AB16955 Sigma-AldrichAnti-DR4 Antibody, CT

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10/Apo2L) holds promise for cancer therapy as it induces apoptosis in a large variety of cancer cells while exerting negligible toxicity in normal ones. However, TRAIL can also induce proliferative and migratory signaling in cancer cells resistant to apoptosis induced by this cytokine. In that regard, the molecular mechanisms underlying the tumor selectivity of TRAIL and those balancing apoptosis versus survival remain largely elusive. We show here that high mRNA levels of PLAU, which encodes urokinase plasminogen activator (uPA), are characteristic of cancer cells with functional TRAIL signaling. Notably, decreasing uPA levels sensitized cancer cells to TRAIL, leading to markedly increased apoptosis. Mechanistic analyses revealed three molecular events taking place in uPA-depleted cells: reduced basal ERK1/2 prosurvival signaling, decreased preligand decoy receptor 2 (DcR2)-death receptor 5 (DR5) interaction and attenuated recruitment of DcR2 to the death-inducing signaling complex upon TRAIL challenge. These phenomena were accompanied by increased FADD and procaspase-8 recruitment and processing, thus guiding cells toward a caspase-dependent cell death that is largely independent of the intrinsic apoptosis pathway. Collectively, our results unveil PLAU mRNA levels as marker for the identification of TRAIL-responsive tumor cells and highlight a key role of uPA signaling in 'apoptosis versus survival' decision-making processes upon TRAIL challenge.

The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

OBJECTIVE: Studies in mice suggest a protective role for the TNF-related apoptosis-inducing ligand (TRAIL) in arthritis. We investigated the role of TRAIL in rheumatoid arthritis (RA) patients.METHODS: In the present study, we compared RA Fibroblast-like synoviocytes (FLS) resistant and RAFLS-sensible to TRAIL-induced apoptosis, including levels of the TRAIL receptors (TRAIL-R) and clinical features of respective patient. Furthermore, we evaluated TRAIL and its soluble decoy receptor osteoprotegerin (OPG) levels in RA patients, osteoarthritis (OA) and spondylarthritis (SpA).RESULTS: Sensitivity to TRAIL-induced apoptosis varied in FLS from different patients and disease severity of RA patients inversely correlated with susceptibility of FLS to TRAIL-induced apoptosis. TRAIL-sensitive cells expressed significantly lower levels of TRAIL-R1 and silencing of TRAIL-R1 increased TRAIL-induced apoptosis in RA FLS. TRAIL levels were elevated in the arthritic joints of patients with established RA and synovial fluids displayed elevated TRAIL levels compared to OA and SpA patients. A low ratio of the soluble decoy receptor OPG to TRAIL in sera of early RA patients at baseline was associated with a better evolution of disease activity, but high serum levels of TRAIL at follow-up were associated with joint damages.CONCLUSION: These findings suggest a dual role for TRAIL in RA and resistance of RA FLS to TRAIL-induced apoptosis is associated with a disease promoting activity of TRAIL in RA.Copyright © 2011 by the American College of Rheumatology.

TRAIL induces apoptosis through two closely related receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Here we show that TRAIL-R1 can associate with TRAIL-R2, suggesting that TRAIL may signal through heteroreceptor signaling complexes. Both TRAIL receptors bind the adaptor molecules FADD and TRADD, and both death signals are interrupted by a dominant negative form of FADD and by the FLICE-inhibitory protein FLIP. The recruitment of TRADD may explain the potent activation of NF-kappaB observed by TRAIL receptors. Thus, TRAIL receptors can signal both death and gene transcription, functions reminiscent of those of TNFR1 and TRAMP, two other members of the death receptor family.

A novel tumor necrosis factor (TNF) family member has been cloned and characterized. This protein, designated TNF-related apoptosis-inducing ligand (TRAIL), consists of 281 and 291 aa in the human and murine forms, respectively, which share 65% aa identity. TRAIL is a type II membrane protein, whose C-terminal extracellular domain shows clear homology to other TNF family members. TRAIL transcripts are detected in a variety of human tissues, most predominantly in spleen, lung, and prostate. The TRAIL gene is located on chromosome 3 at position 3q26, which is not close to any other known TNF ligand family members. Both full-length cell surface expressed TRAIL and picomolar concentrations of soluble TRAIL rapidly induce apoptosis in a wide variety of transformed cell lines of diverse origin.