Clathrin- and dynamin-dependent endocytosis limits canonical NF-?B signaling triggered by lymphotoxin ? receptor.

ABSTRACT: BACKGROUND:Lymphotoxin ? receptor (LT?R) 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-?B and AP-1 pathways. Yet, the intracellular distribution of LT?R, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LT?R internalization to its signaling potential. METHODS:Intracellular localization of LT?R 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 LT?R-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-?B 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 LT?R stimulation was assessed by qRT-PCR analysis. RESULTS:We demonstrated that LT?R 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 LT?R internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-?B signaling represented by increased degradation of I?B? inhibitor and elevated expression of LT?R target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LT?R-triggered activation of the non-canonical branch of the NF-?B pathway. CONCLUSIONS:Our work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LT?R stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli. Video Abstract.

SUBMITTER: Maksymowicz M 

PROVIDER: S-EPMC7640449 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature