Upregulation Of FasL Expression Research Articles

A novel trivalent non-Fc anti-CD3 Collabody preferentially induces Th1 cell apoptosis in vitro and long-lasting remission in recent-onset diabetic NOD mice.

Specific anti-CD3 treatment is deemed to be a promising therapy for allograft rejection and type 1 diabetes (T1D). Fc receptor (FcR) reduced-binding antibodies, by avoiding adverse effects of Fc and FcR interaction, have good therapeutic potential. We generated a trivalent anti-mouse-CD3 Collabody, h145CSA, by using a triplex-forming collagen-like peptide (Gly-Pro-Pro)10 to drive the trimerization of the Fab fragments. Exposure to h145CSA, but not its bivalent counterparts 145-2C11 and h145chIgGAA (FcR reduced-binding format), upregulates FasL expression on Th1 cells and causes Th1 cell apoptosis. Administration of h145CSA invokes minimal mitogenic effects in mice. The ability of multiple dosing of h145CSA to induce splenic CD4+ T-cell depletion is comparable to bivalent antibodies but is characterized by more rapid CD4+ T-cell recovery kinetics. h145CSA is more potent than h145chIgGAA in inducing long-lasting remission in recent-onset diabetic NOD mice. Its therapeutic effect is accompanied by a significantly lower percentage of CD4+IFNγ+ T cells and a higher Treg/Th1 ratio in pancreatic and mesenteric lymph nodes. The results of our study demonstrate that trivalent non-Fc anti-CD3 Collabody has the potential to be used in the treatment of T1D.

A unique subset of CD11c+ B cells upregulate FasL expression in response to IL-12p40 during lethal dose influenza virus (IAV) infections.

Abstract Our recent work has detailed a novel regulation of the IAV-specific CD8 T cell response during lethal dose IAV infections. During such infections, FasL+ plasmacytoid dendritic cells (pDC) within the regional lymph nodes (LN) target and eliminate the developing Fas+ effector CD8 T cell response limiting immunity and driving severe disease. While our studies have shown that pDC are sufficient to drive this mechanism (i.e. transfer of wild type pDC into gld mice results in elimination of activated CD8 T cells), antibody targeted elimination of pDC in wild type mice does not completely reverse CD8 T cell apoptosis. This suggested that an additional cell type might contribute to the overall loss of CD8 T cells during lethal/high pathogenic IAV infections. Here we describe a CD11c+B cell population that shares some common markers with both pDC and several defined B cell subsets, but can be distinguished from both based on unique marker expression. Our data suggests that these CD11c+ B cells are recruited to the LN in greater numbers compared to pDCs and appear to express FasL upon exposure to IL-12p40 homodimer in lethal dose IAV infections. Further our results suggest that FasL+ expression with BST2+ expression within CD11c+ B cells. Our current studies are focused on further determining the factors regulating this CD11c+B cell population and its relative contribution to the overall FasL-dependent control of the magnitude of the CD8 T cell response during lethal dose IAV infections.

Hepatitis B Virus X Protein Modulates Apoptosis in NRK-52E Cells and Activates Fas/FasL Through the MLK3-MKK7-JNK3 Signaling Pathway

Background/Aims: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. Methods: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. Results: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. Conclusions: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway.

Thy-1/β3 Integrin Interaction-Induced Apoptosis of Dermal Fibroblasts Is Mediated by Up-Regulation of FasL Expression

Thy-1/β3 Integrin Interaction-Induced Apoptosis of Dermal Fibroblasts Is Mediated by Up-Regulation of FasL Expression

The Role of Apoptosis in Immune Hyporesponsiveness Following AAV8 Liver Gene Transfer

Gene therapy provides a significant opportunity to treat a variety of inherited and acquired diseases. However, adverse immune responses toward the adeno-associated virus (AAV) antigens may limit its success. The mechanisms responsible for immunity or tolerance toward AAV-encoded transgene products remain poorly defined. Studies in mice demonstrate that AAV2/8 gene transfer to liver is associated with immunological hyporesponsiveness toward both AAV vector and antigenic transgene product. To evaluate the role of activation-induced cell death (AICD) and cytokine withdrawal (intrinsic cell death) in the deletion of mature T lymphocytes, we compared immunological responses in hepatic AAV2/8 transfer in murine recipients lacking the Fas receptor, and recipients overexpressing Bcl-xL, to WT murine counterparts. Prolonged transgene expression was dependent on both Fas signaling and Bcl-xL-regulated apoptosis in T cells. Abrogation of intrinsic cell death enhanced Th1 responses, whereas AICD functioned to limit neutralizing antibody production toward AAV2/8. In addition, immune hyporesponsiveness and stable transgene expression was dependent on upregulation of FasL expression on transduced hepatocytes and a corresponding apoptosis of infiltrating Fas (+) cells. These data provide evidence that both AICD and apoptosis due to cytokine withdrawal of lymphocytes are essential for immune hyporesponsiveness toward hepatic AAV2/8-encoded transgene product in the setting of liver gene transfer.

HSV-2 Regulates Monocyte Inflammatory Response via the Fas/FasL Pathway

Monocytic cells represent important cellular elements of the innate and adaptive immune responses in viral infections. We assessed the role of Fas/FasL in promoting monocyte apoptosis during HSV-2 infection by using an in vitro model based on the murine RAW 264.7 monocytic cell line and an in vivo murine model of HSV-2 infection applied to C57BL6, MRL-Faslpr/J (Fas−/−) and C3-Faslgld/J (FasL−/−) mice. HSV-2 infection of the monocytic cell line led to early induction of apoptosis, with no protective expression of anti-apoptotic Bcl-2. HSV-2 infected monocytes up-regulated Fas and FasL expression early during in vitro infection but were susceptible to Fas induced apoptosis. The vaginal monocytes in the HSV-2 murine model of infection up-regulated FasL expression and were susceptible to Fas induced apoptosis. HSV-2 infection of Fas and FasL- deficient mice led to decreased apoptosis of monocytes and impaired recruitment of NK, CD4+ and CD8+ T cells within the infection sites. The vaginal lavages of HSV-2 infected Fas and FasL- deficient showed decreased production of CXCL9, CXCL10 and TNF-α in comparison to HSV-2 infected wild-type mice strain. The decreased recruitment of immune competent cells was accompanied by delayed virus clearance from the infected tissue. Triggering of the Fas receptor on HSV-2 infected monocytes in vitro up-regulated the expression of CXCL9 chemokines and the cytokine TNF-α. Our study provides novel insights on the role of Fas/FasL pathway not only in apoptosis of monocytes but also in regulating local immune response by monocytes during HSV-2 infection.

Up-regulation of Fas Ligand Expression by Sirtuin 1 in both Flow-restricted Vessels and Serum-stimulated Vascular Smooth Muscle Cells

To study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms. SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell (VSMC) lysate. Smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) C57BL/6 mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (ligation groups) or the ligation-excluded operation (sham groups). The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 (SIRT1H363Y), and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis. SIRT1 was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation (P<0.001) and VSMCs treated with serum (P<0.05 at the transcriptional level, P<0.001 at the protein level). No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL (P<0.001). The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (P<0.001), while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 (P<0.001). Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT1.

The serum miR-21 level serves as a predictor for the chemosensitivity of advanced pancreatic cancer, and miR-21 expression confers chemoresistance by targeting FasL

miR-21 expression in cancer tissue has been reported to be associated with the clinical outcome and activity of gemcitabine in pancreatic cancer. However, resection is possible in only a minority of patients due to the advanced stages often present at the time of diagnosis, and safely obtaining sufficient quantities of pancreatic tumor tissue for molecular analysis is difficult at the unresectable stages. In this study, we investigated whether the serum level of miR-21 could be used as a predictor of chemosensitivity. We tested the levels of serum miR-21 in a cohort of 177 cases of advanced pancreatic cancer who received gemcitabine-based palliative chemotherapy. We found that a high level of miR-21 in the serum was significantly correlated with a shortened time-to-progression (TTP) and a lower overall survival (OS). The serum miR-21 level was an independent prognostic factor for both the TTP and the OS (HR 1.920; 95% CI, 1.274–2.903, p = 0.002 for TTP and HR 1.705; 95% CI, 1.147–2.535, p = 0.008 for OS). The results from a functional study showed that gemcitabine exposure down-regulated miR-21 expression and up-regulated FasL expression. The increased FasL expression following gemcitabine treatment induced cancer cell apoptosis, whereas the ectopic expression of miR-21 partially protected the cancer cells from gemcitabine-induced apoptosis. Additionally, we confirmed that FasL was a direct target of miR-21. Therefore, the serum level of miR-21 may serve as a predictor of chemosensitivity in advanced pancreatic cancer. Additionally, we identified a new mechanism of chemoresistance mediated by the effects of miR-21 on the FasL/Fas pathway.

Adenovirus-mediated expression of mutated forkhead human transcription like-1 suppresses tumor growth in a mouse melanoma xenograft model

Melanoma is generally resistant to chemotherapy, which may be related to defects in death receptor signaling and to defects in induction of apoptosis. Forkhead family transcription factors induce the expression of death receptor ligands such as Fas ligand (Fas-L) resulting in apoptosis. We therefore investigated whether a triple mutant form of forkhead transcription factor FKHRL1 (FKHRL1/TM) can enhance Fas-L mediated-apoptosis in melanoma cells. Two melanoma cells A2058 or DM6 were tested for their sensitivity to agonistic anti-Fas antibody (CH-11); adenovirus expressing FKHRL1/TM (Ad-FKHRL1/TM) was assessed for its capability to induce activation of the caspase pathway; the role of Fas-L in the Ad-FKHRL1/TM mediated-cell death was also assessed in vitro. Ad-FKHRL1/TM antitumor activity in vivo was also evaluated in a mouse melanoma xenograft model. We found that DM6 melanoma cells were more resistant to Fas/Fas-L-mediated apoptosis induced by agonistic anti-Fas antibody than A2058 melanoma cells. Ectopic expression of FKHRL1/TM in melanoma cells upregulated Fas-L expression, decreased procaspase-8 levels, and significantly increased Fas/FasL-mediated cell death in both cells lines; this induced cell death was partially blocked by a Fas/Fas-L antagonist. Importantly, Ad-FKHRL1/TM treatment of subcutaneous melanoma xenografts in mice resulted in approximately 70% decrease in tumor size compared with controls. These data indicate that overexpression of FKHRL1/TM can induce the Fas-L pathway in melanoma cells. Ad-FKHRL1/TM therefore might represent a promising vector for melanoma treatment.

MiR‐21 represses FasL in microglia and protects against microglia‐mediated neuronal cell death following hypoxia/ischemia

A growing body of evidence suggests that microRNA (miRNA) dysregulation contributes to many types of human disease, including central nervous system disorders. In this study, we identified an inverse correlation between the expression of miR-21 and Fas ligand (FasL) during hypoxia-induced microglial activation. Specifically, hypoxia caused the upregulation of FasL expression but the downregulation of miR-21 expression in microglia. Furthermore, we demonstrated that miR-21 suppresses FasL production by directly binding to its 3'-untranslated region. The overproduction of FasL following hypoxic microglial activation induced neuronal apoptosis, whereas the ectopic expression of miR-21 partially protected neurons from cell death caused by hypoxia-activated microglia. Finally, we confirmed that the function of miR-21 in microglia-mediated neuronal injury is dependent on FasL. Our study demonstrates an important role for miRNAs in microglia-mediated neuronal apoptosis, and suggests potential novel therapeutic interventions for cerebral hypoxic diseases associated with microglial activation.

(G2019S) LRRK2 activates MKK4-JNK pathway and causes degeneration of SN dopaminergic neurons in a transgenic mouse model of PD.

(G2019S) mutation of leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of both familial and sporadic Parkinson's disease (PD) cases. Twelve- to sixteen-month-old (G2019S) LRRK2 transgenic mice prepared by us displayed progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurons and parkinsonism phenotypes of motor dysfunction. LRRK2 is a member of mixed lineage kinase subfamily of mitogen-activated protein kinase kinase kinases (MAPKKKs). We hypothesized that (G2019S) mutation augmented LRRK2 kinase activity, leading to overphosphorylation of downstream MAPK kinase (MKK) and resulting in activation of neuronal death signal pathway. Consistent with our hypothesis, (G2019S) LRRK2 expressed in HEK 293 cells exhibited an augmented kinase activity of phosphorylating MAPK kinase 4 (MKK4) at Ser(257), and protein expression of active phospho-MKK4(Ser257) was upregulated in the SN of (G2019S) LRRK2 transgenic mice. Protein level of active phospho-JNK(Thr183/Tyr185) and phospho-c-Jun(Ser63), downstream targets of phospho-MKK4(Ser257), was increased in the SN of (G2019S) LRRK2 mice. Upregulated mRNA expression of pro-apoptotic Bim and FasL, target genes of phospho-c-Jun(Ser63), and formation of active caspase-9, caspase-8 and caspase-3 were also observed in the SN of (G2019S) LRRK2 transgenic mice. Our results suggest that mutant (G2019S) LRRK2 activates MKK4-JNK-c-Jun pathway in the SN and causes the resulting degeneration of SNpc dopaminergic neurons in PD transgenic mice.

Abstract 248: JNK-FoxO3a dependent apoptosis in quercetin treated MDA-MB-231 human breast cancer cells

Abstract FoxO transcription factors are well known as key tumor suppressors in mammalian cells. The suppression of FoxOs in cancer cells is thought to be mainly due to activation of multiple onco-kinases, such as Akt and SGK, by a phosphorylation-ubiquitylation-mediated cascade. On the other hand, phosphorlyation of FoxO by JNK relocalizes it from the cytoplasm to the nucleus allowing transcription of FoxO target genes. In this study, we applied human breast cancer cells to determine whether the activity of FoxO3a is regulated during the quercetin mediated apoptosis. After treatment with the indicated amount of quercetin (5-80uM) for 5days, the growth of human breast cancer cell lines including MDA-MB-231, MDA-MB-486 and MCF-7 cells was reduced in dose- and time-dependent manner. FACS analysis with AnnexinV/PI double staining showed that the proportion of early apoptotic cancer cells were significantly increased in quercetin treated cells. It represents the potent apoptotic effect of quercetin in breast cancer cells. Furthermore, a marked elevation of FoxO3a transcriptional activity was detected at 20μM of quercetin using FHRE-reporter assay, as well as increased protein level of FoxO3a was detected by western blotting. And also it was determined that the activation of FoxO3a lead to the upregulation of FasL expression during the apoptotic process. To identify the signaling mechanism of FoxO3a regulation, we examined the activation of several well known upstream kinases such as Akt, Erk, IKKβ, AMPK and JNK. Notably, a significant increase of JNK phosphorylation was observed in quercetin treated cells. In addition, the inhibition study with pharmacological inhibitor (SP600125) showed that the involvement of JNK for the regulation of FoxO3a activity is critical in quercetin treated breast cancer cells. These results demonstrate that quercetin induces FoxO3a-related apoptosis by activation of JNK in MDA-MB-231 cells, and suggest the possibility that JNK-FoxO3a signaling pathway contributes mainly to the apoptotic process of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 248. doi:1538-7445.AM2012-248

Early Detection of Hepatocellular Carcinoma on top of Liver Cirrhosis: The Fas Receptor and Ligand System

Hepatocyte aberrations, accumulation of chromosomal damage and possibly initiation of hepatic carcinogenesis is thought to be caused by the continued viral replication and the persistent attempt by a less than optimal immune response to eliminate hepatitis C virus (HCV) infected cells. The identification of the “death factors including Fas and its Ligand (Fas-L) as a major regulator of both apoptosis and immune function has provided insight into an attractive mechanism of tumor escape from immune clearance. Aim: To assess the hepatic expression of Fas/Fas-L, the Fas receptor (Fas-R) expression on lymphocyte, and serum soluble Fas (sFas) in an attempt to analyze the role of Fas receptor/ligand system in the multistep process of fibrosis/carcinogenesis and the possible use of the serum marker as possible candidate biomarkers for an early detection of hepatocellular carcinoma (HCC). Material and Method: The current study included 100 samples from cases at Theodor Bilharz Research Institute and Kasr Al Aini Hospital in Egypt. There were 90 cases of chronic hepatitis C (CHC) infection (and negative hepatitis B virus infection). There were 30 cases without liver cirrhosis, 30 cases with liver cirrhosis and 30 cases with HCC. 10 liver biopsies were taken from healthy livers as normal controls. Histopathologic study and immunohistochemistry for detection of hepatic Fas and Fas-L expression were determined for all cases. Electron microscopy (EM) and immunoelectron microscopy (IEM) examination for detection of Fas-R expression on lymphocytes were also done. sFas, liver function tests, serologic markers for viral hepatitis, and serum alpha-fetoprotein level (alpha-FP) were done. Results: The sFas in both HCC and CHC with cirrhosis patients were significantly higher than those of normal controls and CHC without cirrhosis (P<0.01), but there was no significant difference between the cirrhosis and HCC patients. Positive hepatic expression of both Fas and Fas-L were significantly increased in the diseased groups (p<0. 01) compared to the control specimens. A progressive Fas and FasL increase from CHC without cirrhosis to CHC with cirrhosis followed by a decline from the latter to HCC. Apoptotic Fas and Fas-L proteins expression was significantly increased with the necroinflammatory activity and the advancement of fibrosis. There was a non-significant negative correlation between sFas and hepatic Fas. In addition a significant over expression of Fas-R on separated lymphocytes was associated with a higher frequency of apoptotic cell death as detected by EM examination. Conclusion: The Fas receptor/ligand system was significantly involved in the process of liver cirrhosis converting into HCC. Down-regulation of Fas expression, up-regulation of Fas-L expression in hepatocytes and elevation of serum sFas level were important in tumor evasion from immune surveillance and in hepatic carcinogenesis.

Glucuronoxylomannan from Cryptococcus neoformans Down-regulates the Enzyme 6-Phosphofructo-1-kinase of Macrophages

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.

Mannosylated lipoarabinomannan balances apoptosis and inflammatory state in mycobacteria-infected and uninfected bystander macrophages

To assess the role of mannosylated lipoarabinomannan (ManLAM) in the inflammatory and apoptotic response of mycobacteria-infected and uninfected, bystander cells we applied a mouse macrophage model of infection with avirulent strains – Mycobacterium bovis BCG, Mycobacterium tuberculosis (MTB) H37Ra and compared with a virulent MTB H37Rv strain infection. ManLAM contributed to the infection of macrophages by protection from apoptosis with stabilized Bcl-2 expression and down-regulated Bax expression for infected cells (BCG) or with stabilized Bcl-2 expression for uninfected bystander target cells (H37Ra). Additionally, ManLAM up-regulated FasL expression on the infected cells. Active extracellular signal-regulated kinase (ERK1/2) in BCG and H37Rv infection provided an anti-apoptotic effect by stabilization of anti-apoptotic Bcl-2 expression in the infected cells. Inhibitors specific for c-Jun-NH2-terminal kinase or stress-activated kinase (JNK) and p38 kinase decreased apoptosis of infected cells (BCG, H37Ra) and of uninfected bystanders (H37Ra) by down-regulating Bax. ManLAM significantly down-regulated production of pro-inflammatory IL-12 and TNF-alpha and activation of JNK by both avirulent strains. We conclude that by stabilization of Bcl-2 expression, down-regulation of JNK activity and down-regulation of pro-inflammatory cytokines production ManLAM can contribute to suppression of apoptosis and inflammatory reaction of uninfected, bystander cells.

Foxo3a Regulates Apoptosis by Negatively Targeting miR-21

MicroRNAs are a class of small non-coding RNAs and participate in the regulation of apoptotic program. Although miR-21 is able to inhibit apoptosis, its expression regulation and downstream targets remain to be fully elucidated. Here we report that the transcriptional factor Foxo3a initiates apoptosis by transcriptionally repressing miR-21 expression. Our results showed that doxorubicin could simultaneously induce the translocation of Foxo3a to the cell nuclei and a reduction in miR-21 expression. Knockdown of Foxo3a resulted in an elevation in miR-21 levels, whereas enforced expression of Foxo3a led to a decrease in miR-21 expression. In exploring the molecular mechanism by which Foxo3a regulates miR-21, we observed that Foxo3a bound to the promoter region of miR-21 and suppressed its promoter activity. These results indicate that Foxo3a can transcriptionally repress miR-21 expression. In searching for the downstream targets of miR-21 in apoptosis, we found that miR-21 suppressed the translation of Fas ligand (FasL), a pro-apoptotic factor. Furthermore, Foxo3a was able to up-regulate FasL expression through down-regulating miR-21. Our data suggest that Foxo3a negatively regulates miR-21 in initiating apoptosis.

Correlation of Fas/FasL expression to cell apoptosis in Epstein-Barr virus-associated gastric carcinoma (EBVaGC)

Epstein-Barr virus (EBV) has been detected in about 10% of gastric carcinomas. However, the pathogenetic role of EBV in gastric carcinoma is uncertain. This study was to explore the correlation of Fas/FasL expression to the apoptosis of tumor cells and tumor-infiltrating lymphocytes (TIL) in EBV-associated gastric carcinoma (EBVaGC). Fas/FasL expression in 49 specimens of EBVaGC, 20 specimens of EBV-negative gastric carcinoma (EBVnGC) and 12 specimens of normal gastric mucosa was detected by immunohistochemistry. The apoptotic index (AI) of cells was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). The positive rates of Fas were 91.7% in normal gastric mucosa and 76.8% in gastric carcinoma (P < 0.05); those of FasL were 16.7% in normal gastric mucosa and 58% in gastric carcinoma (P < 0.05). The positive rate of Fas was significantly lower in EBVaGC than in EBVnGC (71.4% vs. 90.0%, P < 0.05). The positive rate of FasL in EBVaGC was significantly higher than that in EBVnGC (63.2% vs. 45%, P < 0.05). The AI of EBVaGC cells was significantly lower than that of EBVnGC cells (P = 0.002). The number and AI of TIL in EBVaGC were higher than those in EBVnGC (P < 0.05). The AI of TIL was positively correlated with the level of FasL expression in tumor cells (r=0.237, P = 0.028). Up-regulation of FasL expression and decrease of TIL apoptosis in EBVaGC may facilitate the escape of tumor cells from the host immunosurveillance, and it might contribute to the development and progression of carcinoma.

Cytotoxicity Mediated by the Fas Ligand (FasL)-activated Apoptotic Pathway in Stem Cells

Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. Here, we test the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(-/-) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues.

Lysophosphatidylcholine-induced apoptosis in H19-7 hippocampal progenitor cells is enhanced by the upregulation of Fas Ligand

Lysophospholipids regulate a wide array of biological processes including apoptosis and neutrophil migration. Fas/Apo-1 and its ligand (FasL) participate in neuronal cell apoptosis causing various neurological diseases. Here, we use hippocampal neuroprogenitor cells to investigate how lysophosphatidylcholine (LPC) induces apoptosis in H19-7 hippocampal progenitor cells via Fas/Fas ligand-mediated apoptotic signaling pathway. Exposed cells with LPC presented on apoptotic morphology, positive TUNEL staining, and DNA fragmentation. We found that the expression of FasL was increased after LPC treatment. Furthermore, LPC-induced H19-7 cell apoptosis was decreased by agonistic anti-FasL antibody. In addition to promotion of caspase cascade activity by LPC, the administration of the caspase inhibitor, DEVD-fmk, prevented H19-7 cell apoptosis. LPC also increased the activation of nuclear factor-κB (NF-κB), which in turn, significantly increased FasL mRNA level. The increase in FasL mRNA level by NF-κB transfection was significantly decreased in the presence of IκB-SR, a super-repressor of IκB. Taken together, these results demonstrate that LPC has the ability to induce apoptosis in H19-7 cells through the upregulation of FasL expression via NF-κB activation.

Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells

Fas ligand (FasL/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E2 (PGE2), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE2 increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E2-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE2 positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE2.