Phosphoinositide 3-kinase modulation of β3-integrin represents an endogenous “braking” mechanism during neutrophil transmatrix migration

Abstract

During episodes of inflammation, neutrophils (polymorphonuclear leukocytes[PMNs]) encounter subendothelial matrix substrates that may require additional signaling pathways as directives for movement through the extracellular space. Using an in vitro endothelial and epithelial model, inhibitors of phosphoinositide 3-kinase (PI3K) were observed to promote chemoattractant-stimulated migration by as much as 8 ؎ 0.3-fold. Subsequent studies indicated that PMNs respond in a similar manner to RGD-containing matrix substrates and that PMN-matrix interactions are potently inhibited by antibodies directed against ␤ 3 -but not ␤ 1 -integrin antibodies, and that PI3K inhibitors block ␤ 3 -integrin dependence. Biochemical analysis of intracellular ␤ 3 -integrin uncoupling by PI3K inhibitors revealed diminished ␤ 3 -integrin tyrosine phosphorylation and decreased association with p72 syk . Similarly, the p72 syk inhibitor piceatannol promoted PMN transmatrix migration, whereas HIV-tat peptide-facilitated loading of peptides corresponding to the ␤ 3 -integrin cytoplasmic tail identified the functional tyrosine residues for this activity. These data indicate that PI3K-regulated ␤ 3 -integrin represents a natural "braking" mechanism for PMNs during transit through the extracellular matrix. IntroductionMigration of neutrophils (polymorphonuclear leukocytes [PMNs ]) to sites of inflammation requires the coordinated interplay of soluble mediators, extracellular matrix ligands, and cell surface adhesion molecules. To subserve this function, PMNs must traverse endothelial cells lining the inner lumen of blood vessels. This process of transendothelial migration requires engagement and disengagement of a number of surface molecules and has been extensively studied. 1 After successful transendothelial migration, PMNs encounter subendothelial extracellular matrices in transit to inflammatory sites. Anchorage of cells to the extracellular matrix is mediated in part by integrins, a large family of heterodimeric cell surface proteins that mediate numerous cell functions, including motility, differentiation, and proliferation. 2 Integrin engagement of matrix ligand results in highly regulated signal transduction processes that cooperatively involve both "outside-in" and "insideout" pathways. 3 Importantly, integrin signaling can vary depending on the stimulus and on the cell type, 4 the molecular details of which are not fully understood at the present time.Recent studies have identified an important role for phosphoinositide-3-OH kinase (PI3K) in leukocyte migration. [5][6][7] The 4 known isoforms of PI3K are ␣, ␤, ␥, and ␦, and extracellular ligand coupling through membrane-localized G proteins generates the signaling molecule phosphatidylinositol 3,4,5-triphosphate. 8 PI3K coordinates a number of leukocyte effector functions, including leukocyte migration. [5][6][7]9 Although not fully understood at present, it appears that PI3K is central to stimulated chemotaxis. For instance, mice lacking the catalytic domain of leukocyte-specific PI3...

Ligand-dependent Activation of Integrin αvβ3

“…Although the Syk inhibitor piceatannol blocks RGDinduced phosphorylation of ␤ 3 , it is unlikely that this is the mechanism whereby piceatannol inhibits ␣ v ␤ 3 -mediated adhesion to Fn, because this event is independent of ␤ 3 -tyrosine phosphorylation and yet inhibited by piceatannol. In contrast to another report, we find that wortmannin has no effect on RGD-induced ␤ 3 -tyrosine phosphorylation, and therefore, its inhibitory effects are likely due to inhibition of the increase in PI3K activity and/or the translocation of PI3K to the integrin complex (22). Furthermore, we do not find an effect of wortmannin on the RGD-induced conformational change of ␣ v ␤ 3 (23).…”

Identification of a HoxA10 Activation Domain Necessary for Transcription of the Gene Encoding β3 Integrin during Myeloid Differentiation

“…We chose to study this interaction because fibronectin is an extracellular matrix component involved in enhancing survival of hematopoietic cells (5). Interaction with fibronectin is also implicated in phagocyte rolling (8). In these studies, we found that 48 h of IFN␥ differentiation significantly increased U937 cell adhesion to fibronectin (p Ͻ 0.03, n ϭ 4) (Fig.…”

“…We also investigated activation of Syk protein-tyrosine kinase, a downstream target of ␤3 integrin that mediates cell survival signals (8). For these studies, the blots were serially probed with antibodies to phosphorylated (activated) and total Syk.…”

“…In mature monocytes, ␣v␤3 mediates phagocytosis of apoptotic neutrophils, thereby contributing to down-regulation of the inflammatory response (6,7). In neutrophils, ␣v␤3 provides a "braking" mechanism during transmigration from the vascular space and is also involved in activation of the phagocyte-specific respiratory burst oxidase (8,9). In addition to participation in the innate immune response, ␣v␤3-ligand binding activates Syk protein-tyrosine kinase and may thereby be involved in generation of cell survival signals in phagocytic cells (8).…”

Regulation of Leukocyte Transmigration: Cell Surface Interactions and Signaling Events

“…In humans, inhibitor experiments examining the role of PI3K in PMN chemotaxis have yielded conflicting results. In particular, some reports on treatment of PMN with either wortmannin or LY294002 have shown only minimal inhibitory effects on PMN chemotaxis toward fMLP by under agarose assays (45), whereas others have observed enhanced PMN chemotaxis in Transwell migration assays in the presence of the same inhibitors (31,51). Roles for other PI3Ks in PMN migration have also been inferred.…”