CARD9 Mediates Dectin-2-induced IκBα Kinase Ubiquitination Leading to Activation of NF-κB in Response to Stimulation by the Hyphal Form of Candida albicans

Abstract

The scaffold protein CARD9 plays an essential role in antifungus immunity and is implicated in mediating Dectin-1/Sykinduced NF-B activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-B activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-B 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-B, whereas inhibiting Dectin-1 mainly suppressed zymosan-induced NF-B, indicating that Hyphae-induced NF-B 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-B 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-B activation, they regulate different signaling events in which CARD9 mediates IB␣ kinase ubiquitination, whereas Syk regulates IB␣ kinase phosphorylation. Together, our data demonstrated that CARD9 is selectively involved in Dectin-2-induced NF-B activation in response to C. albicans hyphae challenging.Candida albicans is a major opportunistic fungal pathogen that predominantly causes infection to cancer patients and immunocompromised individuals. During C. albicans infection, macrophages and dendritic cells recognize components from the fungal cell wall through their pattern recognition receptors (1, 2), which triggers a series of signaling cascades leading to activation of various transcription factors including NF-B (1). The activation of NF-B and other transcription factors further induce the expression of various cytokines and chemokines and inflammatory responses. However, the pattern recognition receptors that recognize fungal cell wall components are not fully defined (3).NF-B is a family of transcription factors that control the expression of pro-inflammatory genes in immune cells (4). In resting cells, the activity of NF-B is tightly controlled by the IB family of proteins, which bind to NF-B dimers and keep these dimers in the cytoplasm. The canonical NF-B activation pathway by most of NF-B-inducing stimuli activates the IB␣ kinase (IKK) 2 complex. The IKK complex is controlled by signal-induced phosphorylation of IKK␣ and IKK␤ subunits (5) and signal-induced K63-linked ubiquitination of the regulatory subunit NEMO (6). The activated IKK complex in turn phosphorylates IB␣ proteins on N-terminal conserved serine residues to target them for ubiquitination-dependent degradation (5). This process releases NF-B and allows its translocation into the nucleus for the activation of its target genes (4). Although it has been shown that bacterial and viral infections induce IKK activation by Toll-like receptors (TLR...

NF-κB signaling pathways regulated by CARMA family of scaffold proteins

“…Similarly to CARMA1 and CARMA3, CARD9 is required for activation of the IKK complex [151]. Consistent with this model, CARD9-deficient macrophages and dendritic cells have impaired expression of TNF-α, IL-6, and IL-12 in response to the stimulation with fungal particles [149,151,152,157] and CARD9-deficient mice are more susceptible to infection with the fungus Candida albicans [149,151,152,156,158].…”

“…Because zymosan can activate the signaling pathways induced by Dectin-1, a C-type lectin receptor, it has been proposed that CARD9 mediates Dectin-1-induced NF-κB activation [149]. However, recent studies from our laboratory have surprisingly found that zymosan can still effectively activate NF-κB in macrophages from an independently generated CARD9-deficient mouse strain [151], suggesting that Dectin-1-induced NF-κB activation may also be induced through a CARD9-independent pathway [151]. Therefore, the requirement of CARD9 in Dectin-1-induced NF-κB activation needs to be further investigated.…”

“…The proximal signaling cascade leads to a sequential activation of Syk, CARD9, and IKK following stimulation of these C-type lectin receptors ( Figure 5) [13]. However, the latest work argues that although Syk is required for both Dectin-1-and Dectin-2-induced NF-κB activation, CARD9 is only required for Dectin-2-induced NF-κB activation [151]. In addition, Syk and CARD9 do not seem to function in a linear cascade as Syk mediates IKK phosphorylation, whereas CARD9 controls NEMO polyubiquitination [151].…”

“…Therefore, the requirement of CARD9 in Dectin-1-induced NF-κB activation needs to be further investigated. Besides the Dectin-1 pathway, it has been demonstrated that CARD9 also mediates the signaling induced by other C-type lectin receptors such as Dectin-2 [151,152] and Mincle [153]. These receptors have been implicated to act as the pattern recognition receptors for fungi [154,155].…”

“…It has been suggested that CARD9 functions downstream of the tyrosine kinase Syk and couples Bcl10/ MALT1 to activate classical NF-κB [7,151,152,156,157]. Similarly to CARMA1 and CARMA3, CARD9 is required for activation of the IKK complex [151].…”