Role of NFκB activator Act1 in CD40-mediated signaling in epithelial cells

Biological Reagents and Cell Culture.

Rabbit anti-TAK1 and anti-TAB1 polyclonal Abs were described (17, 19). An Ab to cleaved poly(ADP-ribose) polymerase (PARP) was from Cell Signaling (Beverly, MA). Anti-α-tubulin, anti-IKKα/β, and anti-CD40 were from Santa Cruz Biotechnology. Anti-Act1 was produced by using a purified peptide expressing residues 1–300 of human Act1 as the antigen. HeLa, HT29, and C33A cells were maintained in DMEM, supplemented with 10% FCS, penicillin G (100 μg/ml), and streptomycin (100 μg/ml). A549 cells were maintained in minimum essential media, supplemented with 10% FCS, penicillin G (100 μg/ml), and streptomycin (100 μg/ml). Wild-type primary mouse embryonic fibroblasts (MEFs), IKKα-null, and IKKβ-null MEFs (from I. Verma, Salk Institute, La Jolla, CA) were maintained in DMEM, supplemented with 10% FCS, G418 (400 μg/ml) penicillin G (100 μg/ml), and streptomycin (100 μg/ml). The membrane-bound CD40L prepared from Hi 5 cells infected with baculoviral CD40L was kindly provided by H. Wesche (Tularik, South San Francisco).

Recombinant Plasmids.

The pCMV-Act1 (CMV, cytomegalovirus) expression constructs [flag-tagged and hemagglutinin (HA)-tagged] and pE-selectin-luc have been described (45, 47). Dominant negative TAK1 (DNTAK1-K66W) was a kind gift from K. Matsumoto (Nagoya University, Nagoya, Japan). Mammalian expression constructs for TRAFs 1–6 (flag-tagged) and CD40 (flag-tagged) were kindly provided by H. Wesche.

Coimmunoprecipitation and Immunoblotting.

For coimmunoprecipitations, cells untreated or treated with 100 μg/ml of CD40L were lysed in a Triton-containing buffer (0.5% Triton X-100/20 mM Hepes, pH 7.4/150 mM NaCl/12.5 mM β-glycerophosphate/1.5 mM MgCl₂/10 mM NaF/2 mM DTT/1 mM sodium orthovanadate/2 mM EGTA/20 μM aprotinin/1 mM phenylmethylsulfonyl fluoride). Cell extracts were incubated with 1 μg of Ab or preimmune serum (negative control) for 2 h, followed by incubation for 2 h with 20 μl of protein A-Sepharose beads (prewashed and resuspended in PBS at a 1:1 ratio). After incubations, the beads were washed 4 times with lysis buffer, separated by SDS/PAGE, and analyzed by immunoblotting.

Transfection and Reporter Assays.

For stable transfections, 2 × 10⁵ cells were seeded onto a 10-cm plate and cotransfected the following day by the calcium phosphate method with 10 μg of each expression vector and 1 μg of pBabePuro vector. After 48 h, the cells were selected with 1 μg/ml of puromycin until clones appeared. For reporter assays, 2 × 10⁵ cells were transfected by the same procedure with 1 μg of pE-selectin-luc, 1 μg of pSV₂-β-gal, and 100 ng of each expression construct. After 48 h, the cells were split onto two 35-mm plates and, the next day, were stimulated with CD40L for 4 h before harvest. Luciferase and β-galactosidase activities were determined by using the luciferase assay system and chemiluminescent reagents from Promega.

Gel-Shift Assays.

An NFκB-binding site (5′-GAGCAGAGGGAAATTCCGTAACTT-3′) from the IP-10 gene was used as a probe (48). Complementary oligonucleotides, end-labeled with polynucleotide kinase (Roche Molecular Biochemicals) and [γ-³²P]ATP, were annealed by slow cooling. Approximately 20,000 cpm of probe were used per assay (49). Whole-cell extracts were used for the assay. The binding reaction was carried out at room temperature for 20 min in a total volume of 20 μl containing 20 mM Hepes buffer (pH 7.0), 10 mM KCl, 0.1% Nonidet P-40, 0.5 mM DTT, 0.25 mM phenylmethanesulfonyl fluoride, and 10% glycerol.

Act1 Activates NFκB Through the TAK1 and IKK Signaling Cascade.

Many signaling pathways involving NFκB activation converge on IKK. We have shown that IKK is activated constitutively in cells transfected with pCMV-Act1 (45), indicating that Act1 probably activates NFκB through IKK. Furthermore, Act1 is bound to IKKγ in coimmunoprecipitation experiments, strongly suggesting that it forms a complex with IKK (45). To confirm those interactions, we tested Act1-mediated NFκB activation in IKKα- and IKKβ-null primary MEFs. Act1 activated NFκB in wild-type MEFs but failed to activate it in IKKα- and IKKβ-null MEFs, indicating that both IKKα and IKKβ are required for Act1-mediated NFκB activation (Fig. 1).

TAK1, another mitogen-activated protein kinase kinase kinase, has been implicated in activating IKK (16). TAB1, a TAK1 substrate, is phosphorylated in cells transfected with pCMV-Act1, compared with control cells (Fig. 2A). To determine whether Act1 forms a complex with TAK1, extracts of 293-TK/Zeo cells transfected with flag-tagged Act1 (CMV-Act1-flag) were immunoprecipitated with anti-TAK1 and probed with anti-flag (M2). Act1 was immunoprecipitated with TAK1, strongly suggesting that these two proteins form a complex (Fig. 2B). Moreover, kinase-dead dominant negative TAK1 inhibited Act1-dependent induction of the E-selectin promoter, indicating that Act1 functions upstream of TAK1 (Fig. 2C). Taken together, these results suggest that Act1 activates NFκB through the TAK1 and IKK signaling cascade.

Act1 Interacts with TRAF3.

To find a home for this orphan activator, we examined the interaction of Act1 with known receptor-proximal signaling components in NFκB-dependent signaling pathways. Because Act1 contains several putative TRAF-binding sites, we examined its interaction with TRAFs 1–6. We transfected flag-tagged TRAFs 1–6 into 293 cells already expressing HA-tagged Act1. Cell extracts were coimmunoprecipitated with anti-HA, followed by Western analysis with Ab against the flag-tag (M2). Although HA-tagged Act1 was immunoprecipitated with TRAF3 (Fig. 3), weak interactions of TRAFs 1 and 5 with Act1 were also observed. Because TRAF3 participates in signaling pathways mediated by CD40, LTβR, and several other members of the tumor necrosis factor receptor superfamily, the above result suggests that Act1 may play a role in pathways mediated by different receptors.

Endogenous Act1 Is Recruited to CD40 After Stimulation with CD40L.

We transfected flag-tagged CD40 into 293 cells containing HA-tagged Act1. Extracts of the transfected cells, with or without stimulation, were coimmunoprecipitated with anti-HA and probed with anti-flag. Interestingly, whereas Act1 interacted weakly with CD40 in untreated cells, stimulation with CD40L greatly induced the interaction with CD40 (Fig. 4A). These results suggest that Act1 is recruited to the CD40 receptor after stimulation.

Because the previous experiments were done with overexpressed Act1 and CD40, it was critical to examine whether endogenous Act1 can also interact with the stimulated CD40 receptor. Our recent studies indicate that Act1 is expressed well in epithelial cells of the respiratory and gastrointestinal tracts, and that its expression is augmented by proinflammatory mediators (Z.Z. and X.L., unpublished data). Therefore, we decided to use epithelial cell lines as models to study the functions of Act1. We transfected flag-tagged CD40 stably into HeLa cells, in which endogenous Act1 is highly expressed. Extracts of these transfected cells were immunoprecipitated with anti-flag (M2) and probed with anti-Act1 or vice versa. Endogenous Act1 is clearly recruited to the transfected CD40 after stimulation with CD40L. Furthermore, this recruitment is transient (Fig. 4B).

To test whether endogenous Act1 can be recruited to endogenous CD40, we first identified cell lines in which both endogenous Act1 and CD40 are expressed. By Western analysis, we found that both are expressed at high levels in HeLa and HT29 cells (Figs. 4C and 5B). Extracts of HeLa or HT29 cells, untreated or treated with CD40L, were immunoprecipitated with anti-Act1 and probed with anti-CD40. Importantly, we found that endogenous Act1 does indeed interact with CD40, both in HeLa and HT29 cells stimulated with CD40L (Fig. 4D). The interaction is transient, peaking at 5 min and disappearing after 30 min. Taken together, the above data clearly show that Act1 is recruited to CD40 after stimulation with CD40L, strongly suggesting that Act1 plays a critical role in CD40-mediated signaling.

Expression of Act1 Confers CD40L-Induced NFκB Activation in Act1-Null C33A Cells.

The CD40-mediated signaling pathway has been studied extensively in B cells, but the signaling events in this pathway are poorly understood in epithelial cells. We examined CD40-induced NFκB activation in these epithelial cells in a gel-shift assay. Whereas NFκB is strongly activated by CD40L in HT29 and HeLa cells, where Act1 is highly expressed and slightly activated in A549 cells that express a low level of Act1, CD40L failed to activate NFκB in C33A cells that do not express Act1 (Fig. 5 A and B). It is clear that the level of CD40L-induced NFκB activation correlates well with the levels of Act1 in these cells, suggesting that Act1 might be required for CD40-mediated NFκB activation.

To test this hypothesis, we examined whether the expression of Act1 in the natural Act1-null C33A cells can confer these cells sensitive to CD40L-induced NFκB activation by stably transfecting pCMV-Act1 into them. To avoid overexpression, we selected for clones that express very little Act1 (Fig. 5D). Stable transfection of pCMV-Act1 into C33A cells did render them susceptible to CD40L-induced NFκB activation (Fig. 5C), showing that Act1 can mediate this process.

Expression of Act1 Protects Act1-Null C33A Cells from CD40-Mediated Apoptosis.

Previous studies showed that the ligation of CD40 can lead to apoptosis in some epithelial cells (3). HT29, HeLa, A549, and C33A cells were treated with CD40L for 24 h and analyzed for the activation of caspase 3 by the Western method, with an Ab that recognizes caspase 3-cleaved poly(ADP-ribose) polymerase (PARP). CD40L activates apoptosis in C33A or A549 cells but not in HT29 or HeLa cells (Fig. 5F). The CD40L-induced apoptosis in C33A cells was confirmed by signal-induced DNA fragmentation (Fig. 5E). Taken together, these results show a correlation between the expression of Act1 and protection against apoptosis, which is likely to have resulted from Act1-mediated NFκB activation after stimulation with CD40L. To test this possibility further, we examined C33A cells stably transfected with pCMV-Act1 and found that the expression of Act1 suppressed CD40L-induced apoptosis (Fig. 5 D and F).

TRAF3 Inhibits Act1-Mediated NFκB Activation.

TRAF3 has been implicated in CD40-mediated JNK activation and may have proapoptotic activity but it is not involved in the activation of NFκB (29–33). Furthermore, previous studies showed that overexpression of TRAF3 inhibited CD40-mediated NFκB activation (31, 50, 51). Because we found that Act1 interacts with TRAF3 after stimulation with CD40L, we tested the effect of overexpressing it on Act1-mediated NFκB activation. Increasing amounts of TRAF3 were cotransfected with pCMV-Act1 and E-selectin luciferase into 293 cells, followed by a luciferase reporter assay. Interestingly, the overexpression of TRAF3 effectively inhibited NFκB activation mediated by Act1 (Fig. 6A) but had no effect on IRAK-induced NFκB activation (Fig. 6B).