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FADD, a classical apoptotic signaling adaptor, has recently been reported to exhibit a series of non-apoptotic functions. Here, we report that FADD may play a critical role in the development of renal fibrosis. Neutrophil infiltration in the renal interstitial part, glomerular mesangial cell proliferation, and base-membrane thickening were observed in FADD-D mice by H&E, PAS, and PASM staining. Immunofluorescence analysis revealed that macrophage infiltration was significantly enhanced in FADD-D mice. Renal fibrosis might be induced by IgA nephritis in FADD-D mice as evidenced by increased Ki67 and type IV collagen. Additionally, the levels of α-SMA, Fibronectin, and Vimentin were also found to be elevated. Mechanism study indicated that the TLR4/myD88/NF-κB signaling pathway was activated in FADD-D mice. Moreover, FADD phosphorylation activated the mTOR and TGF-β/Smad pathway and accelerated the process of epithelial mesenchymal transition. Further studies indicated that the TGF-β1 pathway was also activated and the process of EMT was accelerated in both FADD-disrupted HEK293 cells and FADD-deficient MES cells. Thus, we concluded that FADD phosphorylation could lead to IgA nephritis and eventually result in renal fibrosis. Taken together, our study provides evidence, for the first time, that FADD, especially in its phosphorylated form, has an effect on the development of renal fibrosis. Abbreviations: FADD: FAS-associated protein with death domain; DED: death effector domain; DD: death domain; CKD: chronic kidney disease; ECM: extracellular matrix; ESRD: end-stage renal disease; RRT: renal replacement therapy; H&E: hematoxylin and eosin; PASM: periodic acid silver methenamine.

Literatures indicate that microRNA-129-5p (miR-129-5p) or Fas-associated death domain (FADD) is related to intervertebral disc degeneration (IDD), but the effect of miR-129-5p/FADD axis on IDD is not studied. The study aimed to investigate whether miR-129-5p influenced immune privilege and nucleus pulposus (NP) cell apoptosis in rats with IDD via regulating FADD.A rat model with caudal IDD was established, and injected with miR-129-5p agomir or miR-129-5p antagomir to figure out the character of miR-129-5p in the cell apoptosis and inflammation in the nucleus pulposus (NP) tissues of IDD rats. NP cells were grouped as the same ways for determining proliferation, apoptosis, and senescence in NP cells of IDD rats. Annexin V-FITC/PI double staining detected the apoptosis of macrophages and CD8 + cells co-cultured via transfected NP cells. Expression of miR-129-5p, FADD, collagen I, collagen II, aggrecan and Sox-9 in NP tissues and cells were determined.Up-regulated miR-129-5p decreased FADD, collagen I and elevated collagen Ⅱ, aggrecan, and Sox-9 in NP tissues and repressed inflammation in serum and NP tissues in IDD rats. Up-regulated miR-129-5p facilitated proliferation, inhibited senescence, apoptosis, and decreased FADD, collagen I and increased collagen Ⅱ, aggrecan, and Sox-9 in NP cells of IDD rats. Elevated miR-129-5p promoted the apoptosis of macrophages and CD8 + cells.We pronounced that up-regulated miR-129-5p inhibited the apoptosis and facilitated the proliferation of NP cells, as well as the apoptosis of macrophages and CD8 + cells via decreased FADD in IDD, suggesting that miR-129-5p had a protective effect on IDD.

Fas-associated protein with death domain (FADD) plays a key role in extrinsic apoptosis. Here, we show that FADD is SUMOylated as an essential step during intrinsic necrosis. FADD was modified at multiple lysine residues (K120/125/149) by small ubiquitin-related modifier 2 (SUMO2) during necrosis caused by calcium ionophore A23187 and by ischemic damage. SUMOylated FADD bound to dynamin-related protein 1 (Drp1) in cells both in vitro and in ischemic tissue damage cores, thus promoting Drp1 recruitment by mitochondrial fission factor (Mff) to accomplish mitochondrial fragmentation. Mitochondrial-fragmentation-associated necrosis was blocked by FADD or Drp1 deficiency and SUMO-defective FADD expression. Interestingly, caspase-10, but not caspase-8, formed a ternary protein complex with SUMO-FADD/Drp1 on the mitochondria upon exposure to A23187 and potentiated Drp1 oligomerization for necrosis. Moreover, the caspase-10 L285F and A414V mutants, found in autoimmune lymphoproliferative syndrome and non-Hodgkin lymphoma, respectively, regulated this necrosis. Our study reveals an essential role of SUMOylated FADD in Drp1- and caspase-10-dependent necrosis, providing insights into the mechanism of regulated necrosis by calcium overload and ischemic injury., (Copyright © 2017 American Society for Microbiology.)

Purpose: Ultraviolet (UV) radiation-induced apoptosis enabled us to study the mechanism of DNA damage and to investigate how cells avoid consequences of damaged DNA. Cells with extensive DNA damage activate extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway is coupled to a FAS-associated protein with death domain (FADD), an adaptor protein molecule necessary for mediating apoptotic signals through the cell., Materials and Methods: Viability and apoptosis of wild-type and FADD-deficient mouse embryonic fibroblasts were investigated 1, 3, 24 and 48 h after exposure to three doses (50, 75 and 300 J/m(2)) of UVC radiation. Morphological changes were observed using DNA binding dyes (Hoechst and propidium iodide) while biochemical changes were monitored using immunodetection of the poly (ADP-ribose) polymerase (PARP) protein cleavage and caspase-3 activity assay., Results: Results showed that the difference in cell death response between wild-type and FADD-deficient cells depended on dose and incubation time after exposure to UVC radiation. FADD-deficient cells are more sensitive to UVC radiation. Even though FADD-deficient cells lack an adapter protein of apoptotic extrinsic pathway, higher doses of UVC triggered their apoptotic response, while wild-type cells die mainly due to necrosis. A different pattern of caspase 3 activity and PARP cleavage was observed 24 h after radiation between two cell lines confirming higher apoptotic response in FADD-deficient cells., Conclusions: Wild-type cells can execute apoptosis via both, the mitochondrial and the receptor-mediated pathway whereas FADD-deficient cells can only activate the intrinsic pathway. There is a difference in UVC radiation response between two cell lines indicating the role of FADD in the selection of cell death modality.

Signaling by tumor necrosis factor (TNF) receptor 1 (TNF-R1), a prototypic member of the death receptor family, mediates pleiotropic biological outcomes ranging from inflammation and cell proliferation to cell death. Although many elements of specific signaling pathways have been identified, the main question of how these selective cell fate decisions are regulated is still unresolved. Here we identified TNF-induced K63 ubiquitination of TNF-R1 mediated by the ubiquitin ligase RNF8 as an early molecular checkpoint in the regulation of the decision between cell death and survival. Downmodulation of RNF8 prevented the ubiquitination of TNF-R1, blocked the internalization of the receptor, prevented the recruitment of the death-inducing signaling complex and the activation of caspase-8 and caspase-3/7, and reduced apoptotic cell death. Conversely, recruitment of the adaptor proteins TRADD, TRAF2, and RIP1 to TNF-R1, as well as activation of NF-κB, was unimpeded and cell growth and proliferation were significantly enhanced in RNF8-deficient cells. Thus, K63 ubiquitination of TNF-R1 can be sensed as a new level of regulation of TNF-R1 signaling at the earliest stage after ligand binding., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.

It has been known for many years that the protein Fas-associated death domain (FADD) is an essential protein forming the apical portion of the extrinsic apoptosis pathway that permits association of death receptors, e.g., CD95, DR4, DR5 with pro-caspases 8 and 10, thereby facilitating caspase activation (e.g., ref. 1, and references therein). It is also known that FADD can recruit other proteins to regulate NFκB and MAPK pathways which in turn can promote proliferation and cell cycle progression. In NSCLC high expression of FADD has been associated with shorter survival times and lymph node metastasis or oral cancer and worse survival, and the present manuscript in head and neck cancer demonstrates similar findings with respect to lymph node metastasis and survival.(2,3)

FAS-associated death domain (FADD) is a key adaptor protein that bridges a death receptor (e.g., death receptor 5; DR5) to caspase-8 to form the death-inducing signaling complex during apoptosis. The expression and prognostic impact of FADD in head and neck squamous cell carcinoma (HNSCC) have not been well studied. This study focuses on detecting FADD expression and analyzing its prognostic impact in primary and metastatic HNSCCs. We found a significant increase in FADD expression in primary tumors with lymph node metastasis (LNM) in comparison with primary tumors with no LNM. This increase was significantly less in the matched LNM tissues. Both univariate and multivariable analyses indicated that lower FADD expression was significantly associated with better disease-free survival and overall survival in HNSCC patients with LNM although FADD expression did not significantly affect survival of HNSCC patients without LNM . When combined with DR5 or caspase-8 expression, patients with LNM expressing both low FADD and DR5 or both low FADD and caspase-8 had significantly better prognosis than those expressing both high FADD and DR5 or both high FADD and caspase-8. However, the expression of both low FADD and caspase-8 was significantly linked to worse overall survival compared with both high FADD and caspase-8 expression in HNSCC patients without LNM. Hence, we suggest that FADD alone or together with DR5 and caspase-8 participates in metastatic process of HNSCC.

Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-α)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-α-induced pronecrotic RIP1-RIP3 kinase complex, whereas the IκB Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-α-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-κB. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-x(L) protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.

Doxorubicin (Dox) has been employed in cancer chemotherapy for a few decades. However its clinical application became restricted because of dose-dependent cardiomyopathy. Recent studies suggest that Dox-induced cardiomyocyte apoptosis is a primary cause of cardiac damage. Vascular endothelial growth factor (VEGF) is a major factor for endothelial cell survival and angiogenesis. We have previously shown that VEGF165 significantly attenuates oxidative stress-induced cardiomyocytes apoptosis. We hypothesized that VEGF165 will protect the cardiomyocytes from Dox-induced apoptosis. to evaluate our hypothesis, we transfected cardiomyocytes H9c2 with adenovirus expressing VEGF165 24 hours before the cells were challenged with Dox at a concentration of 2 µm. Cardiomyocyte apoptosis was evaluated by Annexin V-FITC staining and by Western blot detection of cleaved caspase-3. The hypothesis was confirmed, and the protective mechanisms involve the inhibition of death receptor-mediated apoptosis and up-regulation of the prosurvival Akt/Nf-κb/bcl-2 signaling pathway.

Disruption of tumor blood supply causes tumor hypoxia. Hypoxia can induce cell death, but cancer cells that remain viable in the absence of oxygen often possess an increased survival potential, and tumors formed by these cells tend to grow particularly aggressively. Thus, developing approaches aimed at increasing the susceptibility of malignant cells to hypoxia-induced death represents a potentially important avenue for cancer treatment. Molecular mechanisms that control the survival of cancer cells under hypoxia are not well understood. In an effort to understand them we found that hypoxia downregulates Beclin 1, a mediator of autophagy, in malignant intestinal epithelial cells. The reversal of this downregulation promoted autophagosome accumulation, enhanced the activation of a pro-apoptotic protease caspase-9 and subsequent caspase-9-dependent activation of two other pro-apoptotic proteases caspases 3 and 7 in these cells. Furthermore, the reversal of hypoxia-induced downregulation of Beclin 1-stimulated caspase-9-dependent apoptosis of the indicated cells under hypoxia. Interestingly, we found that Beclin 1-dependent caspase-9 activation in hypoxic cells was not associated with an increased release of cytochrome c from the mitochondria to the cytoplasm (such release represents a frequently occurring mechanism for caspase-9 activation). We also observed that Beclin 1-dependent apoptosis of hypoxic malignant cells was independent of FADD, a mediator of death receptor signaling. We conclude that hypoxia triggers a feedback mechanism that delays apoptosis of oxygen-deprived malignant intestinal epithelial cells and is driven by hypoxia-induced Beclin 1 downregulation. Thus, approaches aimed at the disruption of this mechanism can be expected to enhance the susceptibility of such cells to hypoxia-induced apoptosis.

Death domain complexes are key protein arrangements in the regulation of various cellular signaling events. One of the most prominent death domain complexes first described in the initiation of apoptosis is formed by the transmembrane receptor Fas, the cytosolic adaptor protein FADD, and caspase-8 and is referred to as the Fas/FADD/caspase-8 death inducing signaling complex (DISC). The recent structure of the Fas/FADD death domain complex reveals how formation of this signaling platform can be stringently regulated utilizing only Fas receptor clustering to form a death domain network. This work reveals that an opening mechanism of Fas is needed to expose binding sites for the FADD death domain and sets the stage for a conditional interaction, which is characterized by weak interactions adapted for a regulatory function. The overall crystal structure reveals a tetrameric arrangement of four primary Fas/FADD complexes. Intriguingly all contacts mediating the tetramer are solely provided through Fas/Fas interactions and are entirely dependent on the open form. These findings are instrumental in depicting a mechanism for DISC regulation where Fas receptor clustering leads to the stabilization of the open Fas death domains which are then capable of binding FADD in a weak interaction. At the same time this mechanism ensures that in the absence of a sufficient stimulus no interaction between Fas and FADD is possible. Therefore the conformation dependent, conditional Fas/FADD death domain interaction represents the regulatory element per se. This interaction contrasts the classic constitutive interactions of adaptor domains, which cannot provide regulatory function themselves. This model portrays how sole death domains are able to mediate signaling upon receptor clustering in the complete absence of enzyme activity.

Islet transplantation is considered a therapeutic option for type 1 diabetes (T1D). However, allorejection is one major barrier for the successful islet transplantation. In the present study, we have tested the feasibility of a deletion construct for Fas-associated death domain protein (FADD; without the death effecter domain) fused with green fluorescent protein (FADDdel-GFP) for blocking the Fas-FasL signaling pathway in prevention of transplanted beta cell destruction by allo-rejection in T1D. In vitro studies have shown that NIT-1 cells with ectopic FADDdel expression were resistant to cytokine-induced apoptosis and CTL-mediated lysis. Diabetic Balb/c mice reached normoglycemia promptly and gained weight after transplantation of NIT-1 cells with ectopic FADDdel-GFP expression. These recipients showed a significant longer survival time than that of recipients transplanted with NIT cells with ectopic GFP expression only. Our results together suggest that FADDdel could be a useful target for the improvement of islet transplantation for T1D.

During an immune response, specific recognition of microbial and tumor antigens leads to the rapid proliferation of lymphocytes. Once the immunological challenge is eliminated, the vast majority of these lymphocytes must be removed via apoptosis. Cell death is also vital for the deletion of autoreactive or chronically activated lymphocytes to prevent the development of autoimmunity in the host. Such processes are highly dependent on death receptors (DRs), molecules of the TNF receptor family. While these DRs promote apoptosis, interference with DR signaling paradoxically interferes with rapid lymphocyte proliferation. Recently, we discovered that T cells lacking Fas-Associated protein with Death Domain (FADD) or caspase-8 (casp8) function, both essential for DR-induced apoptosis, succumb to hyperactivation of autophagy and die through a nonapoptotic form of cell death rather than proliferating after mitogen stimulation. We observed recruitment of FADD, casp8 and serine/threonine kinase RIPK1 to complexes containing Atg5, Atg12 and Atg16L, suggesting that the generation of early autophagosomes leads to the assembly of complexes that activate casp8. Because blockade of RIPK1 or interference with autophagic signaling inhibited this alternative death process, we propose that hyperactive autophagy induced in the absence of caspase activity leads to a necrosis-like form of death that depends on RIPK1 enzymatic function. Herein, we summarize these findings and speculate on the significance and means by which autophagy is normally activated in proliferating lymphocytes.

Lipopolysaccharide (LPS) engages Toll-like receptor 4 (TLR4) on various cells to initiate inflammatory and angiogenic pathways. FADD is an adaptor protein involved in death receptor-mediated apoptosis. Here we report a role for FADD in regulation of TLR4 signals in endothelial cells. FADD specifically attenuates LPS-induced activation of c-Jun NH(2)-terminal kinase and phosphatidylinositol 3'-kinase in a death domain-dependent manner. In contrast, FADD-null cells show hyperactivation of these kinases. Examining physical associations of endogenous proteins, we show that FADD interacts with interleukin-1 receptor-associated kinase 1 (IRAK1) and MyD88. LPS stimulation increases IRAK1-FADD interaction and recruitment of the IRAK1-FADD complex to activated MyD88. IRAK1 is required for FADD-MyD88 interaction, as FADD does not associate with MyD88 in IRAK1-null cells. By shuttling FADD to MyD88, IRAK1 provides a mechanism for controlled and limited activation of the TLR4 signaling pathway. Functionally, enforced FADD expression inhibited LPS- but not vascular endothelial growth factor-induced endothelial cell sprouting, while FADD deficiency led to enhanced production of proinflammatory cytokines induced by stimulation of TLR4 and TLR2, but not TLR3. Reconstitution of FADD reversed the enhanced production of proinflammatory cytokines. Thus, FADD is a physiological negative regulator of IRAK1/MyD88-dependent responses in innate immune signaling.

Trimeric tumor necrosis factor (TNF) binding leads to recruitment of TRADD to TNFR1. In current models, TRADD recruits RIP, TRAF2, and FADD to activate NF-kappaB, Jun N-terminal protein kinase (JNK), and apoptosis. Using stable short-hairpin RNA (shRNA) knockdown (KD) cells targeting these adaptors, TNF death-inducing signaling complex immunoprecipitation demonstrates competitive binding of TRADD and RIP to TNFR1, whereas TRAF2 recruitment requires TRADD. Analysis of KD cells indicates that FADD is necessary for Fas-L- or TRAIL- but not TNF-induced apoptosis. Interestingly, TRADD is dispensable, while RIP is required for TNF-induced apoptosis in human tumor cells. TRADD is required for c-Jun phosphorylation upon TNF exposure. RIP KD abrogates formation of complex II following TNF exposure, whereas TRADD KD allows efficient RIP-caspase 8 association. Treatment with TRAIL also induces formation of a complex II containing FADD, RIP, IKKalpha, and caspase 8 and 10, leading to activation of caspase 8. Our data suggest that TNF triggers apoptosis in a manner distinct from that of Fas-L or TRAIL.

The Fas-associated death domain protein FADD is best known as an adaptor protein that senses a signal received at a death receptor and nucleates the assembly of the death-inducing signaling complex. Recent work reveals unexpected properties for this signaling protein, suggesting new roles for FADD in apoptotic signaling and in non-apoptotic functions linked to chemical modification of the FADD C-terminus. These new studies suggest novel types of high valency complexes may form in the plasma membrane and in the nucleus, raising intriguing questions as to how FADD senses the environment and responds to different signaling inputs to promote a biochemical response. In particular, we discuss the role of FADD in death receptor avidity and examine the relationship between FADD phosphorylation and subcellular localization with respect to various biological functions. Since FADD serves to modulate both apoptosis and cell cycle progression, these new findings promote the concept that differential complex assembly dictates disparate cellular processes mediated by this adaptor molecule.

Fas-associated death domain (FADD)/Mort1 was initially reported as a pro-apoptotic adaptor molecule that recruits the initiator caspases 8 and 10 to promote formation of the death-inducing signal complex (DISC) and mediates receptor induced apoptosis. Recent studies have brought to light ancillary death receptor induced apoptosis-independent activities of FADD that include cell cycle regulation, NF-kappaB activation, cell proliferation and role during embryonic development. We have recently shown that in lung adenocarcinomas increased FADD mRNA and protein are significantly associated with poor survival and that FADD overexpression was not due to gene amplification and/or mutation. In this study we showed that the nuclear localization of FADD and elevated expression of the phosphorylated form of FADD (p-FADD) correlated most closely with an increase in NF-kB activity and poor clinical outcome. These results suggest that levels of p-FADD may be used as a prognostic biomarker for predicting survival of lung cancer patients.