Tumor Necrosis Factor-related Apoptosis-inducing Ligand mRNA Expression Research Articles

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.

Follow-up Analysis of Serum TNF-Related Apoptosis-Inducing Ligand Protein and mRNA Expression in Peripheral Blood Mononuclear Cells from Patients with Ischemic Stroke.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), which is TNF receptor superfamily member, contributes to several diseases pathogenesis. The aim of this research was to investigate the relevance of serum TRAIL protein levels and mRNA expression in peripheral blood mononuclear cells (PBMC) of patients with stroke through 6 months follow-up. We enrolled patients with first-ever acute ischemic stroke (n = 95) and healthy controls (n = 95) in this study. Follow-up blood samples were collected from patients at day 7, 28, and 180 after the onset. The stroke severity was evaluated by National Institutes of Health Stroke Scale score. TRAIL protein levels were quantified by using ELISA kits and TRAIL mRNA expression by quantitative real-time PCR. Our study showed that stroke patients have statistically significant lower levels of serum TRAIL protein (p < 0.0001) and elevated TRAIL mRNA expression (p < 0.0001) in PBMC at the disease onset. Our follow-up study revealed that TRAIL protein levels were increased while mRNA expression levels were downregulated in later periods. Overall, our findings suggest that serum TRAIL levels and mRNA expression in PBMC could reliably serve as a predictor of stroke outcome. Additionally, our study supports that TRAIL plays a role in pathogenesis and progression of ischemic stroke.

Vitamin D Modulation of TRAIL Expression in Human Milk and Mammary Epithelial Cells

The vitamin D levels in mothers affect the health status of both the mother and breastfeeding infant. Vitamin D deficient mothers’ infants are prone to rickets. While tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been implicated in cellular growth/apoptosis, immune cell function and bone-resorbing osteoclast formation, the expression of TRAIL in human milk as a function of vitamin D status in mothers remains unknown. We hypothesized that vitamin D deficiency alters TRAIL protein levels in human breast milk and mammary epithelial cells. Milk from vitamin D deficient mothers showed high levels of TRAIL (α and β) proteins compared to milk from vitamin D replete women. Western blot analysis of total cell lysate obtained from normal human mammary epithelial (HME-1) cells treated with variable doses (0–20 nM) of vitamin D for 24 h demonstrated that low levels (0.5 to 5 nM) significantly increased the TRAIL α but no change in β expression. In contrast, vitamin D at 20 nM concentration suppressed the expression of both TRAIL α and β proteins. Consistently, vitamin D regulated TRAIL mRNA expression in HME-1 cells. Our results indicate that vitamin D status in mothers modulates TRAIL expression in breast milk, which may have implications for both mother and infant health.

TRAIL in CD8+ T cells from patients with severe aplastic anemia.

Severe aplastic anemia (SAA) is an autoimmune disease caused mainly by activated T lymphocytes. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of TNF family, which can induce apoptosis and play a significant role in the pathogenesis of many autoimmune disorders. In this study, we sought to investigate the role of TRAIL in peripheral CD8+ T cells (CTLs) from SAA patients to clarify the autoimmune mechanisms of bone marrow failure in SAA. The expression of TRAIL and TRAIL-R2 in CTLs from SAA patients and normal controls were determined by flow cytometry, real-time PCR, and western blot. Expression of perforin and granzyme B and apoptosis in CTLs were evaluated by flow cytometry. The expression of TRAIL and TRAIL-R2 in SAA patients was significantly decreased compared with controls; however, there was no statistical difference in TRAIL mRNA expression between the two groups. TRAIL expression in CTLs was negatively correlated with the expression of perforin and granzyme B, and negatively correlated with CTLs apoptosis in SAA patients. The TRAIL pathway may be responsible for abnormal CTL activation in SAA patients. Further study of TRAIL and its receptors may elucidate the pathogenesis of SAA.

A ‐1573T&gt;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.

TRAIL gene expression analysis in multiple sclerosis patients.

Multiple sclerosis (MS) as an autoimmune disorder in which the insulating covers of neurons in the Central Nervous System are destructed. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an immunomodulatory molecule to protect against T cells hyper activation. In this Case-control study, we compare TRAIL gene expression in peripheral blood between 50 relapse remitting MS patients and 50 healthy controls by TaqMan Real time PCR. All the patients were negative for HLA-DRB1*15 susceptible allele, normal serum vitamin D, responder to Interferon beta. All the health individuals were matched to patients. Also, we tried to find correlation between TRAIL gene expression and clinical characteristics of patients. No statistically significant difference was found in TRAIL mRNA expression between MS patients and controls (p> 0.05). There was no correlation in the TRAIL expression and age of onset, disease duration and Expanded Disability Status Scale of Kurtzke (EDSS). As IFN-b may have stimulatory effects on immunoregulatory function of TRAIL and all of our patients were treated with interferon beta and were responder, it lead to no significant change in TRAIL expression. We suggest comparing between responders and non-responders should be investigated.

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.

Stimulation of macrophages with the β-glucan produced by aureobasidium pullulans promotes the secretion of tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial β-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.

Role of serum TRAIL level and TRAIL apoptosis gene expression in multiple sclerosis and relation to brain atrophy

One of the presumed pathological mechanisms of multiple sclerosis (MS) is the failure of apoptosis of autoreactive T lymphocytes. This study aimed to determine the relationship of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA gene expression ratio and serum TRAIL levels with MS and brain atrophy. This study was conducted on 53 relapsing-remitting Egyptian MS patients and 25 matched healthy volunteers. The expression of TRAIL in peripheral blood lymphocytes was analyzed by reverse transcription polymerase chain reaction, serum levels of soluble TRAIL (sTRAIL) were determined by enzyme-linked immunosorbent assay and brain MRI measured “black holes” and the bicaudate ratio as a measure of brain atrophy in all patients. The serum TRAIL level was lower in MS patients compared to controls but no difference was seen in the TRAIL mRNA gene expression ratio. No significant correlation was detected between the serum TRAIL level and the TRAIL mRNA expression ratio in either group. No statistically significant correlation was found between serum TRAIL levels or the TRAIL mRNA expression ratio with the number of black holes or the bicaudate ratio on MRI. Apoptosis of T lymphocytes is decreased in MS patients, which could be useful when designing treatments. There was no difference in the TRAIL mRNA gene expression ratio between MS patients and controls.

TRAIL mRNA expression in peripheral blood mononuclear cells of Egyptian SLE patients

Although the definite etiopathogenesis of systemic lupus erythematosus (SLE) remains unclear, many different mechanisms may contribute to its pathogenesis. Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family with pro-apoptotic activity. The accumulation of apoptotic cell debris has been hypothesized to induce the autoimmune inflammation in SLE, and TRAIL may trigger this programmed cell death. We investigated TRAIL mRNA expression levels in peripheral blood mononuclear cells (PBMCs) from 60 SLE patients and 40 controls using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), and we studied the association between the results and clinical and laboratory parameters of the patients. Expression levels of TRAIL mRNAs in SLE patients were significantly higher than in controls (p<0.001). A statistically significant association was detected between TRAIL mRNA expression and SLE activity (p=0.001).

Conditionally replicating adenovirus combined with gene-targeted radiotherapy induces apoptosis via TRAIL death receptors in MDA-MB-231 cells

Malignant tumors are usually treated using monotherapies, which are not always effective. Therefore, combination therapies have gained increasing attention. The aims of this study were to investigate the effects of conditionally replicating adenovirus (CRAd) in combination with X-ray irradiation on the proliferation and apoptosis of MDA-MB-231 cells, as well as to determine the molecular mechanisms involved. MDA-MB-231 cells were treated simultaneously with CRAd and X-ray irradiation. Then, cell viability was measured using Cell Counting Kit-8. Cell apoptosis was assessed by flow cytometry with Annexin V and propidium iodide (PI) double-staining. The expression of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), death receptor 5 (DR5), caspase-3 and caspase-8 mRNA was detected by quantitative polymerase chain reaction. The expression of TRAIL, DR5, caspase-3 and caspase-8 protein was measured by enzyme-linked immunosorbent assay (ELISA) and western blotting, respectively. The results showed that CRAd, in combination with irradiation, inhibited cell proliferation, promoted cell apoptosis and significantly increased the expression of TRAIL, DR5, caspase-3 and caspase-8 mRNA and proteins in MDA-MB-231 cells. Therefore, three aspects, including the targeted killing effect of CRAd, the apoptosis-promoting role of TRAIL and the direct killing effect of ionizing radiation to MDA-MB-231 cells, contribute to the mechanisms of CRAd in combination with irradiation to inhibit the proliferation of MDA-MB-231 cells. The pro-apoptotic effect may involve the interaction between TRAIL, DR5, caspase-3 and caspase-8.

Effects of integrin-targeted photodynamic therapy on pancreatic carcinoma cell

To investigate the effects of photodynamic therapy with quantum dots-arginine-glycine-aspartic acid (RGD) probe as photosensitizer on the proliferation and apoptosis of pancreatic carcinoma cells. Construction of quantum dots-RGD probe as photosensitizer for integrin-targeted photodynamic therapy was accomplished. After cells were treated with photodynamic therapy (PDT), the proliferation of SW1990 cells were measured by methyl thiazolyl tetrazolium assay. Morphologic changes, cell cycle retardance and apoptosis were observed under fluoroscope and flow cytometry. The expression of myeloid cell leukemia-1 (Mcl-1), protein kinase B (Akt) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA were detected by reverse transcription-polymerase chain reaction. The amount of reactive oxygen species were also evaluated by fluorescence probe. The photodynamic therapy with quantum dots-RGD probe as photosensitizer significantly inhibited cell proliferation (P < 0.01). Apoptotic cells and morphologic changes could be found under optical microscope. The FCM revealed PDT group had more significant cell apoptosis rate compared to control cells (F = 130.617, P < 0.01) and cell cycle G0/G1 and S retardance (P < 0.05) compared to control cells. The expression of Mcl-1 and Akt mRNA were down-regulated, while expression of TRAIL mRNA was up-regulated after cells treated with PDT. PDT group had more significant number of cells producing reactive oxygen species compared to control cells (F = 3262.559, P < 0.01). The photodynamic therapy with quantum dots-RGD probe as photosensitizer significantly inhibits cell proliferation and increases apoptosis in SW1990 cells.

Hypoxia-Ischemia Upregulates TRAIL and TRAIL Receptors in the Immature Rat Brain

The immature brain is susceptible to inflammatory injury induced by hypoxia-ischemia (HI) or infection, which causes serious neurodevelopmental disabilities in the survivors of preterm births. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (death receptor DR4/5 and decoy receptor DcR1/2) were reported to mediate various neuroinflammatory responses. However, little information is available regarding the role of TRAIL and its receptors in the immature brain after HI. The purpose of this study was to evaluate the expression of TRAIL and its receptors in the immature brain after HI and relate this expression to neurological function. We performed right common carotid artery ligation followed by hypoxia (6% O₂, 37°C) for 2.5 h to induce HI in postnatal day 3 rats. The distribution of TRAIL and its receptors, caspase-3 and CD68-labeled microglia/macrophages was evaluated 24 h after HI by immunostaining. The protein and mRNA expression of TRAIL and DR5 was measured by Western blot and real-time PCR, respectively. Delayed neuronal loss was evaluated by NeuN and Nissl staining 7 days after HI. Furthermore, neurological deficits were evaluated by a righting reflex test, time of eye opening and T-maze test. The expression of TRAIL, DR5 and DcR1/2 receptors and caspase-3 was more pronounced in the ipsilateral hemisphere compared with the contralateral part and the control group 24 h after HI. DR5/active caspase-3 double-positive cells were observed at 24 h after HI in the ipsilateral hemisphere but not in the contralateral hemisphere. The TRAIL and CD68 double-labeled cells were more pronounced in the ipsilateral cortical regions compared with the corresponding regions of the contralateral part. HI also resulted in a significant increase in TRAIL and DR5 protein and mRNA expression at 24 h, which corresponded to neuronal cell loss 7 days after HI. Furthermore, the HI group displayed impaired neurobehavioral development compared with the control group (p < 0.05). Altogether our results show that the TNF-α superfamily ligand TRAIL is induced on CD68+ microglia/macrophages after perinatal HI and that one of its receptors, DR5, is induced on neocortical neurons and glial cells. That many DR5+ cells were also caspase-3+ strongly supports the conclusion that these signaling molecules are involved in the delayed loss of neurons in the neocortex and in the neurobehavioral deficits that are often seen after perinatal HI.

Proteasome Inhibitor Bortezomib Disrupts Tumor Necrosis Factor-Related Apoptosis-Inducing ligand (TRAIL) Expression and Natural Killer (NK) Cell Killing of TRAIL Receptor-Positive Multiple Myeloma Cells

AbstractAbstract 5021Bortezomib, a potent 26S proteasome inhibitor, is approved for the treatment of multiple myeloma (MM) and clinical trials are under way to evaluate its efficacy in other malignant diseases. However, cytotoxic effects of bortezomib on immune-competent cells have also been observed. The aim of this study was to investigate the putative effects of Bortezomib on activated human natural killer (NK) cell function. In this project, primary lymphocytes or NK cells were isolated or purified from peripheral blood mononuclear cells of healthy donors. Expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by interleukin (IL)-2-activated NK cells was quantified by flow cytometry and real time-PCR method. NK cell degranulation activity and cytotoxicity against MM cell lines were evaluated by CD107a surface translocation and 51Cr release assays, respectively. Our results demonstrate that bortezomib markedly downregulated cell surface expression of TRAIL on primary human IL-2-activated NK cells in a dose-dependent manner. Moreover, bortezomib reduced TRAIL mRNA expression in these cells, suggesting that bortezomib regulates TRAIL expression of activated NK cells at the transcriptional level. Interestingly, pharmacological inhibition of the transcription factor NF-κB also profoundly decreased TRAIL expression both at protein and mRNA levels, indicating a novel role of NF-κB in the regulation of TRAIL expression in activated human NK cells. Furthermore, perforin-independent killing of the human MM cell lines was significantly suppressed following bortezomib treatment. In addition, blocking cell surface-bound TRAIL with a TRAIL antibody impaired lymphokine-activated killer (LAK) cell-mediated lysis of the TRAIL-sensitive MM cell line, RPMI8226. Our study demonstrated that bortezomib decreases TRAIL expression by IL-2 activated NK cells in a dose-dependent manner and disrupts TRAIL-mediated killing of TRAIL-sensitive myeloma cells, suggesting that Bortezomib may potentially hamper NK-dependent immunosurveillance against tumors in patients treated with this drug. Disclosures:No relevant conflicts of interest to declare.

Upregulation of TRAIL in Myeloma Cells Correlates with Skeletal-Related Events.

Abstract 2844Poster Board II-820 Introduction:Skeletal complications including bone fracture, bone pain and hypercalcemia are major clinical events in patients of multiple myeloma (MM). Osteoclastgenesis is known to be induced by free receptor activator of nuclear factor kappa β ligand (RANKL) and inhibited by dimerization of RANKL and osteoprotegerin (OPG). OPG is also known as a soluble inhibitor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); therefore, a possible role of TRAIL as an osteoclast inducer is suggested, although the association of TRAIL with bone lesions in MM is a matter of debate. We thus investigated the expression of TRAIL mRNA in purified MM cells and analyzed its association with skeletal-related events. Patients and Methods:MM cells were purified from bone marrow samples from 40 MM patients by CD138-immunomagnetic beads (Miltenyi Biotech, Paris, France). TRAIL mRNA expression in purified MM cells was analyzed by real time PCR(ABI PRISM 7700 Sequence Detector, Applied Biosystems). Simultaneous analysis of serum TRAIL concentrations, analyzed by ELISA (Diaclone, Cedex, France), and TRAIL mRNA-expression levels was also performed in 23 cases. Each of these patients was given a score called skeletal-related event score (SRE score) according to the skeletal complications (pathological fracture, bone-associated plasmacytoma, >12mg/dL hypercalcemia, and receiving an pathological fracture-related operation or radiation therapy). Results:Significant association (p=0.0006) was seen between TRAIL mRNA expression levels and the SRE score (Fig.1). Serum calcium levels also had significant association to TRAIL mRNA expression levels (p=0.0050). On the other hand, no association of TRAIL mRNA with hemoglobin (p=0.3970) and platelets (p=0.9401) was seen. Serum TRAIL concentrations in MM cases, which were equivalent to those in healthy individuals, did not correlate to TRAIL mRNA expression levels in purified MM cells (p=0.4094). Conclusions:The data suggest that MM patients with high TRAIL expression in MM cells tend to have more skeletal complications, which may be mediated by increased osteoclastgenesis. Since serum TRAIL concentrations did not correlate with TRAIL mRNA levels in MM cells, increased TRAIL expression in bone marrow microenvironment could be important. Despite of previous reports suggesting TRAIL-induced apoptosis of hematopoietic cells, the observed high TRAIL expression did not correlate with anemia or thrombocytopenia in our cases. Although mechanisms regulating TRAIL expression in MM cells and protection from TRAIL-induced apoptosis remain to be determined, our findings may introduce a new strategy targeting TRAIL to reduce skeletal events in MM. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

TRAIL mediates liver injury by the innate immune system in the bile duct–ligated mouse†

The contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand expressed by cells of the innate immune system, to cholestatic liver injury has not been explored. Our aim was to ascertain if TRAIL contributes to liver injury in the bile duct-ligated (BDL) mouse. C57/BL6 wild-type (wt), TRAIL heterozygote (TRAIL(+/-)), and TRAIL knockout (TRAIL(-/-)) mice were used for these studies. Liver injury and fibrosis were examined 7 and 14 days after BDL, respectively. Hepatic TRAIL messenger RNA (mRNA) was 6-fold greater in BDL animals versus sham-operated wt animals (P < 0.01). The increased hepatic TRAIL expression was accompanied by an increase in liver accumulation of natural killer 1.1 (NK 1.1)-positive NK and natural killer T (NKT) cells, the predominant cell types expressing TRAIL. Depletion of NK 1.1-positive cells reduced hepatic TRAIL mRNA expression and serum alanine aminotransferase (ALT) values. Consistent with a role for NK/NKT cells in this model of liver injury, stress ligands necessary for their recognition of target cells were also up-regulated in hepatocytes following BDL. Compared to sham-operated wt mice, BDL mice displayed a 13-fold increase in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and an 11-fold increase in caspase 3/7-positive hepatocytes (P < 0.01). The number of TUNEL and caspase 3/7-positive cells was reduced by >80% in BDL TRAIL knockout animals (P < 0.05). Likewise, liver histology, number of bile infarcts, serum ALT values, hepatic fibrosis, and animal survival were also improved in BDL TRAIL(-/-) animals as compared to wt animals. These observations support a pivotal role for TRAIL in cholestatic liver injury mediated by NK 1.1-positive NK/NKT cells.

The Puzzling Role of TRAIL in Endothelial Cell Biology

To the Editor: In their study published online in Arteriosclerosis, Thrombosis, and Vascular Biology , O’Brien et al demonstrate that activated protein C (APC) decreases tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression in endothelial cells.1 Although the molecular mechanism by which APC decreases the expression of TRAIL mRNA and protein via an Egr-1/Erk-1/2 pathway appears convincing, much less clear is whether the downregulation of TRAIL reflects a physiological relevant decrease of surface TRAIL or TRAIL release by endothelial cells. In fact, we have never detected surface TRAIL by flow cytometry in human umbilical vein endothelial cells (HUVECs), cultured with or without TNF-α (Figure). On the other hand, surface …

Up-regulation of TRAIL mRNA expression in peripheral blood mononuclear cells from patients with active systemic lupus erythematosus

It is recently suggested that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is involved in the pathogenesis of systemic lupus erythematosus (SLE). In this study, we examined whether expression levels of TRAIL depend on SLE activity. To estimate TRAIL mRNA expression levels in peripheral blood mononuclear cells (PBMC), we performed quantitative real-time reverse transcription-polymerase chain reaction analyses of PBMC from 18 SLE patients and 20 healthy subjects. Serum soluble TRAIL (sTRAIL) concentrations were measured by an enzyme-linked immunosorbent assay. The mean TRAIL mRNA expression level and serum sTRAIL concentration in SLE patients were significantly higher than those in healthy controls. Expression levels of TRAIL mRNA correlated with the SLE disease activity index and circulating immune complexes levels, while serum sTRAIL concentrations did not. These results indicate that increased expression of TRAIL mRNA in PBMC closely correlates with SLE activity and suggest an important role for TRAIL in the pathogenesis of SLE.

Apoptosis and expression of protein TRAIL in granulosa cells of rats with polycystic ovarian syndrome

The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the control rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regulating the apoptosis of granulosa cells in PCOS rats.

Tumor necrosis factor-related apoptosis inducing ligand expression and activity in hen granulosa cells

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) represents one of several cytokine members of the tumor necrosis factor superfamily reported to initiate apoptosis in a wide range of transformed, but not most normal, cell types. The present studies were conducted to evaluate the potential for TRAIL to promote apoptotic cell death in differentiated granulosa cells collected from hen preovulatory follicles. While mRNA encoding critical components (including TRAIL) required for a functional extrinsic cell death pathway are expressed in granulosa cells, TRAIL treatment by itself fails to induce either caspase-3 activity or a decrease in cell viability. On the other hand, preculture of cells with the conventional chemotherapeutic, cisplatin, or the 20S proteosome inhibitor, Z-LLF-CHO, sensitizes granulosa cells to TRAIL as evidenced by enhanced caspase-3 activity after 4 h of culture and loss of cell viability after 24 h when compared with either cisplatin or Z-LLF-CHO treatment alone. Moreover, the sensitizing effect of Z-LLF-CHO on TRAIL-induced loss of cell viability is prevented by the selective caspase-8 inhibitor, Z-IETD-FMK. Interestingly, TRAIL mRNA expression is elevated both in prehierarchal follicles undergoing spontaneous atresia and in prehierarchal follicles induced to undergo atresia for 6 h in vitro. In summary, the data demonstrate the presence of a functional TRAIL signaling pathway in hen granulosa cells, and are consistent with the possibility that TRAIL signaling may directly or indirectly participate in the process of follicle atresia in vivo.