House dust mite allergen Der f 1 induces IL-8 in human basophilic cells via ROS-ERK and p38 signal pathways

Der f 1, a major house dust mite allergen and member of the papain-like cysteine protease family, can provoke immune responses with its proteolytic activity. To understand the role of Der f 1 in inflammatory immune responses, we studied the mechanism of the regulation of interleukin (IL)-8 expressions in human basophilic cell KU812 by proteolytically active recombinant Der f 1. Not only production of IL-8 mRNA was induced but also the DNA binding activity of activator protein-1 (AP-1) and phosphorylation of NF-κB p65 were increased in Der f 1-treated KU812. Furthermore, Der f 1 induction of IL-8 expression was sensitive to pharmacological inhibition of ERK and p38 mitogen activated protein kinase (MAPK) pathways. Der f 1 also activated ERK and p38 MAPK phosphorylation and rapidly induced reactive oxygen species (ROS) production. The antioxidant N-acetyl-cysteine (NAC) inhibited phosphorylation of ERK, but not p38, suggesting that secretion of IL-8 in KU812 cells treated with Der f 1 is dependent on ROS, ERK MAPK and p38 MAPK. We describe the mechanism of Der f 1-induced IL-8 secretion from human basophilic cells, which are thought to be important for allergic inflammation independent of IgE antibodies. These findings improve our understanding of the inflammatory immune response in human basophils to protease allergens.     

Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening

Cannabinoid receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, KU-812, were immobilized onto the surface of an open tubular (OT) capillary to create a CB1/CB2–OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2–OT column can be used to determine the binding affinities (K_i values) for a single compound and to screen individual compounds or a mixture of multiple compounds. The CB1/CB2–OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high-affinity phytocannabinoid.     

Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ～20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.     

JNK-dependent NFATc1 pathway positively regulates IL-13 gene expression induced by (–)-epigallocatechin-3-gallate in human basophilic KU812 cells

(–)-Epigallocatechin-3-gallate (EGCG) has been reported to possess a wide range of biological and pharmacological properties. In this study, we investigated the effects of EGCG on IL-13 gene expression in human basophilic KU812 cells. The IL-13 mRNA expression level was dose-dependently increased by treatment with EGCG (5–20 μM) for 1 h and additional incubation in a medium for 23 h. EGCG significantly increased the intracellular peroxide level as detected by the peroxide-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate. A pharmacological experiment using catalase and a structure–activity relationship study revealed that the exogenously produced H₂O₂ significantly, but partially, contributed to the IL-13 expression as well as the intracellular oxidative status. Furthermore, EGCG at the concentration required for IL-13 up-regulation activated c-Jun NH₂-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 mitogen-activated protein kinase in KU812 cells. Transfection of a JNK-specific siRNA as well as treatment with a JNK-specific inhibitor, SP600125, significantly reduced the EGCG-induced IL-13 mRNA expression, by 47.1 and 44.6%, respectively. In addition, we observed the nuclear translocation, mRNA up-regulation, and activation of DNA binding with the IL-13 promoter of nuclear factor of activated T cells (NFATc1) in the EGCG-treated cells. These data provide biological evidence that EGCG induces IL-13 mRNA expression via the JNK-dependent NFATc1 pathway in KU812 cells.     

The impact of the 67kDa laminin receptor on both cell-surface binding and anti-allergic action of tea catechins

Here, we investigated the structure-activity relationship of major green tea catechins and their corresponding epimers on cell-surface binding and inhibitory effect on histamine release. Galloylated catechins; (−)-epigallocatechin-3-O-gallate (EGCG), (−)-gallocatechin-3-O-gallate (GCG), (−)-epicatechin-3-O-gallate (ECG), and (−)-catechin-3-O-gallate (CG) showed the cell-surface binding to the human basophilic KU812 cells by surface plasmon resonance analysis, but their non-galloylated forms did not. Binding activities of pyrogallol-type catechins (EGCG and GCG) were higher than those of catechol-type catechins (ECG and CG). These patterns were also observed in their inhibitory effects on histamine release. Previously, we have reported that biological activities of EGCG are mediated through the binding to the cell-surface 67 kDa laminin receptor (67LR). Downregulation of 67LR expression caused a reduction of both activities of galloylated catechins. These results suggest that both the galloyl moiety and the B-ring hydroxylation pattern contribute to the exertion of biological activities of tea catechins and their 67LR-dependencies.     

Peroxisome proliferator-activated receptor ligands negatively regulate the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells

The high-affinity IgE receptor Fc epsilon RI is expressed on the cell surface of mast cells and basophils, and plays a central role in IgE-mediated inflammatory reactions. Recently, peroxisome proliferator-activated receptors (PPARs) have been implicated in the anti-inflammatory response. To investigate a possible role for PPAR in human basophils, the effect of PPAR ligands on Fc epsilon RI expression in human basophilic KU812 cells was studied. The PPARalpha ligand, leukotriene B(4), did not affect the cell surface expression of Fc epsilon RI. However, prostaglandin (PG) A(1) and 15-deoxy-Delta(12,14) PGJ(2) (15d-PGJ(2)), which are PPARbeta and gamma ligands, respectively, were both able to decrease Fc epsilon RI expression. Treatment with PGA(1) or 15d-PGJ(2) separately also reduced histamine release from KU812 cells in response to cross-linkage of Fc epsilon RI. In addition, RT-PCR analysis showed that KU812 cells expressed the mRNA for PPARalpha, beta, and gamma, indicating that PPARbeta or gamma may negatively regulate the cell activation via Fc epsilon RI. Cells treated with 15d-PGJ(2) expressed lower levels of Fc epsilon RI alpha and gamma mRNA, and PGA(1) treatment decreased the level of Fc epsilon RI gamma mRNA. These results suggest that the suppression of Fc epsilon RI expression by PPARs may be due to the down-regulation of Fc epsilon RI alpha or gamma mRNA.     

A lipid raft-associated 67kDa laminin receptor mediates suppressive effect of epigallocatechin-3-O-gallate on FcepsilonRI expression

(-)-Epigallocatechin-3-O-gallate (EGCG), a major green tea polyphenol, has previously exhibited a suppressive effect on the expression of the high-affinity IgE receptor (FcepsilonRI). This effect has been shown to be elicited by interaction with the plasma membrane microdomain lipid rafts. Recently, we have identified the 67 kDa laminin receptor (67LR) as a cell surface EGCG receptor that mediates an anti-cancer action. Here we show that the 67LR is highly associated with lipid rafts on human basophilic KU812 cells. Experiments using 67LR-enhanced and -reduced cells revealed that the EGCG's ability to downregulate FcepsilonRI expression correlated with the amount of 67LR. Thus, these results suggest that the lipid raft-associated 67LR plays an important role in mediating the FcepsilonRI-suppressive action of EGCG.