The death domain kinase RIP1 links the immunoregulatory CD40 receptor to apoptotic signaling in carcinomas

Abstract

CD40, a tumor necrosis factor (TNF) receptor family member, is widely recognized for its prominent role in the antitumor immune response. The immunostimulatory effects of CD40 ligation on malignant cells can be switched to apoptosis upon disruption of survival signals transduced by the binding of the adaptor protein TRAF6 to CD40. Apoptosis induction requires a TRAF2-interacting CD40 motif but is initiated within a cytosolic death-inducing signaling complex after mobilization of receptor-bound TRAF2 to the cytoplasm. We demonstrate that receptor-interacting protein 1 (RIP1) is an integral component of this complex and is required for CD40 ligand-induced caspase-8 activation and tumor cell killing. Degradation of the RIP1 K63 ubiquitin ligases cIAP1/2 amplifies the CD40-mediated cytotoxic effect, whereas inhibition of CYLD, a RIP1 K63 deubiquitinating enzyme, reduces it. This two-step mechanism of apoptosis induction expands our appreciation of commonalities in apoptosis regulatory pathways across the TNF receptor superfamily and provides a telling example of how TNF family receptors usurp alternative programs to fulfill distinct cellular functions.

Figure 1.

The TRAF2/TRAF3 binding domain of CD40 mediates CD154-induced death signals. (A) Graphical representation of CD40 and its TRAF-binding domains. A double Q²³⁴E²³⁵ → AA mutation, yielding CD40mT6, selectively abolishes the interaction of TRAF6 with CD40, whereas a T²⁵⁴ → A mutation (CD40mT2/T3) inhibits TRAF2 and TRAF3 but not TRAF6 binding to CD40. CD40mT2/T3/T6 combines the aforementioned mutations and perturbs the binding of all TRAFs (Davies et al., 2005b). WT, wild type. (B) The TRAF6-interacting domain of CD40 transduces ERK and Akt signaling. Lysates from CD154-stimulated HeLa/CD40 and HeLa/CD40mT6 cells were analyzed for the expression of phosphorylated, active ERK and Akt, or the total proteins. (C) The TRAF2/TRAF3-interacting domain of CD40 mediates CD154-induced death signals. HeLa clones expressing the WT or mutated CD40 sequences described in A were stimulated with CD154 for 12 h before assessment of apoptosis. Mean values of percentage apoptotic cells from at least three independent experiments are shown with the exception of HeLa/CD40mT6 clone 10, where two determinations were performed. (D and E) The TRAF2/TRAF3-binding domain of CD40 mediates cell death via caspase-8 activation. HeLa/CD40mT6 clone 21 cells were stimulated with CD154 for the indicated time points, and lysates were analyzed for the expression of caspase-8, caspase-3, or β-actin (D). Inhibitors of caspase-8 and -3 but not caspase-9 protect HeLa/CD40mT6 cells from CD154-induced apoptosis assessed by cell death ELISA. Data are expressed as fold increase (±SD; n = 4) in apoptosis induced by CD154 relative to untreated cultures, which was given the arbitrary value of 1. (F) RT-PCR showing up-regulation of TNF mRNA after treatment of HeLa/CD40mT6 cells with CD154. GADPH, glyceraldehyde 3-phosphate dehydrogenase. (G) Early CD154-mediated death signals are independent of autocrine TNF production. HeLa/CD40mT6 cells were exposed to 0.5 µg/ml neutralizing anti-TNF mAb and then treated as described in C before assessment of apoptosis. Error bars indicate SD.

Figure 2.

TRAF2 is required for the transduction of CD40 death signals. HeLa/CD40mT6 cells were transfected with TRAF2, TRAF3, or control siRNA and either lysed for the assessment of TRAF knockdown efficacy by immunoblotting (A) or exposed to 1 µg/ml soluble CD154 before evaluation of apoptosis (B). (C) Knockdown of TRAF2 in EJ bladder carcinoma cells confers resistance to apoptosis induced by soluble CD154 in the presence of 10 µg/ml CHX. Mean values (±SD) from three independent experiments are shown (error bars).

Figure 3.

TRAF2 and RIP1 interact in the context of CD40 signaling. (A) Ectopic expression of CD40 enhances the interaction of RIP1 with endogenous TRAF2. Two million 293 cells were transfected with 0.5 µg FLAG-tagged RIP1 in the presence of increasing amounts of a CD40 expression vector (0, 1, or 2.5 µg). Lysates were immunoprecipitated (IP) with anti-FLAG and immunoblotted (IB) with an anti-TRAF2 or RIP1 antibody as indicated. (B and C) CD40 ligation induces the association of endogenous RIP1 with TRAF2 but not CD40. HeLa/CD40mT6 cells were stimulated with CD154, then lysates were sequentially immunoprecipitated with anti-CD40 (B) and anti-RIP1 (C) and immunoblotted with TRAF2, RIP1, or CD40 antibodies.

Figure 4.

RIP1 is required for CD40-mediated apoptosis in carcinoma cells. (A) Knockdown efficiency of RIP1 siRNA. (B) EJ cells treated with CD154 in the presence of CHX display morphology typical of apoptotic cells (see representative arrowheads), whereas RIP1 siRNA-transfected cultures remain unaffected. Bar, 25 µm. (C and D) RIP1 knockdown rescues EJ cells from CD154 and CHX-induced apoptosis, as determined by propidium iodide staining and immunofluorescence microscopy (C) or cell death ELISA (D). Mean values (±SD) from four independent experiments are shown. (E and F) RIP1 knockdown rescues HeLa/CD40mT6 cells from CD154-induced apoptosis, determined as described in the legend of Fig 4 C. (G and H) Expression of cIAP1/2 (G) and CYLD (H) before and after transfection with the respective siRNAs. (I) Effect of cIAP1/2 and CYLD knockdown on CD154-mediated EJ cell death. (J) Necrostatin (50 µM) inhibits and the pan-caspase inhibitor zVAD-fmk (15 µM) abolishes the pro-apoptotic effects of CD154 in EJ cells. Error bars indicate SD.

Figure 5.

RIP1 associates with caspase-8 and is required for its activation. (A and B) RIP1 knockdown suppresses CD154-induced caspase-8 cleavage in EJ (A) or HeLa/CD40mT6 (B) cells. Lysates were immunoblotted for caspase-8 or, as a control, β-actin or p50 NF-κB. (C) RIP1, TRAF2, FAF1, and caspase-8 interact upon CD40 stimulation. Cells were stimulated with CD154, and lysates were immunoprecipitated with a goat polyclonal against caspase-8. Immunoprecipitants were immunoblotted using rabbit polyclonal antibodies against RIP1, TRAF2, CD40, and FAF1 or a monoclonal anti-caspase-8, as indicated. Results are representative of three independent experiments. The Ig light chains are indicted by asterisks. (D and E) Expression of FADD (D) and FAF1 (E) before and after transfection with the respective siRNAs. (F and G) Crystal violet staining of RNAi-transfected EJ cells treated with CHX in the presence or absence of CD154 or CD95L. (H) FAF1 but not FADD knockdown protects from the pro-apoptotic effects of CD40 ligation. After knockdown, EJ cells were exposed to CD154 and CHX for 5 h before assessment of apoptosis. Error bars indicate SD. (I) Proposed model of CD40-induced death signaling in tumor cells. CD40-mediated apoptosis involves the formation of a secondary cytoplasmic complex of TRAF2, RIP1, FAF1 and caspase-8 (Casp-8). RIP1 is required for caspase-8 activation and cell death, whereas apoptosis is antagonized by survival signals predominantly mediated by the TRAF6-binding domain of CD40, which may operate at the level of the death-inducing signaling complex and/or downstream of it.