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Induction of the alternative NF-?B pathway by lymphotoxin ?? (LT??) relies on internalization of LT? receptor.

ABSTRACT: Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-?B pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin ? receptor (LT?R) as a prototype, we showed that activation of the alternative, but not the classical, NF-?B pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LT?R essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LT?R appeared to be required for the activation of the alternative, but not the classical, NF-?B pathway. In vivo, ligand-induced internalization of LT?R in mesenteric lymph node stromal cells correlated with induction of alternative NF-?B target genes. Thus, our data shed light on LT?R cellular trafficking as a process required for specific biological functions of NF-?B.

SUBMITTER: Ganeff C

PROVIDER: S-EPMC3209329 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Induction of the alternative NF-κB pathway by lymphotoxin αβ (LTαβ) relies on internalization of LTβ receptor.

Ganeff Corinne C Remouchamps Caroline C Boutaffala Layla L Benezech Cécile C Galopin Géraldine G Vandepaer Sarah S Bouillenne Fabrice F Ormenese Sandra S Chariot Alain A Schneider Pascal P Caamaño Jorge J Piette Jacques J Dejardin Emmanuel E

Molecular and cellular biology 20110906 21

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin β receptor (LTβR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTβR essential for its internalizat ...[more]

PMID: 21896778

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Project description:BACKGROUND:Lymphotoxin β receptor (LTβR) plays important roles in the development of the immune system and immune response. At the cellular level, ligand-bound LTβR activates the pro-inflammatory NF-κB pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LTβR and its ligands are promising therapeutic targets. Here, we studied the consequences of perturbed cellular cholesterol content on LTβR-induced NF-κB signaling. METHODS:To modulate cholesterol availability and/or level in lung carcinoma A549 and H2228, and endothelial HUVEC cells different treatment regimens with filipin, methyl-β-cyclodextrin and simvastatin were applied. LTβR localization was studied by confocal microscopy. The activity of LTβR-induced NF-κB pathway was assessed by measuring the levels of NF-κB pathway inhibitor IκBα and phosphorylation of RelA transcription factor by Western blotting. The NF-κB transcriptional response, production of chemokines and adhesion molecules were examined by qRT-PCR, ELISA, and Western blotting, respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LTβR with its protein partners were investigated by immunoprecipitation. RESULTS:We showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-β-cyclodextrin impaired LTβR internalization and potentiated LTβR-dependent activation of the canonical branch of the NF-κB pathway. The latter was manifested by enhanced degradation of IκBα inhibitor, elevated RelA phosphorylation, substantial increase in the expression of NF-κB target genes encoding, among others, cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1, that favored the adhesion of immune cells (NK and T cells, neutrophils) to A549 cells and HUVECs. Mechanistically, we showed that cholesterol depletion stabilized interactions of ligand-stimulated LTβR with modified forms of TRAF2 and NEMO proteins. CONCLUSIONS:Our results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LTβR. Thus, drugs modulating cholesterol levels could potentially improve efficacy of LTβR-based therapies. Video abstract.

Project description:BackgroundLymphotoxin β receptor (LTβR) is a member of tumor necrosis factor receptor (TNFR) superfamily which regulates the immune response. At the cellular level, upon ligand binding, the receptor activates the pro-inflammatory NF-κB and AP-1 pathways. Yet, the intracellular distribution of LTβR, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LTβR internalization to its signaling potential.MethodsIntracellular localization of LTβR in unstimulated and stimulated cells was analyzed by confocal microscopy. Endocytosis impairment was achieved through siRNA- or CRISPR/Cas9-mediated depletion, or chemical inhibition of proteins regulating endocytic routes. The activation of LTβR-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-κB pathway, and the phosphorylation of JNK, Akt, ERK1/2, STAT1 and STAT3 involved in diverse signaling cascades, were measured by Western blotting. A transcriptional response to LTβR stimulation was assessed by qRT-PCR analysis.ResultsWe demonstrated that LTβR was predominantly present on endocytic vesicles and the Golgi apparatus. The ligand-bound pool of the receptor localized to endosomes and was trafficked towards lysosomes for degradation. Depletion of regulators of different endocytic routes (clathrin-mediated, dynamin-dependent or clathrin-independent) resulted in the impairment of LTβR internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-κB signaling represented by increased degradation of IκBα inhibitor and elevated expression of LTβR target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LTβR-triggered activation of the non-canonical branch of the NF-κB pathway.ConclusionsOur work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LTβR stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli. Video Abstract.

Project description:Previous studies suggest that the core protein of hepatitis C virus (HCV) has a pleiotropic function in the replication cycle of the virus. To understand the role of this protein in HCV pathogenesis, we used a yeast two-hybrid protein interaction cloning system to search for cellular proteins physically interacting with the HCV core protein. One such cellular gene was isolated and characterized as the gene encoding the lymphotoxin-beta receptor (LT-betaR). In vitro binding analysis demonstrated that the HCV core protein binds to the C-terminal 98 amino acids within the intracellular domain of the LT-betaR that is involved in signal transduction, although the binding affinity of the full-length HCV core protein was weaker than that of its C-terminally truncated form. Our results also indicated that the N-terminal 40-amino-acid segment of the HCV core protein was sufficient for interaction with LT-betaR and that the core protein could form complexes with the oligomeric form of the intracellular domain of LT-betaR, which is a prerequisite for downstream signaling of this receptor. Similar to other members of the tumor necrosis factor (TNF) receptor superfamily, LT-betaR is involved in the cytotoxic effect of the signaling pathway, and thus we have elucidated the biological consequence of interaction between the HCV core protein and LT-betaR. Our results indicated that in the presence of the synergizing agent gamma interferon, the HCV core protein enhances the cytotoxic effects of recombinant forms of LT-betaR ligand in HeLa cells but not in hepatoma cells. Furthermore, this enhancement of the cytolytic activity was cytokine specific, since in the presence of cycloheximide, the expression of the HCV core protein did not elicit an increase in the cytolytic activity of TNF in both HeLa and hepatoma cells. In summary, the HCV core protein can associate with LT-betaR, and this protein-protein interaction has a modulatory effect on the signaling pathway of LT-betaR in certain cell types. Given the known roles of LT-betaR/LT-alpha1,beta2 receptor-ligand interactions in the normal development of peripheral lymphoid organs and in triggering cytolytic activity and NF-kappaB activation in certain cell types, our finding implies that the HCV core protein may aggravate these biological functions of LT-betaR, resulting in pathogenesis in HCV-infected cells.

Dataset Information

Induction of the alternative NF-?B pathway by lymphotoxin ?? (LT??) relies on internalization of LT? receptor.

Publications

Induction of the alternative NF-κB pathway by lymphotoxin αβ (LTαβ) relies on internalization of LTβ receptor.

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