Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk Is Essential for Toll-like Receptor Activation and Cellular Signaling

Abstract

The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and proinflammatory cytokines in dendritic and macrophage cells. The ␣-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane G␣ i subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR G␣ i -signaling proteins blocks LPS-induced Neu1 activity and NFB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and NFB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling.The mammalian Toll-like receptors (TLRs) 11 are one of the families of sensor receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial infections for innate immune cells; they play important roles in the pathophysiology of infectious, inflammatory, and autoimmune diseases. Thus, the intensity and duration of TLR responses with these diseases must be tightly controlled. It follows that the structural integrity of TLR receptors, their ligand interactions, and their signaling components are important for our understanding of subsequent immunological responses.Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between TLRs and their ligands have remained poorly defined until now. We have recently identified a novel paradigm of TLR activation by its natural ligand, which has not been observed previously (1). This paradigm suggests that ligand-induced TLR activation is tightly controlled by Neu1 activation. The data indicate that Neu1 is already in complex with either TLR2, -3, or -4 receptors and is induced upon ligand binding to their respective receptors. In addition, activated Neu1 specifically hydrolyzes ␣-2,3-sialyl residues linked to ␤-galactosides, which are distant from ligand binding. This desialylation process is proposed to remove steric hindrance to TLR4 dimerization, MyD88-TLR4 complex recruitment, NFB activation, and p...

Neuraminidase-1: A novel therapeutic target in multistage tumorigenesis

“…It is noteworthy that we have reported a striking similarity with this novel receptor signaling platform for nerve growth factor (NGF) TrkA receptors [35], insulin [59, 60] and cell surface TOLL-like receptor (TLR)-4, [34, 61–64] and intracellular TLR7 and TLR9 receptors [65], all of which require receptor dimerization and are regulated by Neu1. Pshezhetsky and Ashmarina [66] have recently summarized the emerging data demonstrating that Neu1, well known for its lysosomal catabolic function, is also localized to the cell surface and assumes the previously unrecognized role as a structural and functional modulator of cellular receptors.…”

“…MMP9 and neuromedin B GPCR (NMBR) are associated with each other on the cell surface, and together form a complex with EGFR [30], TrkA receptors [35], insulin receptor IRβ subunits [59], TLR-4 [34, 61, 62, 64], and TLR7 and −9 [65]. Co-immunoprecipitation experiments using cell lysates from RAW-blue macrophage cells demonstrated that the 80 kDa NMBR isoform forms a complex with the active 88 kDa MMP9 isoform from naïve or lipopolysaccharide (LPS)-stimulated cells [62].…”

“…In addition, the activation of NF-κB in macrophages can be mediated by cell surface and intracellular Toll-like receptors (TLR) [183–186, 188], the process of which has been shown to play major roles in cancer. We have reported for the first time that Neu1 sialidase clearly plays a central role in mediating cell surface and nucleic acid-induced intracellular TLR activation, and the interactions involving NMBR–MMP9–Neu1 cross-talk constitute a novel intracellular TLR signaling platform that is essential for NF-κB activation and pro-inflammatory responses [31, 61, 62, 65]. …”

Lysosomal multienzyme complex: pros and cons of working together

“…Subsequently, TLR dimerization and activation mediates MyD88/TLR4 complex formation and activation of the transcription factor NF-κB, as seen in macrophages treated with the endotoxin LPS. LPS binding to TLR4 apparently induces the interaction of NEU1 with metalloproteinase-9 (MMP9), which in turn stimulates downstream guanine protein-coupled receptor (GPCR) signaling [76]. This NEU1-MMP9 complex was shown to bind to TLR4 on the cell surface of naïve macrophages, but the inhibition of MMP9 and GPCR Gα i -signaling proteins blocked LPS-induced NEU1 activity and downstream NF-κB activation.…”

TRIF-Mediated TLR3 and TLR4 Signaling Is Negatively Regulated by ADAM15

“…Furthermore, ADAM15 has been shown to cleave the ectodomain of MMP-10 (24). Interestingly, it has been proposed that activation of MMP-9 activity may be initiated upon TLR ligand binding (25). Furthermore, TLR3 and TLR2 engagement has been shown to enhance MMP-9 and MMP-10 expression (6,26).…”