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: p110α, p110β, and p110δ. It is unclear to what extent these p110 isoforms have overlapping or distinct biological roles. Mice expressing a catalytically inactive form of p110δ (p110δ D910A ) were generated by gene targeting. Antigen receptor signaling in B and T cells was impaired and immune responses in vivo were attenuated in p110δ mutant mice. They also developed inflammatory bowel disease. These results reveal a selective role for p110δ in immunity.

Phosphoinositide 3-kinases (PI3Ks) signal downstream of multiple cell-surface receptor types. Class IA PI3K isoforms couple to tyrosine kinases and consist of a p110 catalytic subunit (p110alpha, p110beta or p110delta), constitutively bound to one of five distinct p85 regulatory subunits. PI3Ks have been implicated in angiogenesis, but little is known about potential selectivity among the PI3K isoforms and their mechanism of action in endothelial cells during angiogenesis in vivo. Here we show that only p110alpha activity is essential for vascular development. Ubiquitous or endothelial cell-specific inactivation of p110alpha led to embryonic lethality at mid-gestation because of severe defects in angiogenic sprouting and vascular remodelling. p110alpha exerts this critical endothelial cell-autonomous function by regulating endothelial cell migration through the small GTPase RhoA. p110alpha activity is particularly high in endothelial cells and preferentially induced by tyrosine kinase ligands (such as vascular endothelial growth factor (VEGF)-A). In contrast, p110beta in endothelial cells signals downstream of G-protein-coupled receptor (GPCR) ligands such as SDF-1alpha, whereas p110delta is expressed at low level and contributes only minimally to PI3K activity in endothelial cells. These results provide the first in vivo evidence for p110-isoform selectivity in endothelial PI3K signalling during angiogenesis.

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110␣, p110␤, and p110␦) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110␣ and p110␦ to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110␥ class IB PI3K lack SH2 domains and instead couple p110␥ to G protein-coupled receptors (GPCRs). Here, we show, using smallmolecule inhibitors with selectivity for p110␤ and cells derived from a p110␤-deficient mouse line, that p110␤ is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110␤ and p110␥ contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110␤ but not p110␥, p110␤ mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110␥ in these cells reduced the contribution of p110␤ to GPCR signaling. Taken together, these data show that p110␤ and p110␥ can couple redundantly to the same GPCR agonists. p110␤, which shows a much broader tissue distribution than the leukocyterestricted p110␥, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110␥ expression is low or absent.gene targeting ͉ signaling ͉ tyrosine kinase ͉ Akt ͉ insulin T he lipid second messengers generated by phosphoinositide 3-kinases (PI3Ks) regulate a wide variety of cellular functions such as cell growth, proliferation, differentiation, and survival and have been implicated in cancer, inflammation, and diabetes. Mammals have eight isoforms of PI3K, which have been divided in three classes (1). Thus far, attention has focused mainly on the class I PI3Ks that are acutely activated by extracellular ligands. These heterodimers consist of a p110 catalytic subunit in complex with a regulatory subunit and have further been subdivided into class IA and IB PI3Ks. The class IA catalytic subunits (p110␣, p110␤, and p110␦) are in complex with an Src homology 2 (SH2) domain-containing regulatory subunit (of which there are five species, often referred to as p85s) that binds phosphoTyr in intracellular proteins, allowing recruitment of p85/p110 complexes to the membrane. The class IB regulatory subunits (p84, p101) do not have SH2 domains and link the single class IB PI3K catalytic subunit (p110␥) to G protein-coupled receptors (GPCRs).Over the last few years, the cellular signaling contexts and physiological roles of p110␣, p110␦, and p110␥ have become clearer, because of the generation of gene-targeted mice and small-molecule inhibitors for these PI3K isoforms. In contrast, very little is known about p110␤, both at the cellular and organismal level. One confounding factor has been the very early embryonic lethality of the p110␤ KO ...

Many mammalian genes contain overlapping antisense RNAs, but the functions and mechanisms of action of these transcripts are mostly unknown. WT1 is a well-characterized developmental gene that is mutated in Wilms' tumor (WT) and acute myeloid leukaemia (AML) and has an antisense transcript (WT1-AS), which we have previously found to regulate WT1 protein levels. In this study, we show that WT1-AS is present in multiple spliceoforms that are usually expressed in parallel with WT1 RNA in human and mouse tissues. We demonstrate that the expression of WT1-AS correlates with methylation of the antisense regulatory region (ARR) in WT1 intron 1, displaying imprinted monoallelic expression in normal kidney and loss of imprinting in WT. However, we find no evidence for imprinting of mouse Wt1-as. WT1-AS transcripts are exported into the cytoplasm and form heteroduplexes with WT1 mRNA in the overlapping region in WT1 exon 1. In AML, there is often abnormal splicing of WT1-AS, which may play a role in the development of this malignancy. These results show that WT1 encodes conserved antisense RNAs that may have an important regulatory role in WT1 expression via RNA:RNA interactions, and which can become deregulated by a variety of mechanisms in cancer.

Recent studies have identified antisense WT1 mRNAs whose expression is regulated by a promoter located in the first intron of the WT1 gene. Transcription directed by the antisense promoter is positively autoregulated by the WT1 protein implicating the antisense RNA in the control of WT1 gene expression. To elucidate further the biological role of the antisense RNA in the developing kidney, its distribution of expression has been examined relative to WT1 sense mRNA and WT1 protein. Using strand‐specific WT1 riboprobes, the expression of WT1 and the antisense message were examined by in situ hybridization in the developing human fetal kidney at different gestational ages. The expression of the antisense strand was strongest in the podocytes and glomeruli and also in the S‐form nephrons and the condensing blastema in the developing kidney. Expression was also seen in the podocytes of the mature kidney. The WT1 protein and sense mRNA for WT1 also showed a similar pattern, suggesting that the antisense transcript does not function simply as a downregulator of protein production. Expression of antisense WT1 exon 1 in cells constitutively producing high levels of WT1 also demonstrated no downregulation of protein and in most cases actually showed upregulated WT1 protein expression. These results strongly suggest that WT1 antisense transcripts positively modulate WT1 protein levels in vivo. © 1998 John Wiley & Sons, Ltd.