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Toll-like receptors (TLRs) function as pattern-reorganization receptors in mammals and play an essential role in innate immunity. After recognizing certain ligands, TLRs activate a cascade of signal pathways to establish a guard environment. For the first time, we report that in vitro treatment with recombinant calcineurin subunit B (CNB) upregulated several TLR-related genes. Calcineurin subunit B interacted with the ectodomain of TLR4 in vitro. On further investigation, phosphorylation of interferon regulatory factor 3 and degradation of IκB-α were observed by CNB stimulation. In addition to pro-inflammatory cytokines, transcription and production of β-interferon were also enhanced after CNB stimulation. Thus, CNB could be further explored as a cancer and virus immunotherapy drug. (Cancer Sci 2012; 103: 515–521)
Innate immunity provides immediate and efficient defense against microbial infection. Toll-like receptors play an important role in innate immunity. They detect microbes, viruses, and endogenous ligands to mediate the induction of genes encoding inflammatory cytokines, chemokines, interferons, and interferon-related molecules.(1) Toll-like receptor 4 is the only member of the TLR family that functions through both the MyD88 and TRIF signal pathways. After receiving certain kinds of stimulation, TLR4 promotes IRF3 and NF-κB translocation to the nucleus to activate IFN-β and pro-inflammatory cytokines transcription. Long-term IFN-β therapy decreases the recurrence rate of hepatocellular carcinoma after curative therapy.(2) Interferons do not possess the ability to eliminate viruses or tumors; instead, they activate a cascade of signaling pathways to inhibit virus replication or tumor proliferation.(3)
Calcineurin is regulated by Ca2+, which in turn dephosphorylates and induces the nuclear localization of cytoplasmic components of the nuclear factor of activated T cells transcription complexes.(4) Subunit A of calcineurin is a catalytic subunit, whereas CNB is a regulatory subunit that regulates CN’s activity by binding to the CNB binding helix.(5) Subunit B of calcineurin is highly hydrophobic and conserved. CNB−/− mice showed a higher risk for squamous cell carcinoma.(6) The CNB potentiates the activation of procaspase-3 by accelerating its proteolytic maturation.(7) In plants, CNB-like proteins have been associated with many fundamental processes, such as salt tolerance or K+ uptake.(8,9) These results reveal that CNB might possess some biological functions independent of CNA. To further test the function of CNB alone, CNB has been successfully characterized and purified in our laboratory.(10) Suppression of certain types of carcinoma cells was observed by i.p. injection of CNB into mice pre-inoculated with carcinoma.(11) Moreover, CNB activates human PBMC-derived and mice marrow-derived DCs to secrete pro-inflammatory cytokines,(12) but the underlying mechanisms have not been well described.
Here, for the first time, we report that CNB interacted with TLR4 ectodomain in vitro, which in turn led to phosphorylaiton of IRF3 and degradation of IκB-α. Secretion of IFN-β and pro-inflammatory cytokines were induced after CNB stimulation. This provides a molecular mechanism for CNB’s immune booster and anticancer mechanism.
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- Materials and Methods
- Disclosure Statement
Our previous results have shown that the phagocytic activities of peritoneal macrophages and the natural killer activities of murine spleen lymphocytes increased after CNB injection.(27) Subunit B of calcineurin also inhibits the growth of H22 liver ascites cancer cells and S180 solid cancer cells in vivo.(11) Moreover, CNB activates human PBMC-derived and mice marrow-derived DCs to secrete pro-inflammatory cytokines.(12) To elucidate the molecular mechanism, we designed a gene chip assay. From the assay, we found that genes for IKKε, TRAF3, and NF-κB were upregulated by CNB stimulation in U937 cells (Fig. 1). The TRAF3–TBK1–IKKε–IRF3 complex is of great importance to both TLR-dependent and TLR-independent production of type I IFN production.(28,29) IKKε can form a heterodimer with TBK1, and may act as a kinase to phophorylate IRF3.(23,28) Both TRAF3 and IRF3 are uniquely required for the TRIF-dependent interferon response.(29) The regulatory elements for IFN-β included κB sites, IRES sites, and AP-1 sites, which bind NF-κB, IRFs, or c-Jun/ATF2 heterodimer, respectively.(30) Previous reports have also implied that CNB undertakes NF-κB signal pathways to activate DCs to produce several pro-inflammatory cytokines.(12) To further investigate the immune booster mechanism, we carried out experiments to detect NF-κB and IRF3 activation. As expected, CNB promoted degradation of IκB-α in both Raw264.7 cells (Fig. 3) and U937 cells (Wu Wu, Beijing Normal University, Beijing, China, unpublished data). Phosphorylation and dimerization of IRF3 was also observed after CNB stimulation (Fig. 2). Transcription of IFN-β in Raw264.7 cells was enhanced in a TRAF3- and IRF3-dependent way by CNB stimulation (Fig. 4). All of these results indicate that CNB stimulation may lead to IFN-β production as well as pro-inflammatory cytokines.
Stimulation of LPS caused degradation of TRAF3 in Raw264.7 cells and upregulation of RANTES in A549 cells, which has been reported previously.(1,31) Subunit B of calcineurin does not promote degradation of TRAF3, which is different from LPS (Fig. 2). TRAF3 is a key regulator for balancing type I interferon and pro-inflammatory cytokines production. K48 ubiquitination of TRAF3 induced by LPS is designed for proteasomal degradation, whereas K63 ubiquitination of TRAF3 is essential for IFN production.(1) TRAF3−/− cells are defective in type I IFN responses activated by several different TLRs.(28) TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKKε, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. We speculated that cells receiving CNB stimulation might “preferentially” promote K63 ubiquitination of TRAF3 for type I IFN production, which needs further research.
The MKK4/7-JNK or TBK-1/IKKε complex functions as an upstream kinase for IRF3.(23,32) TAK1 forms a complex with TAB 1, TAB 2, and TAB 3. The activated TAK1 complex phosphorylates IκB kinases, which activates NF-κB.(33) Thus, we detected TAK-1 and TBK-1 mRNA levels after CNB stimulation by qPCR (Fig. 5). The mRNA levels for TAK-1 and TBK-1 were both upregulated after addition of CNB. These results implied that the kinases for IRF3 and IκB-α phosphorylation in our CNB stimulation pathways were TBK-1 and TAK-1, respectively.
β-interferon itself does not kill the tumor or virus. Instead, it initiates a cascade of signal pathways to inhibit virus replication or tumor cell proliferation.(3) β-interferon eliminates activated DCs by caspase-3/11 activation, which plays an essential role in re-establishing homeostasis.(34) Antitumor effects mediated by IFN-β have also been reported.(34–36) β-interferon KO mice showed a higher risk for sustained inflammation, cytokine production, and tissue damage with consequent chronic neurological deficits.(37) Injection of CNB upregulated IFN-β production to a mild degree compared to LPS-stimulated mice (Fig. 6). This result explained the efficient antitumor and immune booster effects of CNB.(11,12)
Activation of IRF3 confers a TLR3/4, rather than TLR2/9, specificity.(26) Moreover, from our gene chip assay results, approximately half of the genes changed by twofold or more were TLR-related genes (Fig. 1). To seek the membrane receptor for CNB, we focused mainly on TLRs. An atypical member of the leucine-rich repeats family, TLR4 is composed of N-terminal, central, and C-terminal domains. The primary function of these motifs provides a versatile structural framework for the formation of protein–protein interactions. The leucine-rich repeat domain of TLR4 interacts with a large range of hydrophobic ligands such as HSP70/60, MD-2, fusion protein, envelope protein, and flavolipin.(38,39) The structural basic of TLR4 also provides a possibility for our highly hydrophobic CNB to bind. We validated our speculation by pull-down assay (Fig. 7). Moreover, CNB binds to endogenous TLR4 by IP experiment in U937 cell lines and TAK-242 (TLR4-specific inhibitor) pre-incubation attenuates CNB stimulated signals (Wu Wu, Beijing Normal University, Beijing, China, unpublished data; Fig. 6B). Indeed, CNB interacted with TLR4 and activated a signal cascade.
In conclusion, CNB upregulates IFN-β and several pro-inflammatory cytokines by activation of the IRF3 and NF-κB signal pathways through TLR4 signal pathways. Our findings imply that CNB could be useful for enhancing immune response and preventing cancer and virus infections.