Fas-associated Protein With Death Domain Research Articles

Loss of FADD and Caspases Affects the Response of T-Cell Leukemia Jurkat Cells to Anti-Cancer Drugs

Programmed cell death (PCD) pathways play a crucial role in the response of cancer cells to treatment. Their dysregulation is one of the cancer hallmarks and one of the reasons of drug resistance. Here, we studied the significance of the individual members of PCD signaling pathways in response to treatment with common anti-cancer drugs using the T-cell leukemia Jurkat cells with single or double knockouts of necroptosis and/or apoptosis genes. We identified apoptosis as the primary cell death pathway upon anti-cancer drugs treatment. The cells with knocked out either Fas-associated protein with death domain (FADD) or all executioner caspases were resistant. This resistance could be partially overcome by induction of RIP1-dependent necroptosis through TNFR1 activation using combined treatment with TNF-α and smac mimetic (LCL161). RIP1 was essential for cellular response to TNF-α and smac mimetic, but dispensable for the response to anti-cancer drugs. Here, we demonstrated the significance of FADD and executioner caspases in carrying out programmed cell death upon anti-cancer drug treatments and the ability of combined treatment with TNF-α and smac mimetic to partially overcome drug resistance of FADD and/or CASP3/7/6-deficient cells via RIP1-dependent necroptosis. Thus, a combination of TNF-α and smac mimetic could be a suitable strategy for overcoming resistance to therapy in cells unable to trigger apoptosis.

Effects of Gαi2 and Gαz protein knockdown on alpha2A-adrenergic and cannabinoid CB1 receptor regulation of MEK-ERK and FADD pathways in mouse cerebral cortex.

Alpha2A-adrenergic (α2A-AR) and cannabinoid CB1 (CB1-R) receptors exert their functions modulating multiple signaling pathways, including MEK-ERK (extracellular signal-regulated kinases) and FADD (Fas-associated protein with death domain) cascades. These molecules are relevant in finding biased agonists with fewer side effects, but the mechanisms involving their modulations by α2A-AR- and CB1-R in vivo are unclear. This study investigated the roles of Gαi2 and Gαz proteins in mediating α2A-AR- and CB1-R-induced alterations of MEK-ERK and FADD phosphorylation (p-) in mouse brain cortex. Gαi2 or Gαz protein knockdown was induced in mice with selective antisense oligodeoxinucleotides (ODNs; 3nmol/day, 5days) prior to UK-14,304 (UK or brimonidine; 1mg/kg) or WIN55212-2 (WIN; 8mg/kg) acute treatments. Inactivated (p-T286) MEK1, activated (p-S217/221) MEK1/2, activated (p-T202/Y204) ERK1/2, p-S191 FADD, and the corresponding total forms of these proteins were quantified by immunoblotting. Increased (+ 88%) p-T286 MEK1 cortical density, with a concomitant reduction (-43%) of activated ERK was observed in UK-treated mice. Both effects were attenuated by Gαi2 or Gαz antisense ODNs. Contrastingly, WIN induced Gαi2- and Gαz-independent upregulations of p-T286 MEK1 (+ 63%), p-S217/221 MEK1/2 (+ 86%), and activated ERK (+ 111%) in brain. Pro-apoptotic FADD was downregulated (- 34 to 39%) following UK and WIN administration, whereas the neuroprotective p-S191 FADD was increased (+ 74%) in WIN-treated mice only. None of these latter effects required from Gαi2 or Gαz protein integrity. The results indicate that α2A-AR (UK), but not CB1-R (WIN), agonists use Gαi2 and Gαz proteins to modulate MEK-ERK, but not FADD, pathway in mouse brain cortex.

The lncRNA H19-Derived MicroRNA-675 Promotes Liver Necroptosis by Targeting FADD.

Simple SummaryLiver cancer develops mostly in a chronically inflamed liver. The inflammation process can enhance or suppress the development of liver cancer. H19 is a noncoding RNA that is upregulated in inflamed livers. The role of H19 in liver cancer was intensely investigated, but the reported findings are conflicting. Some reported that H19 is a tumor suppressor and others that it has oncogenic properties. To understand the contribution of H19 to liver cancer development, we investigated miR-675, which is generated from the first exon of the H19 RNA message. Interestingly, we found that miR-675 suppresses liver cancer cell growth by inducing cell death. Following our investigation of the mechanism of this killing effect, we established that miR-675 represses the protein called Fas-associated protein with death domain (FADD) and that this repression leads to the development of a specific type of necrosis named necroptosis. These findings can have future therapeutic implications.The H19-derived microRNA-675 (miR-675) has been implicated as both tumor promoter and tumor suppressor and also plays a role in liver inflammation. We found that miR-675 promotes cell death in human hepatocellular carcinoma (HCC) cell lines. We show that Fas-associated protein with death domain (FADD), a mediator of apoptotic cell death signaling, is downregulated by miR-675 and a negative correlation exists between miR-675 and FADD expression in mouse models of HCC (p = 0.014) as well as in human samples (p = 0.017). We demonstrate in a mouse model of liver inflammation that overexpression of miR-675 promotes necroptosis, which can be inhibited by the necroptosis-specific inhibitor Nec-1/Nec-1s. miR-675 induces the level of both p-MLKL (Mixed Lineage Kinase Domain-Like Pseudokinase) and RIP3 (receptor-interacting protein 3), which are key signaling molecules in necroptosis, and enhances MLKL binding to RIP3. miR-675 also inhibits the levels of cleaved caspases 8 and 3, suggesting that miR-675 induces a shift from apoptosis to a necroptotic cellular pathway. In conclusion, downregulation of FADD by miR-675 promotes liver necroptosis in response to inflammatory signals. We propose that this regulation cascade can stimulate and enhance the inflammatory response in the liver, making miR-675 an important regulator in liver inflammation and potentially also in HCC.

Intrinsic and Extrinsic Cell Apoptotic Pathways in Patients with HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis: A Systematic Review.

We aimed to verify the influence of intrinsic and extrinsic cell apoptotic pathways on the inhibition of cellular apoptosis in patients with tropical spastic paralysis/myelopathy related to human T cell lymphotropic virus type 1. The databases accessed were PubMed, Scopus, Science Direct, and Web of Science. Neither the time of publishing nor the language of the articles was limited. The descriptors used for this systematic literature review were: Tropical Paraparesis, Proto-Oncogenic Protein C, Bcl-2, Bcl-X Protein, Bax protein, Fas ligand (FasL) protein, Fas receptor, TNF-related apoptosis-inducing ligand and Fas-associated protein with death domain (FADD)-like apoptosis regulating. The search resulted in 546 articles from which 9 articles were selected for analysis; ranging from serum levels of Bcl-2, Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) measured by enzyme-linked immunosorbent assay and the levels of cellular expression of Bcl-2 and Bcl-xL the TCD4+ lymphocytes accessed by western blot. Most studies accessed either gene expression or polymorphism of Fas, FasL, and TRAIL in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), whereas one study used flow cytometry and fluorescence to determine Fas expression. Increased Bcl-xL expression inhibited T lymphocyte apoptosis, whereas Bcl-2, serum levels, and cellular expression did not influence T lymphocyte apoptosis and serum levels of Fas were significantly higher and associated with markers of leukocyte activation in patients with HAM/TSP. In addition, Fas polymorphism (FAS-670AA) was associated with higher proviral load. There is a need for additional research on this issue since the number of patients was small and the studies presented higher heterogeneity.

Ancestral role of TNF-R pathway in cell differentiation in the basal metazoan Hydra.

Tumour necrosis factor receptors (TNF-Rs) and their ligands, tumour necrosis factors, are highly conserved proteins described in all metazoan phyla. They function as inducers of extrinsic apoptotic signalling and facilitate inflammation, differentiation and cell survival. TNF-Rs use distinct adaptor molecules to activate signalling cascades. Fas-associated protein with death domain (FADD) family adaptors often mediate apoptosis, and TNF-R-associated factor (TRAF) family adaptors mediate cell differentiation and inflammation. Most of these pathway components are conserved in cnidarians, and, here, we investigated the Hydra TNF-R. We report that it is related to the ectodysplasin receptor, which is involved in epithelial cell differentiation in mammals. In Hydra, it is localised in epithelial cells with incorporated nematocytes in tentacles and body column, indicating a similar function. Further experiments suggest that it interacts with the Hydra homologue of a TRAF adaptor, but not with FADD proteins. Hydra FADD proteins colocalised with Hydra caspases in death effector filaments and recruited caspases, suggesting that they are part of an apoptotic signalling pathway. Regulating epithelial cell differentiation via TRAF adaptors therefore seems to be an ancient function of TNF-Rs, whereas FADD-caspase interactions may be part of a separate apoptotic pathway.

Platelets express adaptor proteins of the extrinsic apoptosis pathway and can activate caspase-8.

BackgroundApoptotic pathways in platelets are important for their survival and function. Platelet apoptosis may be involved in the pathogenesis of immune thrombocytopenia (ITP), an autoimmune-mediated disease. In contrast to the intrinsic apoptosis pathway, not much is known about the extrinsic pathway mechanisms in platelets.ObjectivesTo investigate the expression of proteins involved in the extrinsic apoptosis pathway, including the death receptors, adaptor and regulator proteins in human platelets. To determine a possible trigger of the extrinsic apoptosis pathway in platelets.MethodsTo investigate the expression of key markers of the extrinsic pathway we used targeted immunofluorescence and flow cytometry assays. To study their expression and interaction we performed Western blotting and co-immunoprecipitation. Treated platelets with different apoptosis triggers were subjected to flow cytometry.ResultsWe could identify the protein expression of the pro-apoptotic proteins TRADD (Tumor Necrosis Factor Receptor type 1- Associated DEATH Domain protein), TRAF2/5, (TNF Associated Factor) and DEDAF (Death Effector Domain- Associated Factor), FADD (Fas-Associated protein with death domain) as well as the anti-apoptotic proteins DJ-1 (Deglycase 1) and c-FLIP in human platelets. ABT-737 treatment induced a disruption in the co-localization of DJ-1 with FADD. Platelets treated with ABT-737 showed an activation in caspase-3 and -8. The exposure to TNF (Tumor Necrosis Factor), FasL (Fas ligand), and TWEAK or to plasma derived from ITP patients, did not lead to changes in caspase-3 and -8 activation in platelets.ConclusionsHuman platelets express some proteins of the extrinsic apoptosis pathway which can be modulated only by ABT-737 treatment. However so far, no other apoptosis trigger or interaction with an external receptor have been yet identified.

The cardioprotective effect of microRNA-103 inhibitor against isoprenaline-induced myocardial infarction in mice through targeting FADD/RIPK pathway.

The current study investigates the effect of the innovative phosphorothioate modified backbone locked nucleic acid (LNA) of microRNA-103 (miR-103) specifically designed for systemic delivery in the silencing of miR-103 in experimentally induced myocardial infarction (MI). MicroRNA-103 is a small non-coding RNA which regulates Fas-associated protein with death domain (FADD) gene expression, which is a negative regulator for necroptosis occurs during the progression of MI. Experimental male mice were allocated into three groups; the first group received normal saline, the second group was injected with isoprenaline and served as the infarcted control, while the third group was treated with LNA miR-103 power inhibitor before isoprenaline injection. Blood and heart samples were used for biochemical analysis of miR-103, FADD, receptor-interacting protein kinase (RIPK), nuclear factor-κβ, tumor necrosis factor-α, interleukin-6, troponin-I and creatine kinase-MB (CK-MB) as well as the histological examination of heart tissue. The treated mice showed marked improvement in the troponin-I and CK-MB levels with almost normal histological structure of heart tissue. Significant inhibition of miR-103 accompanied by increased FADD expression and markedly decreased expression of the other biomarkers were observed in the hearts of the treated mice. LNA miR-103 inhibitor is a potent cardioprotective agent and can be a promising treatment against MI through targeting FADD/RIPK pathway.

Overexpression of FADD and Bcl-XS proteins as novel prognostic biomarkers for surgically resected non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most widespread cancer with increasing morbidity and mortality. FAS-associated protein with death domain (FADD) is considered as an essential instrument in cell death, whereas Bcl-XS promotes apoptosis through inhibiting the activity of Bcl-2 and Bcl-XL. We detected the expression of FADD and Bcl-XS in resected NSCLC tissues by immunohistochemistry, and investigated their association with clinicopathological characteristics and prognostic significance of NSCLC patients. Bcl-XS expression was significantly increased in well and moderate differentiated lung SCC (P= 0.004). Lung ADC patients with overexpression of FADD and lung SCC patients with low expression of Bcl-XS had importantly lower overall survival rates by Kaplan-Meier analysis (P= 0.033, P= 0.02, respectively). Multivariate analysis confirmed that elevated expression of FADD was an independent poor prognostic factor for patients with surgically resected lung ADC (P= 0.027) and increased expression of Bcl-XS was an independent good prognostic factor for patients with surgically resected lung SCC (P= 0.016)CONCLUSION: Elevated expression of FADD was identified as independent poor prognostic factor for patients with surgically resected lung ADC, however, increased expression of Bcl-XS was an independent good prognostic biomarker for patients with surgically resected lung SCC.

Cucurbita ficifolia Fruit Extract Induces Tp53/Caspase-Mediated Apoptosis in MCF-7 Breast Cancer Cells.

The second most biggest cancer worldwide is breast cancer. There is an increasing need for safer, effective, and affordable drug candidates from natural sources to treat breast cancer. In the present investigation, the anticancer effect of Cucurbita ficifolia Bouché (C. ficifolia) fruit extract was tested on the human breast cancer cells such as MCF-7. The cells were exposed with different doses of C. ficifolia, for the assessment of IC50 concentrations on the MCF-7 cell lines for 24 hs. The effect of C. ficifolia fruit extract on morphological and apoptotic changes were evaluated by specific fluorescence staining techniques and real-time PCR in a time-dependent manner for 24 hs and 48 hs. The IC50 value for C. ficifolia fruit extract was found to be 90 μg/mL. Morphological alteration and apoptotic distinctiveness aspect like chromatin condensation and nuclear fragmentation were noticed in C. ficifolia extract exposed breast cancer cells. Further, we observed that C. ficifolia extract-induced programmed cell death in the MCF-7 cells were mediated with the elevated expression of the tumor suppressor gene such as p53 and apoptotic markers such as caspase-8, caspase-9, caspase-3, fatty acid synthase (FAS), Fas-associated protein with death domain (FADD), Bcl-2 homologous antagonist/killer (BAK), and Bcl-2-associated X protein (BAX). These observations established that C. ficifolia significantly concealed the cell division and provoked p53/caspase-mediated programmed cell death. Further, we noticed that this cell death in MCF-7 cells is concentration and time dependent. As evaluated through the comet assay, C. ficifolia induced DNA damage; further upon increasing the duration of the treatment, the DNA damage was higher than before. Thus, our study concludes that C. ficifolia could serve as an effective anticancer agent through vital gene modulation.

Bicyclic α-Iminophosphonates as High Affinity Imidazoline I2 Receptor Ligands for Alzheimer's Disease.

Imidazoline I2 receptors (I2-IR), widely distributed in the CNS and altered in patients that suffer from neurodegenerative disorders, are orphans from a structural point of view, and new I2-IR ligands are urgently required for improving their pharmacological characterization. We report the synthesis and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of a new family of bicyclic α-iminophosphonates endowed with relevant affinities for human brain I2-IR. Acute treatment in mice with a selected compound significantly decreased Fas-associated protein with death domain (FADD) in the hippocampus, a key signaling mediator of neuroprotective actions. Additionally, in vivo studies in the familial Alzheimer's disease 5xFAD murine model revealed beneficial effects in behavior and cognition. These results are supported by changes in molecular pathways related to cognitive decline and Alzheimer's disease. Therefore, bicyclic α-iminophosphonates are tools that may open new therapeutic avenues for I2-IR, particularly for unmet neurodegenerative conditions.

A cytosolic heat shock protein 90 and co-chaperone p23 complex activates RIPK3/MLKL during necroptosis of endothelial cells in acute respiratory distress syndrome

Necrosis with inflammation plays a crucial role in acute respiratory distress syndrome (ARDS). Receptor-interacting protein 3 (RIPK3) regulates a newly discovered programmed form of necrosis called necroptosis. However, the underlying mechanism of necroptosis in ARDS remains unknown. Thus, the purpose of this study was to examine the possible involvement of RIPK3 in ARDS-associated necroptosis. RIPK3 protein levels were found to be significantly elevated in the plasma and bronchoalveolar lavage fluid of ARDS patients. Next, we utilised a mouse model of severe ARDS induced with high-dose lipopolysaccharide and found that lung injury was mainly due to RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL)-mediated necroptosis and endothelial dysfunction. The activation of RIPK3-MLKL by tumour necrosis factor receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD) required catalytically active RIPK1 and the inhibition of Fas-associated protein with death domain (FADD)/caspase-8 catalytic activity. We further showed that the molecular chaperone heat shock protein 90 (Hsp90)/p23, as a novel RIPK3- and MLKL-interacting complex, played an important role in RIP-MLKL-mediated necroptosis, inflammation and endothelial dysfunction in the pulmonary vasculature, which resulted in ARDS. Collectively, the results of our study indicate that necroptosis is an important mechanism of cell death in ARDS and the inhibition of necroptosis may be a therapeutic intervention for ARDS. KEY MESSAGES: Lung injury in high-dose LPS-induced severe ARDS is mainly due to RIP3-MLKL-mediated necroptosis and endothelial dysfunction. Chaperone HSP90/p23 is a novel RIP3- and MLKL-interacting complex in HPAECs. HSP90/p23 is a novel RIP3- and MLKL-interacting complex in RIP-MLKL-mediated necroptosis, inflammation and endothelial dysfunction.

Knock down of Fas-Associated Protein with Death Domain (FADD) Sensitizes Osteosarcoma to TNFα-induced Cell Death.

Fas-associated protein with death domain (FADD) was first identified for its role in linking death receptors to the apoptotic signaling pathway with subsequent cell death. Later studies reported non-apoptotic functions for FADD in normal cells and cancer cells. Non-apoptotic functions for FADD in osteosarcoma (OS) have not been reported. In this study, FADD protein expression was knocked down in human CCHOSD, LM7, and SaOS2 OS cell lines followed by assessment of sensitivity to TNFα- or TRAIL-induced cell death. Knock down of FADD significantly increased TNFα-induced cell death in LM7 and SaOS2 cell lines. The mode of TNFα-induced cell death was apoptosis and not necroptosis. Inhibition of nuclear factor kappa B (NFκB) in wildtype cells increased TNFα-induced cell death to similar levels observed in FADD knockdown cells, suggesting a role for FADD in NFκB pro-survival cell signaling. In addition, knock down of FADD increased SMAC mimetic-mediated TNFα-induced cell death in all cell lines studied. The results of this study indicate that FADD has a pro-survival function in OS following TNFα treatment that involves NFκB signaling. The results also indicate that the pro-survival function of FADD is associated with XIAP activity.

DR4-Associated Death Receptor Signal Promotes Cartilage Damage in Patients With Kashin-Beck Disease.

Purpose. To explore the relationship between the death receptor (DR) and the pathological progression of Kashin-Beck disease (KBD). Design. KBD cartilage samples were collected from 15 patients diagnosed according to the "National Diagnostic Criteria of KBD" in China. In vitro monolayer chondrocytes were cultured in complete medium. Caspase-3 and caspase-8 activities in chondrocytes were analyzed using a kit. Nuclear morphology was observed by Hoechst 33258 staining, apoptosis was verified by flow cytometry analysis, and DR molecules were detected using Western blotting and quantitative real-time reverse transcription polymerase chain reaction analysis. Results. Early apoptotic rates of KBD and osteoarthritis (OA) chondrocytes were higher than those of normal control (NC) cells. Excessive apoptotic nuclei were observed in OA and KBD cells after Hoechst 33258 staining. Activities of both caspase-3 and caspase-8 were higher in KBD and OA cells than in NC cells. The average DR4 mRNA level in KBD cells was 3.301-fold higher than that in NC cells, Fas-associating protein with death domain (FADD) transcript level in KBD cells was 2.528-fold higher than that in NC cells. Western blot analyses showed that FAS, DR4, DR5, caspase-3, and FADD were upregulated in the KBD and OA groups compared with the NC group. High expression of caspase-8 in KBD compared with NC was verified, whereas cellular FLICE-inhibitory protein (c-FLIP) in KBD was significantly downregulated. Conclusions. KBD and OA chondrocytes showed obvious FADD-caspase-dependent apoptosis, which is related to the DR pathway. Apoptosis in KBD articular cartilage is mainly related to FAS/DR4-FADD-caspase signaling, and OA is associated with FAS/DR4/DR5-FADD-caspase signaling.

Ganoderma lucidum spore oil induces apoptosis of breast cancer cells in vitro and in vivo by activating caspase-3 and caspase-9.

The mushroom Ganoderma lucidum (G. lucidum) is a traditional Chinese medicine reported to have a variety of pharmacological properties, including anti-cancer activity. G. lucidum spore oil (GLSO) is a lipid substance extracted from sporoderm-broken spore of G. lucidum. However, the effect of GLSO on breast cancer and the underlying molecular mechanism remain unclear. The aim of this study was to identify the effects of GLSO on breast cancer cells in vitro and in vivo as well as to investigate the mechanistic basis for the anticancer effect of GLSO. First, in vitro MDA-MB-231 cells were treated with GLSO (0.2, 0.4, and 0.6 μL/mL). The protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), X-linked inhibitor of apoptosis (XIAP), total poly (ADP-ribose) polymerase (PARP), caspase-3 and caspase-8 were examined using western blotting. The mRNA expression levels of Fas-associated protein with death domain (FADD), TNF receptor-associated factor 2 (TRAF2), caspases-3, -8, -9 and Bax were examined using qRT-PCR. Second, in vivo the anticancer properties of GLSO were assessed by H&E, TUNEL and immunohistochemistry in BALB/c mice injected with 4T1 cells. In addition, the levels of caspase-9/caspase-3 signaling pathway proteins in tumor tissue were evaluated by immunoblotting. Finally, MDA-MB-231 cells were treated with caspase inhibitors to measure cell viability, the protein levels were examined with western blotting. The results in vitro showed that GLSO up-regulated the expression of Bax and caspase-3 in MDA-MB-231 cells, but had no effect on the expression of caspase-8. Moreover, the growth of tumors in vivo was significantly suppressed in the GLSO-treated group. The results of Western blot were consistent with in vitro. In vitro, co-treatment of MDA-MB-231 cells with caspase inhibitors reduced the inhibitory effect of GLSO on cell growth. GLSO inhibits the growth of MDA-MB-231 cells and tumors in vivo by inducing apoptosis, which may be achieved through the mitochondrial apoptotic pathway.

Atrazine induces necroptosis by miR-181–5p targeting inflammation and glycometabolism in carp lymphocytes

Atrazine (ATR) causes environmental problems and damages the health of fish and aquatic animals. MicroRNAs (miRNAs) play important roles in immune regulation. However, the immunotoxicity mechanism of ATR in fish lymphocytes and the role of miRNA in this process remain unclear. To further study these mechanisms, spleen lymphocytes were exposed to 20, 40 and 60 μg/ml ATR for 18 h. Fluorescence staining and flow cytometry showed that the number of necrotic lymphocytes increased after ATR exposure. Compared with the control group, the mRNA expression of miR-181–5p was inhibited and the mRNA levels of TNF-α and HK2 were increased after ATR exposure. Additionally, the NF-κB inflammatory pathway and the levels of glycometabolism-related genes were upregulated. These results suggest that ATR induces inflammation and elevates glycometabolism in lymphocytes. We further found that the mRNA levels of receptor-interacting serine-threonine kinase 1 (RIP1), receptor-interacting serine-threonine kinase 3 (RIP3), mixed lineage kinase domain-like pseudokinase (MLKL), cylindromatosis (CYLD) and Fas-Associated protein with Death Domain (FADD) and the protein levels of RIP3 and MLKL in the treatment groups were significantly increased compared to those in control group, suggesting that ATR causes lymphocyte necroptosis. We conclude that miR-181–5p plays a key role in necroptosis in carp lymphocytes exposed to ATR by downregulating the expression of HK and TNF-α, which increases the level of glycometabolism and induces the inflammatory response, respectively.

Protease Activity in Vascular Disease.

Protease Activity in Vascular Disease.

Protective effect of remifentanil on myocardial ischemia-reperfusion injury through Fas apoptosis signaling pathway.

The aim of this study was to investigate the protective effect of remifentanil (RFT) on myocardial ischemia-reperfusion (IR) injury through Fas apoptosis signaling pathway. A total of 36 Sprague-Dawley (SD) rats were randomly divided into 3 groups, including the sham operation (Sham) group, IR model (IR) group and RFT pretreatment (RFT) group, with 12 rats in each group. Myocardial tissues of rats in each group were collected. Hematoxylin and eosin (H&E) staining was used to examine the pathological differences of the myocardium in the three groups. The levels of lactate dehydrogenase (LDH), creatine kinase (CK), superoxide dismutase (SOD) and malondialdehyde (MDA) in the serum of rats in each group were detected by enzyme-linked immunosorbent assay (ELISA). Meanwhile, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was adopted to detect the apoptosis level of myocardial cells in each group. Furthermore, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting were applied to measure the mRNA and protein expression levels of Fas and its pathway indexes, respectively. Compared with the Sham group, LDH and CK activities and MDA level in the IR group were significantly increased, whereas the level of SOD was remarkably decreased (p<0.05). Compared with the IR group, RFT pretreatment could significantly reduce the release of LDH and CK-muscle/brain (CK-MB), increase SOD level and decrease MDA level (p<0.05). TUNEL results manifested that the apoptosis rate of myocardial cells in the IR group was markedly increased than that of the Sham group (p<0.05). Meanwhile, the apoptosis rate of myocardial cells in the RFT group was notably decreased when compared with that of the IR group (p<0.05). ELISA results demonstrated that the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) proteins in the RTF group were significantly lower than those of the IR group (p<0.05). RT-PCR and Western blotting results indicated that the expressions of Fas, Fas ligand (FasL), and Fas-associated protein with death domain (FADD) in IR and RFT groups were significantly higher than those of the Sham group (p<0.05). However, RTF pretreatment could markedly reduce the levels of Fas, FasL, and FADD (p<0.05). RFT can reduce the apoptosis of myocardial cells as well as IR-induced oxidative stress and inflammation by inhibiting the Fas/FasL signal transduction pathway.

ZBP1 mediates interferon-induced necroptosis.

Interferons (IFNs) play an important role in immunomodulatory and antiviral functions. IFN-induced necroptosis has been reported in cells deficient in receptor-interacting protein kinase 1 (RIPK1), Fas-associated protein with death domain (FADD), or caspase-8, but the mechanism is largely unknown. Here, we report that the DNA-dependent activator of IFN regulatory factors (ZBP1, also known as DAI) is required for both type I (β) and type II (γ) IFN-induced necroptosis. We show that L929 fibroblast cells became susceptible to IFN-induced necroptosis when RIPK1, FADD, or Caspase-8 was genetically deleted, confirming the antinecroptotic role of these proteins in IFN signaling. We found that the pronecroptotic signal from IFN stimulation depends on new protein synthesis and identified ZBP1, an IFN-stimulated gene (ISG) product, as the de novo synthesized protein that triggers necroptosis in IFN-stimulated cells. The N-terminal domain (ND) of ZBP1 is important for ZBP1-ZBP1 homointeraction, and its RHIM domain in the C-terminal region interacts with RIPK3 to initiate RIPK3-dependent necroptosis. The antinecroptotic function of RIPK1, FADD, and caspase-8 in IFN-treated cells is most likely executed by caspase-8-mediated cleavage of RIPK3, since the inhibitory effect on necroptosis was eliminated when the caspase-8 cleavage site in RIPK3 was mutated. ZBP1-mediated necroptosis in IFN-treated cells is likely physiologically relevant, as ZBP1 KO mice were significantly protected against acute systemic inflammatory response syndrome (SIRS) induced by TNF + IFN-γ.

Molecular cloning and characterization of FADD from the manila clam Ruditapes philippinarum

Fas-associated protein with death domain (FADD) is an essential element in cell death, and also implicates in cell cycle progression, inflammation and innate immunity. In the study, an FADD (designated as RpFADD) was identified and characterized from manila clam, Ruditapes philippinarum. Multiple alignments and phylogenetic analysis strongly suggested that RpFADD was a new member of the FADD family. The RpFADD transcripts were constitutively expressed in a wide range of tissues, and dominantly expressed in hemocytes. After challenged with Vibrio anguillarum or Micrococcus luteus, the expression level of RpFADD transcripts was significantly induced and reached the maximum level at 72 h and 48 h, respectively. Knockdown of RpFADD down-regulated the transcript levels of RpIKK, RpTAK1 and RpNF-κB with the exception of RpIκB. Moreover, RpFADD primarily localized in the cell cytoplasm, and its over-expression promoted the apoptosis of HeLa cells. These results revealed that RpFADD perhaps regulated the NF-κB signaling pathways positively, which provided a better understanding of RpFADD in innate immunity.

FADD-deficient mouse embryonic fibroblasts undergo RIPK1-dependent apoptosis and autophagy after NB-UVB irradiation

Sun or therapy-related ultraviolet B (UVB) irradiation induces different cell death modalities such as apoptosis, necrosis/necroptosis and autophagy. Understanding of mechanisms implicated in regulation and execution of cell death program is imperative for prevention and treatment of skin diseases. An essential component of death-inducing complex is Fas-associated protein with death domain (FADD), involved in conduction of death signals of different death modalities. The purpose of this study was to enlighten the role of FADD in the selection of cell death mode after narrow-band UVB (NB-UVB) irradiation using specific cell death inhibitors (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (zVAD-fmk), Necrostatin-1 and 3-Methyladenine) and FADD-deficient (FADD−/−) mouse embryonic fibroblasts (MEFs) and their wild type (wt) counterparts. The results imply that lack of FADD sensitized MEFs to induction of receptor–interacting protein 1 (RIPK1)-dependent apoptosis by the generation of reactive oxygen species (ROS), but without activation of the proteins p53, Bax and Bcl-2 as well as without the enrolment of calpain-2. Autophagy was established as a contributing factor to NB-UVB-induced death execution. By contrast, wt cells triggered intrinsic apoptotic pathway that was resistant to the inhibition by zVAD-fmk and Necrostatin-1 pointing to the mechanism overcoming the cell survival. These findings support the role of FADD in prevention of autophagy-dependent apoptosis.