RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation

Abstract

Background: Programmed necrosis/necroptosis is an emerging form of cell death that plays important roles in mammalian development and the immune system. The pro-necrotic kinases in the receptor interacting protein (RIP) family are crucial mediators of programmed necrosis. Recent advances in necrosis research have been greatly aided by the identification of chemical inhibitors that block programmed necrosis. Necrostatin-1 (Nec-1) and its derivatives were previously shown to target the pro-necrotic kinase RIP1/RIPK1. The protective effect conferred by Nec-1 and its derivatives in many experimental model systems was often attributed to the inhibition of RIP1 function.

Methodology/principal findings: We compared the effect of Nec-1 and siRNA-mediated silencing of RIP1 in the murine fibrosarcoma cell line L929. Treatment of L929 cells with the pan-caspase inhibitor zVAD-fmk or exogenous TNF induces necrosis. Strikingly, we found that siRNA-mediated silencing of RIP1 inhibited zVAD-fmk induced necrosis, but not TNF-induced necrosis. TNF-induced cell death in RIP1 knocked down L929 cells was inhibited by Nec-1, but not the caspase inhibitor zVAD-fmk. We found that PKA-C§ expression, but not Jnk or Erk activation, was moderately inhibited by Nec-1. Moreover, we found that Nec-1 inhibits proximal T cell receptor signaling independent of RIP1, leading to inhibition of T cell proliferation.

Conclusions/significance: Our results reveal that besides RIP1, Nec-1 also targets other factors crucial for necrosis induction in L929 cells. In addition, high doses of Nec-1 inhibit other signal transduction pathways such as that for T cell receptor activation. These results highlight the importance to independently validate results obtained using Nec-1 with other approaches such as siRNA-mediated gene silencing. We propose that some of the previous published results obtained using Nec-1 should be re-evaluated in light of our findings.

Figure 1. Nec-1 inhibits necrosis in L929 cells in a dose-dependent manner.

(A) Nec-1 inhibits TNF-induced necrosis in L929 cells. L929 cells were treated with 10 µM zVAD-fmk, 20 µM Nec-1 and 10 ng/ml mouse TNF (mTNF) as indicated. Cell death was determined by MTS assay as described in materials and methods. (B) L929 cells were treated with the indicated doses of Nec-1, followed by stimulation with 10 ng/ml mTNF. Cell Death was analyzed as in (A). (C) Nec-1 did not alter RIP1 or RIP3 protein expression in L929 cells.

Figure 2. Nec-1 inhibits TNF-induced necrosis independent of RIP1 in L929 cells.

(A–C) RIP1 is required for zVAD-fmk, but not TNF-induced necrosis in L929 cells. (A) L929 cells were transfected with the indicated siRNA oligonucleotides with HiPerfect (Qiagen). TR4 is the control siRNA against TRAIL-R4 . Cells were treated with (A) 50 µM zVAD-fmk or (B) 10 ng/ml mTNF and cell death was measured by MTS assay. (C) Western blot shows RIP1 and RIP3 expression in cells transfected with the indicated siRNAs. (D–E) RIP1 is required for TNF-induced necrosis in 3T3 fibroblasts. NIH 3T3 fibroblasts were transfected with the indicated siRNA. (D) Expression of RIP1, RIP3 and ß-actin in cells transfected with the indicated siRNA was determined by Western blot. (E) Necrosis was induced with 10 µM zVAD-fmk, 0.5 µg/ml cycloheximide and 10 ng/ml mTNF for 20 hours. Cell death was assessed as in (A).

Figure 3. Knock-down of RIP1 expression in L929 cells did not result in caspase-dependent apoptosis.

L929 cells were transfected with siRNA against (A) RIP1 or (B) TR4. Transfected cells were treated with zVAD-fmk, Nec-1 and TNF as indicated. Cell death was determined by MTS assay. (C) L929 cells transfected with the indicated siRNA or Jurkat 4E3 cells were treated with TNF. PARP-1 cleavage was determined by Western blot. (D) Cell lysates from (C) were tested for active caspase 3 as measured by cleavage of the substrate DEVD-AMC. Release of AMC was determined by increases in AMC fluorescence as described in materials and methods.

Figure 4. Effects of Nec-1 on Jnk, Erk and PKA-Cβ.

(A) L929 cells were treated with TNF in the presence of 20 µM Nec-1 or 50 µM of the Erk inhibitor U0126. Cell death was determined by MTS assay. (B) The effect of Nec-1 on Erk or Jnk phosphorylation. L929 cells treated with 10 ng/ml of mTNF in the presence or absence of 30 µM Nec-1 were analyzed for Erk activation (p-p54 and p-p46) or Jnk activation by Western blot. (C) Erk inhibitor U0126 and Nec-1 blocks TNF-induced necrosis in Jurkat cells. Jurkat 4E3 cells were treated with Erk inhibitor (50 µM U0126), Jnk inhibitor (30 µM Jnk inhibitor II), p38 inhibitor (50 µM SB203580), or 10 µM Nec-1 and stimulated with 100 ng/ml recombinant human TNF plus 10 µM zVAD-fmk for 14 hours. Cell death was determined by MTS assay. (D) Jurkat 4E3 cells were stimulated with 10 ng/ml PMA for 10 minutes in the presence of 30 µM Nec-1 or 50 µM U0126. Phospho-Erk was examined by Western blot as indicated. (E) Jurkat 4E3 cells were treated with the indicated doses of Nec-1 and 100 nM calyculin A. The expression of the two different PKA isoforms and phosphorylation of protein kinase A substrates was determined by Western blot with specific μantibodies as described in materials and methods.

Figure 5. High doses of Nec-1 inhibit T cell proliferation.

(A) Low dose Nec-1 inhibits TCR-induced necrosis, but high dose Nec-1 inhibits T cell proliferation. Purified T cells from wild type or FADD^−/− mice were stimulated with plate-bound anti-CD3, anti-CD28 and increasing doses of Nec-1. Three days later, cell proliferation was measured by incorporation of [³H]-thymidine. Results shown are mean ± SEM. (B) Nec-1 did not compromised T cell viability. Naïve CD3⁺ T cells were purified from the spleen of wild type C57BL/6 mice and incubated at 37°C for 24 hours in the presence or absence of 50 µM Nec-1. Viability of the cells was determined by flow cytometry using propidium iodide (PI) uptake as an indication of cell death. Note that Nec-1 increased the baseline fluorescence of the T cells. (C) Nec-1 inhibits T cell division. Purified CD3⁺ primary T cells were labeled with CellTracer Violet fluorescent dye and stimulated with 1 µg/ml plate-bound anti-CD3 and 200 ng/ml anti-CD28 antibodies. Three days later, cell division was analyzed using a BD LSR2 flow cytometer. The numbers above each peak represent the number of cell division the cells had undergone. The numbers on the left represent the percentages of cells in each peak. (D) Nec-1 inhibits T cell blast formation. Purified CD3⁺ T cells were similarly activated as in (C). Three days later, formation of T cell blast as measured by forward scatter was determined by flow cytometry. (E) FADD^−/−RIP1^−/− DKO T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in the absence or presence of Nec-1 were measured for cell proliferation as in (C).

Figure 6. Nec-1 blocks sustained phosphorylation of LAT during T cell activation.

(A) Whole cell extracts (WCE) were examined from T cells stimulated with anti-CD3 antibody in the presence or absence of 50 µM Nec-1. Western blot was performed with phospho-tyrosine specific antibody. Short and long exposures were shown to highlight the different phosphorylated species. (B) Nec-1 impairs TCR-induced LAT phosphorylation. Total lysates from activated T cells were subjected to immunoprecipitation with phospho-tyrosine antibody. The resulting immune complex was evaluated for presence of LAT and Lck by Western blot. (C) Nec-1 does not affect Erk phosphorylation. Purified T cells were stimulated as in (A). Western blot was performed with phospo-Erk or total Erk specific antibodies.