We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3K␥ in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3K␥-mediated oxidant signaling in the mechanism of NF-B activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110␥ catalytic subunit of PI3K␥. Tumor necrosis factor (TNF) ␣ challenge of wild type ECs caused p110␥ translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110␥ ؊/؊ ECs. ROS production was the result of TNF␣ activation of Ser phosphorylation of NADPH oxidase subunit p47 phox and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110␥ ؊/؊ ECs. Four mammalian phosphatidylinositol 3-kinase (PI3K) 2 type 1 isoforms, p110␣, p110␤, p110␥, and p110␦, have been identified (1), and of these, p110␥ has distinct properties. Type 1A PI3Ks, p110␣, p110␤, and p110␦, associate with one of the five regulatory subunits: p50␣, p55␣, and p85␣ (products of alternative splicing of a single gene) and p55␥ and p85␤ (2). In contrast, type 1B PI3K (or PI3K␥), the catalytic subunit p110␥ binds to the p101 adaptor molecule (3) or the G␤␥-activated regulatory subunit p84 (4). Type 1A PI3Ks are activated by interactions with tyrosine-phosphorylated molecules, whereas p110␥ is activated by heterotrimeric G proteins G␣ and G␤␥ that bind to the pleckstrin homology domain found in the NH 2 -terminal region of PI3K␥ (3, 5). p110␥ is also activated by pro-inflammatory cytokines such as TNF␣ (6). Expression of PI3K␥ is largely confined to leukocytes, and there is a growing appreciation of its important role in immunity and host defense (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Studies also demonstrated the presence of the PI3K␥ isoform in endothelial cells (ECs) (19,20), but its function remains unclear.PI3Ks catalyze the conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), which is involved in the recruitment and activation of a variety of regulatory proteins via interactions with their pleckstrin homology and phox homology domains (21). phox domains, present in two subunits of the NADPH oxidase complex, p47 phox and p40 phox , bind to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate (both breakdown products of PIP 3 ) (21, 22). Degradation of PIP 3 occurs by either PTEN (3Ј-phosphatase and tensin homolog deleted on chromosome 10) or SH2-containing phosphatidyl inositol phosphatases (SHIP-1 and SHIP-2) (7,23,24).NADPH oxidase is a tightly regulated membrane-bound enzyme complex catalyzing the one-electron reduction of oxygen to superoxide with the simultaneous oxidation of cytosolic NADPH (25). We showed that TNF␣-induced oxidant generation in ECs requires activation of PKC (26,27). PKC associates with and phosphorylates p47 ph...

We tested the hypothesis that TNF-alpha induces early-onset endothelial adhesivity toward PMN by activating the constitutive endothelial cell surface ICAM-1, the beta2-integrin (CD11/CD18) counter-receptor. Stimulation of human pulmonary artery endothelial cells with TNF-alpha resulted in phosphorylation of ICAM-1 within 1 minute, a response that was sustained up to 15 minutes after TNF-alpha challenge. We observed that TNF-alpha induced 10-fold increase in PMN adhesion to endothelial cells in an ICAM-1-dependent manner and that this response paralleled the rapid time course of ICAM-1 phosphorylation. We also observed that the early-onset TNF-alpha-induced endothelial adhesivity was protein synthesis-independent and associated with cell surface ICAM-1 clustering. Pretreatment of cells with the pan-PKC inhibitor, chelerythrine, prevented the activation of endothelial adhesivity. As PKCzeta, an atypical PKC isoform abundantly expressed in endothelial cells, is implicated in signaling TNF-alpha-induced ICAM-1 gene transcription, we determined the possibility that PKCzeta was involved in mediating endothelial adhesivity through ICAM-1 expression. We observed that TNF-alpha stimulation of endothelial cells induced PKCzeta activation and its association with ICAM-1. Inhibition of PKCzeta by pharmacological and genetic approaches prevented the TNF-alpha-induced phosphorylation and the clustering of the cell surface ICAM-1 as well as activation of endothelial adhesivity. Thus, TNF-alpha induces early-onset, protein synthesis-independent expression of endothelial adhesivity by PKCzeta-dependent phosphorylation of cell surface ICAM-1 that precedes the de novo ICAM-1 synthesis. The rapid ICAM-1 expression represents a novel mechanism for promoting the stable adhesion of PMN to endothelial cells that is needed to facilitate the early-onset transendothelial migration of PMN.

The Src homology 2-containing inositol phosphatase SHIP1 functions in hemopoietic cells to limit activation events mediated by PI3K products, including Akt activation and cell survival. In contrast to the limited cellular expression of SHIP1, the related isoform SHIP2, is widely expressed in both parenchymal and hemopoietic cells. The goal of this study was to determine how SHIP2 functions to regulate M-CSF signaling. We report that 1) SHIP2 was tyrosine-phosphorylated in M-CSF-stimulated human alveolar macrophages, human THP-1 cells, murine macrophages, and the murine macrophage cell line RAW264; 2) SHIP2 associated with the M-CSF receptor after M-CSF stimulation; and 3) SHIP2 associated with the actin-binding protein filamin and localization to the cell membrane, requiring the proline-rich domain, but not on the Src homology 2 domain of SHIP2. Analyzing the function of SHIP2 in M-CSF-stimulated cells by expressing either wild-type SHIP2 or an Src homology 2 domain mutant of SHIP2 reduced Akt activation in response to M-CSF stimulation. In contrast, the expression of a catalytically deficient mutant of SHIP2 or the proline-rich domain of SHIP2 enhanced Akt activation. Similarly, the expression of wild-type SHIP2 inhibited NF-κB-mediated gene transcription. Finally, fetal liver-derived macrophages from SHIP2 gene knockout mice enhanced activation of Akt in response to M-CSF treatment. These data suggest a novel regulatory role for SHIP2 in M-CSF-stimulated myeloid cells.