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

Abstract

Background Lymphotoxin 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. Methods Intracellular 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. Results We 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. Conclusions Our 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. Graphical abstract

SARS‐CoV‐2–host proteome interactions for antiviral drug discovery

“…It can be speculated that these receptors regulate the immune response during infection. Predictably, the same set of host receptors was involved in the clathrin‐related endocytosis process (Appendix Fig S8 ), which is activated during the internalization of a large number of viruses (e.g., influenza virus, SARS coronavirus, Hepatitis B virus) (Brodsky et al , 1992 ; Veiga & Cossart, 2006 ; Maksymowicz et al , 2020 ). Moreover, domain‐based enrichment analysis showed that adaptin domains were overrepresented in the interactomes of several receptors (TOP3B, CLEC4D, CLEC4E, TMPRSS2, IFITM1, IFITM3), indicating their roles in cellular trafficking (Appendix Fig S9 ).…”

Understanding nanoparticle endocytosis to improve targeting strategies in nanomedicine

“… 205 CRISPR-Cas9 is a novel efficient method for gene knockout and involves the Cas9 protein, an enzyme responsible for DNA cutting and a single guided RNA (sgRNA), which guides Cas9 to a specific location in the DNA sequence. 204,205 This approach has been used to prepare AP2M1, 206 CAV1, 207 dynamin 208 and Rac 209 knockout cells. Patel et al , studied the intracellular delivery of mRNA from lipid NPs.…”

PmAP2-β depletion enhanced activation of the Toll signaling pathway during yellow head virus infection in the black tiger shrimp Penaeus monodon

“…It is possible that lack of Pm AP2-β may impair clathrin-mediated endocytosis, resulting in alteration of signaling. It was previously reported that clathrin and dynamin-deficient cells showed enhanced activation of canonical NF-κB signaling 25 .…”

“…The disruption of endocytosis and endosomal sorting results in increased LPS signaling. In addition, the impairment of clathrin internalization enhances expression of lymphotoxin β receptor (LTβR) and activation of canonical NF-κB signaling 25 . These evidences suggest that clathrin-dependent endocytosis could regulate several signaling pathways.…”