Abstract
Dectin-1 is a C-type lectin that recognizes beta-glucan in the cell walls of fungi and plays an important role in anti-fungal immunity. It signals via tyrosine kinase Syk and adaptor protein Card9 to activate NF-kappaB leading to proinflammatory cytokine production in dendritic cells (DCs). Other than this, not much else is known of the mechanism of Dectin-1 signaling. We demonstrate here that stimulation of DCs with zymosan triggers an intracellular Ca2+ flux that can be attenuated by a blocking anti-Dectin-1 antibody or by pre-treatment of cells with the phospholipase C (PLC) gamma-inhibitor U73122, suggesting that Dectin-1 signals via a PLCgamma pathway to induce Ca2+ flux in DCs. Interestingly, treatment of DCs with particulate curdlan, which specifically engages Dectin-1, results in the phosphorylation of both PLCgamma1 and PLCgamma2. However, we show that PLCgamma2 is the critical enzyme for Dectin-1 signaling in DCs. PLCgamma2-deficient DCs have drastic impairment of Ca2+ signaling and are defective in their secretion of interleukin 2 (IL-2), IL-6, IL-10, IL-12, IL-23, and tumor necrosis factor alpha. PLCgamma2-deficient DCs also exhibit impaired activation of ERK and JNK MAPKs and AP-1 and NFAT transcription factors in response to Dectin-1 stimulation. In addition, PLCgamma2-deficient DCs are also impaired in their activation of NF-kappaB upon Dectin-1 engagement due to defective assembly of the Card9-Bcl10-Malt1 complex and impaired IKKalpha/beta activation and IkappaBalpha degradation. Thus, our data indicate that pattern recognition receptors such as Dectin-1 could elicit Ca2+ signaling and that PLCgamma2 is a critical player in the Dectin-1 signal transduction pathway.
Highlights
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Dectin-1 Engagement Induces Ca2ϩ Flux and Activates phospholipase C (PLC)␥ in Bone marrow-derived DCs (BMDCs)—It has been speculated that Dectin-1 could signal in a manner analogous to the lymphocyte antigen receptor as it possesses an ITAM-like motif in its cytoplasmic tail and activates Syk when stimulated by its ligands [11, 16, 17]
It was readily apparent that Pam3Csk could trigger Ca2ϩ flux in BMDCs but the transient nature of the Ca2ϩ flux elicited by Pam3Csk was quite different from the sustained pattern of Ca2ϩ flux elicited by zymosan
Summary
The costs of publication of this article were defrayed in part by the payment of page charges. Because Ca2ϩ mobilization is one of the most important signaling events after BCR or TCR engagement, we first investigated if the activation of Dectin-1 could induce Ca2ϩ flux in DCs. stimulation of BMDCs by zymosan, a yeast derivative that is commonly used as an agonist of Dectin-1 [8, 10, 15], led to an induction of intracellular Ca2ϩ flux in these cells.
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