Stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) are important regulators of the development of the dentate gyrus (DG). Both SDF-1 and CXCR4 are also highly expressed in the adult DG. We observed that CXCR4 receptors were expressed by dividing neural progenitor cells located in the subgranular zone (SGZ) as well as their derivatives including doublecortinexpressing neuroblasts and immature granule cells. SDF-1 was located in DG neurons and in endothelial cells associated with DG blood vessels. SDF-1-expressing neurons included parvalbumin-containing GABAergic interneurons known as basket cells. Using transgenic mice expressing an SDF-1-mRFP1 (monomeric red fluorescence protein 1) fusion protein we observed that SDF-1 was localized in synaptic vesicles in the terminals of basket cells together with GABA-containing vesicles. These terminals were often observed to be in close proximity to dividing nestin-expressing neural progenitors in the SGZ. Electrophysiological recordings from slices of the DG demonstrated that neural progenitors received both tonic and phasic GABAergic inputs and that SDF-1 enhanced GABAergic transmission, probably by a postsynaptic mechanism. We also demonstrated that, like GABA, SDF-1 was tonically released in the DG and that GABAergic transmission was partially dependent on coreleased SDF-1. These data demonstrate that SDF-1 plays a novel role as a neurotransmitter in the DG and regulates the strength of GABAergic inputs to the pool of dividing neural progenitors. Hence, SDF-1/ CXCR4 signaling is likely to be an important regulator of adult neurogenesis in the DG.

SUMMARY In developing limb skin, peripheral nerves provide a spatial template that controls the branching pattern and differentiation of arteries. Our previous studies indicate that nerve-derived VEGF-A is required for arterial differentiation but not for nerve-vessel alignment. In this study, we demonstrate that nerve-vessel alignment depends on the activity of Cxcl12-Cxcr4 chemokine signaling. Genetic inactivation of Cxcl12-Cxcr4 signaling perturbs nerve-vessel alignment, and abolishes arteriogenesis. Further in vitro assays allow us to uncouple nerve-vessel alignment and arteriogenesis, revealing that nerve-derived Cxcl12 stimulates endothelial cell migration, while nerve-derived VEGF-A is responsible for arterial differentiation. These findings suggest a coordinated sequential action in which nerve-Cxcl12 functions over a distance to recruit vessels to align with nerves and subsequent arterial differentiation presumably requires a local-action of nerve-VEGF-A in the nerve-associated vessels.

Grb2 is an adaptor molecule that mediates Ras-MAPK activation induced by various receptors. Here we show that conditional ablation of Grb2 in thymocytes severely impairs both thymic positive and negative selections. Strikingly, the mutation attenuates T-cell antigen receptor (TCR) proximal signaling, including tyrosine phosphorylation of multiple signaling proteins and Ca 2+ influx. The defective TCR signaling can be attributed to a marked impairment in Lck activation. Ectopic expression of a mutant Grb2 composed of the central SH2 and the C-terminal SH3 domains in Grb2 −/− thymocytes fully restores thymocyte development. Thus, Grb2 plays a pivotal role in both thymic positive and negative selection. It amplifies TCR signaling at the top end of the tyrosine phosphorylation cascade via a scaffolding function.T lymphopoiesis is a sequential developmental process regulated by multiple environmental signals. The final outcome is the generation of different lineages of T cells with a diverse antigen receptor repertoire (1-4). Diversification of the T-cell antigen receptor (TCR) is initially generated through successive rearrangements of the V-(D)-J genes in CD4 and CD8 doublenegative (DN) T precursors. Successful rearrangement of the TCR β gene allows DN T cells to express pre-TCR and to develop into CD4 and CD8 double-positive (DP) cells, at which stage the TCR repertoire is further shaped by thymic selection. Cells expressing a TCR with too strong or too weak an affinity/avidity for MHC-peptide complexes will die via apoptosis, the processes termed negative selection or neglect. In contrast, cells bearing a TCR with moderate avidity toward MHC-peptide complexes will survive the selection (positive selection) and become mature CD4 + or CD8 + single positive (SP) thymocytes (5-8).The importance of TCR signaling in thymic positive and negative selection has been demonstrated in a variety of experimental models. However, it remains to be determined precisely how TCR signaling is initiated and propagated intracellularly during thymic selection. The first step in initiating the TCR signaling cascade is thought to be the phosphorylation of tyrosine residues in immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3ζ chain by the Src family tyrosine kinase Lck (9-11). Phosphorylated ITAMs of the CD3ζ chain then recruit tyrosine kinase Zap70 to the TCR complexes, where it can be activated by autophosphorylation. Upon activation, Zap70 phosphorylates the membrane anchor protein linker for activation of T cells (LAT), which in turn assembles signaling complexes containing multisignaling molecules, including Grb2, SLP-76, Vav, Gads, PLCγ-1, and Itk. The coordination between these signaling components in the complexes determines multiple downstream cellular responses, including Ca 2+ mobilization, cytoskeleton reorganization, and activation of nuclear transcription factors, which eventually define various T-cell development programs (9, 12-14).Grb2 is a positive regulator of Ras signaling downstream of many growth fa...

Macrophage-derived chemokine (MDC/CC chemokine ligand 22 (CCL22)) mediates its cellular effects principally by binding to its receptor CCR4, and together they constitute a multifunctional chemokine/receptor system with homeostatic and inflammatory roles in the body. We report the CCL22-induced accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3) in the leukemic T cell line CEM. CCL22 also had the ability to chemoattract human Th2 cells and CEM cells in a pertussis toxin-sensitive manner. Although the PI(3,4,5)P3 accumulation along with the pertussis toxin-susceptible phosphorylation of protein kinase B were sensitive to the two phosphoinositide 3-kinase inhibitors, LY294002 and wortmannin, cell migration was unaffected. However, cell migration was abrogated with the Rho-dependent kinase inhibitor, Y-27632. These data demonstrate that although there is PI(3,4,5)P3 accumulation downstream of CCR4, phosphoinositide 3-kinase activity is a dispensable signal for CCR4-stimulated chemotaxis of Th2 cells and the CEM T cell line.

Grb2 (growth-factor receptor-bound protein-2) is a signaling adaptor that interacts with numerous receptors and intracellular signaling molecules. However, its role in B-cell development and function remains unknown. Here we show that ablation of Grb2 in B cells results in enhanced B-cell receptor signaling; however, mutant B cells do not form germinal centers in the spleen after antigen stimulation. Furthermore, mutant mice exhibit defects in splenic architecture resembling that observed in B-cell-specific lymphotoxin-β-deficient mice, including disruption of marginal zone and follicular dendritic cell networks. We find that grb2 −/− B cells are defective in lymphotoxin-β expression. Although lymphotoxin can be up-regulated by chemokine CXCL13 and CD40 ligand stimulation in wild-type B cells, elevation of lymphotoxin expression in grb2 −/− B cells is only induced by anti-CD40 but not by CXCL13. Our results thus define Grb2 as a nonredundant regulator that controls lymphoid follicle organization and germinal center reaction. Loss of Grb2 has no effect on B-cell chemotaxis to CXCL13, indicating that Grb2 executes this function by connecting the CXCR5 signaling pathway to lymphotoxin expression but not to chemotaxis.B-cell signaling and activation | follicular dendritic cell development | germinal center development | regulation of lymphotoxin expression | chemokine signaling

Macrophage-derived chemokine [CC chemokine ligand 22 (CCL22)] and thymus- and activation-regulated chemokine (CCL17) mediate cellular effects, principally by binding to their receptor CC chemokine receptor 4 (CCR4) and together, constitute a multifunctional chemokine/receptor system with homeostatic and inflammatory roles within the body. This study demonstrates that CCL22 and CCL17 stimulate pertussis toxin-sensitive elevation of intracellular calcium in the CEM leukemic T cell line and human peripheral blood-derived T helper type 2 (Th2) cells. Inhibition of phospholipase C (PLC) resulted in the abrogation of chemokine-mediated calcium mobilization. Chemokine-stimulated calcium responses were also abrogated completely by the inhibition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor-mediated calcium release. Chemotactic responses of CEM and human Th2 cells to CCL17 and CCL22 were similarly abrogated by inhibition of PLC and inhibition of novel, Ca2+-independent/diacylglycerol-dependent protein kinase C (PKC) isoforms. Inhibition of Ins(1,4,5)P3 receptor-mediated calcium release from intracellular stores had no effect on chemotactic responses to CCR4 ligands. Taken together, this study provides compelling evidence of an important role for PLC and diacylglycerol-dependent effector mechanisms (most likely involving novel PKC isoforms) in CCL17- and CCL22-stimulated, directional cell migration. In this regard, CCL22 stimulates phosphatidylinositol-3 kinase-independent phosphorylation of the novel delta isoform of PKC at threonine 505, situated within its activation loop--an event closely associated with increased catalytic activity.

CXCR4 regulates cell proliferation, enhances cell survival and induces chemotaxis, yet molecular mechanisms underlying its signaling remain elusive. Like all other G-protein coupled receptors (GPCRs), CXCR4 delivers signals through G-protein-dependent and -independent pathways, the latter involving its serine-rich cytoplasmic tail. To evaluate the signaling and biological contribution of this G-protein-independent pathway, we generated mutant mice that express cytoplasmic tail-truncated CXCR4 (ΔT) by a gene knock-in approach. We found that ΔT mice exhibited multiple developmental defects, with not only G-protein-independent but also G-protein-dependent signaling events completely abolished, despite ΔT's ability to still associate with G-proteins. These results reveal an essential positive regulatory role of the cytoplasmic tail in CXCR4 signaling and suggest the tail is crucial for mediating G-protein activation and initiating crosstalk between G-protein-dependent and G-protein-independent pathways for correct GPCR signaling.

The NF-KB/Rel transcription factors play an important role in the expression of genes involved in B cell development, differentiation and function. Although it is clear that BCR stimulation leads to the activation of NF-KB and despite the importance of NF-KB to B cell function, little is known about the signaling pathways leading to NF-KB activation. We will address the role of PI 3-kinase in BCRand LPS-induced NF-KB activation in primary murine B cells. Using PI 3-kinase inhibitors, we demonstrate that PI 3-kianse activity is vital for BCR-induced NF-KB DNA-binding activity.Furthermore, we show that this is achieved via PKCdependent degradation of I K B~. Similarly, we show that PI 3-kinase is also required for LPS-induced NF-KB-DNA binding activity and I K B~ degradation. Interestingly, blocking PKC activity did not affect the degradation of I K B~ after LPS stimulation. This suggests the involvement of PI 3-kinase in at least two distinct signaling pathways leading to activation of NF-KB in B cells.90 Ma1 (MyD88 adapter-like) is a novel Toll IL-1 receptor (TIR)