CARD9 mediates dectin-2-induced IkappaBalpha kinase ubiquitination leading to activation of NF-kappaB in response to stimulation by the hyphal form of Candida albicans

Abstract

The scaffold protein CARD9 plays an essential role in anti-fungus immunity and is implicated in mediating Dectin-1/Syk-induced NF-kappaB activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-kappaB activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-kappaB activation induced by the hyphal form of C. albicans hyphae (Hyphae) but not by its heat-inactivated unicellular form. Our data show that inhibiting Dectin-2 expression selectively blocked Hyphae-induced NF-kappaB, whereas inhibiting Dectin-1 mainly suppressed zymosan-induced NF-kappaB, indicating that Hyphae-induced NF-kappaB activation is mainly through Dectin-2 and not Dectin-1. Consistently, we find that the hyphae stimulation induces CARD9 association with Bcl10, an adaptor protein that functions downstream of CARD9 and is also involved in C. albicans-induced NF-kappaB activation. This association is dependent on Dectin-2 but not Dectin-1 following the hyphae stimulation. Finally, we find that although both CARD9 and Syk are required for Hyphae-induced NF-kappaB activation, they regulate different signaling events in which CARD9 mediates IkappaBalpha kinase ubiquitination, whereas Syk regulates IkappaBalpha kinase phosphorylation. Together, our data demonstrated that CARD9 is selectively involved in Dectin-2-induced NF-kappaB activation in response to C. albicans hyphae challenging.

FIGURE 1.

CARD9 and Bcl10 are required for NF-κB activation induced by C. albicans but not activation induced by zymosan and curdlan. A, wild-type (WT) and Card9^−/− (KO) BMDMs were stimulated with C. albicans (MOI = 1), zymosan (50 μg/ml), curdlan (50 μg/ml), or LPS (100 ng/ml) for the indicated times. The nuclear extracts were prepared from these cells and subjected to EMSA using ³²P-labeled NF-κB or Oct-1 probe. B and C, WT and Card9^−/− (KO) BMDMs were stimulated with live C. albicans (MOI = 1) and LPS (100 ng/ml) (B) or zymosan (50 μg/ml) and LPS (100 ng/ml) (C) for the indicated times. The cell lysates were subjected to immunoblotting analysis using the indicated antibodies. D, WT and Bcl10^−/− (KO) BMDMs were stimulated with C. albicans (MOI = 1), zymosan (50 μg/ml), curdlan (50 μg/ml), or LPS (100 ng/ml) at the indicated times. The nuclear extracts were prepared from these cells and subjected to EMSA using ³²P-labeled NF-κB or Oct-1 probe. A set of representative results from three independent experiments was presented.

FIGURE 2.

Both hyphal and yeast forms of C. albicans can induce NF-κB activation. A, wild-type (WT) and Card9^−/− BMDMs were stimulated with C. albicans at MOI = 1 for the indicated time points. Nuclear extracts were prepared and subjected to EMSA using ³²P-labeled NF-κB and Oct-1 probes. B, wild-type BMDMs were stimulated with heat-inactivated C. albicans (H.I.C.a) or live C. albicans (C.a) for the indicated time points. The nuclear extracts were prepared and subjected to EMSA as in A. C, cell lysates from the samples in B were subjected to immunoblotting analysis using the indicated antibodies. The results are representative of three independent experiments. D, the morphological changes of both heat-inactivated and live C. albicans in the condition for mammalian cell culture at the indicated time points were examined with microscopy. E, wild-type and Card9^−/− BMDMs stimulated with hyphae or the heat-inactivated form of C. albicans at MOI = 1 or zymosan (50 μg/ml) for 1 h. The nuclear extracts were prepared from these cells and subjected to EMSA using ³²P-labeled NF-κB and Oct-1 probes. Stim., unstimulated; Unsti., unstimulated; KO, knock-out.

FIGURE 3.

Dectin-2 but not Dectin-1 is required for NF-κB activation induced by C. albicans hyphae. A, BMDMs with stable knockdown of Dectin-1, Dectin-2, or green fluorescent protein (shGFP) control (Mock) were stimulated with hyphae or heat-inactivated (Yeast) C. albicans (MOI = 1), or zymosan (50 μg/ml) for 1 h. The nuclear extracts were prepared and subjected to EMSA using ³²P-labeled NF-κB or Oct-1 probes. B, lysates prepared from the cells of A were subjected to immunoblotting analysis using the indicated antibodies. C, the levels of tumor necrosis factor tumor necrosis factor α (TNF-α), IL-12p40, and IL-10 in the supernatants from BMDMs with stable knockdown of Dectin-1, Dectin-2, or vector control were stimulated with C. albicans hyphae (MOI = 1), C. albicans yeast (MOI = 1), zymosan (50 μg/ml), or LPS (100 g/ml) overnight (error bars, standard deviation of triplicate each sample). Unsti., unstimulated.

FIGURE 4.

Expression of Dectin-2 significantly enhanced C. albicans hyphae-induced NF-κB activation. A, THP-1 cells stably expressing Dectin-1, Dectin-2, or control vector were stimulated with C. albicans hyphae, C. albicans yeast, or zymosan for 1 h. The nuclear extracts were prepared and subjected to EMSA as described in A. B, lysates prepared from samples in the A were subjected to immunoblotting analysis using the indicated antibodies. Unsti., unstimulated.

FIGURE 5.

Dectin-2 is required for the CARD9-Bcl10 interaction upon the stimulation by C. albicans hyphae. A, wild-type BMDMs were challenged with C. albicans hyphae at the indicated time points. The cell lysates were collected and subjected to the immunoprecipitation (IP) using CARD9 antibodies. The precipitates and lysates were subjected to immunoblotting analysis using the indicated antibodies. B, BMDMs with stable knockdown of Dectin-1, Dectin-2, or vector control were stimulated with C. albicans hyphae for the indicated time points. Cell lysates from these samples were subjected to immunoprecipitation analysis as in A. C, THP-1 cells stably expressing Dectin-1, Dectin-2, or its control vector were challenged with C. albicans hyphae at the indicated time points. Immunoprecipitation analysis was performed as in A and B.

FIGURE 6.

CARD9 and Syk function independently regulates IKK phosphorylation and ubiquitination. A, wild-type (WT) BMDMs were stimulated with C. albicans hyphae (Hyphae), zymosan (50 μg/ml), heat-inactivated C. albicans (Yeast), or LPS (100 ng/ml) for 1 h with or without pretreatment of the Syk inhibitor piceatanol for 0.5 h. The nuclear extracts were prepared and subjected to EMSA using ³²P-labeled NF-κB or Oct-1 probe. The cell lysates were subjected to immunoblotting analysis using the indicated antibodies. B, wild-type and Card9^−/− (KO) BMDMs, as well as wild-type BMDMs pretreated with piceatanol, were stimulated with C. albicans hyphae (MOI = 1) or LPS (L) for the indicated time points. The cell lysates from these samples were prepared and subjected to immunoblotting analysis using the indicated antibodies. C, wild-type and Card9^−/− (KO) BMDMs and wild-type BMDMs pretreated with piceatanol were stimulated with C. albicans hyphae (MOI = 1). The cell lysates from these samples were subjected to immunoprecipitation using NEMO/IKKγ antibodies. The immunoprecipitates and cell lysates were subjected to immunoblot analysis using ubiquitin or NEMO antibodies. D, wild-type BMDMs were treated with or without piceatanol and then challenged with C. albicans hyphae at the indicated time points. The cell lysates were collected and subjected to the immunoprecipitation using CARD9 antibodies. The precipitates and lysates were subjected to immunoblotting analysis using the indicated antibodies. Unsti., unstimulated; Pic., piceatanol.

FIGURE 7.

The model for C. albicans-induced signaling pathways leading to NF-κB activation. The hyphal form of C. albicans recognizes Dectin-2, whereas yeast and β-glucans, such as zymosan, activate Dectin-1. Although both Dectin-1 and Dectin-2 pathways are required for Syk that regulates IKK phosphorylation, CARD9 and Bcl10 are selectively involved in Dectin-2-induced signaling cascades leading to regulation of NEMO ubiquitination. The phosphorylation and ubiquitination of IKK in the Dectin-2-induced signaling pathway are required for activation of the IKK complex for NF-κB activation. LPS and other TLR ligands activate NF-κB through a MyD88-dependent but CARD9-independent pathway.