E3 ubiquitin ligases are important in both innate and adaptive immunity. Here we report that Nrdp1, an E3 ubiquitin ligase, inhibited the production of proinflammatory cytokines but increased interferon-beta production in Toll-like receptor-triggered macrophages by suppressing adaptor MyD88-dependent activation of transcription factors NF-kappaB and AP-1 while promoting activation of the kinase TBK1 and transcription factor IRF3. Nrdp1 directly bound and polyubiquitinated MyD88 and TBK1, which led to degradation of MyD88 and activation of TBK1. Knockdown of Nrdp1 inhibited the degradation of MyD88 and the activation of TBK1 and IRF3. Nrdp1-transgenic mice showed resistance to lipopolysaccharide-induced endotoxin shock and to infection with vesicular stomatitis virus. Our data suggest that Nrdp1 functions as both an adaptor protein and an E3 unbiquitin ligase to regulate TLR responses in different ways.

T raditionally, heat shock proteins (HSP) 5 are regarded as chaperones assisting protein folding and translocation. However, HSP can also serve as cytokines that can stimulate dendritic cells (DC) and macrophages to produce proinflammatory cytokines and chemokines (1-5). More importantly, HSP derived from tumor cells are capable of chaperoning tumor Ags to DC and then cross-presenting the Ags to T cells (6). HSP70 proteins, including the constitutively expressed cognate HSP70 (HSC70 or HSP73), the stress-inducible HSP70 (HSP70i or HSP72), and the mitochondrial HSP70 (HSP75), constitute the most conserved class of all HSP. Previous reports have shown that HSP can be released from various cells via passive (e.g., HSP released during cell injury conditions, such as surgery, excessive exercise, and necrosis) and active (e.g., translocation of HSP to plasma membrane and subsequent secretion) pathways (3-5). However, the roles of HSP70 proteins released from tumor cells in the induction of antitumor immunity and the underlying mechanisms have not been fully elucidated.Hyperthermia (HT) has been reported to enhance the immunogenecity of cancer cells concomitantly with expression of HSP (7,8). Recent reports demonstrate that heat stress (HS) can induce the cell surface expression and the release of HSP70, HSP90, and gp96 (glucose-regulated protein 94; Grp94) (1-5). However, the underlying mechanisms that local HT can initiate antitumor immunity via released HSP and via subsequent activation of DC still lack direct evidence and thus need to be further investigated.During the investigations of local HT (42-43°C)-elicited antitumor immunity, we find that infiltration of DC and T cells within heat-stressed tumor is significantly increased. We thus hypothesize that chemokines, induced by HS, may be involved in the initiation of HT-elicited antitumor immunity by chemoattraction and activation of DC. Our studies show that HSP70 proteins simultaneously released by tumor cells can serve as autocrine and paracrine cytokines inducing the production of various chemokines by tumor cells and the activation of DC via TLR4 signaling pathway. Our data provide direct evidence for the important roles of releasable HSP in the initiation of antitumor immunity during local HT.

Exosomes derived from dendritic cells or tumor cells are a population of nanometer-sized membrane vesicles that can induce specific antitumor immunity. During investigation of the effects of hyperthermia on antitumor immune response, we found that exosomes derived from heat-stressed tumor cells (HS-TEX) could chemoattract and activate dendritic cells (DC) and T cells more potently than that by conventional tumor-derived exosomes. We show that HS-TEX contain chemokines, such as CCL2, CCL3, CCL4, CCL5, and CCL20, and the chemokine-containing HS-TEX are functionally competent in chemoattracting CD11c+ DC and CD4+/CD8+ T cells both in vitro and in vivo. Moreover, the production of chemokine-containing HS-TEX could be inhibited by ATP inhibitor, calcium chelator, and cholesterol scavenger, indicating that the mobilization of chemokines into exosomes was ATP- and calcium-dependent and via a lipid raft-dependent pathway. We consistently found that the intracellular chemokines could be enriched in lipid rafts after heat stress. Accordingly, intratumoral injection of HS-TEX could induce specific antitumor immune response more efficiently than that by tumor-derived exosomes, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice more significantly. Therefore, our results demonstrate that exosomes derived from HS-TEX represent a kind of efficient tumor vaccine and can chemoattract and activate DC and T cells, inducing more potent antitumor immune response. Release of chemokines through exosomes via lipid raft-dependent pathway may be a new method of chemokine exocytosis.

Migration of dendritic cells (DCs) into tissues and secondary lymphoid organs plays a crucial role in the initiation of innate and adaptive immunity. In this article, we show that cyclosporin A (CsA) impairs the migration of DCs both in vitro and in vivo. Exposure of DCs to clinical concentrations of CsA neither induces apoptosis nor alters development but does impair cytokine secretion, chemokine receptor expression, and migration. In vitro, CsA impairs the migration of mouse bone marrow–derived DCs toward macrophage inflammatory protein-3β (MIP-3β) and induces them to retain responsiveness to MIP-1α after lipopolysaccharide (LPS)–stimulated DC maturation, while in vivo administration of CsA inhibits the migration of DCs out of skin and into the secondary lymphoid organs. CsA impairs chemokine receptor and cyclooxygenase-2 (COX-2) expression normally triggered in LPS-stimulated DCs; administration of exogenous prostaglandin E2 (PGE2) reverses the effects of CsA on chemokine receptor expression and DC migration. Inhibition of nuclear factor–κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway signaling by CsA may be responsible for the CsA-mediated effects on the regulation of chemokine receptor and cyclooxygenase-2 (COX-2) expression. Impairment of DC migration due to inhibition of PGE2 production and regulation of chemokine receptor expression may contribute, in part, to CsA-mediated immunosuppression.

Promoting apoptosis is a strategy for cancer drug discovery. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide range of malignant cells. However, several cancers, including human hepatocellular carcinoma (HCC), exhibit a major resistance to TRAIL-induced cell death. Melittin, a water-soluble 26-amino acid peptide derived from bee venom of Apis mellifera, can exert toxic or inhibitory effects on many types of tumor cells. Here we report that melittin can induce apoptosis of HCC cells by activating Ca 2؉ / calmodulin-dependent protein kinase, transforming growth factor-␤-activated kinase 1 (TAK1), and JNK/p38 MAPK. We show that melittin-induced apoptosis can be inhibited by calcium chelator, by inhibitors for Ca 2؉ /calmodulin-dependent protein kinase, JNK and p38, and by dominant negative TAK1. In the presence of melittin, TRAIL-induced apoptosis is significantly increased in TRAIL-resistant HCC cells, which may be attributed to melittin-induced TAK1-JNK/p38 activation and melittin-mediated inhibition of IB␣ kinase-NFB. Our data suggest that melittin can synergize with TRAIL in the induction of HCC cell apoptosis by activating the TAK1-JNK/p38 pathway but inhibiting the IB␣ kinase-NFB pathway. Therefore, the combination of melittin with TRAIL may be a promising therapeutic approach in the treatment of TRAIL-resistant human cancer.At present about 20 different ligands that belong to the tumor necrosis factor (TNF) 4 superfamily have been identified, among which TNF␣, lymphotoxin ␣, Fas ligand, apo3L, and TNF-related apoptosis-inducing ligand (TRAIL) have been characterized as major mediators of apoptosis (1, 2). TRAIL, in its soluble form, is emerging as an attractive anticancer agent because of its cancer cell specificity and potent antitumor activity (1, 2).TRAIL signals by interacting with its receptors (1). Thus far, five receptors (Fas ligand) have been identified, namely the two agonistic receptors, TRAIL-R1 and TRAIL-R2, and the three antagonistic receptors TRAIL-R3, TRAIL-R4, and osteoprotegerin (3-6). Binding of TRAIL to the extracellular domain of agonistic receptors results in the trimerization of the receptors and clustering of the intracellular death domains, which lead to the recruitment of the adaptor molecule Fas-associated protein with death domain (FADD) (1). Subsequently, FADD recruits and activates initiator caspase-8 and caspase-10, leading to cellular disassembly (1). Meanwhile, TRAIL-initiated apoptotic signaling requires an amplification loop by mitochondrial pathway engagement through impairment of the mitochondrial membrane permeability regulated by Bcl-2 family members, which sequentially leads to cytochrome c or Smac/DIABLO (second mitochondrial activator of caspases/direct IAP-binding protein with low isoelectric point (pI)) release, apoptosome formation, and the final DNA fragmentation (7).Similar to TNF-induced activation of the nuclear factor B (NFB) transcription factor and the MAPK pathway (1, 2), TRAIL can also initiate the activation of ...

The key molecular mechanisms that control signaling via T cell antigen receptors (TCRs) remain to be fully elucidated. Here we found that Nrdp1, a ring finger-type E3 ligase, mediated Lys33 (K33)-linked polyubiquitination of the signaling kinase Zap70 and promoted the dephosphorylation of Zap70 by the acidic phosphatase-like proteins Sts1 and Sts2 and thereby terminated early TCR signaling in CD8(+) T cells. Nrdp1 deficiency significantly promoted the activation of naive CD8(+) T cells but not that of naive CD4(+) T cells after engagement of the TCR. Nrdp1 interacted with Zap70 and with Sts1 and Sts2 and connected K33 linkage of Zap70 to Sts1- and Sts2-mediated dephosphorylation. Our study suggests that Nrdp1 terminates early TCR signaling by inactivating Zap70 and provides new mechanistic insights into the non-proteolytic regulation of TCR signaling by E3 ligases.

Resistance to chemotherapeutic treatment, which is indirectly responsible for many cancer deaths, is normally associated with an aggressive phenotype including increased cell motility and acquisition of invasive properties. Here we describe how breast cancer cells overcome doxorubicin-induced senescence and become drug resistant by overexpression of the microRNA (miR)-106bB25 cluster. Although all three miRs in the cluster contribute to the generation of doxorubicin resistance, miR-25 is the major contributor to this phenotype. All three miRs in this cluster target EP300, a transcriptional activator of E-cadherin, resulting in cells acquiring a phenotype characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT), including an increase in both cell motility and invasion, as well as the ability to proliferate after treatment with doxorubicin. These findings provide a novel drug resistance/EMT regulatory pathway controlled by the miR-106bB25 cluster by targeting a transcriptional activator of E-cadherin.

In mouse macrophages and dendritic cells, the CHIP E3 ubiquitin ligase is needed for transduction of signals initiated by TLR4 and TLR9 stimulation.