Selectins mediate leukocyte rolling, trigger ␤ 2 -integrin activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to activation of an immunoreceptor tyrosinebased activation motif (ITAM)-dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to integrin activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) ␥2-and phosphoinositide 3-kinase (PI3K) ␥-dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP 3 ), and inducing E-selectin-mediated slow rolling. Inhibition of this signal-transduction pathway diminished G␣ i -independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. G␣ i -independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk ؊/؊ and Plcg2 ؊/؊ mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by Eselectin engagement. (Blood. 2010;115(15): 3118-3127) IntroductionLeukocyte recruitment into inflamed tissue is required for host defense and proceeds in a coordinated sequence of different steps. The first contact of neutrophils with the endothelium is mediated by selectins and their counter-receptors, followed by rolling and integrin-mediated arrest. While rolling, neutrophils collect different inflammatory signals that can activate several pathways and mediate integrin activation, arrest, crawling, and extravasation of leukocytes into inflamed tissue. 1 E-selectin is expressed on inflamed endothelial cells and can bind to different glycosylated ligands on leukocytes, including CD44, 2 P-selectin glycoprotein ligand-1 (PSGL-1), 3 CD43, 4 Eselectin ligand-1 (ESL-1), 5 macrophage antigen-1 (Mac-1; ␣ M ␤ 2 ), 6 and other unknown ligands. E-selectin engagement induces the activation of a receptor-proximal Src family immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor proteinSyk signaling pathway, which induces lymphocyte functionassociated antigen-1 (LFA-1)-dependent slow rolling in vitro and in vivo. 7,8 Selplg Ϫ/Ϫ and Syk Ϫ/Ϫ neutrophils cannot reduce their rolling velocity when rolling on E-selectin and intercellular adhesion molecule-1 (ICAM-1) in flow chamber experiments. 8 In neutrophils from Tyrobp Ϫ/Ϫ Fcrg Ϫ/Ϫ (DAP12-and FcR␥-deficient) mice, E-selectin engagement fails to phosphorylate Syk and does not induce slow rolling. 7 Binding of neutrophils to E-selectin under shear induces the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), 7 and blocking of p38 MAPK elevates the rolling velocity on E-selectin and ICAM-1 compare...

Rolling leukocytes are exposed to different adhesion molecules and chemokines. Neutrophil rolling on E-selectin induces integrin αLβ2-mediated slow rolling on intercellular adhesion molecule-1 by activating a phospholipase C (PLC)γ2- and a separate phosphoinositide-3-kinase (PI3K)γ-dependent pathway. E-selectin-signaling cooperates with chemokine signaling to recruit neutrophils into inflamed tissues. However, the distal signaling pathway linking PLCγ2 (Plcg2) to αLβ2-activation is unknown. To identify this pathway, we used different TAT-fusion-mutants and gene-deficient mice in intravital microscopy, autoperfused flow chamber, peritonitis, and biochemical studies. We found that the small GTPase Rap1 is activated following E-selectin engagement and that blocking Rap1a in Pik3cg−/− mice by a dominant-negative TAT-fusion mutant completely abolished E-selectin mediated slow rolling. We identified CalDAG-GEFI (Rasgrp2) and p38 MAPK as key signaling intermediates between PLCγ2 and Rap1a. Gαi-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster muscle were completely abolished in Rasgrp2−/− mice. The physiologic importance of CalDAG-GEFI in E-selectin-dependent integrin activation is shown by complete inhibition of neutrophil recruitment into the inflamed peritoneal cavity of Rasgrp2−/− leukocytes treated with pertussis toxin to block Gαi-signaling. Our data demonstrate that Rap1a activation by p38 MAPK and CalDAG-GEFI is involved in E-selectin-dependent slow rolling and leukocyte recruitment.

Acute kidney injury (AKI) is a common clinical problem in critically ill patients and increases in-hospital mortality. Acute loss of renal function (ALRF) reduces leukocyte recruitment into inflamed tissue, but the underlying molecular mechanisms remain unknown. In this study, we investigated the effects of ALRF on the different steps of the leukocyte recruitment cascade by using intravital microscopy, flow chamber experiments, and biochemistry assays. ALRF abolished selectin-induced slow leukocyte rolling on E-selectin/ICAM-1 and P-selectin/ICAM-1 and also reduced transmigration without affecting chemokine-induced arrest. A reduced phosphorylation of spleen tyrosine kinase (Syk), Akt, phospholipase C (PLC) γ2, and p38 MAPK, but not altered expression levels of adhesion molecules on the surface of neutrophils, was responsible for the abolished selectin-mediated slow leukocyte rolling. The results observed in the murine system could be reproduced in flow chamber experiments with human blood. Samples from critically ill patients with sepsis-induced AKI showed a significantly higher rolling velocity on E-selectin/ICAM-1 and P-selectin/ICAM-1 compared to patients with sepsis alone or to healthy volunteers. In conclusion, these data suggest that ALRF inhibits selectin-mediated slow leukocyte rolling by reducing phosphorylation of Syk, Akt, PLCγ2, and p38 MAPK and transmigration.

Tryptophan hydroxylase-2 (TPH2) is the rate-limiting enzyme in raphe serotonin biosynthesis, and polymorphisms of TPH2 are implicated in vulnerability to psychiatric disorders. Dynamic transcription regulation of TPH2 may underlie differences in vulnerability. We identified a transcription element in the TPH2 promoter that resembles the binding motif for RE-1 silencer of transcription (REST; also known as NRSF) transcription factor. REST limits tissue expression of non-neuronal genes through a canonical 21-bp motif called the NRSE (neuron-restrictive silencing element). The NRSE in TPH2 is a novel bipartite variant interrupted by a 6-base insertion. We confirmed that this bipartite NRSE permits transcriptional repression by REST identical to canonical NRSE in rat C6-glioma cells. Synthetic permutations of the motif revealed considerable flexibility in the juxtaposition of the two halves of bipartite NRSE. Computational analysis revealed many bipartite NRSE variants conserved between mouse and human genomes. A subgroup of these was found to bind REST by chromatin immunoprecipitation. Messenger RNAs for TPH2 and potassium channel H6, another gene with a bipartite NRSE, were up-regulated by dominant-negative REST in C6-glioma cells. These findings, which indicate that TPH2 expression is part of the developmental program regulated by REST and suggest that many previously unrecognized genes may be regulated by REST through the novel motif, have implications for the mechanism of REST action.Serotonin levels are regulated by the rate-limiting enzyme tryptophan hydroxylase, of which a neuronal isoform (NCBI Entrez Gene ID: TPH2) 3 was recently identified (1). TPH2 gene expression in the brain is restricted to raphe neurons, and TPH2 is likely the gene coding for the majority of tryptophan hydroxylase in these cells (1, 2). Serotonin modulates a variety of complex behaviors (3), and its signaling is implicated in the mechanism for a subset of antidepressants (4), but its precise role in mood regulation is unclear. A number of manipulations, including restriction of the essential dietary precursor tryptophan (5), stimulant drugs of abuse (6), or adverse developmental experiences (7), result in low brain serotonin levels and may underlie associated depression or anxiety phenotypes. Functional variants of TPH2 in mice are associated with decreased brain serotonin levels (8), and a TPH2 polymorphism in humans arguably (9) confers vulnerability to major depression (10). Associations with TPH2 polymorphisms are also reported for suicide (11), obsessivecompulsive disorder (12), and attention deficit disorder (13)(14)(15).In contrast to the growing number of genetic association studies, little is known about the regulation of TPH2 expression. Limited studies suggest that TPH2 mRNA levels are decreased following stress corticosteroids (16), unchanged (16) or increased by ovarian steroids (17), and increased by hypotensive stress (18) and one recent report suggests that POU3F2 may regulate TPH2 through a functional single-nucleoti...