Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type–Specific Manner

Abstract

The clathrin-dependent endocytic pathway is crucial for endosomal TLR3- and TLR4-mediated Toll–IL-1R domain–containing adaptor molecule-1 (TICAM-1) signaling. TLR4 uses a different signaling platform, plasma membrane and endosomes, for activation of TIRAP-MyD88 and TICAM-2–TICAM-1, respectively. LPS-induced endocytosis of TLR4 is mandatory for TICAM-1–mediated signaling including IFN-β production. Several molecules/mechanisms such as CD14, clathrin, and phosphatidylinositol metabolism have been reported to act as inducers of TLR4 translocation. However, the molecular mechanism of spatiotemporal regulation of TLR4 signaling remains unresolved. We have previously shown that Raftlin is essential for clathrin-dependent endocytosis of TLR3 ligand in human epithelial cells and myeloid dendritic cells (DCs). In this article, we demonstrate that Raftlin also mediated LPS-induced TLR4 internalization and TICAM-1 signaling in human monocyte-derived DCs and macrophages (Mo-Mϕs). When Raftlin was knocked down, LPS-induced TLR4-mediated IFN-β promoter activation, but not NF-κB activation, was decreased in HEK293 cells overexpressing TLR4/MD-2 or TLR4/MD-2/CD14. LPS-induced IFN-β production by monocyte-derived DCs and Mo-Mϕs was significantly decreased by knockdown of Raftlin. Upon LPS stimulation, Raftlin moved from the cytoplasm to the plasma membrane in Mo-Mϕs, where it colocalized with TLR4. Raftlin associated with clathrin-associated adaptor protein–2 in resting cells and transiently bound to TLR4 and clathrin at the cell surface in response to LPS. Thus, Raftlin appears to modulate cargo selection as an accessary protein of clathrin-associated adaptor protein–2 in clathrin-mediated endocytosis of TLR3/4 ligands.

Cell type-specific role of Raftlin in the regulation of endosomal TLR signaling

“…Hence, Raftlin is recruited to the plasma membrane when poly(I:C) bound to the uptake receptor, where associates with clathrin to modulate cargo sorting and delivery. In steady state, Raftlin interacts with clathrin adaptor AP-2 -subunit [17] , suggesting that Raftlin regulates clathrin-dependent endocytosis of poly(I:C) as an accessory molecule of AP-2 through recognition of poly(I:C)-uptake receptor. Notably, internalization of transferin is independent of Raftlin irrespective of its dependency of clathrin [16] .…”

“…Importantly, Raftlin homolog, Raftlin2, is expressed in a species-and cell type-specific manner [16,17,19] . Human myeloid DCs and epithelial cells express only Raftlin, while mouse conventional DCs express Raftlin2 in addition to Raftlin.…”

“…We have studied about the role of Raftlin in LPS-induced TLR4-mediated signaling and found that LPS-induced IFN- promoter activation, but not NF-B activation, was impaired in Raftlin knockdown HEK293 cells overexpressing human TLR4/MD-2 or TLR4/MD-2/CD14 [17] . The LPS-induced IFN- production was also decreased in Raftlin knockdown human monocyte-derived macrophages and DCs.…”

“…The LPS-induced IFN- production was also decreased in Raftlin knockdown human monocyte-derived macrophages and DCs. Raftlin was resided in the cytoplasm in human macrophages and moved to the plasma membrane upon LPS stimulation, where it colocalized with TLR4 followed by internalization along with TLR4 [17] . Raftlin physically associates with TLR4 and clathrin in response to LPS stimulation, suggesting the involvement in the Raftlin-and clathrin-dependent TLR4 endocytosis ( Figure 2).…”

“…Thus, innate immune system utilizes endocytic pathway to detect extracellular microbial components, but precise molecular mechanisms of PAMP loading remain unknown. Recently, we have reported that the cytosolic protein Raftlin that possesses membrane-anchoring motif mediates cellular uptake of poly(I:C), CpG ODNs and LPS in human dendritic cells (DCs), macrophages and epithelial cells through association with clathrin-AP-2 complex [16,17] . In this review, we will summarize the molecular mechanisms of exNA internalization and focus on the role of Raftlin in the regulation of endosomal TLR signaling.…”