Phosphoinositide 3-kinase (PI3K) is thought to contribute to the pathogenesis of asthma by effecting the recruitment, activation, and apoptosis of inflammatory cells. We examined the role of class IA PI3K in antigen-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of Δp85 protein, a dominant negative form of the class IA PI3K regulatory subunit, p85α, which was fused to HIV-TAT (TAT-Δp85). Intraperitoneal administration of TAT-Δp85 caused time-dependent transduction into blood leukocytes, and inhibited activated phosphorylation of protein kinase B (PKB), a downstream target of PI3K, in lung tissues in mice receiving intranasal FMLP. Antigen challenge elicited pulmonary infiltration of lymphocytes, eosinophils and neutrophils, increase in mucus-containing epithelial cells, and airway hyperresponsiveness to methacholine. Except for modest airway neutrophilia, these effects all were blocked by treatment with 3–10 mg/kg of TAT-Δp85. There was also significant reduction in IL-5 and IL-4 secretion into the BAL. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Δp85. We conclude that PI3K has a regulatory role in Th2-cell cytokine secretion, airway inflammation, and airway hyperresponsiveness in mice.
In this study, we determined the functional significance and mechanism of the exogenous hVPLA 2 -induced arachidonic acid (AA) release and leukotriene C 4 (LTC 4 ) synthesis in isolated human peripheral blood eosinophils. As low a concentration as 10 nM exogenous hVPLA 2 was able to elicit the significant release of AA and LTC 4 from unstimulated eosinophils, which depended on its ability to act on phosphatidylcholine membranes. hVPLA 2 also augmented the release of AA and LTC 4 from eosinophils activated with formyl-Met-Leu-Phe ؉ cytochalasin B. A cellular fluorescent PLA 2 assay showed that hVPLA 2 had a lipolytic action first on the outer plasma membrane and then on the perinuclear region. hVPLA 2 also caused the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and a 2-fold increase in 5-lipoxygenase activity. However, hVPLA 2 induced neither the increase in intracellular calcium concentration nor cPLA 2 phosphorylation; consequently, cPLA 2 activity was not affected by hVPLA 2 . Pharmacological inhibition of cPLA 2 and the hVPLA 2 -induced activation of eosinophils derived from the cPLA 2 -deficient mouse corroborated that hVPLA 2 mediates the release of AA and leukotriene in a cPLA 2 -independent manner. As such, this study represents a unique example in which a secretory phospholipase induces the eicosanoid formation in inflammatory cells, completely independent of cPLA 2 activation.
G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.
The Forkhead box f1 (Foxf1) transcription factor is expressed in mesenchymal cells of the lung, liver, and gallbladder. Although Foxf1 deficiency causes severe abnormalities in the development of these organs, the molecular mechanisms underlying Foxf1 function remain uncharacterized. In this study we inactivated
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