Caspase inhibition sensitizes inhibitor of NF-kappaB kinase beta-deficient fibroblasts to caspase-independent cell death via the generation of reactive oxygen species

Abstract

Cells lacking functional NF-kappaB die after ligation of some tumor necrosis factor (TNF) receptor family members through failure to express NF-kappaB-dependent anti-apoptotic genes. NF-kappaB activation requires the IkappaB kinase (IKK) complex containing two catalytic subunits named IKKalpha and IKKbeta that regulate distinct NF-kappaB pathways. IKKbeta is critical for classical signaling that induces pro-inflammatory and anti-apoptotic gene profiles, whereas IKKalpha regulates the non-canonical pathway involved in lymphoid organogenesis and B-cell development. To determine whether IKKalpha and IKKbeta differentially function in rescuing cells from death induced by activators of the classical and non-canonical pathways, we analyzed death after ligation of the TNF and lymphotoxin-beta receptors, respectively. Using murine embryonic fibroblasts (MEFs) lacking each of the IKKs, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, and dominant negative Fas-associated death domain protein, we found that deletion of these kinases sensitized MEFs to distinct cell death pathways. MEFs lacking IKKalpha were sensitized to death in response to both cytokines that was entirely caspase-dependent, demonstrating that IKKalpha functions in this process. Surprisingly, death of IKKbeta-/- MEFs was not blocked by caspase inhibition, demonstrating that IKKbeta negatively regulates caspase-independent cell death (CICD). CICD was strongly activated by both TNF and lymphotoxin-beta receptor ligation in IKKbeta-/- MEFs and was accompanied by loss of mitochondrial membrane potential and the generation of reactive oxygen species. CICD was inhibited by the anti-oxidant butylated hydroxyanosole and overexpression of Bcl-2, neither of which blocked caspase-dependent apoptosis. Our findings, therefore, demonstrate that both IKKalpha and IKKbeta regulate cytokine-induced apoptosis, and IKKbeta additionally represses reactive oxygen species- and mitochondrial-dependent CICD.

FIGURE 1. TNF and LTα1β2 induce cell death in IKKα^−/− and IKKβ^−/− MEFs

A, MEFs were untreated (shaded) or treated with TNF (10 ng/ml; solid line) or LTα1β2 (100 ng/ml; dotted line) for 18 h. Cells were harvested, stained with Alexa Fluor-conjugated annexin V, and analyzed by FACS. The percentage of positively stained cells gated in region M1 were determined and plotted in the histogram shown in B. Ctr, control. C, MEFs were treated as in A then incubated in PBS containing PI and analyzed by FACS. The percentage of membrane-permeable cells that took up PI gated in region M1 was determined, and these data are shown in the histogram in panel D.

FIGURE 2. Caspase inhibition blocks cytokine-induced cell death in HUVEC and IKKα^−/− MEFs

A, HUVEC were untreated (shaded) or treated with TNF (10 ng/ml; dashed line), cycloheximide (2.5 μg/ml; solid line), or a combination of TNF plus cycloheximide (dotted line). Each treatment was performed in the absence of presence of zVADfmk (25 μM). Cells were harvested, incubated in PBS containing PI, and analyzed by FACS. The population percentage that was membrane-permeable and took up PI was determined from region M1 and illustrated in B. Ctl, control. C, HUVEC were treated as described in A, then the percentage of the population with hypodiploid DNA was determined by FACS. D, WT and IKKα^−/− MEFs were either untreated (shaded) or treated with LTα1β2 (solid line) either alone or in combination with zVADfmk (25 μM) as shown. Cells were harvested, incubated in PBS containing PI, and analyzed by FACS. The membrane-permeable population percentage that took up PI was determined from region M1 and is shown in the histogram in panel E.

FIGURE 3. Caspase inhibition does not block TNF- and LTα1β2-induced cell death in IKKβ^−/− MEFs

A, WT or IKKβ^−/− MEFs were either untreated (shaded) or treated overnight with TNF (10 ng/ml; solid line) or LTα1β2 (100 ng/ml; dotted line) in the absence (control (Ctr)) or presence of zVADfmk at the concentrations indicated (left). Cells were harvested, incubated in PBS containing PI, and then analyzed by FACS. Membrane-permeable cells that took up PI were determined in region M1. The % PI-positive MEFs treated with 25 μM zVADfmk are shown in panel B (+) compared with cells that received no inhibitor (−). C, WT or IKKβ^−/− MEFs were either untreated or treated with TNF in the presence (+) or absence (−) of zVADfmk (25 μM). Cells were harvested, fixed, permeabilized, and incubated with PI, then the percentage of the population with hypodiploid DNA was determined by FACS.

FIGURE 4. Repression of CICD by IKKβ does not require NEMO

A, WT and IKKβ^−/− MEFs transduced with LZRS or IKKβ as shown (parentheses) were either untreated (shaded) or treated overnight with TNF (10 ng/ml; unshaded) in the presence of zVADfmk (25 μM). Cells were harvested, incubated in PBS containing PI, and then analyzed by FACS. Membrane-permeable cells that took up PI were determined in region M1, and the percentage PI-positive MEFs is shown in panel B. C, WT and NEMO-deficient MEFs were either untreated (shaded) or treated overnight with TNF (10 ng/ml; solid line) in the absence (Control) or presence of zVADfmk (25 μM). PI uptake was determined as described for panel A, and the percentage of PI-positive cells is shown in D.

FIGURE 5. FADD^DN blocks cell death in IKKα^−/− but not IKKβ^−/− MEFs

A, WT, IKKα^−/−, or IKKβ^−/− MEFs were either untreated (shaded) or treated overnight with TNF (10 ng/ml; unshaded) in the absence (Control) or presence of zVADfmk (25 μM). FADD ^DN-transduced IKKα^−/− and IKKβ^−/− MEFs were also either untreated or treated overnight with TNF. Cells were harvested, incubated in PBS containing PI, and then analyzed by FACS. Membrane-permeable cells that took up PI were determined in region M1, and the percentage of PI-positive cells is shown in B.

FIGURE 6. CICD in IKKβ^−/− MEFs results from mitochondrial dysfunction

A, WT and IKKβ^−/− MEFs were either untreated (shaded) or treated overnight with TNF (10 ng/ml; solid line) in the absence (Ctr) or presence of zVADfmk (25 μM). Cells were harvested and loaded with JC-1 (10 μg/ml), and mitochondrial membrane potential (ΔΨ) was determined by FACS. Loss of ΔΨ is indicated by a shift in the histogram to the right. B, LZRS and Bcl-2 transduced IKKβ^−/− MEFs were either untreated (Ctr) or treated with etoposide (50 μM) overnight. Cells were harvested, incubated in PBS containing PI, and analyzed by FACS. The percentage of the population that was PI-positive is shown. C, LZRS or Bcl-2 transduced MEFs were either untreated (shaded), or treated overnight with either LTα1β2 (100 ng/ml; dotted line) or TNF (10 ng/ml; solid line) in the absence (Control) or presence of zVADfmk (25 μM). Cells were harvested, incubated in PBS containing PI, and analyzed by FACS, and the percentage that took up PI was determined from region M1. The data from the TNF with and without zVAD-treated MEFs are summarized in D.

FIGURE 7. The death of IKKβ^−/− cells treated with TNF and zVADfmk is dependent on the generation of ROS

A, WT and IKKβ^−/− cells were either untreated (shaded) or treated overnight with TNF (10 ng/ml; solid line) in the absence (Control) or presence of zVADfmk (25 μM). After treatment, cells were harvested, and ROS levels were determined by FACS using H2DCFDA (10 μM. The generation of ROS relative to the levels observed in untreated WT or IKKβ^−/− cells was determined and is shown in panel B. C, WT or IKKβ^−/− MEFs were either untreated (Control) or pretreated for 60 min with BHA at the concentrations indicated (left) before either no further treatment (shaded) or overnight treatment with TNF (10 ng/ml; dashed line), zVADfmk (25 μM; dotted line), or TNF plus zVADfmk (solid line). Cells were harvested, incubated in PBS containing PI, and analyzed by FACS. The percentage of the population that were PI positive was determined from region M1 and is shown in panel D.

FIGURE 8. Effects of TNF and zVADfmk on Mn-SOD and catalase levels in WT and IKKβ^−/− MEFs

WT and IKKβ^−/− MEFs were either untreated or incubated overnight with TNF (10 ng/ml) in the absence or presence of zVADfmk (25 μM) as shown. Cell lysates were separated by SDS-PAGE (10%), and the resulting immunoblots were probed using either anti-Mn-SOD (A) or anti-catalase (B). Anti-tubulin was used to ensure equal protein loading.

FIGURE 9. Model of apoptosis and CICD in WT and IKKβ^−/− MEFs

In this model activation is denoted by an arrowhead and inhibition by a straight line. A, in WT MEFs, TNF and LTα1β2 activate IKKβ that inhibits both caspase-dependent apoptosis and CICD. Caspases are blocked by the classical NF-κB-dependent induction of caspase inhibitors including c-FLIP and c-IAP2, and ROS are blocked by the up-regulation of antioxidant enzymes such as Mn-SOD (Fig. 8A). B, in the absence of IKKβ cytokines activate caspase-dependent apoptosis. ROS levels are increased possibly via a lack of induced Mn-SOD expression (Fig. 8A), and mitochondrial membrane potential is lost (Fig. 6A). However, in the presence of active caspases, CICD does not occur, and neither Bcl-2 (Figs. 6, C and D) nor BHA (Figs. 7, C and D) can block cell death. C, caspase-dependent apoptosis is blocked in IKKβ^−/− MEFs stimulated with cytokines in the presence of zVADfmk. These cells die by CICD that can be inhibited by mitochondrial regulation via Bcl-2 overexpression (Figs. 6, C and D) or by ROS scavenging via BHA treatment (Figs. 7, C and D).