Human T-cell leukemia virus type 1 Tax-deregulated autophagy pathway and c-FLIP expression contribute to resistance against death receptor-mediated apoptosis

Abstract

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein is considered to play a central role in the process that leads to adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 Tax-expressing cells show resistance to apoptosis induced by Fas ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). The regulation of Tax on the autophagy pathway in HeLa cells and peripheral T cells was recently reported, but the function and underlying molecular mechanism of the Tax-regulated autophagy are not yet well defined. Here, we report that HTLV-1 Tax deregulates the autophagy pathway, which plays a protective role during the death receptor (DR)-mediated apoptosis of human U251 astroglioma cells. The cellular FLICE-inhibitory protein (c-FLIP), which is upregulated by Tax, also contributes to the resistance against DR-mediated apoptosis. Both Tax-induced autophagy and Tax-induced c-FLIP expression require Tax-induced activation of IκB kinases (IKK). Furthermore, Tax-induced c-FLIP expression is regulated through the Tax-IKK-NF-κB signaling pathway, whereas Tax-triggered autophagy depends on the activation of IKK but not the activation of NF-κB. In addition, DR-mediated apoptosis is correlated with the degradation of Tax, which can be facilitated by the inhibitors of autophagy.

Importance: Our study reveals that Tax-deregulated autophagy is a protective mechanism for DR-mediated apoptosis. The molecular mechanism of Tax-induced autophagy is also illuminated, which is different from Tax-increased c-FLIP. Tax can be degraded via manipulation of autophagy and TRAIL-induced apoptosis. These results outline a complex regulatory network between and among apoptosis, autophagy, and Tax and also present evidence that autophagy represents a new possible target for therapeutic intervention for the HTVL-1 related diseases.

FIG 1

HTLV-1 Tax increases autophagosome accumulation. (A) Detection of LC3-I and LC3-II expression. U251 cells (2 × 10⁵ cells) were infected with LV-EGFP or LV-Tax for 2 to 4 days. Forty micrograms total protein was loaded onto SDS-PAGE gels and further analyzed by Western blotting to detect the conversion of LC3-I to LC3-II and Tax expression. (B) GFP-LC3 aggregation was visualized via fluorescence microscopy. U251 cells expressing GFP-LC3 were infected with LV-Empty (empty expression cassette) or LV-Tax; after 4 days, the GFP-LC3 aggregations in the cells were assessed via fluorescence microscopy. Representative images are shown (top), and the percentage of cells with GFP-LC3 puncta (% GFP-LC3^vac cells) is presented (bottom; values are means and standard deviations [SD]; *, P < 0.01). (C) Autophagic vacuoles were detected via transmission electron microscopy. U251 cells expressing EGFP or Tax were processed 4 days after lentiviral infection for transmission electron microscopy. Representative images are shown, and the arrows indicate autophagic vacuoles. Bars, 0.5 μm. (D and E) Tax induces autophagosome accumulation in primary human astrocytoma cells. Astrocytes (5 × 10⁵) were infected with LV-EGFP or LV-Tax (MOI = 3), and after 4 and 5 days of infection, 40 μg total protein was used in Western blot assays to detect the conversion of LC3-I to LC3-II (D). GFP-LC3 dots were analyzed using confocal microscopy 4 days after infection. Astrocytes expressing GFP-LC3 were infected with LV-Empty or LV-Tax. The GFP-LC3 aggregations were assessed via confocal microscopy after 4 days of infection. Cell nuclei were stained with Hoechst 33258. Representative images are shown (E).

FIG 2

Tax suppresses TRAIL-induced apoptosis in U251 astroglioma cells. U251 cells were infected with LV-EGFP or LV-Tax (MOI = 3) for 4 days and then treated with recombinant soluble TRAIL or the anti-DR5 agonistic monoclonal antibody AD5-10 at the indicated concentrations for 0 to 12 h. (A) The cell viability after TRAIL or AD5-10 treatment for 12 h was determined using MTS assays (mean ± SD; n = 3). (B and C) Apoptosis in cells treated with 50 ng/ml TRAIL or PBS (control) for 6 h was detected through fluorescence microscopy analysis of Hoechst 33258-stained cells (B) and flow cytometry analysis of PE-conjugated Annexin V-stained cells (C). The mean percentages of Annexin V-positive cells from three independent experiments are shown (mean ± SD; *, P < 0.01 compared with TRAIL-treated EGFP-expressing cells). (D) The activation of caspase-8, -3, and -9 in EGFP- and Tax-expressing cells treated with PBS, 50 ng/ml TRAIL, or 100 ng/ml AD5-10 for 1 and 6 h was analyzed by Western blotting. GAPDH was used as a protein loading control.

FIG 3

Inhibition of autophagy pathway sensitizes Tax-expressing U251 cells to TRAIL-induced apoptosis. (A and B) U251 cells were infected with LV-EGFP or LV-Tax for 4 days, pretreated with 3-MA (5 mM or 10 mM), LY294002 (20 μM) (A), or 100 μM chloroquine (B) for 3 h, and then cultured for 12 h in the presence or absence of 50 ng/ml TRAIL. Cell viability was determined using MTS assays (mean ± SD; n = 3; *, P < 0.01 compared with TRAIL-treated Tax-expressing cells). (C) U251 cells were infected as for panels A and B, pretreated with 10 mM 3-MA for 3 h, and then treated with 50 ng/ml TRAIL for 6 h. The cell lysates were analyzed via Western blotting to determine the expression of c-FLIP, LC3, and Tax and the activation of caspase-8 and caspase-3. GAPDH was used as a protein loading control. Protein quantification was performed via densitometry, and the ratios of LC3-II to GAPDH are shown under the respective blots. (D) U251 cells were infected with LV-EGFP or LV-Tax for 12 h and then with LVs encoding Beclin-1 shRNA (shBECN1) or no shRNA (shCo) for another 12 h (MOI = 3). After 4 days, the cells were treated with 50 ng/ml TRAIL or PBS (control) for 12 h. Cell viability was determined via MTS assays (mean ± SD; n = 3; *, P < 0.01 compared with TRAIL-treated cells expressing Tax and shCo). The efficacies of BECN1 knockdown and LC3 expression are shown in the bottom panel. U251 cells were infected with LV-shCo or LV-shBECN1 for 4 days. The cell lysates were analyzed by Western blotting to determine the expression of BECN1 and LC3.

FIG 4

Tax-induced c-FLIP expression also protects U251 cells from TRAIL-induced apoptosis. (A) The expression of c-FLIP_L and c-FLIP_S mRNA in U251 cells infected with LV-EGFP or LV-Tax for the indicated times was assessed via real-time quantitative PCR. Data are presented as fold change in gene expression relative to EGFP-expressing cells (mean ± SD; n = 3). (B) Tax upregulates the level of c-FLIP protein expression. Western blot analysis of c-FLIP_L, c-FLIP_S, and Tax expression in U251 cells infected with the LVs for 2 to 4 days was carried out. GAPDH expression was used as a loading control. (C) Primary human astrocytoma cells were infected with LV-EGFP or LV-Tax (MOI = 3), and then the cell lysates of the primary human astrocytoma cells were analyzed 4 and 5 days after infection by Western blotting to detect the expressions of the c-FLIP and Tax-Flag proteins. (D and E) U251 cells were infected with LV-EGFP or LV-Tax for 24 h and then with LVs encoding c-FLIP shRNA (shFLIP) or no shRNA (shCo) (MOI = 3) for 3 days, after which they were treated with 50 ng/ml TRAIL for 6 h (D) or 12 h (E). The expression of c-FLIP and Tax and the activation of caspase-8 and caspase-3 were analyzed by Western blotting. The bands of procaspase-8 and cleaved caspase-8 were cut from two films with different exposure times. GAPDH was used as a loading control (D). Cell viability was detected via MTS assays (E) (mean ± SD; n = 3; *, P < 0.01 compared with TRAIL-treated cells expressing Tax and shCo). (F) Tax-expressing U251 cells were infected with LV-shFLIP or LV-shCo (MOI = 1) for 3 days, pretreated with 3-MA (10 mM) for 3 h, and then cultured for 12 h in the presence or absence of 20 ng/ml TRAIL. Cell viability was determined using MTS assays (mean ± SD; n = 3; *, P < 0.01 compared with TRAIL-only-treated Tax-shFLIP cells).

FIG 5

The Tax mutant M22 is unable to induce c-FLIP expression and autophagosome accumulation. (A) M22 has no effect on c-FLIP and LC3-II expression. U251 cells were infected with the indicated LVs; 4 days after the infection, the levels of c-FLIP_L, c-FLIP_S, and LC3 expression were analyzed by Western blotting. Protein quantification was performed via densitometry, and the ratios of LC3-II to GAPDH are shown under the respective blots. (B) U251 cells were infected as for panel A and processed for the detection of autophagic vacuoles through transmission electron microscopy. Representative images are shown (scale bars, 0.5 μm). (C) U251 cells expressing GFP-LC3 were infected with LV-Empty, Tax, M22, or M47 and analyzed using confocal microscopy 4 days after infection. Cell nuclei were stained with Hoechst 33258. Representative images are shown (left), and the number of GFP-LC3 dots per cell is reported (right) (mean ± SD; 20 cells; *, P < 0.01 compared with LV-Empty-infected cells). (D and E) M22 is unable to induce resistance to TRAIL-induced apoptosis. U251 cells were infected with the indicated LVs for 4 days and then treated with 50 ng/ml TRAIL for 6 h. The lysates were analyzed by Western blotting to assess caspase activation and the expression of Tax-Flag, M22-Flag, and M47-Flag (D). Alternatively, LV-infected U251 cells were treated with 50 ng/ml TRAIL or the anti-DR5 agonistic monoclonal antibody AD5-10 (100 ng/ml) for 12 h, after which cell viability was determined via MTS assays (mean ± SD; n = 3; *, P < 0.01 compared with TRAIL- or AD5-10-treated EGFP-expressing cells) (E).

FIG 6

Activation of NF-κB is necessary for Tax-induced c-FLIP expression but not for Tax-increased autophagosome accumulation. (A to C) U251 cells were infected with LVs encoding no shRNA sequence (shCo), an shRNA targeting IKKα (A), IKKβ (C), or p65 (B), or IκB-DN mutant genes (C) for 3 days and then infected with LV-EGFP or LV-Tax. The knockdown efficacy, IκBα degradation, and c-FLIP and LC3-II expression levels were analyzed by Western blotting. (D and E) IKKα (D), p65 (D), and IκBα (E) contribute to Tax-induced TRAIL resistance. After the infection of the cells with LVs as described for panels A to C, the U251 cells were cultured in the presence or absence of 50 ng/ml TRAIL for 12 h. Cell viability was then determined via MTS assays (mean ± SD; n = 3; *, P < 0.01 compared with TRAIL-treated cells expressing Tax and shCo [D] or Tax and Empty [E]). (F) AMPKα and mTOR are involved in Tax-increased autophagosome accumulation. U251 cells were infected with LVs; 4 days after infection, the cell lysates were analyzed by Western blotting to determine IKK and AMPKα activation, mTOR inhibition, and c-FLIP and LC3-II expression levels.

FIG 7

Inhibition of autophagy promotes TRAIL-induced Tax degradation. (A and B) 3-MA promotes TRAIL-induced degradation of Tax and IKKα/β. U251 cells were infected with LV-EGFP (A) or LV-Tax (B) for 4 days, pretreated without (control) or with 10 mM 3-MA for 3 h, and then treated with 50 ng/ml TRAIL for the indicated times. The caspase-3 activation and the expression levels of the IKK complex, IκBα, and Tax were determined using Western blot assays. (C) The pan-caspase inhibitor blocks the Tax degradation induced by the combination of 3-MA and TRAIL. U251 cells were infected with LV-Tax for 4 days, pretreated with or without 10 mM 3-MA, 50 μM MG-132, 1 mM PS-341, 10 μM z-VAD-fmk, or 200 μM chloroquine for 3 h, and then treated with 50 ng/ml TRAIL for 3 h. The level of Tax expression was analyzed using Western blot assays. Protein quantification was performed via densitometry, and the ratios of Tax to GAPDH are shown for the respective blots. (D) Summarized interactions between Tax, autophagy, and TRAIL-mediated apoptosis.