Journal: Molecular and Cellular Biology
Article Title: Differences and Similarities in TRAIL- and Tumor Necrosis Factor-Mediated Necroptotic Signaling in Cancer Cells
doi: 10.1128/MCB.00941-15
Figure Lengend Snippet: Overview of similarities and differences in signaling pathways of TRAIL- and TNF-mediated necroptosis in cancer cells. Death receptors such as TRAIL-R1/2 or TNF-R1 under caspase-compromised conditions (and, for some cancer cell lines, after sensitization with protein synthesis inhibitors) are able to mediate necroptosis. During necroptosis death receptors may be (for TNF-R1) internalized in a clathrin-dependent or clathrin-independent manner followed by receptosome formation or (for TRAIL-R1/R2) signaled without receptor internalization directly through formation of a necrosomal initiation complex. In the course of the first steps of necroptosis, membranes and membrane-bound molecules (e.g., Atg5 for TNF-R1 and TRAIL-R1/R2 and Atg16L1 for TNF-R1) are recruited to the necrosomal initiation complex consisting of, e.g., FADD (indispensable for TRAIL-R1/2 but not for TNF-R1) and, crucial for the execution of both TNF- and TRAIL-mediated necroptosis, proteins RIPK1 and RIPK3 (not shown), which are assembled in a filamentous fibril-like manner. Deficiency in some membrane-bound proteins such as Atg5 may inhibit the execution of TRAIL- and TNF-induced necroptosis. However, deficiency in other proteins such as Atg16L1 or inhibition of clathrin-dependent and -independent events reduces the level of TNF-induced necroptosis but enhances or has no influence, respectively, on TRAIL-induced necroptosis. Further phosphorylation events for RIPK1, RIPK3, and its downstream effector MLKL are necessary to execute necroptosis (not shown). The lack of the adaptor protein FADD within the necrosomal initiation complex abrogates the execution of TRAIL-induced necroptosis (Fig. 1G), but it potentiates the execution of TNF-induced necroptosis (88). As a consequence of necroptosis initiation, secondary messengers (i.e., ceramide) are produced by A-SMase and N-SMase as intracellular signals to promote necroptosis. As a result, a myriad of executive mechanisms in various cellular compartments is promoted to accomplish the execution of necroptosis. Inhibition of prosurvival pathways such as PARP-1, tankyrases (analyzed here only for TRAIL), and p38α leads to enhancement of TRAIL- and TNF-induced necroptosis. The executive, necroptotic pathway is built up by some common mechanisms, shared by both TRAIL- and TNF-induced necroptosis, that result in the same outcome, while they have been modulated (e.g., through inhibition, deficiency, or overexpression). However, among those executive mechanisms, some differences existed in TRAIL- or TNF-induced necroptosis. While for TNF-induced necroptosis inhibition of certain executive components led to a decrease in the level of cell death, e.g., through inhibition of lysosomal acidification and inhibition of UCH-L1, for TRAIL-induced necroptosis, on the contrary, modulation of those components led to an increase in or had no influence on the level of necroptosis. Along the way, some unique executive mechanisms were identified to play a role exclusively in TNF-induced necroptosis, such as monoubiquitination of UCH-L1. Moreover, overexpression of Bcl-XL did not influence TNF-induced necroptosis, but it reduced TRAIL-mediated necroptosis. The involvement of particular signaling molecules in the promotion or inhibition of TRAIL- and TNF-mediated necroptosis is described in detail in Discussion.
Article Snippet: Reactive proteins were detected by using antibodies specific for Atg16L1 (D5D6, catalogue number 8089; Cell Signaling), β-actin (catalogue number A1978; Sigma), Bcl-XL (2H12, catalogue number 551020; BD), Bcl-2 (catalogue number sc-509; Santa Cruz), IκBα (Ser32) (14D4, catalogue number 2859; Cell Signaling), IκBα (C-21, catalogue number sc-371G; Santa Cruz), p-p65 (Ser536) (93H1, catalogue number 3033; Cell Signaling), p65 (C22B4, catalogue number 4764; Cell Signaling), HtrA2/Omi (catalogue number ab32092; Abcam), LC3 (catalogue number 0231-100/LC3-5F10; nanoTools), PAR (catalogue number 551813, component 51-8114KC; BD Pharmingen), PARP-1 (catalogue number 9542; Cell Signaling), p38α (5F11, catalogue number 9217; Cell Signaling), RIPK1 (catalogue number 610459; BD Biosciences), murine RIPK3 (catalogue number PRS2283; Sigma), human RIPK3 (catalogue number PAB0287; Abnova), UCH-L1 (polyclonal antibody [PAb] CL95101; Cedarlane), and UCH-L1 (monoclonal antibody [MAb] described previously [ 9 ]) and the LumiGLO chemiluminescent substrate (Cell Signaling, Danvers, MA) and captured on Amersham Hyperfilm ECL (GE Healthcare, Munich, Germany).
Techniques: Inhibition, Produced, Over Expression
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![TRAIL- and TNF-induced necroptosis are mediated by ceramide that is generated by A-SMase and N-SMase. (A to D) Cells were pretreated for 2 h with the indicated concentrations of inhibitors of A-SMase (ARC39, zoledronic acid, TP064/14e, D609, desipramine, and imipramine), N-SMase (3-OMS, spiroepoxide, and GW4869), and ceramide synthase (fumonisin B1), with the subsequent addition of 30 ng/ml killerTRAIL and 20 μM zVAD for 14 h (L929Ts) (A), 100 ng/ml hrTNF in combination with 20 μM zVAD for 5 h (L929Ts) (B), 100 ng/ml killerTRAIL and 20 μM zVAD for 16 h (NIH 3T3) (C), and 100 ng/ml hrTNF and 20 μM zVAD for 16 h (NIH 3T3) (D). (E) Inhibitors of A-SMase and N-SMase protect human Jurkat I.42 cells (FADD deficient and TNF-R2 positive) from TNF-mediated necroptosis. Cells were treated with the indicated concentrations of inhibitors and stimulated afterwards with 100 ng/ml hrTNF in combination with 50 μM zVAD for 6 h. (F) Inhibitors of A-SMase, HtrA2/Omi, <t>UCH-L1,</t> and vacuolar H+-ATPase protect the human pancreas adenocarcinoma cell line A818-6 from TRAIL-mediated necroptosis. Cells were pretreated for 2 h (or 3 h for LDN57444) with the indicated concentrations of inhibitors with the subsequent addition of 100 ng/ml killerTRAIL in combination with 50 μM zVAD for 24 h. Morphological changes of human A818-6 pancreas adenocarcinoma cells after induction of TRAIL-mediated necroptosis in combination with inhibitors of A-SMase, HtrA2/Omi, and vacuolar H+-ATPase were observed. Cells were pretreated for 2 h with 10 μM ARC39, 25 μM Ucf-101, or 10 μM BafA1 with the subsequent addition of 100 ng/ml killerTRAIL in combination with 50 μM zVAD for 24 h. Arrowheads in micrographs show typical necroptotic morphologies. Bar, 100 μm. Shown are means ± standard deviations (n = 3 [A to D] and n = 9 [E and F]), with differences being considered significant at P values of <0.05 (*), <0.01 (**), and <0.001 (***) (as determined by a t test).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8143/pmc05038143/pmc05038143__zmb9991013280003.jpg)
![Ubiquitinated, active UCH-L1 is not involved in the execution of TRAIL-mediated necroptosis. (A) Cells were prestimulated for 3 h with the indicated concentrations of the UCH-L1 inhibitor LDN57444 (top) or LDN91946 (bottom), with the subsequent addition of either 30 ng/ml killerTRAIL and 20 μM zVAD for 14 h; 100 ng/ml hrTNF and 20 μM zVAD for 5 h (L929Ts); 50 ng/ml killerTRAIL, 50 μM zVAD, and 2 μg/ml CHX for 20 h (Jurkat); or 30 ng/ml killerTRAIL, 20 μM zVAD, and 5 μg/ml CHX for 16 h (HT-29). (B) L929Ts cells were treated as described above for panel A, and afterwards, necroptotic changes in morphology (arrowheads) were observed by microscopy. Bar, 100 μm. (C) Cells were transfected with siRNAs specific for murine UCH-L1 (siRNA) or a nontargeting control siRNA (NT). At 24 h posttransfection, cells were stimulated with 30 ng/ml killerTRAIL and 20 μM zVAD for 14 h. Measurement of intracellular ATP levels served as an indicator of cell viability. Shown are means ± standard deviations (n = 4 [A] and n = 3 [C], each with five repetitions, relative to untreated cells), with statistical significance at P values of <0.05 (*), <0.01 (**), and <0.001 (***) (as determined by a t test). n.s., nonsignificant. Control Western blots show downregulation of endogenous murine UCH-L1 (PAb) and β-actin as a loading control. (D) MEFs deficient for HtrA2/Omi and their wild-type counterparts were left untreated or treated with 100 ng/ml hrTNF or 30 ng/ml killerTRAIL, 20 μM zVAD, and 1 μg/ml CHX for 16 h. * and *** indicate the disappearance of the main form of UCH-L1 (MAb and PAb, respectively). ** indicates the appearance of an active, ubiquitinated form of UCH-L1 (PAb). (E) Cells were treated as described above for panel D, omitting hrTNF, for the indicated times. Further PAbs for UCH-L1 were used to analyze the appearance of the active form of UCH-L1. WB, Western blot.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8143/pmc05038143/pmc05038143__zmb9991013280006.jpg)
