The Study On The Function Of PI3K In The Signaling Of L-selectin And PSGL-1

Under numerous pathological or physiological conditions, leukocytes will transmigrate through the endothelium clefts to the underlying tissue, and this process is called leukocyte transendothelium migtation (TEM). Till now, it is known that many adhesion molecules on leukocytes and endothelium, and the intracellular signals which regulate adhesion and chemotaxis play parts in the transendothelium migtation of leukocytes. On one hand, the integrity of endothelium needs to be regulated in the process. One the other, the leukocytes in the blood need to be caught and roll on the endothelium, adhere to and subsequently traverse the endothelium and finally reach the inflammation sites.There are mainly two kinds of adhesion molecules which are involved in leukocyte TEM, namely the selectin and integrin families, among which L-selectin and PSGL-1 are a kind of selectin and selectin ligand, respectively. They are both expressed constitutively on the tips of leukocyte microvilli, and are the first ones to get in touch with the endothelium during leukocyte TEM. Therefore, L-selectin and PSGL-1 function crucially during the initial adhesion of leukocyte TEM. In fact L-selectin and PSGL-1 can not only recognize and bind the corresponding ligands expressed on activated endothelium, which speeds down the leukocytes in the fast-running blood. They can also function as signal receptors to transduce and amply inflammation signals which prepare for the stable adhesion between leukocytes and endothelium.There are quite a large number of researches on L-selectin and PSGL-1 signaling. By comparing the signaling pathways initiated from L-selectin and PSGL-1, we found that there are great similarities between them, although they are different signal molecules, and the amino acid sequences of their cytoplasmic domain are of low homology. Both their cytoplasmic domains are connected to cytoskeleton by ERM. Both of them recruit signal complexes through their cytoplasmic domains and upregulate CSF-1 gene expression. Under some conditions, L-selectin and PSGL-1 can even function synergicly to compensate each other's physiologic function. However, till now it is yet unknown how L-selectin and PSGL-1 initiate similar signaling pathways, nor any crosslinking between the two pathways has been found. During investigating which kinase integrates the L-selectin and PSGL-1 signaling pathways, we notified PI3K, a kind of critical lipid kinase,which phosphorylates PtIns (4, 5)P2 into the lipid second messenger PtIns (3, 4, 5)P3, and thereby involves in the regulation of cytoskeleton and gene transcription. The studies in this article demonstrate that during leukocyte TEM, L-selectin and PSGL-1 act as the ligands for E-selectin, and induce F-actin assembly and redistribution, and thereby support leukocyte rolling on E-selectin. PI3K activation, peaking within 5 minutes is induced rapidly upon L-selectin and PSGL-1 stimulation with E-selectin, and can be detected until 10 minute. After PI3K activation, Vav1 (the pivotal downstream effector of PI3K signaling pathway involved in cytoskeleton regulation) is activated. Furthermore, the F-actin rearrangement and assembly mediated by ligation with E-selectin were blocked by LY294002, a PI3K specific inhibitor. Additional experiments showed that PI3K activity was involved in neutrophil rolling on E-selectin. However, Syk/Zap70, the well-known upstream kinase of PI3K, was not involved in this event. These data suggest that PI3K is required for the F-actin-based cytoskeleton changes during neutrophil rolling on E-selectin, which may consequently regulate the rolling event. And LY294002, the PI3K specific inhibitor can block the activation of Vav1 upon E-selectin stimulation. PI3K plays a part in neutrophil rolling on E-selectin after its activation by regulate F-actin assembly and redistribution. But the typical upstream PI3K kinase Syk/Zap70 is not involved in the activation of PI3K. Additionally, using Jurkat T cells, we demonstrated by cotransfection and Real time PCR that L-selectin and PSGL-1 can also induce the expression of IL-18 and PI3K is also required for the regulation of IL-18 gene expression.Taken together, our exprements imply for the first time that L-selectin and PSGL-1 can use PI3K as the crosslinker of their signaling pathways to regulate cytoskeleton and the expression of the pre-inflammation factor IL-18, and therefore play crucial parts in leukocyte transendothelium migration and leukocyte activation.