Transduction of phosphatase and tensin homolog deleted on chromosome 10 into eosinophils attenuates survival, chemotaxis, and airway inflammation.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is part of a complex signaling system that affects a variety of important cell functions. PTEN antagonizes the action of PI3K by dephosphorylating the signaling lipid phosphatidylinositol 3,4,5-triphosphate. In the present study, we used a TAT fusion protein transduction system to elucidate the role of PTEN in eosinophils and airway inflammation. A small region of the HIV TAT protein (YGRKKRRQRRR), a protein transduction domain known to enter mammalian cells efficiently, was fused to the N terminus of PTEN. Flow cytometric analysis of annexin V- and propidium iodide-stained cells was used to assess eosinophil survival. A chemotaxis assay was performed using a Boyden chamber. Cell analysis in bronchoalveolar lavage fluid and histological examinations were performed using OVA-challenged A/J mice. We found that TAT-PTEN was successfully internalized into eosinophils and functioned as a phosphatase in situ. TAT-PTEN, but not a TAT-GFP control protein, blocked the ability of IL-5 to prevent the apoptosis of eosinophils from allergic subjects. The eotaxin-induced eosinophil chemotaxis was inhibited by TAT-PTEN in a dose-dependent manner. Intranasal pretreatment with TAT-PTEN, but not TAT-GFP, significantly inhibited the OVA-induced eosinophil infiltration in bronchoalveolar lavage fluid. Histological examination of the lung, including H&E and Alcian blue/periodic acid-Schiff staining, revealed that TAT-PTEN, but not TAT-GFP, abrogated eosinophilic inflammation and mucus production. Our results suggest that PTEN negatively regulates eosinophil survival, chemotaxis, and allergic inflammation. The pharmacological targeting of PTEN may constitute a new strategy for the treatment of eosinophilic disorders.

The role of platelet-derived growth factor receptor in eotaxin signaling of eosinophils.

BACKGROUND: Receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) are capable of eliciting kinase activity after ligand binding. In several cells, RTKs are activated via the G-protein-coupled receptor independent of the ligand-RTK interaction. We have previously found that EGFR is transactivated via CC chemokine receptor 3 in bronchial epithelial cells and that this pathway is important for mitogen-activated protein (MAP) kinase activation and cytokine production. It has recently been suggested that hypereosinophilic syndrome results from the fusion tyrosine kinase FIP1L1-PDGFRA. Although it is possible that the PDGFR signal is involved in eosinophil function, the details are still unclear. METHODS: Blood eosinophils were purified using Percoll and anti-CD16 antibody-coated magnetic beads. Expression of PDGFR mRNA was examined by RT-PCR. After stimulating eosinophils with eotaxin, the phosphorylation of MAP kinases was examined by Western blotting with the antiphosphospecific MAP kinase antibody. The eotaxin-induced eosinophil chemotaxis was studied using Boyden chambers. RESULTS: Eosinophils expressed PDGFRbeta mRNA in 4 out of 8 donors, while PDGFRalpha mRNA was expressed in only 1 donor. Protein expression of PDGFR was also detectable in eosinophils from some donors. AG1295, a specific inhibitor of PDGFR, showed dose-dependent inhibition of eotaxin-induced MAP kinase phosphorylation in the eosinophils expressing PDGFRbeta mRNA. The chemotaxis of these eosinophils was significantly inhibited by AG1295 (n = 3). CONCLUSIONS: Our results suggest that PDGFR modifies the CCR3-MAP kinase signaling pathway and chemotactic response in some donors. The pharmacological targeting of PDGFR may be a new strategy to treat eosinophilic disorders.

Analysis of the comprehensive effects of polyunsaturated fatty acid on mRNA expression using a gene chip.

To investigate the comprehensive effects of polyunsaturated fatty acids (PUFA) on gene expression, we analyzed changes of mRNA expression in PUFA-treated HepG2 cells using a DNA micro array. We incubated HepG2 cells for 24 h with or without 0.25 mM oleic acid (OA), arachidonic acid (AA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and then compared the expression profiles of thousands of genes using a GeneChip. PUFA influenced the expression of various genes related to cell proliferation, growth and adhesion, as well as for many transcription factors including sterol regulatory element binding proteins (SREBP). Treatments with AA, EPA, and DHA repressed the expression of genes related to cholesterol and lipid metabolism. Moreover, data from gene chip analysis proved that PUPA reduced the expression ofprostasin, which is a serine protease. By measuring the mRNA levels of SREBPs, mevalonate pyrophosphatase and prostasin using quantitative RT-PCR, we confirmed the effect of PUFA revealed by gene chip analysis. These data might provide useful clues with which to explore novel functions of PUPA.

[Reye syndrome and drug induced encephalopathy].

Reye syndrome, characterized by acute encephalopathy, selective liver damages, a fatty degeneration in visceral organs and miserable prognosis, is probably caused by various drugs, especially antipyretic such as acetylsalicylate. The incidence of the disease has been decreased by prohibition of administration of aspirin for children with high fever, especially caused by influenza infection in western countries, also in Japan. The pathophysiology of the disease has extensively studied, however, still being unknown to be dissolved. Our previous study of lipid analysis of brain from experimental measles encephalitis revealed an increase of cholesterol ester and decrease of sphingomyelin. As cholesterol ester is synthesized from cholesterol and fatty acids catalyzed by acylCoA-acyltransferase(ACAT), ACAT activity can be increased in the experimental animal brain. In the present report, ACAT m-RNA could not be expressed in control brain but in the experimental animal brain, so ACAT may play a role in pathogenesis of Reye syndrome.