidation of CaM methionines drastically reduces CaM affinity for FADD and TRADD suggesting that oxidation might regulate CaM-FADD and CaM-TRADD interactions. Finally, using Met-to-Leu CaM mutants and binding assays we show that both the N- and C-terminal domains of CaM are important for binding

Functional complementation between FADD and RIP1 in embryos and lymphocytes.

FADD is a common adaptor shared by several death receptors for signalling apoptosis through recruitment and activation of caspase 8 (refs 1-3). Death receptors are essential for immune homeostasis, but dispensable during embryogenesis. Surprisingly, Fadd(-/-) mice die in utero and conditional deletion of FADD leads to impaired lymphocyte proliferation. How FADD regulates embryogenesis and lymphocyte responses has been a long-standing enigma. FADD could directly bind to RIP1 (also known as RIPK1), a serine/threonine kinase that mediates both necrosis and NF-κB activation. Here we show that Fadd(-/-) embryos contain raised levels of RIP1 and exhibit massive necrosis. To investigate a potential in vivo functional interaction between RIP1 and FADD, null alleles of RIP1 were crossed into Fadd(-/-) mice. Notably, RIP1 deficiency allowed normal embryogenesis of Fadd(-/-) mice. Conversely, the developmental defect of Rip1(-/-) lymphocytes was partially corrected by FADD deletion. Furthermore, RIP1 deficiency fully restored normal proliferation in Fadd(-/-) T cells but not in Fadd(-/-) B cells. Fadd(-/-)Rip1(-/-) double-knockout T cells are resistant to death induced by Fas or TNF-α and show reduced NF-κB activity. Therefore, our data demonstrate an unexpected cell-type-specific interplay between FADD and RIP1, which is critical for the regulation of apoptosis and necrosis during embryogenesis and lymphocyte function

cMet and fas receptor interaction inhibits death-inducing signaling complex formation in endothelial cells

Fas receptor is constitutively expressed on endothelial cells; however, these cells are highly resistant to Fas-mediated apoptosis. In this study, we examined death-inducing signaling complex (DISC) formation in endothelial cells after Fas receptor stimulation. Nonfunctional DISC formation was observed in human umbilical vein endothelial cells (HUVECs). Fas-associated death domain (FADD) and large amounts of FADD-like interleukin-1–converting enzyme–inhibitory protein-L were recruited to the receptor; however, no caspase 8 recruitment was observed. A role for the cell surface molecule cMet in controlling Fas sensitivity in endothelial cells was observed. Here, we report that Fas is associated with cMet in HUVECs. Such an interaction may inhibit self-association of Fas in these cells, as suggested by the fact that monomeric Fas is expressed in these cells. Endothelial cells undergoing cell matrix detachment, anoikis, are sensitive to Fas-mediated apoptosis. Despite upregulating the level of Fas receptor, endothelial cells undergoing anoikis have reduced cMet/Fas interaction, in part because of cMet being cleaved in these cells. Dimeric Fas was observed on anoikis cells. These data suggest that cMet/Fas interaction may inhibit self-association of Fas receptor such that reduced DISC formation occurs in these cells after Fas receptor ligation. cMet/Fas interaction may help explain why endothelial cells are resistant to Fas-mediated apoptosis

Kinase-independent function of RIP1, critical for mature T-cell survival and proliferation.

The death receptor, Fas, triggers apoptotic death and is essential for maintaining homeostasis in the peripheral lymphoid organs. RIP1 was originally cloned when searching for Fas-binding proteins and was later shown to associate also with the signaling complex of TNFR1. Although Fas exclusively induces apoptosis, TNFR1 primarily activates the pro-survival/pro-inflammatory NF-κB pathway. Mutations in Fas lead to lymphoproliferative (lpr) diseases, and deletion of TNFR1 results in defective innate immune responses. However, the function of RIP1 in the adult lymphoid system has not been well understood, primarily owing to perinatal lethality in mice lacking the entire RIP1 protein in germ cells. This current study investigated the requirement for RIP1 in the T lineage using viable RIP1 mutant mice containing a conditional and kinase-dead RIP1 allele. Disabling the kinase activity of RIP1 had no obvious impact on the T-cell compartment. However, T-cell-specific deletion of RIP1 led to a severe T-lymphopenic condition, owing to a dramatically reduced mature T-cell pool in the periphery. Interestingly, the immature T-cell compartment in the thymus appeared intact. Further analysis showed that mature RIP1(-/-) T cells were severely defective in antigen receptor-induced proliferative responses. Moreover, the RIP1(-/-) T cells displayed greatly increased death and contained elevated caspase activities, an indication of apoptosis. In total, these results revealed a novel, kinase-independent function of RIP1, which is essential for not only promoting TCR-induced proliferative responses but also in blocking apoptosis in mature T cells

Autophosphorylation at serine 166 regulates RIP kinase 1-mediated cell death and inflammation

Receptor interacting protein kinase 1 (RIPK1) regulates cell death and inflammatory responses downstream of TNFR1 and other receptors, and has been implicated in the pathogenesis of inflammatory and degenerative diseases. RIPK1 kinase activity induces apoptosis and necroptosis, however the mechanisms and phosphorylation events regulating RIPK1-dependent cell death signaling remain poorly understood. Here we show that RIPK1 autophosphorylation at serine 166 plays a critical role for the activation of RIPK1 kinase-dependent apoptosis and necroptosis. Moreover, we show that S166 phosphorylation is required for RIPK1 kinase-dependent pathogenesis of inflammatory pathologies in vivo in four relevant mouse models. Mechanistically, we provide evidence that trans autophosphorylation at S166 modulates RIPK1 kinase activation but is not by itself sufficient to induce cell death. These results show that S166 autophosphorylation licenses RIPK1 kinase activity to induce downstream cell death signaling and inflammation, suggesting that S166 phosphorylation can serve as a reliable biomarker for RIPK1 kinase-dependent pathologies

PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors.

Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA, a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-α and enhanced NF-κB activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD-deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo

Cell death and life in cancer: mathematical modeling of cell fate decisions

Tumor development is characterized by a compromised balance between cell life and death decision mechanisms, which are tighly regulated in normal cells. Understanding this process provides insights for developing new treatments for fighting with cancer. We present a study of a mathematical model describing cellular choice between survival and two alternative cell death modalities: apoptosis and necrosis. The model is implemented in discrete modeling formalism and allows to predict probabilities of having a particular cellular phenotype in response to engagement of cell death receptors. Using an original parameter sensitivity analysis developed for discrete dynamic systems, we determine the critical parameters affecting cellular fate decision variables that appear to be critical in the cellular fate decision and discuss how they are exploited by existing cancer therapies