Proteasome inhibition results in TRAIL sensitization of primary keratinocytes by removing the resistance-mediating block of effector caspase maturation

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-kappaB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-kappaB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.

FIG. 1.

Proteasome inhibitor MG115 sensitizes primary keratinocytes to TRAIL-induced apoptosis. (A) Primary keratinocytes were treated with 50 ng of TRAIL per ml for 16 h. Caspase-dependent cytotoxicity of TRAIL (sub-G₀ DNA analysis) is only detectable in the presence of the proteasome inhibitor MG115 (10 μM). zVAD-FMK (40 μM) completely abolishes TRAIL-induced apoptosis. (B) Dose-dependent effect of MG115 on TRAIL-induced cell death in primary keratinocytes was determined by crystal violet staining 16 h after addition of the indicated concentrations of TRAIL.

FIG. 2.

TRAIL dose-dependently enhances NF-κB binding in primary keratinocytes. (A) Primary keratinocytes were treated with 5 to 1,000 ng of TRAIL per ml for 6 h and analyzed for NF-κB DNA binding by electrophoretic mobility shift assay with nuclear extracts. Induction of p65/p50 heterodimers (upper arrow) or p50 homodimers (lower arrow) is shown. As a control for the specificity of DNA binding, control experiments with an excess of unlabeled probe were performed (not shown). Cell viability was determined in parallel experiments by crystal violet staining and is indicated as a percentage of the control value. (B) Primary keratinocytes were exposed to 250 ng of TRAIL per ml for 6 h in the presence or absence of 40 μM zVAD-FMK. Survival of cells was determined in parallel experiments as described for A. (C) Composition of TRAIL-induced nuclear NF-κB complexes in primary keratinocytes. Following incubation of primary keratinocytes with TRAIL (250 ng/ml) for 4 h, supershift electrophoretic mobility shift analysis of inducible NF-κB-specific complexes was performed with nuclear extracts. Each of the Rel-specific antibodies (1 μl of antibody [Ab]; see the text) was added to nuclear protein extracts. Control supershift electrophoretic mobility shift assays (EMSAs) were performed with normal rabbit serum (not shown). Arrows indicate supershifted complexes. (D) MG115 leads to dose-dependent inhibition of TRAIL-induced NF-κB activation (upper panel) and IκBα degradation (lower panel). Membranes were rehybridized with antitubulin monoclonal antibodies to confirm comparable loading of cytoplasmic proteins.

FIG. 3.

Overexpression of dominant negative mutant of IKK2 (IKK2-KD) or transdominant IκBα (IκBα-TD) inhibits TRAIL-induced degradation of IκBα without modulation of TRAIL-induced apoptosis. (A) Primary human keratinocytes were retrovirally transduced with IKK2-KD, IκBα-TD, or control vector and subsequently analyzed for expression of mutant proteins as well as degradation of endogenous IκBα following stimulation with 500 ng of TRAIL per ml for 1 h by Western blotting. Membranes were rehybridized with antitubulin monoclonal antibodies to confirm comparable loading of cellular proteins. (B) Primary keratinocytes overexpressing IKK2-KD or IκBα-TD as described for A were either left untreated or incubated with 25, 50, or 125 ng of LZ-TRAIL per ml for 6 h. Thereafter, the percentage of dead green fluorescent protein-positive cells was determined by tetramethylrhodamine ethyl ester staining. Data are shown as mean ± standard deviation of two independent experiments.

FIG. 4.

Caspases 8 and 10 are recruited and cleaved at the native TRAIL DISC independent of MG115. (A) Primary keratinocytes were treated with 10 μM MG115 or diluent for 6 h and subsequently analyzed for surface expression of TRAIL-R1 and TRAIL-R2 by fluorescence-activated cell sorting. MG115 does not modulate TRAIL-R1 or TRAIL-R2 surface expression on primary keratinocytes. (B) Analysis of the native TRAIL DISC in primary keratinocytes. Cells were treated for the indicated times with MG115 or diluent alone. The stimulated TRAIL DISC (+) or unstimulated receptors (−) were precipitated with Flag-TRAIL precomplexed with 2 μg of anti-Flag antibodies per ml (clone M2). The resulting protein complexes were separated by SDS-PAGE and analyzed by Western blot for components of the TRAIL DISC. The adaptor protein FADD was detectable only when stimulated receptors were precipitated. Also, recruitment of the two isoforms of caspase 8, caspase 8a and 8b, was detected only following stimulation of TRAIL receptors. In addition, the intermediate cleavage products, p43/p41, of the two isoforms of caspase 8 were detectable in the DISC. For caspase 10, the full-length forms caspase 10d (p59) and caspase 10a (p55) as well as the cleavage products p47/43 and the p25 fragment, corresponding to the prodomain (Prodom.), were detectable in the immunoprecipitates. Analysis of cFLIP levels revealed that both isoforms, cFLIP_L and cFLIP_S, were present in the TRAIL DISC, with comparable levels in control cells and MG115-treated cells. For cFLIP_L, only the p43 cleavage product was detectable in the DISC, indicative of proteolytic processing by active caspases in the DISC. The migration position of the IgG heavy and light chains of the precipitating anti-Flag M2 antibody as well as that of protein G cross-reacting with the antibody used for Western blot detection (caspase 10, lower part) are indicated by asterisks.

FIG. 5.

Proteasome inhibitor MG115 does not influence early TRAIL-induced caspase 8, caspase 10, and cFLIP cleavage. Primary keratinocytes were incubated for 60 min with 10 μM MG115 or diluent alone and subsequently treated with 50 ng of TRAIL per ml for the indicated times. TRAIL treatment led to cleavage of caspase 8 (p43/p41/p18), caspase 10 (p47/43/25), and cFLIP (p43) under both conditions, as determined by Western blot analysis. Membranes were rehybridized with antitubulin monoclonal antibodies to confirm comparable loading of proteins.

FIG. 6.

Full processing of caspase 3 to a 17-kDa fragment in TRAIL-treated primary keratinocytes upon proteasome inhibition. (A) Primary keratinocytes were incubated for 60 min with 10 μM MG115 (right part) or diluent alone (left part) and subsequently treated with 50 ng of TRAIL per ml for the indicated time intervals. TRAIL leads to substantial cleavage of caspase 3 to a 20-kDa fragment (p20), whereas only in the presence of the proteasome inhibitor is a 17-kDa fragment (p17) detectable. No cleavage fragments were detected in the presence of zVAD-FMK or after incubation with MG115 alone. Membranes were rehybridized with antitubulin monoclonal antibodies to confirm comparable loading of proteins. (B) TRAIL-induced effector caspase activity with affinity labeling with biotinylated DEVD-AOMK (see Materials and Methods). Activity of cleaved effector caspases by 50 ng of TRAIL per ml requires the presence of the proteasome inhibitor MG115. Primary keratinocytes were treated as above, and DEVD-specific caspase activity in cellular lysates was analyzed 3 h after TRAIL stimulation. DEVDase activity of cleaved fragments (p17/p15, upper panel) was only detectable in cells treated with TRAIL in the presence of MG115, whereas TRAIL or MG115 alone did not induce detectable caspase activity. Slight labeling of full-length caspase 3 was detected in cells treated with TRAIL alone. Subsequent rehybridization of the blot with caspase 3-specific antibody confirmed that the slower-migrating caspase 3 species (p20) did not have detectable enzymatic activity in primary keratinocytes (middle panel). In line, poly(ADP-ribose) polymerase (PARP) cleavage was only detected upon treatment with TRAIL in the presence of MG115 (lower panel). Membranes were rehybridized with antitubulin monoclonal antibodies to confirm comparable loading of proteins.

FIG. 7.

Caspase 3 activation pattern in primary and transformed keratinocytes. (A) DEVD-specific caspase activity in cellular lysates was analyzed 3 h after TRAIL stimulation in primary keratinocytes (PK) and transformed HaCaT keratinocytes (TK). DEVDase activity of p19, p17, and p15 (upper panel) was detectable in transformed keratinocytes treated with TRAIL, whereas the same concentrations of TRAIL did not induce detectable DEVDase activity of cleaved fragments of caspase 3 in primary keratinocytes. Induction of apoptosis was monitored in parallel plates by hypodiploidy analysis. Higher concentrations of TRAIL sufficient to induce partial cell death in primary keratinocytes led to a dose-dependent increase in full-length caspase 3 labeling, indicative of some enzymatic activity of full-length caspase 3. Subsequent reprobing of blots with caspase 3-specific antibody confirmed that low concentrations of TRAIL were sufficient to induce the slower-migrating caspase 3 form (p20) in primary keratinocytes. In contrast, mainly the mature cleaved forms p17 and a smaller fragment (p15) were detected in transformed keratinocytes, even at low concentrations of TRAIL. Ponceau Red staining of the membrane confirmed comparable loading of proteins. HRP, horseradish peroxidase.

FIG. 8.

XIAP confers resistance to TRAIL-induced apoptosis in primary keratinocytes. (A) XIAP is expressed in primary keratinocytes (PK) but not in transformed keratinocytes (TK) and can be coimmunoprecipitated under native conditions with caspase 3 following TRAIL treatment. Total cellular lysates from primary keratinocytes and transformed keratinocytes were prepared, and equal amounts of protein were either analyzed by Western blotting (25 μg) with XIAP monoclonal antibodies (lysate, left and right side) or subjected to immunoprecipitation (500 μg). A 57-kDa protein representing full-length XIAP was detected in primary (right) but not transformed (left) keratinocytes. An additional band of roughly 65 kDa (asterisks) detected in total lysates was shown to be a nonspecific band (immunoprecipitation: XIAP, right lanes). Following immunoprecipitation with caspase 3 antiserum, full-length but not cleaved XIAP was detected only in immunoprecipitates of TRAIL-treated primary keratinocytes PK (immunoprecipitation: caspase 3). (B) Transfection of transformed keratinocytes with XIAP leads to decreased cleavage of full-length caspase 3 and increased detection of caspase 3 p20. Transformed keratinocytes were transiently transfected with 3 μg of pEBB-XIAP vector or pEBB control vector as indicated in Materials and Methods. Then 125 ng of TRAIL per ml was added to the six-well plates, and cellular lysates were collected 3 h later. Primary keratinocytes were treated in parallel plates. The same membrane was first incubated with monoclonal antibodies to XIAP and subsequently reprobed with caspase 3 antibodies. Rehybridization with antitubulin monoclonal antibodies confirmed comparable loading of cellular proteins. Shown is a representative experiment of a total of three independent experiments. (C) XIAP overexpression leads to relative resistance of transformed keratinocytes to TRAIL-induced apoptosis. Cells were transfected as described for B and treated with the indicated concentrations of TRAIL, and viability was determined 6 h later with annexin/propidium iodide staining. Shown are means and standard deviations for two independent experiments. (D) Primary keratinocytes were incubated for 60 min with 10 μM MG115 (right part) or diluent alone (left part) and subsequently treated with 50 ng of TRAIL per ml for the indicated time periods. TRAIL treatment resulted in cleavage of XIAP to a 29-kDa fragment (p29), with similar cleavage at early time points in the presence or absence of the proteasome inhibitor. No cleavage fragments were detected in the presence of zVAD-FMK or after incubation with MG115 alone for 4 h, at which time XIAP levels were comparable to those found under control conditions.

FIG. 9.

MG115 sensitizes primary keratinocytes upstream or at the mitochondrial level. (A) Cytochrome c, Smac/DIABLO, and HtrA2/Omi release after TRAIL treatment of primary keratinocytes. Cells were treated for the indicated time intervals with 50 ng of TRAIL per ml in the presence or absence of 10 μM MG115. Cytoplasmic lysates were subsequently prepared as described in Materials and Methods and analyzed for cytochrome c, Smac/DIABLO, and HtrA2/Omi by Western blot analysis. Equal loading of cytoplasmic proteins was determined by reprobing of blots with antitubulin antibodies. (B) TRAIL-induced loss of mitochondrial transmembrane potential ΔΨ_m requires the presence of the proteasome inhibitor MG115. Cells were incubated for 60 min with either diluent alone or 10 μM MG115, followed by treatment with 50 ng of TRAIL per ml. Cells were analyzed for ΔΨ_m by staining with 40 nM tetramethylrhodamine ethyl ester 4 h later. Quantitative disruption of ΔΨ_m was only seen in cells treated with TRAIL in the presence of the proteasome inhibitor MG115.

FIG. 10.

Model of action of proteasome inhibitors mediating sensitization of primary keratinocytes to TRAIL. TRAIL activates caspases 8 and 10 at the DISC. Initial processing of caspase 3 produces a p20/p12 intermediate of caspase 3 which is inhibited by XIAP. Proteasome inhibitors interfere with caspase 8-mediated activation of mitochondrial pathways of apoptosis, leading to the caspase 8-activated release of Smac/DIABLO and HtrA2/Omi and subsequent removal of the caspase 3 prodomain (hatched bars). This activation of mitochondrial pathways is necessary for inactivation of XIAP and subsequent full caspase 3 activity in primary keratinocytes.