Clathrin- and dynamin-dependent endocytosis limits canonical NF-κB signaling triggered by lymphotoxin β receptor

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