Fas-mediated Signaling Research Articles

CD14 is a decision-maker between Fas-mediated death and inflammation

Signaling through classical death receptor Fas was mainly appreciated as a pro-death pathway until recent reports characterized pro-inflammatory outcomes of Fas-mediated activation in pathological contexts. How Fas signaling can switch to pro-inflammatory activation is poorly understood. Herein, we report that in macrophages and neutrophils, the Toll-like receptor (TLR) adapter CD14 determines the inflammatory output of Fas-mediated signaling. Our findings propose CD14 as a crucial chaperone of Fas receptor internalization in macrophages and neutrophils, resulting in Cd14-/- myeloid cells that are protected from FasL-induced apoptosis, activate nuclear factor κB (NF-κB), and release cytokines in response. As in TLR signaling, CD14 is also required for Fas to signal through the adaptor TRIF (TIR-domain-containing adapter-inducing interferon-β) and induce a pro-death complex. Our findings demonstrate that CD14 availability can determine the switch between Fas-mediated pro-death and pro-inflammatory outcomes by internalizing the receptor.

Indicaxanthin prevents eryptosis induced by cigarette smoke extract by interfering with active Fas-mediated signaling.

A physiological mechanism of programmed cell death called eryptosis occurs in aged or damaged red blood cells (RBCs). Dysregulated eryptosis contributes to abnormal microcirculation and prothrombotic risk. Cigarette smoke extract (CSE) induces a p38 MAPK-initiated, Fas-mediated eryptosis, activating the death-inducing signaling complex (DISC). Indicaxanthin (Ind) from cactus pear fruits, is a bioavailable dietary phytochemical in humans and it is able to incorporate into RBCs enhancing their defense against numerous stimuli. This in vitro work shows that Ind, at concentrations that mimic plasma concentrations after a fruit meal, protects erythrocytes from CSE-induced eryptosis. CSE from commercial cigarettes was prepared in aqueous solution using an impinger air sampler and nicotine content was determined. RBCs were treated with CSE for 3 h in the absence or presence of increasing concentrations of Ind (from 1 to 5 μM). Cytofluorimetric measurements indicated that Ind reduced CSE-induced phosphatidylserine externalization and ceramide formation in a concentration-dependent manner. Confocal microscopy visualization and coimmunoprecipitation experiments revealed that Ind prevented both CSE-triggered Fas aggregation and FasL/FADD/caspase 8 recruitment in the membrane, indicating inhibition of DISC assembly. Ind inhibited the phosphorylation of p38 MAPK, caspase-8/caspase-3 cleavage, and caspase-3 activity induced by CSE. Finally, Ind reduced CSE-induced ATP depletion and restored aminophospholipid translocase activity impaired by CSE treatment. In conclusion, Ind concentrations comparable to nutritionally relevant plasma concentrations, can prevent Fas-mediated RBC death signaling induced by CSE, which suggests that dietary intake of cactus pear fruits may limit the deleterious effects of cigarette smoking.

Eye Drop with Fas-Blocking Peptide Attenuates Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), characterized by macular retinal degeneration, poses a significant health concern due to the lack of effective treatments for prevalent dry AMD. The progression of AMD is closely linked to reactive oxygen species and Fas signaling, emphasizing the need for targeted interventions. In this study, we utilized a NaIO3-induced retinal degeneration mouse model to assess the efficacy of Fas-blocking peptide (FBP). Intravitreal administration of FBP successfully suppressed Fas-mediated inflammation and apoptosis, effectively arresting AMD progression in mice. We developed a 6R-conjugated FBP (6R-FBP) for eye drop administration. 6R-FBP, administered as an eye drop, reached the retinal region, attenuating degeneration by modulating the expression of inflammatory cytokines and blocking Fas-mediated apoptosis in rodent and rabbit NaIO3-induced retinal degeneration models to address practical concerns. Intravitreal FBP and 6R-FBP eye drops effectively reduced retinal degeneration and improved retinal thickness in rodent and rabbit models. This study highlights the therapeutic potential of FBP, particularly 6R-FBP as an eye drop, in inhibiting Fas-mediated cell signaling and protecting against retinal cell death and inflammation in dry AMD. Future investigations should explore the translational prospects of this approach in primates with eye structures comparable to those of humans.

Development of adoptive immunotherapy with KK-LC-1-specific TCR-transduced γδT cells against lung cancer cells.

The present study analyzed the antitumor effect of γδT cells transduced with the TCR of cancer‐specific CTLs to establish forceful cancer‐specific adoptive immunotherapy. We cloned the TCRαβ genes from CTLs showing HLA‐B15 restricted recognition of Kita‐Kyushu lung cancer antigen‐1 (KK‐LC‐1), a cancer/germline gene antigen, identified in a lung adenocarcinoma case (F1121). The TCRαβ and CD8 genes were transduced into γδT cells induced from PBLs of healthy volunteers stimulated with zoledronate and IL‐2. The KK‐LC‐1‐specific TCRαβ‐CD8 γδT cells showed cytotoxic activity against the KK‐LC‐1 positive lung cancer cell line F1121L and produced IFN‐γ against F1121L and KK‐LC‐1 peptide‐pulsed F1121 EBV‐B cells. These responses were blocked by HLA class I and HLA‐B/C antibodies. An in vivo assay using NOD/SCID mice with xenotransplantation of human lung cancer cells was performed, and the TCRαβ‐CD8 transduced γδT cells (TCRαβ‐CD8 γδT cells) were intravenously injected. Growth inhibition of KK‐LC‐1+, HLA‐B15+ lung cancer cells was confirmed in mice with injection of the TCRαβ‐CD8 γδT cells from 1 wk after xenotransplantation of cancer cells but not in those treated 2 wk after xenotransplantation. The resected specimens of the tumor, 2 wk after xenotransplantation, highly expressed FasL but not programmed death ligand‐1 (PD‐L1) by immunohistochemical staining. FasL highly expressed cancer cells xenotransplanted 2 wk ago were resistant to TCRαβ‐CD8 γδT cells injection. These results suggested that apoptosis of Fas‐positive TCRαβ‐CD8 γδT cells may be induced by a Fas‐mediated signal after interacting with FasL‐positive cancer cells.

Diplatin, a novel water-soluble platinum complex, inhibits lung cancer growth via augmentation of Fas-mediated apoptosis

Platinum drugs, such as cisplatin (DDP) and carboplatin (CBP), are the main drugs for the treatment of lung cancer, but their practical clinical application is limited by severe toxicity and acquired drug resistance. Our previous study has indicated that diplatin, [2-(4-(diethyl-amino)butyl)malonate-O,O’]-[(1R,2R)-cyclohexane-1,2-diamine N,N’] platinum (II) phosphate, a novel water-soluble platinum complex, could overcome DDP-resistant cells and was less toxic than comparable platinum drugs. In the present study, the effects and mechanisms of diplatin were further evaluated for its development as a novel anti-lung cancer platinum drug. Here, we found diplatin down-regulated the viability of H460 and LTEP-A-2 cells in a dose-dependent manner. Nude mice administrated with diplatin (30–120 mg/kg) via tail vein injection dose-dependently inhibited the growth of H460 and LTEP-A-2 xenograft tumors, whose action mainly correlated with the induction of tumor apoptosis. Particularly, the exposure of lung cancer cells or xenograft tumors to diplatin resulted in elevated Fas level, and knockdown of Fas ameliorated diplatin-induced cells apoptosis. Overall, we suggest that diplatin has potent anti-tumor activity, which probably acts through Fas-mediated signaling pathway.

Phenotypic alterations in liver cancer cells induced by mechanochemical disruption

Hepatocellular carcinoma (HCC) is a highly fatal disease recognized as a growing global health crisis worldwide. Currently, no curative treatment is available for early-to-intermediate stage HCC, characterized by large and/or multifocal tumors. If left untreated, HCC rapidly progresses to a lethal stage due to favorable conditions for metastatic spread. Mechanochemical disruption of cellular structures can potentially induce phenotypic alterations in surviving tumor cells that prevent HCC progression. In this paper, HCC response to mechanical vibration via high-intensity focused ultrasound and a chemical disruptive agent (ethanol) was examined in vitro and in vivo. Our analysis revealed that mechanochemical disruption caused a significant overproduction of reactive oxygen species (ROS) in multiple HCC cell lines (HepG2, PLC/PRF/5, and Hep3B). This led to a decrease in cell viability and long-term proliferation due to increased expression and activity of death receptors TNFR1 and Fas. The cells that survived mechanochemical disruption had a reduced expression of cancer stem cell markers (CD133, CD90, CD49f) and a diminished colony-forming ability. Mechanochemical disruption also impeded HCC migration and their adhesion to vascular endothelium, two critical processes in hematogenous metastasis. The HCC transformation to a non-tumorigenic phenotype post mechanochemical disruption was confirmed by a lack of tumor spheroid formation in vitro and complete tumor regression in vivo. These results show that mechanochemical disruption inhibits uncontrolled proliferation and reduces tumorigenicity and aggressiveness of HCC cells through ROS overproduction and associated activation of TNF- and Fas-mediated cell death signaling. Our study identifies a novel curative therapeutic approach that can prevent the development of aggressive HCC phenotypes.

Continuous gibberellin A3 exposure from weaning to sexual maturity induces ovarian granulosa cell apoptosis by activating Fas-mediated death receptor signaling pathways and changing methylation patterns on caspase-3 gene promoters

Information on the effects of gibberellic acid (gibberellin A3, GA3) on ovarian follicle development is limited. In our present study, 21-day-old female Wistar rats were exposed to GA3 by gavage (25, 50, and 100 mg/kg body weight, once per day) for eight weeks to evaluate the influence of GA3 on ovarian follicle development. After treatment, significant (P < 0.05) increases (to 40.17 % and 44.5 %, respectively) in atretic follicle proportions and significant decreases (to 19.49 % and 17.86 %, respectively) in corpus luteum proportions were observed in the 50 and 100 mg/kg treatment groups compared to the control group. Significant (P < 0.05) increases (to 31.3 % and 42.0 %, respectively) in follicle apoptosis were observed in the 50 and 100 mg/kg treatment groups by transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Significantly increased expression of caspase-3, caspase-8, caspase-9 and Fas was observed by real-time PCR and Western blotting. Bisulfite sequencing PCR (BSP) revealed obviously decreased total methylation percentages of the caspase-3 promoter region in the two treatment groups. Real-time quantitative PCR also showed significantly decreased mRNA expression of DNA methyltransferase (Dnmt) 3a and Dnmt3b. Further in vitro studies showed that a DNA methylation inhibitor could enhance the GA3-induced increase in the mRNA expression of caspase-3. Overall, our present study indicates that GA3 administration from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis and suggests that signaling through the Fas-mediated apoptotic pathway may be an important underlying mechanism of this apoptosis. In addition, GA3-induced aberrant DNA methylation patterns might be partly responsible for upregulation of caspase-3 gene expression.

Apoptotic signalling targets the post-endocytic sorting machinery of the death receptor Fas/CD95

Fas plays a major role in regulating ligand-induced apoptosis in many cell types. It is well known that several cancers demonstrate reduced cell surface levels of Fas and thus escape a potential control system via ligand-induced apoptosis, although underlying mechanisms are unclear. Here we report that the endosome associated trafficking regulator 1 (ENTR1), controls cell surface levels of Fas and Fas-mediated apoptotic signalling. ENTR1 regulates, via binding to the coiled coil domain protein Dysbindin, the delivery of Fas from endosomes to lysosomes thereby controlling termination of Fas signal transduction. We demonstrate that ENTR1 is cleaved during Fas-induced apoptosis in a caspase-dependent manner revealing an unexpected interplay of apoptotic signalling and regulation of endolysosomal trafficking resulting in a positive feedback signalling-loop. Our data provide insights into the molecular mechanism of Fas post-endocytic trafficking and signalling, opening possible explanations on how cancer cells regulate cell surface levels of death receptors.

FAS receptor regulates HNSCC cancer stemness ability and pulmonary metastasis through Notch pathway

Up to 80% of head and neck squamous cell carcinoma (HNSCC) distance metastasis is occurring in lung through seeding of circulating cancer stem cells. It is crucial to understand the genes that are involved in HNSCC pulmonary colonization which may provide an important therapy niche in treating HNSCC patients successfully. Here, we found the FAS receptor knockout SAS cells could diminished the in vitro stemness ability and completely abolished the HNSCC pulmonary colonization in the immunodeficiency NSG mice. Even pretreated with single dose of FAS neutralize antibody could also significantly reduce the lung colonies forming numbers in vivo. FAS knockout could also prevent the in vitro cell migration and invasion abilities in the Boyden chamber assay. Using the reporter assays of common oncogenic signaling pathways, our results showed that Notch, HIF1A, RhoA, and STAT1 were suppressed in the FAS knockout cells. Moreover, the microarray analysis revealed that FAS knockout could also decrease stemness gene expressions, such as XIST, WDR66, HMGA2, and the ligand of Notch, Jag1 expressions. These genes and pathways reversal and complementation assays were performed and confirmed that indeed Notch pathway is one of the major downstream pathways of FAS‐mediated HNSCC stemness signaling to promote lung cancer metastasis. Taken together, our study found the novel FAS‐XIST axis might be mediated through Jag1/Notch pathway that may be contributing to HNSCC patient's lung metastasis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of MiR-98 and Its Underlying Mechanisms in Systemic Lupus Erythematosus.

T-lymphocyte apoptosis plays a critical role in the pathogenesis of systemic lupus erythematosus (SLE). However, the underlying regulatory mechanisms of apoptosis in SLE remain unclear. The aim of this study was to explore the role of miR-98 in SLE and its underlying mechanisms. Western blotting and quantitative reverse transcription PCR (qRT-PCR) were used to analyze miR-98 and Fas expression. Luciferase reporter assays were performed to identify miR-98 targets. To modify miRNA levels, miR-98 mimics and inhibitor were transfected into cells. A lentiviral construct was used to overexpress the level of Fas in SLE CD4+ T cells. Gene and protein expression were determined by qRT-PCR and Western blotting. Apoptosis levels were evaluated by annexin V staining and flow cytometry. Compared to those of healthy donors, miR-98 was downregulated in SLE CD4+ T cells, whereas Fas mRNA and protein expression were upregulated. Upregulation of miR-98 by mimic transfection protected Jurkat cells against Fas-mediated apoptosis at both mRNA and protein levels, while miR-98 inhibitor induced the completely opposite effect. Luciferase reporter assays demonstrated that miR-98 directly targeted Fas mRNA. Further, miR-98 inhibitor induced apoptosis in primary healthy CD4+ T cells through the Fas-caspase axis, while upregulation of miR-98 in SLE CD4+ T cells led to the opposite effect. The current study revealed that downregulation of miR-98 induces apoptosis by modulating the Fas-mediated apoptotic signaling pathway in SLE CD4+ T cells. These results suggest that miR-98 might serve as a potential target for SLE treatment.

Fas- and Mitochondria-Mediated Signaling Pathway Involved in Osteoblast Apoptosis Induced by AlCl3.

Aluminum (Al) is known to induce apoptosis of osteoblasts (OBs). However, the mechanism is not yet established. To investigate the apoptotic mechanism of OBs induced by aluminum trichloride (AlCl3), the primary OBs from the craniums of fetal Wistar rats were exposed to 0mg/mL (control group, CG), 0.06mg/mL (low-dose group, LG), 0.12mg/mL (mid-dose group, MG), and 0.24mg/mL (high-dose group, HG) AlCl3 for 24h, respectively. We observed that AlCl3 induced OB apoptosis with the appearance of apoptotic morphology and increase of apoptosis rate. Additionally, AlCl3 treatment activated mitochondrial-mediated signaling pathway, accompanied by mitochondrial membrane potential (ΔΨm) depolarization, release of cytochrome c from the mitochondria to the cytoplasm, as well as survival signal-related factor caspase-9 and caspase-3 activation. AlCl3 exposure also activated Fas/Fas ligand signaling pathway, presented as Fas, Fas ligand, and Fas-associated death domain expression enhancement and caspase-8 activation, as well as the hydrolysis of Bid to truncated Bid, suggesting that the Fas-mediated signaling pathway might aggravate mitochondria-mediated OB apoptosis through hydrolyzing Bid. Furthermore, AlCl3 exposure inhibited Bcl-2 protein expression and increased the expressions of Bax, Bak, and Bim in varying degrees. These results indicated that AlCl3 exposure induced OB apoptosis through activating Fas- and mitochondria-mediated signaling pathway and disrupted B-cell lymphoma-2 family proteins.

Role of Fas and RANKL Signaling in Peripheral Immune Tolerance

The death receptor, Fas, has been well-characterized and is a critical factor in apoptosis in immune cells. Fas also has an important role in maintaining immune tolerance as demonstrated in the autoimmune-prone MRL/lpr mouse strain which carries a defect in Fas-mediated apoptosis of T cells. However, the role of Fas-independent apoptosis remains to be characterized in autoimmune diseases. In dendritic cells (DCs), binding of receptor activator of nuclear factor-κB ligand (RANKL) to RANK perpetuates the survival of mature DCs. However, cross-talk between the RANK/RANKL pathway and Fas-mediated signaling during the function or activation of DCs has not been well-studied. This short communication review describes a mechanism involving interactions between activated DCs and T cells in the autoimmune response of MRL/lpr mice and a novel Fas-independent apoptosis pathway in T cells that maintains peripheral tolerance, and controls autoimmunity in MRL/lpr mice.

The Long Form of Cellular FLIP Renders NK-Cell Lymphoma Cells Resistant to Fas-Mediated Apoptosis

Activation-induced cell death mediated by Fas/Fas ligand (FasL) system plays a key role in regulating immune response. Although normal NK cells employ this system for their homeostasis, malignant NK cells derived from extranodal NK/T-cell lymphoma, nasal type (ENKL) seem to disrupt the process. ENKL cells usually express both Fas andFasL, whereas they seldom undergo apoptosis. Previous studies showed that loss-of-function mutations of Fas gene are considered a major mechanism of resistance to Fas-mediated apoptosis in ENKL (Shen et al., Am J Pathol 2002; Takakuwa et al., Oncogene 2002). In addition, recent findings indicated that the long form of cellular FLIP (c-FLIPL) is a crucial modulator of procaspase-8 and has the potency to interfere with the Fas-mediated apoptotic pathway in several cancer cells (reviewed by Lavrik and Krammer, Cell Death Differ 2012). We focused on c-FLIPL and examined whether down-regulation of c-FLIPL leads to efficient cell death in ENKL-derived cells.We used two ENKL-derived cell lines, NK-YS and Hank1, and Fas-sensitive Jurkat as a control. To determine the expression of Fas and FasL, flow cytometry (FCM) was performed and showed the co-expression Fas and FasL in both NK-cell lines. Western blot analysis detected the components of the death-inducing signaling complex (DISC), including FADD, procaspase-8, and c-FLIPL in the three lines. We next confirmed that NK-YS and Hank1 were resistant to the apoptotic stimuli induced by direct Fas cross-linking. Annexin V staining assay by FCM detected that above 40% of Jurkat cells were positive for annexin V, whereas most NK-YS and Hank1 cells failed to show apoptotic changes after Fas ligation.To determine whether Fas-mediated signaling is functional, we investigated the DISC formation in the NK-cell lines. After Fas ligation, FADD and cleaved caspase-8 were coimmunoprecipitated with Fas in NK-YS and Hank1 as well as in Jurkat. Confocal fluorescence imaging also detected fusion signals showing colocalization between Fas and caspase-8 in the three lines. In contrast, cleaved c-FLIPL was coimmunoprecipitated only in NK-YS and Hank1. Confocal microscopy detected that the colocalization of Fas and c-FLIPL was significantly increased in the NK-cell lines, compared with Jurkat. These results showed that c-FLIPL plays a critical role in inhibition of the proximal Fas-mediated signaling pathway in NK-YS and Hank1. Therefore, we next performed RNA interference for c-FLIPL in the NK-cell lines. Although the cells transfected control siRNA hardly showed apoptotic change even after Fas ligation, knockdown of c-FLIPL slightly increased apoptosis in NK-YS and Hank1 (Figure 1). Furthermore, the suppression of c-FLIPL remarkably sensitized the NK cell lines to the Fas stimulus. FCM detected that above 50% of cells came to be positive for annexin V in both NK-cell lines (Figure 1). Here, we demonstrated another mechanism of resistance to Fas-mediated apoptosis in ENKL cells.Our findings indicate that two ENKL-derived cell lines are similar to activated normal NK cells in being primed to undergo Fas-mediated apoptosis. However, they constitutively express and exploit c-FLIPL, which prevents their Fas-mediated apoptosis. The specific down-regulation of c-FLIPL can induce the autonomous death of ENKL cells. c-FLIPL may be a therapeutic target against ENKL. [Display omitted] DisclosuresKomatsu:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Speakers Bureau.

The effects of agonistic α-4-1BB antibody on natural killer cell function

Abstract 4-1BB is a member of the Tumor Necrosis Factor Receptor (TNFR) family. The TNFR family regulates a variety of functions including immune response, inflammation, and cell life/death. 4-1BB is a co-stimulatory molecule known best for its ability to increase survival, proliferation, anti-viral/tumor effects on T cells, and thus is currently undergoing clinical trials. However, the effects of 4-1BB stimulation in other cell types, such as natural killer cells (NK), are not well studied. Using a murine cytomegalovirus (MCMV) infected mouse model, we investigated the effects of agonistic α-4-1BB antibody stimulation on NK cell function and its impact on viral resistance. We hypothesized that 4-1BB stimulation will induce robust activation and proliferation of NK cells, resulting in an increased resistance against MCMV. To the contrary, treatment of the α-4-1BB antibody increased viral burden on 4 days post infection (p.i.) with highly increased NK cell proportion in spleens and livers. Further studies showed higher viral burden on day 1.5 p.i. with a reduced NK cell frequency. This suggests that an uncontrolled viral replication due to low NK numbers on early days of MCMV infection allows rapid expansion of residual NK cells by day 4. In vitro, treatment with α-4-1BB along with α-FasL antibodies rescued NK cell reduction, indicating that the NK cell loss is induced by Fas-mediated death signaling. Taken together, our data demonstrates that 4-1BB stimulation induces cell death signaling in the NK cell population, and results in an impaired anti-viral response. Further studies will provide a more complete view of the effects of 4-1BB stimulation as a cancer therapeutic, and also into the mechanism of NK cell life/death through TNFR signaling.

Fas Regulates Macrophage Polarization and Fibrogenic Phenotype in a Model of Chronic Ethanol-Induced Hepatocellular Injury

The role of Fas-mediated apoptosis and its effect on proinflammatory cytokine production in early alcoholic liver disease has not been addressed. Wild-type mice (C57Bl/6) or mice with a functional mutation in the Fas ligand (B6.gld) were given either high-fat control diet or ethanol diet by intragastric cannulation for 2 or 4 weeks. Liver injury, hepatic lipid accumulation, and proinflammatory cytokine production associated with chronic ethanol consumption were largely prevented in B6.gld mice compared with wild-type mice. Conversely, B6.gld mice given ethanol exhibited increases in collagen deposition, hepatic collagen gene expression, and profibrogenic cytokines (eg, transforming growth factor-β and IL-13) and alterations in matrix remodeling proteins (eg, matrix metalloproteinases and tissue inhibitor of metalloproteinases) compared with wild-type mice. Hepatic F4/80(+) macrophage populations were increased significantly in B6.gld mice compared with wild-type mice; hepatic CD3(+) cell populations were not significantly different. Importantly, a shift toward the expression of M2/Th2 cytokines (eg, IL-4 and IL-13) after ethanol exposure was observed in B6.gld mice compared with classical M1 cytokine expression in wild-type mice under similar conditions. In isolated macrophages, stimulation of Fas receptor minimally enhances lipopolysaccharide-induced M1 cytokine production and significantly limits M2 cytokine production. These data support the hypothesis that Fas-mediated signaling is important for an early ethanol-induced proinflammatory response but limits the profibrogenic response, regulating collagen production in response to chronic ethanol.

An Evolution-Guided Analysis Reveals a Multi-Signaling Regulation of Fas by Tyrosine Phosphorylation and its Implication in Human Cancers.

Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive “Fas apoptosis” to “Fas multisignals” paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases.

Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death

Long noncoding RNAs (lncRNAs) interact with other RNAs, DNA and/or proteins to regulate gene expression during development. Erythropoiesis is one developmental process that is tightly controlled throughout life to ensure accurate red blood cell production and oxygen transport to tissues. Thus, homeostasis is critical and maintained by competitive outcomes of pro- and anti-apoptotic pathways. LncRNAs are expressed during blood development; however, specific functions are largely undefined. Here, a culture model of human erythropoiesis revealed that lncRNA Fas-antisense 1 (Fas-AS1 or Saf) was induced during differentiation through the activity of essential erythroid transcription factors GATA-1 and KLF1. Saf was also negatively regulated by NF-κB, where decreasing NF-κB activity levels tracked with increasing transcription of Saf. Furthermore, Saf over-expression in erythroblasts derived from CD34+ hematopoietic stem/progenitor cells of healthy donors reduced surface levels of Fas and conferred protection against Fas-mediated cell death signals. These studies reveal a novel lncRNA-regulated mechanism that modulates a critical cell death program during human erythropoiesis.

Molecular manipulation of keratin 8/18 intermediate filaments: modulators of FAS-mediated death signaling in human ovarian granulosa tumor cells.

BackgroundGranulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence. Keratin intermediate filaments expressed in these tumors are a diagnostic marker, yet paradoxically, may also constitute a target for therapeutic intervention. In the current study, we evaluated keratin 8/18 (K8/18) filament expression as a mechanism of resistance to apoptosis in GCT, specifically focusing on regulation of the cell surface death receptor, Fas (FAS).MethodsThe GCT cell line, KGN, was transiently transfected with siRNA to KRT8 and KRT18 to reduce K8/18 filament expression. Expression of K8/18, FAS, and apoptotic proteins (PARP, cleaved PARP) were evaluated by fluorescence microscopy, flow cytometric analysis, and immunoblotting, respectively. The incidence of FAS-mediated apoptosis in KGN cells was measured by caspase 3/7 activity. All experiments were performed independently three to six times, using a fresh aliquot of KGN cells for each experiment. Quantitative data were analyzed by one- or two-way analysis of variance (ANOVA), followed by a Tukey’s post-test for multiple comparisons; differences among means were considered statistically significant at P < 0.05.ResultsControl cultures of KGN cells exhibited abundant K8/18 filament expression (~90 % of cells), and minimal expression of FAS (<25 % of cells). These cells were resistant to FAS-activating antibody (FasAb)-induced apoptosis, as determined by detection of cleaved PARP and measurement of caspase 3/7 activity. Conversely, siRNA-mediated knock-down of K8/18 filament expression enhanced FAS expression (> 70 % of cells) and facilitated FasAb-induced apoptosis, evident by increased caspase 3/7 activity (P < 0.05). Additional experiments revealed that inhibition of protein synthesis, but not MEK1/2 or PI3K signaling, also prompted FasAb-induced apoptosis.ConclusionsThe results demonstrated that K8/18 filaments provide resistance to apoptosis in GCT by impairing FAS expression. The abundance of keratin filaments in these cells and their role in apoptotic resistance provides a greater mechanistic understanding of ovarian tumorgenicity, specifically GCT, as well as a clinically-relevant target for potential therapeutic intervention.

A55: Changes in peripheral blood lymphocyte subpopulations implicated in regulatory and cytotoxic immune reactions mirror the process of immunoediting in cervical pre-invasive cancer

Background The ability to promote an immunosuppressive microenvironment has been recognized as one of the “next generation” cancer hallmarks, being most apparent in case of HPV-induced carcinogenesis, since it is a prerequisite to the virus persistence and proliferation of keratinocytes expressing HPV-antigens. With respect to HPV-associated cervical cancer, it is commonly known that initial pre-cancerous intraepithelial lesions frequently undergo reverse development due to effective recognition and eradication by the immunocompetent cells. Both innate (e.g. NK) and adaptive (helper and cytotoxic O-cells) arms of immunity have been implicated in the regression of neoplastic cells, with NK T-cells considering as a connecting link between two branches. Progression of intraepithelial neoplasia leads to the establishment of pre-invasive cancer, which can be regarded as the result of a completed immunoediting of the tumor locus, because further, with 100% probability, it turns into invasive metastatic form. We assumed that the signs of immune activation, on the one hand, and the signs of immunosuppression, on the other hand, despite the locality of pre-invasive cervical cancer, can be found in the circulation and can be relevant to the development of tolerogenic microenvironment. The aim of the present work was to investigate the number of functionally different subpopulations of T- and NK-lymphocytes in the blood of patients with pre-invasive cervical cancer. Material and methods Blood samples were taken from 35 HPV(+) patients diagnosed with cancer in situ. The control group consisted of 30 healthy HPV(−) women with no pathology of the cervix. Lymphocytes were immunophenotyped by MACSQuant flow cytometer. The percentage of the following populations were measured: CD3+CD95+/high, CD3+CD4+, CD3+CD4+CD95+/high, CD3+CD8+, CD3+CD8+CD95+/high, CD4+CD25+, CD4+CD25high, CD4+CD25+CD127dim/neg, CD4+CD25+FoxP3+ (CD4 Tregs), CD8+CD25+, CD8+CD25+CD127dim/neg, CD8+CD25+FoxP3+ (CD8 Tregs), CD3−CD16+, CD3−CD56+, CD3−(CD16±)CD56bright, CD3−CD16+CD56+, CD3−CD16low/negCD56+, CD3−CD16+CD56- (NK subpopulations), CD3+CD56+, CD3+CD16+CD56+ (NK T-lymphocytes). Results Increase in the number of CD4 Tregs that are known to exert immunosuppressive effect was found in cancer patients’ group, namely the increased quantity of cells with CD4+CD25+ (especially, CD4+CD25high fraction), CD4+CD25+CD127dim/neg, or CD4+CD25+FoxP3+ phenotypes was shown. At the same time, the portion of CD4+FoxP3+ lymphocytes didn’t differ between the studied groups. A rare population of CD8+ Tregs (CD8+CD25+CD127dim/negFoxP3+) also exhibited a tendency to accumulate in the blood of cancer patients compared with the control. Analysis of the major subpopulation of NK cells performing cytotoxic reactions didn’t reveal significant difference between the examined groups. Similar result was obtained in relation to the other functional groups of NK cells, specifically CD3−CD16±CD56bright (cytokine-producing NKs with low cytotoxicity), CD3−CD16dim/−CD56+ (NKs with potential anti-tumor activity), and CD3−CD16+CD56 – (NKs associated with viral infection or NK-precursors). However, an increase of NK T-lymphocyte amounts was established, arguing both activation of anti-tumor response and its inhibition, in view of functional polymorphism of this population. Similarly, the increased number of CD3+CD95+/high cells and CD3+CD4+CD95+/high T-helpers may have dual significance, taking into account that the mid-early T-cell activation marker CD95/Fas presents the key death-receptor (earlier we have revealed the elevated activity of caspases involved in the Fas-mediated signaling in circulating lymphocytes of cervical cancer patients). The total numbers of CD3+CD4+ T-helpers and CD3+CD8+ T-killers were generally comparable between the groups analyzed, with T-killers displaying slightly decreased percentage in the patient group. Conclusion The processes supporting establishment of immunosuppressive microenvironment extend beyond the local level during the earliest steps of cervical cancer progression. The observed changes may reflect the shift of equilibrium between immune activation and suppression, which is the essence of immunoediting. Further phenotypic analysis of cell subpopulations reported here is required for understanding the functional meaning of these changes. The study was supported by the grants NK-1404-32098, 11.G34.31.0052, the Program of strategic development of PetrSU for 2012-2016, and the State task for R&D.

Loss of α(E)-catenin promotes Fas mediated apoptosis in tubular epithelial cells.

The aging kidney undergoes structural and functional alterations which make it more susceptible to drug-induced acute kidney injury (AKI). Previous studies in our lab have shown that the expression of α(E)-catenin is decreased in aged kidney and loss of α(E)-catenin potentiates AKI-induced apoptosis, but not necrosis, in renal tubular epithelial cells (NRK-52E cells). However, the specific apoptotic pathway underlying the increased AKI-induced cell death is not yet understood. In this study, cells were challenged with nephrotoxicant cisplatin to induce AKI. A ~5.5-fold increase in Fas expression in C2 (stable α(E)-catenin knockdown) relative to NT3 (non-targeted control) cells was seen. Increased caspase-8 and -9 activation was induced by cisplatin in C2 as compared to NT3 cells. In addition, decreased Bcl-2 expression and increased BID cleavage and cytochrome C release were detected in C2 cells after cisplatin challenge. Treating the cells with cisplatin, in combination with a Bcl-2 inhibitor, decreased the viability of NT3 cells to the same level as C2 cells after cisplatin. Furthermore, caspase-3/-7 activation is blocked by Fas, caspase-8, caspase-9 and pan-caspase inhibitors. These inhibitors also completely abolished the difference in viability between NT3 and C2 cells in response to cisplatin. These results demonstrate a Fas-mediated apoptotic signaling pathway that is enhanced by the age-dependent loss of α(E)-catenin in renal tubule epithelial cells.