Phosphoinositide 3-kinase (PI3K) activity is crucial for leukocyte function, but the roles of the four receptor-activated isoforms are unclear. Mice lacking heterotrimeric guanine nucleotide-binding protein (G protein)-coupled PI3Kgamma were viable and had fully differentiated neutrophils and macrophages. Chemoattractant-stimulated PI3Kgamma-/- neutrophils did not produce phosphatidylinositol 3,4,5-trisphosphate, did not activate protein kinase B, and displayed impaired respiratory burst and motility. Peritoneal PI3Kgamma-null macrophages showed a reduced migration toward a wide range of chemotactic stimuli and a severely defective accumulation in a septic peritonitis model. These results demonstrate that PI3Kgamma is a crucial signaling molecule required for macrophage accumulation in inflammation.

Despite development of novel agents targeting oncogenic pathways, matching targeted therapies to the genetic status of individual tumors is proving to be a daunting task for clinicians. To improve the clinical efficacy and to reduce the toxic side effects of treatments, a deep characterization of genetic alterations in different tumors is required. The mutational profile often evidences a gain of function or hyperactivity of phosphoinositide 3-kinases (PI3Ks) in tumors. These enzymes are activated downstream tyrosine kinase receptors (RTKs) and/or G proteins coupled receptors (GPCRs) and, via AKT, are able to induce mammalian target of rapamycin (mTOR) stimulation. Here, we elucidate the impact of class I (p110α, β, γ, and δ) catalytic subunit mutations on AKT-mediated cellular processes that control crucial mechanisms in tumor development. Moreover, the interrelation of PI3K signaling with mTOR, ERK, and RAS pathways will be discussed, exploiting the potential benefits of PI3K signaling inhibitors in clinical use.

Pentraxins are a superfamily of conserved proteins that are characterized by a cyclic multimeric structure. The classical short pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are acute-phase proteins produced in the liver in response to inflammatory mediators. Short pentraxins regulate innate resistance to microbes and the scavenging of cellular debris and extracellular matrix components. In contrast, long pentraxins have an unrelated, long amino-terminal domain coupled to the carboxy-terminal pentraxin domain, and differ, with respect to short pentraxins, in their gene organization, chromosomal localization, cellular source, and in their stimuli-inducing and ligand-recognition ability. To investigate the in vivo function of the long pentraxin PTX3, we generated mice deficient in Ptx3 by homologous recombination. Ptx3-null mice were susceptible to invasive pulmonary aspergillosis. Ptx3 binds selected microbial agents, including conidia of Aspergillus fumigatus, and we found that susceptibility of Ptx3-null mice was associated with defective recognition of conidia by alveolar macrophages and dendritic cells, as well as inappropriate induction of an adaptive type 2 response. Thus, the long pentraxin Ptx3 is a secreted pattern-recognition receptor that has a non-redundant role in resistance to selected microbial agents, in particular to the opportunistic fungal pathogen Aspergillus fumigatus.

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.

The PTEN/PI3K signaling pathway regulates a vast array of fundamental cellular responses. We show that cardiomyocyte-specific inactivation of tumor suppressor PTEN results in hypertrophy, and unexpectedly, a dramatic decrease in cardiac contractility. Analysis of double-mutant mice revealed that the cardiac hypertrophy and the contractility defects could be genetically uncoupled. PI3Kalpha mediates the alteration in cell size while PI3Kgamma acts as a negative regulator of cardiac contractility. Mechanistically, PI3Kgamma inhibits cAMP production and hypercontractility can be reverted by blocking cAMP function. These data show that PTEN has an important in vivo role in cardiomyocyte hypertrophy and GPCR signaling and identify a function for the PTEN-PI3Kgamma pathway in the modulation of heart muscle contractility.

Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P). How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.

The G protein-coupled, receptor-activated phosphoinositide 3-kinase gamma (PI3Kgamma) mediates inflammatory responses and negatively controls cardiac contractility by reducing cAMP concentration. Here, we report that mice carrying a targeted mutation in the PI3Kgamma gene causing loss of kinase activity (PI3KgammaKD/KD) display reduced inflammatory reactions but no alterations in cardiac contractility. We show that, in PI3KgammaKD/KD hearts, cAMP levels are normal and that PI3Kgamma-deficient mice but not PI3KgammaKD/KD mice develop dramatic myocardial damage after chronic pressure overload induced by transverse aortic constriction (TAC). Finally, our data indicate that PI3Kgamma is an essential component of a complex controlling PDE3B phosphodiesterase-mediated cAMP destruction. Thus, cardiac PI3Kgamma participates in two distinct signaling pathways: a kinase-dependent activity that controls PKB/Akt as well as MAPK phosphorylation and contributes to TAC-induced cardiac remodeling, and a kinase-independent activity that relies on protein interactions to regulate PDE3B activity and negatively modulates cardiac contractility.

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3–/– mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3–/– mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-α-trypsin inhibitor is normal in Ptx3–/– cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor α-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.