Biomedical subjects
Publications and source records attributed to Bart Vanhaesebroeck.
When it comes to silencing genes in mice, not all approaches are equal. An example published in this issue of Cell (Patrucco et al., 2004) suggests that caution should be used when validating potential drug targets by genetic disruption.
Atheroma formation involves the movement of vascular smooth muscle cells (VSMC) into the subendothelial space. The aim of this study was to determine the involvement of PI3K and MAPK pathways and the importance of cross-talk between these pathways, in glucose-potentiated VSMC chemotaxis to serum factors. VSMC chemotaxis occurred in a serum gradient in 25 mmol/L glucose (but not in 5 mmol/L glucose) in association with increased phosphorylation (activation) of Akt and ERK1/2 in PI3K and MAPK pathways, respectively. Inhibitors of these pathways blocked chemotaxis, as did an mTOR inhibitor. VSMC expressed all class IA PI3K isoforms, but microinjection experiments demonstrated that only the p110beta isoform was involved in chemotaxis. ERK1/2 phosphorylation was reduced not only by MAPK pathway inhibitors but also by PI3K and mTOR inhibitors; when PI3K was inhibited, ERK phosphorylation could be induced by microinjected activated Akt, indicating important cross-talk between the PI3K and ERK1/2 pathways. Glucose-potentiated phosphorylation of molecules in the p38 and JNK MAPK pathways inhibited these pathways but did not affect chemotaxis. The statin, mevinolin, blocked chemotaxis through its effects on the MAPK pathway. Mevinolin-inhibited chemotaxis was restored by farnesylpyrophosphate but not by geranylgeranylpyrophosphate; in the absence of mevinolin, inhibition of farnesyltransferase reduced ERK phosphorylation and blocked chemotaxis, indicating a role for the Ras family of GTPases (MAPK pathway) under these conditions. In conclusion, glucose sensitizes VSMC to serum, inducing chemotaxis via pathways involving p110beta-PI3K, Akt, mTOR, and ERK1/2 MAPK. Cross-talk between the PI3K and MAPK pathways is necessary for VSMC chemotaxis under these conditions.
Phosphoinositide 3-kinases (PI3Ks) play an important role in a variety of cellular functions, including phagocytosis. PI3Ks are activated during phagocytosis induced by several receptors and have been shown to be required for phagocytosis through the use of inhibitors such as wortmannin and LY294002. Mammalian cells have multiple isoforms of PI3K, and the role of the individual isoforms during phagocytosis has not been addressed. The class I PI3Ks consist of a catalytic p110 isoform associated with a regulatory subunit. Mammals have three genes for the class IA p110 subunits encoding p110alpha, p110beta, and p110delta and one gene for the class IB p110 subunit encoding p110gamma. Here we report a specific recruitment of p110beta and p110delta (but not p110alpha) isoforms to the nascent phagosome during apoptotic cell phagocytosis by fibroblasts. By microinjecting inhibitory antibodies specific to class IA p110 subunits, we have shown that p110beta is the major isoform required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by primary mouse macrophages. Macrophages from mice expressing a catalytically inactive form of p110delta showed no defect in the phagocytosis of apoptotic cells and IgG-opsonized particles, confirming the lack of a major role for p110delta in this process. Similarly, p110gamma-deficient macrophages phagocytosed apoptotic cells normally. Our findings demonstrate that p110beta is the major class I catalytic isoform required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by primary macrophages.
Class IA phosphoinositide 3'-kinases (PI3Ks) regulate many cellular processes downstream of tyrosine kinases and Ras. Despite a clear implication of PI3K in cancer, little is known about the distribution of the different PI3K isoforms in malignant cells. We screened a large panel of tissues and cell lines for expression of class IA PI3Ks, and document a ubiquitous expression of the p110alpha and p110beta isoforms but a variable and more restricted tissue distribution of the p110delta isoform. Originally found in WBCs, p110delta was also detected in some nonhematopoietic cell types especially those of breast or melanocytic origin, both in the untransformed and transformed state. Isoform-specific neutralization of PI3K isoforms in breast cancer cell lines (by PI3K antibody microinjection or a p110delta-selective pharmacological inhibitor) demonstrated that p110delta is the most important class IA PI3K in the regulation of epidermal growth factor-driven motility in vitro, controlling the directionality and, to a lesser extent, the speed of migration. In contrast, p110beta was required for the direction but not the speed of migration, whereas p110alpha did not impact on either of these parameters. These results show a nonredundant function of PI3K isoforms downstream of the epidermal growth factor receptor and indicate that the presence of p110delta may confer breast cancer cells with selective migratory capacities. The potential clinical implications of p110delta expression in non-WBC-derived tumors are discussed.
Phosphoinositide 3-kinases (PI3Ks) regulate numerous biological processes, including cell growth, differentiation, survival, proliferation, migration and metabolism. In the immune system, impaired PI3K signalling leads to immunodeficiency, whereas unrestrained PI3K signalling contributes to autoimmunity and leukaemia. New insights into the role of PI3Ks in lymphocyte biology have been derived from gene-targeting studies, which have identified the PI3K subunits that are involved in B-cell and T-cell signalling. In particular, the catalytic subunit p110delta seems to be adapted to transmit antigen-receptor signalling in B and T cells. Additional recent work has provided new insights into the molecular interactions that lead to PI3K activation and the signalling pathways that are regulated by PI3K.
Eight human isoforms of phosphoinositide 3-kinases (PI3Ks) exist, but their individual functions remain poorly understood. Here, we show that different human small cell lung carcinoma (SCLC) cell lines overexpress distinct subsets of class I(A) and II PI3Ks, which results in striking differences in the signalling cascades activated by stem cell factor (SCF). Over expression of class I(A) p85/p110alpha in SCLC cells increased SCF-stimulated protein kinase B (PKB) activation and cell growth, but did not affect extracellular signal-regulated kinase (Erk) or glycogen synthase kinase-3 (GSK-3). This effect was selective, since it was not observed in SCLC cell lines overexpressing p85/p110beta or p85/p110delta. The SCF receptor associated with both class I(A) p85 and class II PI3KC2beta, and both enzymes contributed to SCF-stimulated PKB activity. A dominant-negative PI3KC2beta blocked both PKB activation and SCLC cell growth in response to SCF. Together our data provide novel insights into the specificity and functional significance of PI3K signalling in human cancer.
The phosphoinositide 3-kinase (PI3K) catalytic subunit p110delta, the most recently discovered member of the heterodimeric Class IA PI3K family, has been detected uniquely in leukocytes, but not in one member of the leukocyte family: platelets. We have examined freshly prepared isolates of human platelets for the presence of this enzyme, realizing that p110delta is highly susceptible to proteolytic degradation. We have utilized p110delta-directed Western blotting, RT-PCR, PI3K activity assays, and immunoprecipitations of PI3K Class IA subunits p85alpha, p85beta, and p110delta from lysed human platelets, as well as Triton X-100-insoluble cytoskeletal preparations from resting and thrombin receptor-activated platelets. We report that p110delta is present in association with p85alpha and p85beta in platelets, both in cytosolic and cytoskeletal fractions. The latter finding is consistent with the proposed role of p110delta in cytoskeletal function.
Class IA phosphoinositide 3-kinases (PI3Ks) are a family of p85/p110 heterodimeric lipid kinases that generate second messenger signals downstream of tyrosine kinases, thereby controlling cell metabolism, growth, proliferation, differentiation, motility, and survival. Mammals express three class IA catalytic subunits: p110alpha, p110beta, and p110delta. It is unclear to what extent these p110 isoforms have overlapping or distinct biological roles. Mice expressing a catalytically inactive form of p110delta (p110delta(D910A)) were generated by gene targeting. Antigen receptor signaling in B and T cells was impaired and immune responses in vivo were attenuated in p110delta mutant mice. They also developed inflammatory bowel disease. These results reveal a selective role for p110delta in immunity.
We have investigated the function of the p110delta catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in platelets using p110delta knock-out (p110delta(-/-)) mice and p110delta knock-in (p110delta(D910A/D910A)) mice, which express a catalytically inactive form of the enzyme. Aggregation to threshold concentrations of the GPVI-specific agonist, CRP, was partially reduced in p110delta(-/-) and p110delta(D910A/D910A) platelets. This inhibition was overcome by higher concentrations of CRP. The degree of inhibition was considerably weaker than that induced by LY294002 and wortmannin, which inhibit all isoforms of PI 3-kinase. p110delta(-/-) platelets showed decreased spreading on fibrinogen- or von Willebrand factor (VWF)-coated surfaces under static conditions, whereas they spread normally on collagen. LY294002 had a more pronounced inhibitory effect on spreading on all three surfaces. Adhesion and aggregate formation of p110delta(-/-) platelets to collagen or fibrinogen/VWF at intermediate/high rates of shear were normal. This study demonstrates a minor role for the p110delta catalytic subunit in mediating platelet activation by the collagen receptor GPVI and integrin alphaIIbeta3. The more pronounced inhibitory effect of LY294002 and wortmannin indicates that other isoforms of PI 3-kinase play a more significant role in signalling by the two platelet glycoprotein receptors.