No abstract

The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of naïve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC(50) of 1.2 microM compared to an IC(50) of 1015 microM for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NFkappaB and the production of nitric oxide and pro-inflammatory IL-6 and TNFalpha cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines.

The pH gradient in normal cells is tightly controlled by the activity of various pH-regulatory membrane proteins including the isoform protein of the Na þ /H þ exchanger (NHE1). NHE1 is constitutively active in a neoplastic microenvironment, dysregulating pH homeostasis and altering the survival, differentiation, and proliferation of cancer cells, thereby causing them to become tumorigenic. Cytoplasmic alkalinization in breast cancer cells occurs as a result of increased NHE1 activity and, while much is known about the pathophysiologic role of NHE1 in tumor progression with regard to ion flux, the regulation of its activity on a molecular level is only recently becoming evident. The membrane domain of NHE1 is sufficient for ion exchange. However, its activity is regulated through the phosphorylation of key amino acids in the cytosolic domain as well as by its interaction with other intracellular proteins and lipids. Here, we review the importance of these regulatory sites and what role they may play in the disrupted functionality of NHE1 in breast cancer metastasis. Cancer Res; 73(4); 1259-64. Ó2013 AACR.

Dysregulation of Na+ /H+ exchanger isoform one (NHE1) activity is a hallmark of cells undergoing tumorigenesis and metastasis, the leading cause of patient mortality. The acidic tumor microenvironment is thought to facilitate the development of resistance to chemotherapy drugs and to promote extracellular matrix remodeling leading to metastasis. Here, we investigated NHE1 as a co-adjuvant target in paclitaxel chemotherapy of metastatic breast cancer. We generated a stable NHE1-knockout of the highly invasive, triple-negative, MDA-MB-231 breast cancer cells. The NHE1-knockout cells proliferated comparably to parental cells, but had markedly lower rates of migration and invasion in vitro. In vivo xenograft tumor growth in athymic nude mice was also dramatically decreased compared to parental MDA-MB-231 cells. Loss of NHE1 expression also increased the susceptibility of knockout cells to paclitaxel-mediated cell death. NHE1 inhibition, in combination with paclitaxel, resulted in a dramatic decrease in viability, and migratory and invasive potential of triple-negative breast cancer cells, but not in hormone receptor-positive, luminal MCF7 cells. Our data suggest that NHE1 is critical in triple-negative breast cancer metastasis, and its chemical inhibition boosts the efficacy of paclitaxel in vitro, highlighting NHE1 as a novel, potential co-adjuvant target in breast cancer chemotherapy.

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...