Reactive Oxygen Species as Signaling Molecules in Neutrophil Chemotaxis

Neutrophil chemotaxis is a critical component in innate immunity. Recently, using a small-molecule functional screening, we identified NADPH-oxidase-dependent Reactive Oxygen Species (ROS) as key regulators of neutrophil chemotactic migration. Neutrophils depleted of ROS form more frequent multiple pseudopodia and lost their directionality as they migrate up a chemoattractant concentration gradient. Here, we further studied the role of ROS in neutrophil chemotaxis and found that multiple pseudopodia formation induced by NADPH inhibitor diphenyleneiodonium chloride (DPI) was more prominent in relatively shallow chemoattractant gradient. It was reported that, in shallow chemoattractant gradients, new pseudopods are usually generated when existing ones bifurcate. Directional sensing is mediated by maintaining the most accurate existing pseudopod, and destroying pseudopods facing the wrong direction by actin depolymerization. We propose that NADPH-mediated ROS production may be critical for disruption of misoriented pseudopods in chemotaxing neutrophils. Thus, inhibition of ROS production will lead to formation of multiple pseudopodia

Phosphoinositide Lipid Posphatase SHIP1 and PTEN Coordinate to Regulate Cell Migration and Adhesion

The second messenger phosphatidylinositol$(3,4,5)P_3 (PtdIns(3,4,5)P_3)$ is formed by stimulation of various receptors, including G protein–coupled receptors and integrins. The lipid phosphatases PTEN and SHIP1 are critical in regulating the level of PtdIns$(3,4,5)P_3$ during chemotaxis. Observations that loss of PTEN had minor and loss of SHIP1 resulted in a severe chemotaxis defect in neutrophils led to the belief that SHIP1 rather than PTEN acts as a predominant phospholipid phosphatase in establishing a PtdIns$(3,4,5)P_3$ compass. In this study, we show that SHIP1 regulates PtdIns$(3,4,5)P_3$ production in response to cell adhesion and plays a limited role when cells are in suspension. $SHIP1^{−/−}$ neutrophils lose their polarity upon cell adhesion and are extremely adherent, which impairs chemotaxis. However, chemo­taxis can be restored by reducing adhesion. Loss of SHIP1 elevates Akt activation following cell adhesion due to increased PtdIns$(3,4,5)P_3$ production. From our observations, we conclude that SHIP1 prevents formation of top-down PtdIns$(3,4,5)P_3$ polarity to facilitate proper cell attachment and detachment during chemotaxis

Cancer Cell-Derived Clusterin Modulates the Phosphatidylinositol 3'-Kinase-Akt Pathway through Attenuation of Insulin-Like Growth Factor 1 during Serum Deprivation

Cancer cells in their respective microenvironments must endure various growth-constraining stresses. Under these conditions, the cancer cell-derived factors are thought to modulate the signaling pathways between cell growth and dormancy. Here, we describe a cancer cell-derived regulatory system that modulates the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway under serum deprivation stress. Through the use of biochemical purification, we reveal that cancer cell-secreted insulin-like growth factor 1 (IGF-1) and clusterin, an extracellular stress protein, constitute this regulatory system. We show that secreted clusterin associates with IGF-1 and inhibits its binding to the IGF-1 receptor and hence negatively regulates the PI3K-Akt pathway during serum deprivation. This inhibitory function of clusterin appears to prefer IGF-1, as it fails to exert any effects on epidermal growth factor signaling. We demonstrate furthermore that the constitutive activation of oncogenic signaling downstream of IGF-1 confers insensitivity to the inhibitory effects of clusterin. Thus, the interplay between cancer cell-derived clusterin and IGF-1 may dictate the outcome of cell growth and dormancy during tumorigenic progression

Small-Molecule Screen Identifies Reactive Oxygen Species as Key Regulators of Neutrophil Chemotaxis

Neutrophil chemotaxis plays an essential role in innate immunity, but the underlying cellular mechanism is still not fully characterized. Here, using a small-molecule functional screening, we identified NADPH oxidase–dependent reactive oxygen species as key regulators of neutrophil chemotactic migration. Neutrophils with pharmacologically inhibited oxidase, or isolated from chronic granulomatous disease (CGD) patients and mice, formed more frequent multiple pseudopodia and lost their directionality as they migrated up a chemoattractant concentration gradient. Knocking down NADPH oxidase in differentiated neutrophil-like HL60 cells also led to defective chemotaxis. Consistent with the in vitro results, adoptively transferred CGD murine neutrophils showed impaired in vivo recruitment to sites of inflammation. Together, these results present a physiological role for reactive oxygen species in regulating neutrophil functions and shed light on the pathogenesis of CGD

Non-Classical Roles of NADPH-Oxidase Dependent Reactive Oxygen Species in Phagocytes

Phagocytes are classically known to utilize the NADPH-oxidase dependent Reactive Oxygen Species (ROS) release during host defense for clearance of pathogenic organisms. Insufficient activation of the oxidase, as in Chronic Granulomatous Disease, can lead to inadequate elimination of pathogens and can cause severe, life threatening infections. In this review, we present and discuss non-classical functions of the NADPH oxidase and ROS in phagocytes. We describe mechanisms by which the NADPH oxidase and ROS play regulatory roles in intracellular signal transduction, affecting various immune cell functions such as migration, survival, cytokine / chemokine secretion and cell adhesion. Specifically, we discuss redox dependent post-translational protein modifications such as glutathionylation and oxidation, which affect function of various proteins including actin, protein tyrosine kinases and phosphatases, Ras, and transcription factors such as NFκB, and hence can modulate cell signaling and function. In addition, we describe novel mechanisms by which the electrogenic function of the NADPH oxidase can regulate phagocyte signaling via modulating membrane charge potential and controlling membrane localization of charge sensitive enzymes and proteins. Finally, implications of these mechanisms on the pathogenesis of Chronic Granulomatous Disease are discussed

Cancer Cell-Derived Clusterin Modulates the Phosphatidylinositol 3′-Kinase-Akt Pathway through Attenuation of Insulin-Like Growth Factor 1 during Serum Deprivation▿ †

Cancer cells in their respective microenvironments must endure various growth-constraining stresses. Under these conditions, the cancer cell-derived factors are thought to modulate the signaling pathways between cell growth and dormancy. Here, we describe a cancer cell-derived regulatory system that modulates the phosphatidylinositol 3′-kinase (PI3K)-Akt pathway under serum deprivation stress. Through the use of biochemical purification, we reveal that cancer cell-secreted insulin-like growth factor 1 (IGF-1) and clusterin, an extracellular stress protein, constitute this regulatory system. We show that secreted clusterin associates with IGF-1 and inhibits its binding to the IGF-1 receptor and hence negatively regulates the PI3K-Akt pathway during serum deprivation. This inhibitory function of clusterin appears to prefer IGF-1, as it fails to exert any effects on epidermal growth factor signaling. We demonstrate furthermore that the constitutive activation of oncogenic signaling downstream of IGF-1 confers insensitivity to the inhibitory effects of clusterin. Thus, the interplay between cancer cell-derived clusterin and IGF-1 may dictate the outcome of cell growth and dormancy during tumorigenic progression

Myeloid-Specific Deletion of Tumor Suppressor PTEN Augments Neutrophil Transendothelial Migration during Inflammation

Phosphatidylinositol 3,4,5-trisphosphate $(PIP_3)$ is a second messenger that is involved in a number of cell activities including cell growth, proliferation, and motility. $PIP_3$ is produced by PI3K and regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP lipid phosphatases. Evidence from our experiments shows that enhanced $PIP_3$ production results in elevated neutrophil recruitment under inflammatory conditions. However, the mechanism of this elevation is not well understood. We used intravital video microscopy to investigate neutrophil recruitment in the cremaster venules of wild-type and PTEN knockout (KO) mice. Neutrophil transmigration was augmented in PTEN KO mice 4 h after TNF-α intrascrotal injection. PTEN KO neutrophils also showed significantly enhanced transmigration 2 h after MIP-2 intrascrotal injection, an effect that dramatically decreased when PI3K or Src kinase inhibitor treatments preceded MIP-2 stimulation. Similarly, fMLP superfusion of the cremaster muscle lead to enhanced emigration in PTEN KO mice. The observed elevation in neutrophil emigration was likely caused by increased speed of crawling, crossing the venular wall, and migrating through the muscular tissue in PTEN KO mice because the effect of PTEN depletion on neutrophil rolling or adhesion was minimal. Interestingly, chemoattractant-induced release of gelatinase and elastase was also elevated in PTEN null neutrophils, providing a potential mechanism for the enhanced neutrophil migration in the PTEN KO mice. Collectively, these results demonstrate that PTEN deletion in neutrophils enhances their invasivity and recruitment to inflamed sites more likely by raising the cell physical capability to cross the vascular and tissue barriers