TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-B (NF-B) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-B-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-Bmediated gene expression in cells transfected with the TLR4 gene in a dose-and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dosedependent manner (IC 50 ϳ30 nM). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-B activation and subsequent cellular events. Lipopolysaccharide (LPS),1 a component of the outer membrane of Gram-negative bacteria, is a potent activator of a variety of mammalian cell types (1, 2). Activation by LPS constitutes the first step in a cascade of events believed to lead to the manifestation of Gram-negative sepsis, a condition that results in approximately 20,000 annual deaths in the United States (3). Activation of LPS-responsive cells, such as monocytes and macrophages, occurs rapidly after LPS interacts with circulating LPS-binding protein and CD14, a glycosylphosphatidylinositol-linked cell surface glycoprotein necessary for sensitive responses to LPS (1, 2). LPS has been shown to initiate multiple intracellular signaling events (4), including the activation of NF-B, which ultimately leads to the synthesis and release of a number of proinflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-␣ (1). However, since CD14 is not a transmembrane protein, it lacks the ability to transduce cytoplasmic signals (2), and before the recent discovery of Toll-like receptors (TLRs), the identity of a transmembrane protein that could relay LPS-induced signals across the cell-surface membrane remained elusive.Toll is a transmembrane receptor in Drosophila that is involved in dorsal-ventral patterning in embryos and in the induction of an anti-fungal response (5, 6). Activation of the Toll receptor by its ligand Spä tzle results in the interaction and stimulation of several signaling molecules that are homologous to proteins involved in NF-B activation by the IL-1 receptor in mammalian cells (7,8). The cloning of a family of human receptors structurally related to Drosophila Toll revealed five proteins that have extracellular domains that contain multiple leucine-rich repeats and cytoplasmic domains with sequence homology to the intracellular portion of the IL-1 receptor (9). Furthermore, constitutively active mutants of TLR2, TLR4, and TLR5 can induce the activation of NF-B (10, 11)...

Cellular fibronectin, which contains an alternatively spliced exon encoding type III repeat extra domain A (EDA), is produced in response to tissue injury. Fragments of fibronectin have been implicated in physiological and pathological processes, especially tissue remodeling associated with inflammation. Because EDAcontaining fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), we examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. We found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response.

␣-D-Glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-␤-D-glucopyranosyl]-2- [(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt (E5564) is a second-generation synthetic lipodisaccharide designed to antagonize the toxic effects of endotoxin, a major immunostimulatory component of the outer cell membrane of Gram negative bacteria. In vitro, E5564 dose dependently (nanomolar concentrations) inhibited lipopolysaccharide (LPS)-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood as measured by production of tumor necrosis factor-␣ and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and has no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 M. E5564 blocked LPSmediated activation of nuclear factor-B in toll-like receptor 4/MD-2-transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar, another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose-and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for durations likely sufficient to permit clinical application. These results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγHigh/RXRαS427F/Y impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic “immuno-oncogenes” that modulate the immune microenvironment of cancer.

Insulin-like growth factor-I (IGF-I) receptors activate divergent signaling pathways by phosphorylating multiple cellular proteins, including insulin receptor substrate-1 (IRS-1) and the Shc proteins. Following hormone binding, IGF-I receptors cluster into clathrincoated pits and are internalized via an endocytotic mechanism. This study investigates the relationship between IGF-I receptor internalization and signaling via IRS-1 and Shc. A mutation in the C terminus of the IGF-I receptor decreased both the rate of receptor internalization and IGF-I-stimulated Shc phosphorylation by more than 50%, but did not affect IRS-1 phosphorylation. Low temperature (15°C) decreased IGF-I receptor internalization and completely inhibited Shc phosphorylation. Although receptor and IRS-1 phosphorylation were decreased in accordance with delayed binding kinetics at 15°C, the ratio of IRS-1 to receptor phosphorylation was increased more than 2-fold. Dansylcadaverine decreased receptor internalization and Shc phosphorylation, but did not change receptor or IRS-1 phosphorylation. Consistent with these findings, dansylcadaverine inhibited IGF-I-stimulated Shc-Grb2 association, mitogen-activated protein kinase phosphorylation, and p90 ribosomal S6 kinase activation, but did not affect the association of phosphatidylinositide 3-kinase with IRS-1 or activation of p70 S6 kinase. These data support the concept that Shc/mitogen-activated protein kinase pathway activation requires IGF-I receptor internalization, whereas the IRS-1 pathway is activated by both cell surface and endosomal receptors.