Phosphatase and tensin homologue deleted on chromosome ten (PTEN) is part of a complex signaling system that affects a variety of important cell functions. PTEN blocks the action of PI3K by dephosphorylating the signaling lipid phosphatidylinositol 3,4,5-triphosphate. We have used a mouse model for asthma to determine the effect of PI3K inhibitors and PTEN on allergen-induced bronchial inflammation and airway hyperresponsiveness. PI3K activity increased significantly after allergen challenge. PTEN protein expression and PTEN activity were decreased in OVA-induced asthma. Immunoreactive PTEN localized in epithelial layers around the bronchioles in control mice. However, this immunoreactive PTEN dramatically disappeared in allergen-induced asthmatic lungs. The increased IL-4, IL-5, and eosinophil cationic protein levels in bronchoalveolar lavage fluids after OVA inhalation were significantly reduced by the intratracheal administration of PI3K inhibitors or adenoviruses carrying PTEN cDNA (AdPTEN). Intratracheal administration of PI3K inhibitors or AdPTEN remarkably reduced bronchial inflammation and airway hyperresponsiveness. These findings indicate that PTEN may play a pivotal role in the pathogenesis of the asthma phenotype.

Isocyanate chemicals, including toluene diisocyanate (TDI), are currently the most common causes of occupational asthma. Although considerable controversy remains regarding its pathogenesis, TDI-induced asthma is characterized by hyperresponsiveness and inflammation of the airways. One of the histological hallmarks of inflammation is angiogenesis, but the possible role of vascular endothelial growth factor (VEGF), a potent angiogenic cytokine, in TDI-induced asthma is unknown. We developed a murine model to investigate TDI-induced asthma by performing two courses of sensitization with 3% TDI and one challenge with 1% TDI using ultrasonic nebulization to examine the potential involvement of VEGF in that disease. These mice develop the following typical pathophysiological features: airway hyperresponsiveness, airway inflammation, and increased VEGF levels in the airway. Administration of VEGFR inhibitors reduced all these pathophysiological symptoms. These results suggest that VEGF is one of the major determinants of TDI-induced asthma and that the inhibition of VEGF may be a good therapeutic strategy.

Lysophosphatidic acid (LPA) is elevated in ascites of ovarian cancer patients and stimulates growth and other activities of ovarian cancer cells in vitro. Tissue hypoxia is a critical factor for tumor aggressiveness and metastasis in cancers. We tested whether the ascites of ovarian cancer is hypoxic and whether hypoxia influences the effects of LPA on ovarian cancer cells. We found that ovarian ascitic fluids were hypoxic in vivo. Enhanced cellular responsiveness to LPA, including migration and/or invasion of ovarian cancer cells, was observed under hypoxic conditions. This enhancement could be completely blocked by geldanamycin or a small interfering RNA targeting hypoxia-inducible factor 1A (HIF1A). LPA-induced cell migration required cytosolic phospholipase A 2 (cPLA 2 ) and LPA stimulates cPLA 2 phosphorylation in a HIF1A-dependent manner under hypoxia conditions. Furthermore, we show for the first time that exogenous LPA enhances tumor metastasis in an orthotopic ovarian cancer model and HIFA expression in tumors. 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (an inhibitor of the heat shock protein 90) effectively blocked LPA-induced tumor metastasis in vivo. Together, our data indicate that hypoxic conditions are likely to be pathologically important for ovarian cancer development. HIF1A plays a critical role in enhancing and/or sensitizing the role of LPA on cell migration and invasion under hypoxic conditions, where cPLA 2 is required for LPAinduced cell migration. (Cancer Res 2006; 66(16): 7983-90)

The RNA world hypothesis presumes that RNA will be competent for varied essential cellular functions. One such indispensable cell function is regulation of membrane permeability. Though this was not a known RNA activity, selection-amplification yielded RNAs that bound phosphatidylcholine:cholesterol liposomes. At least eight distinct, Ϸ95-mer sequences bind well to the outside of the lipid bilayer, though randomized sequences had no such activity. No distinct sequence motif for lipid binding was found. However, truncation of one such RNA shows that a smaller, 44-nucleotide irregular RNA hairpin is an active membrane binding domain. Bound RNA increases the permeability of liposomes to 22 Na ؉ . In addition, using voltage clamp technique, four individual RNAs increase the ion permeability of the plasma membrane of cultured human cells. The existence of multiple sequences that bind membranes and provoke permeability changes suggests that these may be elementary RNA functions that could be selected in vivo.Cells communicate with their environments across phospholipid bilayer membranes. However, the permeability of such bilayers (relatively permeable to water, but virtually impermeable to polar molecules, even to small ions) is dramatically mismatched to cellular needs. Cellular life therefore absolutely requires facilitation of transport through phospholipid boundaries. The RNA-world hypothesis (1) posits ancestral cells in which RNA plays many of the roles taken by modern proteins. Might RNAs serve membrane functions?We have approached this question, first, by isolating RNAs that bind to pure phospholipid membranes by using selectionamplification (2, 3). In this technique, novel RNA activities are isolated by selecting infrequent, perhaps unique active molecules from large pools of transcripts (Ϸ10 14 different molecules) with randomized sequences. Repetition of the selection (purification) is made possible by nucleic acid amplification (replication) applied to the partially purified pools, so that large cumulative purifications are possible after multiple cycles of selection-amplification. Ultimately, cDNA cloning and in vitro transcription make available single pure active RNAs for study. Second, we have reintroduced purified membranebinding RNAs from selection-amplification into several membrane systems to measure their effects on permeability, as has long been done for channel proteins (4).To act in membranes, RNA must interact with membrane constituents. Phospholipids, the main components of the biological membrane, are chemically tripartite. They consist of a polar head group, glycerol phosphate, and fatty acids. RNAs should easily interact with polar head groups, particularly cationic ones. Glycerol phosphate (which resembles the RNA backbone) also presents easily used hydrogen-bonding opportunities to an RNA. In contrast, fatty acids might be thought of as improbable RNA ligands. However, it has previously been shown that RNAs fold to form specifically shaped, hydrophobic sites that interact favorab...