The recruitment and activation of phosphatidylinositol 4-phosphate 5-kinases α critically regulate CD28-dependent signaling responses

CD28 costimulatory receptor is a crucial determinant of the outcome of T lymphocyte activation. The engagement of CD28 by its natural ligands, B7.1/CD80 or B7.2/CD86, expressed on the surface of professional APC, lowers T cell receptor (TCR) activation threshold, thus leading to the enhancement of early signalling events necessary for efficient cytokine production, cell cycle progression, survival and regulation of T cells effector responses. CD28 is also able to act as a unique signalling receptor and to deliver TCR-independent autonomous signals, which account for its critical role in the regulation of pro-inflammatory cytokine/chemokine production and T cell survival. Most of the CD28-dependent signalling functions are initiated by the recruitment and activation of class IA phosphatidylinositol 3-kinase (PI3K), The intracytoplasmic domain of CD28 contains a N-terminal YMNM motif that following phosphorylation binds the p85 subunit of phosphatidylinositol 3-kinase (PI3K). Once activated, PI3K catalyzes the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) and generates the docking sites for key signalling proteins. PIP2 plays a critical role in the regulation of both cytoskeleton dynamics and second messenger generation. Indeed, PIP2 is the common source for two major distinct signalling cascades involving PI3K and PLCγ1 that often colocalize in the same signalling complexes competing for the common pool of substrate. Consequently, PIP2 levels decrease following receptor activation, thus suggesting that stimulation of PIP2 synthesis may be an essential regulatory step to sustain the activation of both PI3K and PLCγ1 following CD28 engagement. The main biosynthetic pathway of PIP2 involves phosphorylation of phosphatidylinositol 4-monophosphate (PI4P) at the D5 position of the inositol ring by PIP5K. Three PIP5K isoforms (α, β and γ) have been identified. Several data obtained in different cell systems evidenced differential subcellular localizations of each isoform. PIP5Kα, for instance, is localized at the plasma membrane, where it guarantees the local availability of PIP2. Here we show that CD28 stimulation by both B7.1/CD80 or agonistic Abs induces the recruitment and activation of PIP5Kα in human primary CD4+ T lymphocytes. This event leads to the neo-synthesis of PIP2 that is consumed by CD28-activated PI3K. By either small interference RNA (siRNA)-driven cell silencing or overexpressing a kinase dead mutant, we evidenced that PIP5Kα activation is required for both CD28 autonomous signals regulating IL-8 gene expression as well as for CD28/TCR-induced Ca2+ mobilization, NF-AT nuclear translocation and IL-2 gene transcription. Our findings identify PIP5Kα as a critical mediator of CD28-dependent responses

The RhoA transcriptional program in pre-T cells

The GTPase RhoA is essential for the development of pre-T cells in the thymus. To investigate the mechanisms used by RhoA to control thymocyte development we have used Affymetrix gene profiling to identify RhoA regulated genes in T cell progenitors. The data show that RhoA plays a specific and essential role in pre-T cells because it is required for the expression of transcription factors of the Egr-1 and AP-1 families that have critical functions in thymocyte development. Loss of RhoA function in T cell progenitors causes a developmental block that pheno-copies the consequence of losing pre-TCR expression in Recombinase gene 2 (Rag2) null mice. Transcriptional profiling reveals both common and unique gene targets for RhoA and the pre-TCR indicating that RhoA participates in the pre-TCR induced transcriptional program but also mediates pre-TCR independent gene transcription

The PtdIns 3-Kinase/Akt Pathway Regulates Macrophage-Mediated ADCC against B Cell Lymphoma

Macrophages are important effectors in the clearance of antibody-coated tumor cells. However, the signaling pathways that regulate macrophage-induced ADCC are poorly defined. To understand the regulation of macrophage-mediated ADCC, we used human B cell lymphoma coated with Rituximab as the tumor target and murine macrophages primed with IFNγ as the effectors. Our data demonstrate that the PtdIns 3-kinase/Akt pathway is activated during macrophage-induced ADCC and that the inhibition of PtdIns 3-kinase results in the inhibition of macrophage-mediated cytotoxicity. Interestingly, downstream of PtdIns 3-kinase, expression of constitutively active Akt (Myr-Akt) in macrophages significantly enhanced their ability to mediate ADCC. Further analysis revealed that in this model, macrophage-mediated ADCC is dependent upon the release of nitric oxide (NO). However, the PtdIns 3-kinase/Akt pathway does not appear to regulate NO production. An examination of the role of the PtdIns 3-kinase/Akt pathway in regulating conjugate formation indicated that macrophages treated with an inhibitor of PtdIns 3-kinase fail to polarize the cytoskeleton at the synapse and show a significant reduction in the number of conjugates formed with tumor targets. Further, inhibition of PtdIns 3-kinase also reduced macrophage spreading on Rituximab-coated surfaces. On the other hand, Myr-Akt expressing macrophages displayed a significantly greater ability to form conjugates with tumor cells. Taken together, these findings illustrate that the PtdIns 3-kinase/Akt pathway plays a critical role in macrophage ADCC through its influence on conjugate formation between macrophages and antibody-coated tumor cells

Phospholipase D signaling: orchestration by PIP2 and small GTPases

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions

Malattie allergiche

Con lo studio del capitolo il lettore sarà in grado di: 1. conoscere i meccanismi fisiopatologici che hanno ispirato l'approccio diagnostico delle malattie allergiche mediate da IgE; conoscere gli algoritmi diagnostici delle malattie allergiche anche in riferimento ai più moderni avanzamenti tecnologici