Fas Ligand-induced Apoptosis Research Articles

Varicella-Zoster Virus ORF63 Protects Human Neuronal and Keratinocyte Cell Lines from Apoptosis and Changes Its Localization upon Apoptosis Induction.

There are many facets of varicella-zoster virus (VZV) pathogenesis that are not fully understood, such as the mechanisms involved in the establishment of lifelong latency, reactivation, and development of serious conditions like postherpetic neuralgia (PHN). Virus-encoded modulation of apoptosis has been suggested to play an important role in these processes. VZV open reading frame 63 (ORF63) has been shown to modulate apoptosis in a cell-type-specific manner, but the impact of ORF63 on cell death pathways has not been examined in isolation in the context of human cells. We sought to elucidate the effect of VZV ORF63 on apoptosis induction in human neuron and keratinocyte cell lines. VZV ORF63 was shown to protect differentiated SH-SY5Y neuronal cells against staurosporine-induced apoptosis. In addition, VZV infection did not induce high levels of apoptosis in the HaCaT human keratinocyte line, highlighting a delay in apoptosis induction. VZV ORF63 was shown to protect HaCaT cells against both staurosporine- and Fas ligand-induced apoptosis. Confocal microscopy was utilized to examine VZV ORF63 localization during apoptosis induction. In VZV infection and ORF63 expression alone, VZV ORF63 became more cytoplasmic, with aggregate formation during apoptosis induction. Taken together, this suggests that VZV ORF63 protects both differentiated SH-SY5Y cells and HaCaT cells from apoptosis induction and may mediate this effect through its localization change during apoptosis. VZV ORF63 is a prominent VZV gene product in both productive and latent infection and thus may play a critical role in VZV pathogenesis by aiding neuron and keratinocyte survival.IMPORTANCE VZV, a human-specific alphaherpesvirus, causes chicken pox during primary infection and establishes lifelong latency in the dorsal root ganglia (DRG). Reactivation of VZV causes shingles, which is often followed by a prolonged pain syndrome called postherpetic neuralgia. It has been suggested that the ability of the virus to modulate cell death pathways is linked to its ability to establish latency and reactivate. The significance of our research lies in investigating the ability of ORF63, a VZV gene product, to inhibit apoptosis in novel cell types crucial for VZV pathogenesis. This will allow an increased understanding of critical enigmatic components of VZV pathogenesis.

Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation.

Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration.

Regenerative and immunogenic characteristics of cultured nucleus pulposus cells from human cervical intervertebral discs.

Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.

Immunogenic Properties of Cultured Human Nucleus Pulposus Cells for Regenerative Treatment Approaches

Introduction Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc diseases. Potential alterations of immunogenic properties along intervertebral disc (IVD) cell isolation and expansion and in three-dimensional (3D) culture with various biomaterials, however, have been scarcely addressed in the literature. Therefore, our aim was to evaluate the immunogenic properties of mild and severely degenerated nucleus pulposus (NP) cells with regard to cell surface markers and cocultures with autologous or allogenic peripheral blood mononuclear cells (PBMC) as well as changes of theses immunogenic properties after 3D culture in a polymer-based porous biomaterial. Materials and Methods Ten patients with cervical disc herniations, five with severe and five with mild grades of IVD degeneration according to the Miyazaki MRI classification, were operated, and tissue from the NP compartment was carefully selected. NP cells were isolated, expanded, and cultured in 3D fibrin/poly(lactic-coglycolic) acid transplants for 21 days. NP cells were evaluated for induction of immune responses in coculture with PBMC from the same donor or from an unrelated healthy volunteer, using a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay. Surface marker detection and evaluation of cytokines was performed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS) analysis. Results NP cells from donors with severe degeneration tended to have higher expression of the adhesion molecules ICAM and VCAM, as well as Fas ligand, though only VCAM was significantly increased. All cells expressed HLA-ABC and CD95 (Fas), but lacked expression of HLA-DR and CD24. NP cells after monolayer culture did not provoke a significant proliferation of T cells, NK cells, or B cells from either the same donor or from allogenic PBMC. Proliferating capacity was verified with the mitogenic stimulator ConA. When NP cells were incorporated into a 3D matrix, the immune cell proliferation was provoked to a greater extent than for NP cells preseeded as a monolayer. 3D specimens containing NP cells from patients with severe degeneration tended to provoke a larger response, especially for CD3+CD8+ and CD3+CD4+ T cells, than those with mild degeneration, though these differences were not significant. In general, the cytokine release was low or undetectable, especially for the inflammatory cytokines TNF-α and IFN-gamma. Conclusion NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, NK cells, or B cells, not only with donor PBMC but also with allogenic PBMC. In conjunction with low inflammatory cytokine expression, a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D culture with a fibrin/poly(lactic-coglycolic) acid matrix, however, we found higher immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor on cultured NP cells could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T cell activity acting by Fas ligand-induced apoptosis. Disclosure of Interest None declared

The significance of interferon-γ on Fas ligand-induced apoptosis of vascular smooth muscle cells

Objective To investigate the effect of interferon-γ （IFN-γ on Fas ligand （FasL） -in duced apoptosis of rat vascular smooth muscle cells （VSMCs） and explore the underlying mechanism. Methods VSMCs isolated from rat aorta were cultured in vitro and treated with IFN-γor FasL alone, or both in combination, with or without the pretreatment of Tunicamycin. Hoechst 33342 staining and flow cyto metric analysis of Annexin V-fluoresceine isothiocyanate （FITC）/propidium iodide （ PI ） staining were em ployed to define the effect of IFN-γon FasL-induced VSMCs apoplosis. Protein levels of surface Fas, cyto plasmic Fas and total Fas were detected by Western blotting in order to observe the expression and distribu tion of Fas. Results As shown by both Hoeehst 33342 staining, apoptosis of VSMCs was only slightly in duced by treatment with IFN-γ or FasL alone [ apoptotie index of （7.4 ± 1.9 ） % vs. （6. 8 ± 2. 5 ） % ], but significantly induced by IFN-γ and FasL in combination [ （20. 1 ± 2. 2）%, both P 〈 O. 01 ]. Increased sur face Fas （ relative level of O. 266 ± 0. 042 vs. O. 042 ± O. 017, P 〈 O. O1 ） , decreased cytoplasmic Fas （ O. 193 ±0. 038 vs. O. 426 ±0. 074, P 〈0. O1 ） but unchanged total Fas expression （0. 469 ＋0. 129/0. 383 ±0. 045 for 36 h/72 h vs. 0. 402 ± 0. 031 for 0 h, both P 〉 O. 05 ） were detected by Western blotting after IFN-γ treatment. And pretreatment with membrane trafficking inhibitor Tunicamycin significantly diminished FasL-indueed VSMCs apoptosis promoted by IFN-γ. The apoptotic index [ （6. 1 ± 0. 8）% ] were less than that of control [ （23.0 ± 1.0）% , P 〈0. 011- Flow eytometric analysis also confirmed the results of Ho echst 33342 staining. Conclusion Instead of inducing VSMCs apoptosis directly, IFN-γchanges the Fas distribution by promoting translocation of Fas receptor to the cell membrane, which significantly enhances the sensitivity of VSMCs to FasL-indueed apoptosis. Key words: Interferon-γ; Vascular smooth muscle cells; Fas ligand; Fas; Cell apoptosis

Abstract 028: CCN1 Enables Fas Ligand-Induced Apoptosis by Elevating Fas Expression in Primary Cardiomyocytes or by Increasing Cytoplasmic Smac in Cardiomyoblast H9c2 Cells

Fas/Fas ligand (FasL) is implicated in cardiac ischemia/reperfusion injury. However, cardiomyocytes in culture are resistant to FasL-induced apoptosis, suggesting that additional factor(s) are required for FasL-induced apoptosis. Matricellular protein CCN1 has been demonstrated to promote cytotoxicity of FasL in human skin fibroblasts. CCN1 is induced in a variety of cardiac pathologies. We assessed the hypothesis that CCN1 may be involved in the regulation of FasL-induced apoptosis in cardiomyocytes. We found that either FasL or CCN1 did not induce cell death in neonatal rat ventricular cardiomyocytes (NRVM). Interestingly, the combination of FasL+CCN1 generated 2-fold induction of apoptosis (vs. control p<0.001). An integrin-α 6 β 1 -binding defective mutant CCN1, CCN1-DM failed to exert synergy with FasL to induce apoptosis, indicating a critical role of α 6 β 1 . The engagement between CCN1 and α 6 β 1 instigated the elevation of cellular reactive oxygen species (ROS), the activation of mitogen activated protein kinase p38, and followed by the induction of cell surface display of Fas, thereby sensitizing NRVM to FasL-induced apoptosis. Pretreatment of the p38 inhibitor SB202190 abolished the CCN1-induced cell-surface Fas expression and the apoptosis induced by FasL+CCN1. In addition, we tested the interaction between CCN1 and FasL on the cardiomyoblast H9c2 cells. We found that FasL or CCN1 alone did not cause apoptosis in H9c2, and required the combination of FasL+CCN1 to induced apoptosis (vs. control p<0.001) in H9c2 cells, reminiscent of the observation in NRVM. Mechanistically, CCN1 acted through binding to integrin α 6 β 1 , ROS generation, and p38 activation, however, did not increase the expression of cell surface Fas for its synergy with FasL in H9c2 cells. Instead, CCN1 induced Bax translocation to mitochondria, which in turn led to the release of Smac from mitochondria to cytosol. The cytosolic Smac functions to neutralize XIAP. Smac is critical for CCN1 action, because the knockdown of Smac blunted the apoptotic activities of CCN1. In conclusion, CCN1 may play a detrimental role in a stressed heart to both the differentiated cardiomyocytes and the proliferative cardioblasts through distinct signaling mechanisms.

Mo1772 Kinase Inhibition Normalizes Mutation-Mediated Keratin Filament Disruption and Predisposition to Apoptosis and Liver Injury

Objective: Mutations in members of the cytoskeletal intermediate filament proteins (IFs), keratin polypeptides 8 and 18 (K8/K18), predispose their carriers to acute and chronic liver disease progression. Keratin associated diseases are among .70 human diseases that are caused or predisposed to by IF mutations, with the defining feature in many of these diseases being disruption of the normal IF organization within the cytoplasm. In contrast to microfilaments and microtubules, no drugs are available for IFs that can modulate their organization and potentially provide a sorely lacking therapy. Methods: We used an imagingbased drug screening platform consisting of lung carcinoma A549 cells transduced with lentiviruses containing GFP-tagged K18 R90C and wild-type K8. The cells were screened against several compound libraries, and a select compound was further tested in cell culture and in livers of mice that express mutant K18 in order to define its mechanism of action. Results: Screening of a 320-compound kinase inhibitor library showed that the inhibitors of protein kinase C (PKC) and tyrosine kinases, particularly MIDOSTAURIN, led to significant correction of the defective organization of K18 R90C puncta towards normal filaments after 24 h with a maximal effect at 48 h. The findings were confirmed using multiple MIDOSTAURIN concentrations and in another cell line, Hacat, that expresses mutant K14. While MIDOSTAURIN diminished the expression of the mutant keratin 14 in Hacat cells, the effect in the A549 cells was independent of K18 expression but causes changes in K18 phosphorylation that are known to regulate filament formation. MIDOSTAURIN significantly prevented interferon and FAS ligand-induced apoptosis in A549 cells. The protective effect occurs after 48 h but not after 1 h treatment with MIDOSTAURIN, suggesting that the drug effect is more likely to be related to the correction of filament organization. In mice, administration of MIDOSTAURIN is well tolerated and its administration prior to exposure to FAS results in dramatic protection from liver injury. Conclusion: The kinase inhibitor MIDOSTAURIN alleviates cytoskeletal structural defects caused by K18 R90C mutation and also protects from apoptosis in cultured and in the livers of mice exposed to FAS ligand. MIDOSTAURIN may be a potential therapeutic for IF diseases and, notably, it is presently undergoing clinical trials in patients with acute myeloid leukemia and gastrointestinal stromal tumors.

Mo1775 Epigenetic Inactivation of Claudin 3 in Hepatocellular Carcinoma and Its Functional Consequences

Objective: Mutations in members of the cytoskeletal intermediate filament proteins (IFs), keratin polypeptides 8 and 18 (K8/K18), predispose their carriers to acute and chronic liver disease progression. Keratin associated diseases are among .70 human diseases that are caused or predisposed to by IF mutations, with the defining feature in many of these diseases being disruption of the normal IF organization within the cytoplasm. In contrast to microfilaments and microtubules, no drugs are available for IFs that can modulate their organization and potentially provide a sorely lacking therapy. Methods: We used an imagingbased drug screening platform consisting of lung carcinoma A549 cells transduced with lentiviruses containing GFP-tagged K18 R90C and wild-type K8. The cells were screened against several compound libraries, and a select compound was further tested in cell culture and in livers of mice that express mutant K18 in order to define its mechanism of action. Results: Screening of a 320-compound kinase inhibitor library showed that the inhibitors of protein kinase C (PKC) and tyrosine kinases, particularly MIDOSTAURIN, led to significant correction of the defective organization of K18 R90C puncta towards normal filaments after 24 h with a maximal effect at 48 h. The findings were confirmed using multiple MIDOSTAURIN concentrations and in another cell line, Hacat, that expresses mutant K14. While MIDOSTAURIN diminished the expression of the mutant keratin 14 in Hacat cells, the effect in the A549 cells was independent of K18 expression but causes changes in K18 phosphorylation that are known to regulate filament formation. MIDOSTAURIN significantly prevented interferon and FAS ligand-induced apoptosis in A549 cells. The protective effect occurs after 48 h but not after 1 h treatment with MIDOSTAURIN, suggesting that the drug effect is more likely to be related to the correction of filament organization. In mice, administration of MIDOSTAURIN is well tolerated and its administration prior to exposure to FAS results in dramatic protection from liver injury. Conclusion: The kinase inhibitor MIDOSTAURIN alleviates cytoskeletal structural defects caused by K18 R90C mutation and also protects from apoptosis in cultured and in the livers of mice exposed to FAS ligand. MIDOSTAURIN may be a potential therapeutic for IF diseases and, notably, it is presently undergoing clinical trials in patients with acute myeloid leukemia and gastrointestinal stromal tumors.

Mo1774 BCL6B Inhibits Hepatocellular Carcinoma Metastases In Vitro and in Mice

Objective: Mutations in members of the cytoskeletal intermediate filament proteins (IFs), keratin polypeptides 8 and 18 (K8/K18), predispose their carriers to acute and chronic liver disease progression. Keratin associated diseases are among .70 human diseases that are caused or predisposed to by IF mutations, with the defining feature in many of these diseases being disruption of the normal IF organization within the cytoplasm. In contrast to microfilaments and microtubules, no drugs are available for IFs that can modulate their organization and potentially provide a sorely lacking therapy. Methods: We used an imagingbased drug screening platform consisting of lung carcinoma A549 cells transduced with lentiviruses containing GFP-tagged K18 R90C and wild-type K8. The cells were screened against several compound libraries, and a select compound was further tested in cell culture and in livers of mice that express mutant K18 in order to define its mechanism of action. Results: Screening of a 320-compound kinase inhibitor library showed that the inhibitors of protein kinase C (PKC) and tyrosine kinases, particularly MIDOSTAURIN, led to significant correction of the defective organization of K18 R90C puncta towards normal filaments after 24 h with a maximal effect at 48 h. The findings were confirmed using multiple MIDOSTAURIN concentrations and in another cell line, Hacat, that expresses mutant K14. While MIDOSTAURIN diminished the expression of the mutant keratin 14 in Hacat cells, the effect in the A549 cells was independent of K18 expression but causes changes in K18 phosphorylation that are known to regulate filament formation. MIDOSTAURIN significantly prevented interferon and FAS ligand-induced apoptosis in A549 cells. The protective effect occurs after 48 h but not after 1 h treatment with MIDOSTAURIN, suggesting that the drug effect is more likely to be related to the correction of filament organization. In mice, administration of MIDOSTAURIN is well tolerated and its administration prior to exposure to FAS results in dramatic protection from liver injury. Conclusion: The kinase inhibitor MIDOSTAURIN alleviates cytoskeletal structural defects caused by K18 R90C mutation and also protects from apoptosis in cultured and in the livers of mice exposed to FAS ligand. MIDOSTAURIN may be a potential therapeutic for IF diseases and, notably, it is presently undergoing clinical trials in patients with acute myeloid leukemia and gastrointestinal stromal tumors.

Removal of Syndecan-1 Promotes TRAIL-Induced Apoptosis in Myeloma Cells

Syndecan is the major transmembrane proteoglycan in cells. Of the four syndecans, syndecan-1 is the dominant form expressed in multiple myeloma and is an indicator of poor prognosis. In the current study, we observed that early TRAIL-induced apoptotic processes were accompanied by cleavage of syndecan-1 intracellular region, and explored the possibility whether removal of syndecan-1 promotes apoptotic processes. We found that syndecan-1 knockdown by specific small interfering RNA in multiple myeloma enhanced TRAIL-induced apoptosis, even though the expression of TRAIL receptors and several apoptosis-associated molecules was unaffected. The enhanced TRAIL-mediated apoptosis in syndecan-1-deficient cells was not due to a decrease in surface heparan sulfate or a reduction in TRAIL receptor endocytosis. The increase in TRAIL-induced cell death was accompanied by an elevated caspase-8 activation and an enhanced formation of death-inducing signaling complexes, which could be attributed to an increased expression of TRAIL receptor O-glycosylation enzyme in syndecan-1-deficient cells. We also found that in H9 lymphoma and Jurkat cells, knockdown of the predominant syndecan member also led to an increase in Fas ligand-induced apoptosis. Our results demonstrate that syndecan plays a negative role in death receptor-mediated cell death, suggesting potential application of syndecan downregulation in the treatment of myeloma in combination with TRAIL.

The Ribonucleotide Reductase R1 Subunits of Herpes Simplex Virus 1 and 2 Protect Cells against Poly(I · C)-Induced Apoptosis

We recently provided evidence that the ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 (HSV-1 and -2) protect cells against tumor necrosis factor alpha- and Fas ligand-induced apoptosis by interacting with caspase 8. Double-stranded RNA (dsRNA) is a viral intermediate known to initiate innate antiviral responses. Poly(I · C), a synthetic analogue of viral dsRNA, rapidly triggers caspase 8 activation and apoptosis in HeLa cells. Here, we report that HeLa cells after HSV-1 and HSV-2 infection were quickly protected from apoptosis caused by either extracellular poly(I · C) combined with cycloheximide or transfected poly(I · C). Cells infected with the HSV-1 R1 deletion mutant ICP6Δ were killed by poly(I · C), indicating that HSV-1 R1 plays a key role in antiapoptotic responses to poly(I · C). Individually expressed HSV R1s counteracted caspase 8 activation by poly(I · C). In addition to their binding to caspase 8, HSV R1s also interacted constitutively with receptor-interacting protein 1 (RIP1) when expressed either individually or with other viral proteins during HSV infection. R1(1-834)-green fluorescent protein (GFP), an HSV-2 R1 deletion mutant protein devoid of antiapoptotic activity, did not interact with caspase 8 and RIP1, suggesting that these interactions are required for protection against poly(I · C). HSV-2 R1 inhibited the interaction between the Toll/interleukin-1 receptor domain-containing adaptor-inducing beta interferon (IFN-β) (TRIF) and RIP1, an interaction that is essential for apoptosis triggered by extracellular poly(I · C) plus cycloheximide or TRIF overexpression. TRIF silencing reduced poly(I · C)-triggered caspase 8 activation in mock- and ICP6Δ-infected cells, confirming that TRIF is involved in poly(I · C)-induced apoptosis. Thus, by interacting with caspase 8 and RIP1, HSV R1s impair the apoptotic host defense mechanism prompted by dsRNA.

IL-27 increases intestinal epithelial cell proliferation and migration and its mRNA expression is up-regulated in active Crohn's disease

Background & Aims: Interleukin (IL)-17F is a novel proinflammatory cytokine which is a member of the IL-17 cytokine family. IL-17F mediates signal transduction via the heterodimeric IL-17RA/IL-17RC receptor complex. IL-17F is closely related to IL-17A. Both cytokines are secreted by Th17 cells which play a major role in the pathogenesis of Crohn's disease (CD) and other autoimmune diseases. Previous studies demonstrated that IL-17RA, the specific receptor for IL-17A, is expressed in intestinal epithelial cells (IECs). We therefore analyzed expression, signal transduction and potential biological functions of IL-17F in IECs. Methods: Expression studies were performed by RT-PCR, Western blot analysis, and immunohistochemical analysis. IL-17F mediated signaling was analyzed in Western blot experiments. IEC restitution was analyzed by wounding assays in IEC monolayer cultures. Cell proliferation was measured in MTS assays. The effect of IL-17F on apoptosis was analyzed by flow cytometry. Results: Using RT-PCR, we demonstrated expression of IL17RA and IL-17RC in several colorectal cancer (CRC)-derived IEC lines. High IL-17RC expression was found especially in DLD-1 cells which was also confirmed by Western blot analysis and immunohistochemistry. DLD-1 cells were therefore used in the following experiments. IL-17F mediated the activation of ERK-1/2 and p38 MAP kinases and of Akt but had no significant effect on the phosphorylation levels of SAPK/JNK. IL-17F mediated signals resulted in significantly increased cell proliferation (p=0.01) and an increase of IEC restitution in wounding assays (p<0.0001). However, IL-17F had no significant effect on Fas ligand-induced apoptosis. Importantly, colonic IL-17F mRNA expression was significantly increased in inflamed colonic lesions in CD. Conclusions: Here, we demonstrate for the first time expression of the functional IL-17F receptor complex in CRC-derived IECs. IL-17F mediates similar functions as the Th17 cytokine IL-22 including up-regulation of IEC migration and proliferation suggesting tumor promoting effects in CRC. Considering that previous studies demonstrated that IL-23 promotes tumour incidence and growth (Nature 2006; 442:461-5), we propose that some of these effects may be mediated via IL-23Rexpressing Th17 cells promoting IL-17F secretion. Given the increased IL-17F expression in CD, we currently analyze whether the IL-17F receptor complex is also expressed in primary IECs modulating epithelial barrier function in intestinal inflammation.

S1721 The Selective Glucocorticoid Receptor Agonist CpdA Offers Anti-Inflammatory Action Without Affecting TGF-β Mediated Intestinal Epithelial Wound Healing

Background & Aims: Interleukin (IL)-17F is a novel proinflammatory cytokine which is a member of the IL-17 cytokine family. IL-17F mediates signal transduction via the heterodimeric IL-17RA/IL-17RC receptor complex. IL-17F is closely related to IL-17A. Both cytokines are secreted by Th17 cells which play a major role in the pathogenesis of Crohn's disease (CD) and other autoimmune diseases. Previous studies demonstrated that IL-17RA, the specific receptor for IL-17A, is expressed in intestinal epithelial cells (IECs). We therefore analyzed expression, signal transduction and potential biological functions of IL-17F in IECs. Methods: Expression studies were performed by RT-PCR, Western blot analysis, and immunohistochemical analysis. IL-17F mediated signaling was analyzed in Western blot experiments. IEC restitution was analyzed by wounding assays in IEC monolayer cultures. Cell proliferation was measured in MTS assays. The effect of IL-17F on apoptosis was analyzed by flow cytometry. Results: Using RT-PCR, we demonstrated expression of IL17RA and IL-17RC in several colorectal cancer (CRC)-derived IEC lines. High IL-17RC expression was found especially in DLD-1 cells which was also confirmed by Western blot analysis and immunohistochemistry. DLD-1 cells were therefore used in the following experiments. IL-17F mediated the activation of ERK-1/2 and p38 MAP kinases and of Akt but had no significant effect on the phosphorylation levels of SAPK/JNK. IL-17F mediated signals resulted in significantly increased cell proliferation (p=0.01) and an increase of IEC restitution in wounding assays (p<0.0001). However, IL-17F had no significant effect on Fas ligand-induced apoptosis. Importantly, colonic IL-17F mRNA expression was significantly increased in inflamed colonic lesions in CD. Conclusions: Here, we demonstrate for the first time expression of the functional IL-17F receptor complex in CRC-derived IECs. IL-17F mediates similar functions as the Th17 cytokine IL-22 including up-regulation of IEC migration and proliferation suggesting tumor promoting effects in CRC. Considering that previous studies demonstrated that IL-23 promotes tumour incidence and growth (Nature 2006; 442:461-5), we propose that some of these effects may be mediated via IL-23Rexpressing Th17 cells promoting IL-17F secretion. Given the increased IL-17F expression in CD, we currently analyze whether the IL-17F receptor complex is also expressed in primary IECs modulating epithelial barrier function in intestinal inflammation.

Acidic Mammalian Chitinase Regulates Epithelial Cell Apoptosis via a Chitinolytic-Independent Mechanism

Acidic mammalian chitinase (AMCase) is produced during and plays an important role in the pathogenesis of Th2-mediated diseases and antiparasite responses. However, the effector responses of AMCase in these settings have not been adequately defined and the relationship(s) between its chitinolytic and other biologic properties have not been investigated. In these studies, we demonstrate that AMCase protects airway epithelial cells from Fas ligand- and growth factor withdrawal-induced apoptosis. This cytoprotection was associated with Akt phosphorylation and abrogated when the PI3K/Akt pathway was inhibited. Comparable cytoprotection was also seen in experiments comparing wild-type AMCase and mutant AMCase that lacked chitinolytic activity. Importantly, the apoptosis-inhibiting effect of enzymatically active and inactive AMCase was abrogated by treatment with allosamidin. These studies demonstrate that secreted AMCase feeds back in an autocrine and/or paracrine manner to protect pulmonary epithelial cells from growth factor withdrawal- and Fas ligand-induced apoptosis. They also demonstrate that the cytoprotection is mediated via a PI3K/Akt-dependent and allosamidin-sensitive pathway that is independent of the chitinolytic activity of this chitinase.

C/EBPα or C/EBPα oncoproteins regulate the intrinsic and extrinsic apoptotic pathways by direct interaction with NF-κB p50 bound to the bcl-2 and FLIP gene promoters

CCAAT/enhancer binding protein α (C/EBPα) is mutated in 10% of acute myeloid leukemias, resulting in either a truncated protein or an altered leucine zipper (C/EBPαLZ) that prevents DNA-binding. C/EBPα induces bcl-2 in cooperation with NF-κB p50 to inhibit apoptosis. We now demostrate that C/EBPα or a C/EBPαLZ oncoprotein bind the bcl-2 P2 promoter in chromatin immunoprecipitation assays and induce the promoter dependent on the integrity of a κB site. C/EBPα expressed as a transgene in B cells binds and activates the bcl-2 promoter, but not in nfkb1−/− mice lacking NF-κB p50. Bcl-2 is central to the intrinsic apoptotic pathway, while FLICE inhibitory protein (FLIP) modulates caspase-8, the initiator caspase of the extrinsic pathway. C/EBPα and C/EBPαLZ also bind the FLIP promoter and induce its expression dependent upon NF-κB p50. Moreover, induction of FLIP by C/EBPα protects splenocytes from Fas ligand-induced apoptosis, but only if p50 is present. We also demonstrate direct interaction between bacterially produced C/EBPα and NF-κB p50, mediated by the C/EBPα basic region. These findings indicate that C/EBPα or its oncoproteins activate the bcl-2 and FLIP genes by tethering to their promoters via bound NF-κB p50. Targeting their interaction may favor apoptosis of transformed cells.

Open Reading Frame 2, Encoded by the Latency-Related Gene of Bovine Herpesvirus 1, Has Antiapoptotic Activity in Transiently Transfected Neuroblastoma Cells

The latency-related (LR) RNA encoded by bovine herpesvirus 1 is abundantly expressed in the trigeminal ganglia of latently infected calves. Expression of LR proteins is necessary for reactivation from latency and the protection of infected neurons from apoptosis. In this study, we demonstrated that an LR-encoded protein, open reading frame 2 (ORF-2), or ORF-2 fusion proteins encoded by alternatively spliced LR transcripts inhibit cold shock or Fas ligand-induced apoptosis in mouse neuroblastoma (neuro-2A) cells. Frameshift mutants of ORF-2 do not inhibit apoptosis, which suggests that protein expression, not LR RNA expression, mediates the antiapoptotic activity of the LR gene in transfected neuro-2A cells.

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cε

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cε (PKCε) but not PKCα. In the absence of NO production PKCε interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCε using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCε plays an important role in the regulation of Fas-induced apoptosis.

Mithramycin A activates Fas death pathway in leukemic cell lines

Mithramycin A (MMA, trade name Plicamycin) can facilitate TNFalpha- (Tumor Necrosis Factor) and Fas ligand-induced apoptosis. Besides, several drugs play their anticancer effect through Fas apoptotic pathway. So we investigated the effect of MMA on Fas signaling. In this study we show that MMA induces apoptosis in Fas sensitive Jurkat cells and Fas resistant KG1a cells. This effect involves Fas apoptotic pathway: cell exposure to MMA leads to Fas clustering at the cell surface, DISC (Death Inducing Signaling Complex) formation and caspase cleavage. This phenomenon is independent of Fas ligand/Fas interaction and blockade of Fas death pathway partially inhibits MMA-induced apoptosis. Moreover the activation of Fas apoptotic pathway by MMA is correlated to the modulation of c-Flip(L) expression. Finally, pre-treatment with sub-lethal doses of MMA sensitizes KG1a cells to chemotherapeutic agents. Thus all these results may have important implications to improve clinical treatments.

Transgenic Expression of Dominant-Negative Fas-Associated Death Domain Protein in β Cells Protects against Fas Ligand-Induced Apoptosis and Reduces Spontaneous Diabetes in Nonobese Diabetic Mice

In type 1 diabetes, many effector mechanisms damage the beta cell, a key one being perforin/granzyme B production by CD8(+) T cells. The death receptor pathway has also been implicated in beta cell death, and we have therefore generated NOD mice that express a dominant-negative form of the Fas-associated death domain protein (FADD) adaptor to block death receptor signaling in beta cells. Islets developed normally in these animals, indicating that FADD is not necessary for beta cell development as it is for vasculogenesis. beta cells from the transgenic mice were resistant to killing via the Fas pathway in vitro. In vivo, a reduced incidence of diabetes was found in mice with higher levels of dominant-negative FADD expression. This molecule also blocked signals from the IL-1R in culture, protecting isolated islets from the toxic effects of cytokines and also marginally reducing the levels of Fas up-regulation. These data support a role for death receptors in beta cell destruction in NOD mice, but blocking the perforin/granzyme pathway would also be necessary for dominant-negative FADD to have a beneficial clinical effect.

CEACAM1 (CD66a) mediates delay of spontaneous and Fas ligand-induced apoptosis in granulocytes

Granulocytes form the first and fastest line of defense against pathogenic infections. Their survival is limited by apoptosis, a process that is critical for the resolution of inflammation. Pro-apoptotic and pro-inflammatory cytokines, as well as several receptors, can alter the lifespan of granulocytes. Here we report that the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CD66a) is involved in the regulation of granulocyte survival. Until now CEACAM1 is described to control cell proliferation, cell migration, tumor growth, angiogenesis and diverse leukocyte functions. However, very little is known about its role in granulocytes. We found that CEACAM1 expression in resting rat granulocytes is significantly higher than in other leukocyte subtypes. Stimulation led to a strongly increased CEACAM1 cell surface expression and to release of soluble CEACAM1. DNA fragmentation assays and annexin V staining revealed that binding of CEACAM1-specific antibodies, Fab fragments and soluble CEACAM1-Fc constructs to cell surface-expressed CEACAM1 causes a delay of spontaneous and Fas ligand (CD95L)-induced apoptosis. Tyrosine phosphorylation of CEACAM1-L, its association with SHP-1, the activation of Erk1/2 and caspase-3 appeared to be crucial for the CEACAM1-mediated anti-apoptotic effect. These findings provide evidence that CEACAM1 influences the resolution of inflammation by prolonging the survival of rat granulocytes.