TNF-related Apoptosis-inducing Ligand Promoter Research Articles

Snoopligase-catalyzed molecular glue enables efficient generation of hyperoligomerized TRAIL variant with enhanced antitumor effect

Clinical application of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is predominantly limited by its inefficient apoptosis induction in tumor cells, which might be improved by using molecular superglue-mediated hyperoligomerization to increase its valency. Here, the minimal superglue peptide pairs, including Snoopligase-catalyzed SnoopTagJr/SnoopDogTag and SpyStapler-catalyzed SpyTag/SpyBDTag, were individually fused at the N- or C-terminus of the TRAIL promoter to produce superglue-fusion TRAIL variants. Similar to native trivalent TRAIL, these superglue-fusion TRAIL variants were highly expressed in Escherichia coli (E. coli) and spontaneously trimerized. In the presence of Snoopligase or SpyStapler, the trivalent superglue-fusion TRAIL variants were predominantly crosslinked into hexavalent TRAIL variants. Nevertheless, Snoopligase was more efficient than SpyStapler in the production of hexavalent TRAIL variants. In particular, Snoopligase-catalyzed trivalent TRAIL variants with N-terminal fusion of SnoopTagJr/SnoopDogTag produced hexavalent SnHexaTR with the highest yield (∼70%). The in vitro cytotoxicity of SnHexaTR was 10–40 times greater than that of TRAIL in several tumor cells. In addition, compared to trivalent TRAIL, hexavalent SnHexaTR showed a longer serum half-life and greater tumor uptake, which resulted in eradication of 50% of tumor xenografts of TRAIL-sensitive COLO 205. In mice bearing TRAIL-resistant HT-29 tumor xenografts, hexavalent SnHexaTR combined with bortezomib encapsulated in liposomes also showed robust tumor growth suppression, indicating that hyperoligomerization mediated by minimal molecular superglue significantly increased the cytotoxicity and antitumor effect of TRAIL. As a novel anticancer agent candidate, the hexavalent SnHexaTR has great potential for clinical application in cancer therapy.

Tumor necrosis factor‑related apoptosis‑inducing ligand is a novel transcriptional target of runt‑related transcription factor1.

Runt-related transcription factor 1 (RUNX1), which is also known as acute myeloid leukemia 1 (AML1), has been frequently found with genomic aberrations in human leukemia. RUNX1 encodes a transcription factor that can regulate the expression of hematopoietic genes. In addition, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) performs an important function for malignant tumors in immune surveillance. However, the regulatory mechanism of TRAIL expression remain to be fully elucidated. In the present study, tetradecanoylphorbol 13-acetate-treated megakaryocytic differentiated K562 cells was used to examine the effect of RUNX1 on TRAIL expression. Luciferase assay series of TRAIL promoters for the cells co-transfected with RUNX1 and core-binding factor β (CBFβ) expression vectors were performed to evaluate the nature of TRAIL transcriptional regulation. Electrophoresis mobility shift assay of the RUNX1 consensus sequence of the TRAIL promoter with recombinant RUNX1 and CBFβ proteins was also performed. BloodSpot database analysis for TRAIL expression in patients with acute myeloid leukemia were performed. The expression of TRAIL, its receptor Death receptor 4 and 5 and RUNX1 in K562 cells transfected with the RUNX1 expression vector and RUNX1 siRNA were evaluated by reverse transcription-quantitative PCR (RT-qPCR). TRAIL and RUNX1-ETO expression was also measured in Kasumi-1 cells transfected with RUNX1-ETO siRNA and in KG-1 cells transfected with RUNX1-ETO expression plasmid, both by RT-qPCR. Cell counting, lactate dehydrogenase assay and cell cycle analysis by flow cytometry were performed on Kasumi-1, KG-1, SKNO-1 and K562 cells treated with TRAIL and HDAC inhibitors sodium butyrate or valproic acid. The present study demonstrated that RUNX1 is a transcriptional regulator of TRAIL. It was initially found that the induction of TRAIL expression following the megakaryocytic differentiation of human leukemia cells was RUNX1-dependent. Subsequently, overexpression of RUNX1 was found to increase TRAIL mRNA expression by activating its promoter activity. Additional analyses revealed that RUNX1 regulated the expression of TRAIL in an indirect manner, because RUNX1 retained its ability to activate this promoter following the mutation of all possible RUNX1 consensus sites. Furthermore, TRAIL expression was reduced in leukemia cells carrying the t(8;21) translocation, where the RUNX1-ETO chimeric protein interfere with normal RUNX1 function. Exogenous treatment of recombinant TRAIL proteins was found to induce leukemia cell death. To conclude, the present study provided a novel mechanism, whereby TRAIL is a target gene of RUNX1 and TRAIL expression was inhibited by RUNX1-ETO. These results suggest that TRAIL is a promising agent for the clinical treatment of t(8;21) AML.

Dihydrotestosterone Increases Cytotoxic Activity of Macrophages on Prostate Cancer Cells via TRAIL.

Although androgen deprivation therapy (ADT) and immunotherapy are potential treatment options in men with metastatic prostate cancer (CaP), androgen has conventionally been proposed to be a suppressor of the immune response. However, we herein report that DHT activates macrophages. When the murine macrophage cell line (RAW 264.7), human monocyte cell line (THP-1), and human peripheral blood monocytes were cultured with androgen-resistant CaP cell lines, DHT increased cytotoxicity of macrophages in a concentration-dependent manner. Further studies revealed that DHT induced M1 polarization and increased the expression levels of TNF-related apoptosis-inducing ligand (TRAIL) in macrophages and that this effect was abrogated when TRAIL was neutralized with a blocking antibody or small interfering RNA. Subsequent experiments demonstrated that induction of TRAIL expression was regulated by direct binding of androgen receptor to the TRAIL promoter region. Finally, an in vivo mouse study demonstrated that castration enhanced the growth of an androgen-resistant murine CaP tumor and that this protumorigenic effect of castration was blocked when macrophages were removed with clodronate liposomes. Collectively, these results demonstrate that DHT activates the cytotoxic activity of macrophages and suggest that immunotherapy may not be optimal when combined with ADT in CaP.

A ‐1573T>C SNP within the human TRAIL promoter determines TRAIL expression and HCC tumor progression

The cytokine tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) induces apoptosis in liver cancer cells but not in normal liver cells. Therefore, TRAIL got credited to play a role in hepatocellular carcinoma (HCC) development and progression. Impaired expression of TRAIL in HCC cells and sequence variations in the TRAIL promoter may facilitate development, growth, and spread . The TRAIL promoter was sequenced from liver tissue of 93 patients undergoing partial liver resection (PRT) or liver transplantation (LT) for HCC. TRAIL mRNA expression was investigated by quantitative real‐time PCR. A variant ‐1573T>C (single‐nucleotide polymorphism; C, cytosine) SNP was characterized by electron mobility shift assay and supershift assays. Functionality of the ‐1573T>C SNP was analyzed in reporter gene assays and cell migration assays. In approximately 30% of HCC samples, a loss‐of‐function shift of the binding pattern due to a ‐1573T>C SNP was found within the human TRAIL promoter. Correlation analysis revealed significantly lower TRAIL expression in HCC samples with the ‐1573C sequence (P ≤ 0.05). Reporter gene assays revealed significantly reduced inducibility of the TRAIL promoter due to the ‐1573C sequence. The variant ‐1573C sequence impaired not only binding of transcription factors but also expression of TRAIL. Interestingly, this impairment resulted in enhanced migration activity and colony formation of the liver tumor cells. Our findings suggest that loss of function of the human TRAIL promoter due to the ‐1573T>C SNP leads to reduced expression and impaired inducibility of TRAIL, with the consequence of enhanced growth and migration of tumor cells, ultimately resulting in the progression of the HCC.

Pro-inflammatory sebocyte growth and survival signalling in acne vulgaris are reversed by pro-apoptotic isotretinoin signalling.

Pro-inflammatory sebocyte growth and survival signalling in acne vulgaris are reversed by pro-apoptotic isotretinoin signalling.

Insulin promotes vascular smooth muscle cell proliferation and apoptosis via differential regulation of tumor necrosis factor-related apoptosis-inducing ligand.

Insulin regulates glucose homeostasis but can also promote vascular smooth muscle (VSMC) proliferation, important in atherogenesis. Recently, we showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) stimulates intimal thickening via accelerated growth of VSMCs. The aim of the present study was to determine whether insulin-induced effects on VSMCs occur via TRAIL. Expression of TRAIL and TRAIL receptor in response to insulin and glucose was determined by polymerase chain reaction. Transcriptional activity was assessed using wild-type and site-specific mutations of the TRAIL promoter. Chromatin immunoprecipitation studies were performed. VSMC proliferation and apoptosis was measured. Insulin and glucose exposure to VSMC for 24 h stimulated TRAIL mRNA expression. This was also evident at the transcriptional level. Both insulin- and glucose-inducible TRAIL transcriptional activity was blocked by dominant-negative specificity protein-1 (Sp1) overexpression. There are five functional Sp1-binding elements (Sp1-1, Sp1-2, Sp-5/6 and Sp1-7) on the TRAIL promoter. Insulin required the Sp1-1 and Sp1-2 sites, but glucose needed all Sp1-binding sites to induce transcription. Furthermore, insulin (but not glucose) was able to promote VSMC proliferation over time, associated with increased decoy receptor-2 (DcR2) expression. In contrast, chronic 5-day exposure of VSMC to 1 µg/mL insulin repressed TRAIL and DcR2 expression, and reduced Sp1 enrichment on the TRAIL promoter. This was associated with increased cell death. The findings of the present study provide a new mechanistic insight into how TRAIL is regulated by insulin. This may have significant implications at different stages of diabetes-associated cardiovascular disease. Thus, TRAIL may offer a novel therapeutic solution to combat insulin-induced vascular pathologies.

The DNA demethylating agent decitabine activates the TRAIL pathway and induces apoptosis in acute myeloid leukemia

Although epigenetic drugs have been approved for use in selected malignancies, there is significant need for a better understanding of their mechanism of action. Here, we study the action of a clinically approved DNA-methyltransferase inhibitor – decitabine (DAC) – in acute myeloid leukemia (AML) cells. At low doses, DAC treatment induced apoptosis of NB4 Acute Promyelocytic Leukemia (APL) cells, which was associated with the activation of the extrinsic apoptotic pathway. Expression studies of the members of the Death Receptor family demonstrated that DAC induces the expression of TNF-related apoptosis-inducing ligand (TRAIL). Upregulation of TRAIL, upon DAC treatment, was associated with specific epigenetic modifications induced by DAC in the proximity of the TRAIL promoter, as demonstrated by DNA demethylation, increased DNaseI sensitivity and histone acetylation of a non-CpG island, CpG-rich region located 2kb upstream to the transcription start site. Luciferase assay experiments showed that this region behave as a DNA methylation sensitive transcriptional regulatory element. The CpG regulatory element was also found methylated in samples derived from APL patients. These findings have been confirmed in the non-APL, AML Kasumi cell line, suggesting that this regulatory mechanism may be extended to other AMLs. Our study suggests that DNA methylation is a regulatory mechanism relevant for silencing of the TRAIL apoptotic pathway in leukemic cells, and further elucidates the mechanism by which epigenetic drugs mediate their anti-leukemic effects.

Sp1, acetylated histone‐3 and p300 regulate TRAIL transcription: Mechanisms of PDGF‐BB‐mediated VSMC proliferation and migration

We recently reported that TNF-related apoptosis-inducing ligand (TRAIL) is important in atherogenesis, since it can induce vascular smooth muscle cell (VSMC) proliferation and arterial thickening following injury. Here we show the first demonstrate that TRAIL siRNA reduces platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMC proliferation and migration. PDGF-BB-inducible VSMC proliferation was completely inhibited in VSMCs isolated from aortas of TRAIL(-/-) mice; whereas inducible migration was blocked compared to control VSMCs. TRAIL transcriptional control mediating this response is not established. TRAIL mRNA, protein and promoter activity was increased by PDGF-BB and subsequently inhibited by dominant-negative Sp1, suggesting that the transcription factor Sp1 plays a role. Sp1 bound multiple Sp1 sites on the TRAIL promoter, including two established (Sp1-1 and -2) and two novel Sp1-5/6 and -7 sites. PDGF-BB-inducible TRAIL promoter activity by Sp1 was mediated through these sites, since transverse mutations to each abolished inducible activity. PDGF-BB stimulation increased acetylation of histone-3 (ac-H3) and expression of the transcriptional co-activator p300, implicating chromatin remodelling. p300 overexpression increased TRAIL promoter activity, which was blocked by dominant-negative Sp1. Furthermore, PDGF-BB treatment increased the physical interaction of Sp1, p300 and ac-H3, while chromatin immunoprecipitation studies revealed Sp1, p300 and ac-H3 enrichment on the TRAIL promoter. Taken together, our studies demonstrate for the first time that PDGF-BB-induced TRAIL transcriptional activity requires the cooperation of Sp1, ac-H3 and p300, mediating increased expression of TRAIL which is important for VSMC proliferation and migration. Our findings have the promising potential for targeting TRAIL as a new therapeutic for vascular proliferative disorders.

Abstract 241: A novel function of thyroid hormone receptors in promoting metastasis of human hepatoma cells via regulation of TRAIL

Abstract Thyroid hormone (T3) and its binding receptors (TRs) are potent regulators mediating cell metabolism, proliferation, and differentiation. Accumulating evidence has confirmed an important role of TRs in tumor progression. However, the specific functions of TRs in carcinogenesis have yet to be clarified. Experiments from the present study have shown that tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is upregulated by T3 in TR-overexpressing hepatoma cell lines. The T3-responsive element of the TRAIL promoter was localized to nucleotides at positions −1967 to −1977 upstream of the transcription start site. Interestingly, TRAIL acts as an apoptosis inducer in several cancer cell types, but also triggers non-apoptotic signals favoring tumorigenesis in apoptosis-resistant cancer cells. In our experiments, TR-overexpressing hepatoma cells treated with T3 were apoptosis-resistant, even under conditions where TRAIL was upregulated. This finding may be attributed to the simultaneous upregulation of Bcl-xL, an anti-apoptosis gene, by T3. Thus, overexpression of Bcl-xL in hepatoma cells possibly protects against apoptotic death triggered by TRAIL, consequently leading to TRAIL-promoted metastasis in hepatoma cells. Moreover, T3-enhanced metastasis was repressed by the treatment of TRAIL-blocking antibody. Notably, TRAIL was highly expressed in tumor cells of HCC patients, and this high expression was significantly correlated with the high TR expression as examined in 65 HCC tissues. Collectively, our findings indicate that TR directly binds to the TRAIL promoter to induce TRAIL expression, which acts in concert with the simultaneously activated anti-apoptotic factor, Bcl-xL, to promote metastasis, rather than apoptosis. Our findings provide a novel mechanistic link for increased TR and TRAIL in HCC. 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 241. doi:1538-7445.AM2012-241

PKC-δ mediates interferon-α-induced apoptosis through c-Jun NH2-terminal kinase activation

BackgroundInterferon-α (IFN-α) exerts an anti-tumor effect at least through induction of apoptosis in a variety of types including B lymphoma cells. We recently found that IFN-α induced a sustained activation of c-Jun NH2-terminal kinase1 (JNK1), which is implicated in activation of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter. In the present study, we explored upstream component(s) of the prolonged IFN-α-initiated activation of JNK1.ResultsIFN-α caused activation of PKC-δ in Daudi B lymphoma cells and myeloma U266 cells, as detected by Western blotting using a monoclonal antibody specific for the phosphorylated form of PKC-δ. The dominant-negative form of mutant PKC-δ (dnPKC-δ) reduced the IFN-α-induced JNK1 activation, TRAIL promoter activity, loss of mitochondrial membrane potential (ΔΨm), and increase in propidium iodide (PI) positive cells. The IFN-α-induced activation of JNK1 and the TRAIL promoter was also attenuated by the PKC-δ inhibitor rottlerin. Moreover, a constitutively active form of mutant PKC-δ enhanced the IFN-α-induced TRAIL promoter activity and loss of ΔΨm in Daudi B lymphoma cells. In addition, IFN-α-induced Ser727 phosphorylation of Stat1 was also abrogated by dnPKC-δ.ConclusionsIFN-α induced JNK1 activation via PKC-δ, leading to upregulation of TRAIL. The interaction of the consequent enhanced TRAIL expression with TRAIL-receptor results in a loss of ΔΨm and increase in PI positive cells. The IFN-α-induced apoptotic events may also be affected by the Ser727-Stat1 induced by PKC-δ-mediated signaling component(s).

Pigment Epithelium-derived Factor (PEDF) Promotes Tumor Cell Death by Inducing Macrophage Membrane Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)

Pigment epithelium-derived factor (PEDF) is an intrinsic anti-angiogenic factor and a potential anti-tumor agent. The tumoricidal mechanism of PEDF, however, has not been fully elucidated. Here we report that PEDF induces the apoptosis of TC-1 and SK-Hep-1 tumor cells when they are cocultured with bone marrow-derived macrophages (BMDMs). This macrophage-mediated tumor killing is prevented by blockage of TNF-related apoptosis-inducing ligand (TRAIL) following treatment with the soluble TRAIL receptor. PEDF also increases the amount of membrane-bound TRAIL on cultured mouse BMDMs and on macrophages surrounding subcutaneous tumors. PEDF-induced tumor killing and TRAIL induction are abrogated by peroxisome proliferator-activated receptor γ (PPARγ) antagonists or small interfering RNAs targeting PPARγ. PEDF also induces PPARγ in BMDMs. Furthermore, the activity of the TRAIL promoter in human macrophages is increased by PEDF stimulation. Chromatin immunoprecipitation and DNA pull-down assays confirmed that endogenous PPARγ binds to a functional PPAR-response element (PPRE) in the TRAIL promoter, and mutation of this PPRE abolishes the binding of the PPARγ-RXRα heterodimer. Also, PPARγ-dependent transactivation and PPARγ-RXRα binding to this PPRE are prevented by PPARγ antagonists. Our results provide a novel mechanism for the tumoricidal activity of PEDF, which involves tumor cell killing via PPARγ-mediated TRAIL induction in macrophages.

TRAIL Promotes VSMC Proliferation and Neointima Formation in a FGF-2–, Sp1 Phosphorylation–, and NFκB-Dependent Manner

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is well reported as an inducer of apoptosis in tumor models; however, its role and function in vivo in atherosclerosis and vascular injury has not been established. We sought to study the function of TRAIL in cardiovascular pathology and its regulation in vivo. Here, we show that TRAIL was upregulated in medial vascular smooth muscle cells (VSMCs) 24 hours following perivascular cuff placement around femoral arteries of mice. We also show that TRAIL mRNA and promoter activity was induced in VSMCs following in vitro mechanical injury. Intimal thickening 15 days after cuff placement was reduced 2- to 3-fold in TRAIL(-/-) compared to wild-type mice and was reversible by administration of recombinant TRAIL. Additionally, reduced VSMC proliferation was observed in injured arteries of TRAIL(-/-) mice. Fibroblast growth factor (FGF)-2, a potent growth factor released following vascular injury, was also reduced in arteries of TRAIL(-/-) mice, and VSMCs isolated from these animals did not respond to FGF-2 in vitro. Injury and FGF-2 regulated TRAIL transcriptional activity via 2 specificity protein (Sp)1 elements in the proximal TRAIL promoter, a binding site also shared by nuclear factor (NF)kappaB. Mutational studies confirmed a role for Sp1 in injury- and FGF-2-inducible TRAIL transcription. Furthermore, increased NFkappaB expression after injury transactivated the TRAIL promoter. Interestingly, following mechanical injury, Sp1 phosphorylation (Thr453) and an increase in the physical interaction of p-Sp1(Thr453) with NFkappaB was observed. We conclude that TRAIL induction involves FGF-2, Sp1-phosphorylation and NFkappaB and that TRAIL promotes VSMC proliferation and neointima formation after arterial injury.

PU.1 induces apoptosis in myeloma cells through direct transactivation of TRAIL

We earlier reported that PU.1 was downregulated in myeloma cell lines and myeloma cells in a subset of myeloma patients, and that conditional PU.1 expression in PU.1-negative myeloma cell lines, U266 and KMS12PE, induced growth arrest and apoptosis. To elucidate the molecular mechanisms of the growth arrest and apoptosis, we performed DNA microarray analyses to compare the difference in gene expression before and after PU.1 induction in U266 cells. Among cell cycle-related genes, cyclin A2, cyclin B1, CDK2 and CDK4 were downregulated and p21 was upregulated, although among apoptosis-related genes, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) was found highly upregulated. When TRAIL was knocked down by small interference RNAs, apoptosis of PU-1-expressing cells was inhibited, suggesting that TRAIL has a critical role in PU.1-induced apoptosis in both U266 and KMS12PE myeloma cells. In both U266 and KMS12PE cells expressing PU.1, PU.1 directly bound to a region 30 bp downstream of the transcription start site of the TRAIL gene. Upregulation of PU.1-induced transactivation of the TRAIL promoter in reporter assays, and disruption of the PU.1-binding site in the TRAIL promoter eliminated this transactivation. Therefore, we conclude that PU.1 is capable of inducing apoptosis in certain myeloma cells by direct transactivation of TRAIL.

Transforming Growth Factor-β–Mediated Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Expression and Apoptosis in Hepatoma Cells Requires Functional Cooperation between Smad Proteins and Activator Protein-1

Transforming growth factor-beta (TGF-beta) has been shown to induce apoptotic cell death in normal and transformed hepatocytes. We recently identified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as an important mediator of TGF-beta-induced apoptosis in hepatoma cells. In this study, we have further explored the mechanism by which TGF-beta up-regulates TRAIL expression. The 5'-flanking region of the TRAIL gene was isolated and characterized. Deletion mutants of the 5'-untranslated region of the TRAIL gene revealed a region comprising nucleotides -1950 to -1100 responsible for TRAIL induction following treatment with TGF-beta. Within this region, we have identified an activator protein-1 (AP-1) site indispensable for TGF-beta-mediated induction of TRAIL. Activation of this AP-1 site is mediated by a JunD.FosB heterodimer. Expression of DNSmad4, DNJunD, or DNFosB significantly impairs TGF-beta-mediated activation of the TRAIL promoter. Furthermore, with tRNA interference targeting Smad4, junD, FosB, we could abolish TRAIL expression and, subsequently, TGF-beta-induced TRAIL-mediated apoptosis in hepatoma cells. Our results reveal a new AP-1 site within the TRAIL promoter functionally involved in TGF-beta-induced TRAIL expression and apoptosis in hepatomas and thus provide evidence for the underlying mechanism by which TGF-beta might regulate cell death in liver cancer.

Absence of Gene Mutation in TRAIL Receptor 1 (TRAIL-R1) and TRAIL Receptor 2 (TRAIL-R2) in Chronic Myelogenous Leukemia and Myelodysplastic Syndrome, and Analysis of mRNA Expressions of TRAIL and TRAIL-Related Genes in Chronic Myelogenous Leukemia

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an interferon (IFN)-induced molecule with apoptotic activity. We examined gene mutations in the death domains of TRAIL receptor 1 (TRAIL-R1) and TRAIL receptor 2 (TRAIL-R2), and in the TRAIL gene promoter in 46 chronic myelogenous leukemia (CML) patients. In 23 of the 46 patients, all the coding regions of TRAIL-R2 were also examined. However, no mutation or loss of heterozygosity was found. Furthermore, no mutation in the death domains of TRAIL-R1 and TRAIL-R2 genes, which causes amino acid change, was found in 18 myelodysplastic syndrome (MDS) patients. Ribonuclease protection assay (RPA) and real-time quantitative polymerase chain reaction using polymorphonuclear neutrophils of five new CML patients showed that the TRAIL mRNA expression was very low before in vitro IFN-α stimulation and markedly upregulated after IFN-α stimulation. FAS mRNA was also upregulated with IFN-α stimulation but the fold induction was far lower than that of TRAIL mRNA. In addition, RPA revealed that the ratio of (TRAIL-R1 plus TRAIL-R2) to TRAIL-R3 was also increased after IFN-α stimulation. Taken together, gene mutations of TRAIL-R1, TRAIL-R2 are infrequent in patients with CML and MDS. Andso is the TRAIL promoter for CML. These mutations seem unrelated to tumorigenesis, disease progression, and response to IFN-α therapy in CML. A markedly high induction of TRAIL mRNA by IFN-α may have some relevance to IFN-α action in CML patients.

Expression of TNF-related apoptosis-inducing ligand (TRAIL) in megakaryocytes and platelets

Platelets are known to play an important role in hemostasis, thrombosis, wound healing, and inflammation. Platelet-induced modulation of inflammation and adaptive immune responses are mediated in part through tumor necrosis factor (TNF) family member ligands, including CD154, Fas ligand, and TNFalpha, that are expressed upon platelet activation. The present study investigated whether platelets and megakaryocytes also express TNF-related apoptosis-inducing ligand (TRAIL), another pro-apoptotic member of the TNF superfamily. Immunoprecipitation, enzyme-linked immunosorbent assay, and flow cytometry were used to assess TRAIL protein expression on isolated platelets, in vitro-derived megakaryocytes and premegakaryocyte cell lines. Reverse-transcription polymerase chain reaction and transient transfection of TRAIL promoter/reporter constructs were used to elucidate mechanisms of TRAIL regulation during megakaryocyte differentiation. TRAIL-dependent cytotoxicity assays were performed to determine if platelet-derived TRAIL induces apoptosis of TRAIL sensitive target cells. Activated platelets expressed both membrane-bound and soluble TRAIL. TRAIL was also expressed by megakaryocytes, and in vitro studies showed that TRAIL expression was induced upon megakaryocyte differentiation. TRAIL expression was mediated by increased transcriptional activity of the TRAIL promoter, suggesting lineage-specific regulation of TRAIL during megakaryocyte differentiation. Abundant detergent-extractable, full-length TRAIL protein was observed in the lysates of platelets and megakaryocytes, but only low concentrations of TRAIL were released by nondetergent extraction methods. The data reported herein show that platelets express TRAIL that is synthesized by megakaryocytes and was expressed by activated platelets. While these data expand the spectrum of TNF family proteins expressed in platelets, the function of platelet-derived TRAIL is not known.

Identification and functional characterization of a highly polymorphic region in the human TRAIL promoter in multiple sclerosis

TNF-related apoptosis-inducing ligand (TRAIL) is not only involved in cell death but also in other immunoregulatory mechanisms. So far, the regulation of the TRAIL pathway in physiologic and pathologic conditions remains unclear. Due to the implication in brain damage and the elevated expression in peripheral immune cells of patients with multiple sclerosis (MS), an autoimmune disease of the central nervous system, TRAIL might play a central role in the pathology of this disease. Here, we have identified a highly polymorphic region in the TRAIL promoter. Using single-strand conformation polymorphism analysis, we found four single nucleotide polymorphisms (SNPs) within 111 base pairs. One of these SNPs is located in a binding site for the transcription factor AP-1. However, the RNA and protein expression of TRAIL revealed no obvious differences in relation to the genotypes. Furthermore, investigating samples from both MS patients and healthy controls we could not detect any association of these newly described polymorphisms to the clinical disease pattern. Thus, the TRAIL promoter contains a highly polymorphic area which has, however, no impact on molecule expression, and is neither directly related to increased risk of developing MS nor associated with a certain course of this heterogeneous disease in our population.

TGF-beta regulates the expression of TRAIL in hepatoma cells– regulation of programmed cell death in the liver

TGF-beta is a pleiotropic cytokine that is involved in the development of liver cirrhosis and of tumors in different organs. TGF-beta suppresses the immune response and supports immune escape of certain tumors. Moreover, TGF-beta has been shown to induce apoptosis in normal or transformed hepatocytes. However, the mechanism by which apoptosis is induced is largely unknown. We examined whether death receptor-ligand systems play a role in TGF-beta induced apoptosis of hepatoma cells. We examined a potential role of various death receptor/ligand systems in TGF-beta-induced apoptosis and demonstrate, that the TNF-related apoptosis inducing ligand (TRAIL) is critically involved in TGF-beta-induced apoptosis in liver cells. TGF-beta- induced apoptosis is significantly impaired upon blockage of TRAIL in TRAIL sensitive hepatoma and immune cells. We show that TRAIL is upregulated in hepatoma cells upon treatment with TGF-beta, while TRAIL receptor levels remain unchanged. The TRAIL promoter is a direct target of TGF-beta signaling, as it is activated through the action of the TGF-beta signaling molecules Smad2, Smad3 and Smad4. Furthermore, we demonstrate that an AP-1 element in the TRAIL promoter is crucial for TGF-beta-induction of TRAIL expression. Our results provide evidence that the TRAIL system is critically involved in TGF-beta-induced cell death in liver pathology.

Antiviral response by natural killer cells through TRAIL gene induction by IFN-alpha/beta.

Natural killer (NK) cells play an important role in early defense against viral infection. The cytotoxic activity of NK cells is increased by interferon-alpha/beta (IFN-alpha/beta), produced en masse in virally infected cells. However, the mechanism(s) by which IFN-alpha/beta contribute to the NK-cell-mediated antiviral response is not well understood. Here we provide evidence that the cytotoxicity of NK cells is enhanced by IFN-alpha/beta through induction of TNF-related apoptosis-inducing ligand (TRAIL). Isolation and analysis of the murine TRAIL promoter revealed the presence of an IFN-stimulated response element (ISRE), which binds to the transcription factor ISGF3 (interferon stimulated gene factor-3). This promoter is indeed activated by IFN-beta in ISGF3-dependent manner. We also show that virally infected cells, but not uninfected cells, are susceptible to TRAIL-mediated cytotoxicity in vitro, and that the TRAIL expressed in NK cells is indeed crucial in limiting virus replication in vivo. Thus, our study reveals a new molecular link between IFN-alpha/beta signaling and activation of NK cells in antiviral response of the host.