Wayne Pearce

Essential role for the p110δ phosphoinositide 3-kinase in the allergic response

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (FcɛRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110δ isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen–IgE-induced degranulation and cytokine release. Inactivation of p110δ protects mice against anaphylactic allergic responses. These results identify p110δ as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.

Critical role for the p110α phosphoinositide-3-OH kinase in growth and metabolic regulation

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110α, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110α kinase activity. Homozygosity for this kinase-dead p110α led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110α derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110β, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110α was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110α in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110α in a variety of cancers.

Angiogenesis selectively requires the p110α isoform of PI3K to control endothelial cell migration

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 (p110α, p110β or p110δ), 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 p110α activity is essential for vascular development. Ubiquitous or endothelial cell-specific inactivation of p110α led to embryonic lethality at mid-gestation because of severe defects in angiogenic sprouting and vascular remodelling. p110α exerts this critical endothelial cell-autonomous function by regulating endothelial cell migration through the small GTPase RhoA. p110α activity is particularly high in endothelial cells and preferentially induced by tyrosine kinase ligands (such as vascular endothelial growth factor (VEGF)-A). In contrast, p110β in endothelial cells signals downstream of G-protein-coupled receptor (GPCR) ligands such as SDF-1α, whereas p110δ 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.

Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells.

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-β. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110δ PI3K (p110δD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110δD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110δD910A/D910A T cells failed to protect against experimental colitis. The identification of p110δ as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.

Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer

Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8+ cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology.

Isoform-Specific Functions of Phosphoinositide 3-Kinases: p110δ but Not p110γ Promotes Optimal Allergic Responses In Vivo

The leukocyte-enriched p110γ and p110δ isoforms of PI3K have been shown to control in vitro degranulation of mast cells induced by cross-linking of the high affinity receptor of IgE (FcεRI). However, the relative contribution of these PI3K isoforms in IgE-dependent allergic responses in vivo is controversial. A side-by-side comparative analysis of the role of p110γ and p110δ in mast cell function, using genetic approaches and newly developed isoform-selective pharmacologic inhibitors, confirms that both PI3K isoforms play an important role in FcεRI-activated mast cell degranulation in vitro. In vivo, however, only p110δ was found to be required for optimal IgE/Ag-dependent hypersensitivity responses in mice. These observations identify p110δ as a key therapeutic target among PI3K isoforms for allergy- and mast cell-related diseases.

Control of Axonal Growth and Regeneration of Sensory Neurons by the p110δ PI 3-Kinase

The expression and function of the 8 distinct catalytic isoforms of PI 3-kinase (PI3K) in the nervous system are unknown. Whereas most PI3Ks have a broad tissue distribution, the tyrosine kinase-linked p110δ isoform has previously been shown to be enriched in leukocytes. Here we report that p110δ is also highly expressed in the nervous system. Inactivation of p110δ in mice did not affect gross neuronal development but led to an increased vulnerability of dorsal root ganglia neurons to exhibit growth cone collapse and decreases in axonal extension. Loss of p110δ activity also dampened axonal regeneration following peripheral nerve injury in adult mice and impaired functional recovery of locomotion. p110δ inactivation resulted in reduced neuronal signaling through the Akt protein kinase, and increased activity of the small GTPase RhoA. Pharmacological inhibition of ROCK, a downstream effector of RhoA, restored axonal extension defects in neurons with inactive p110δ, suggesting a key role of RhoA in p110δ signaling in neurons. Our data identify p110δ as an important signaling component for efficient axonal elongation in the developing and regenerating nervous system.

Airway inflammation: chemokine-induced neutrophilia and the class I phosphoinositide 3-kinases

Class I phosphoinositide 3‐kinases (PI3K) are known to play a significant role in neutrophil chemotaxis. However, the relative contributions of different PI3K isoforms, and how these impact on lung inflammation, have not been addressed. In vitro studies using wild‐type and PI3Kγ knockout neutrophils demonstrated the major role of the γ isoform in chemotactic but not chemokinetic events. This was confirmed by a model of direct chemokine instillation into the airways in vivo. Within all studies, a low yet significant degree of neutrophil movement in the absence of PI3Kγ could be observed. No role for the δ isoform was demonstrated both in vitro and in vivo using PI3Kδ kinase‐dead knock‐in mice. Moreover, further studies using the broad‐spectrum PI3K inhibitors wortmannin or LY294002 showed no other class I PI3K isoforms to be involved in these chemotactic processes. Here, we identify a contributory PI3K‐independent mechanism of neutrophil movement, yet demonstrate PI3Kγ as the pivotal mediator through which the majority of neutrophils migrate into the lung in response to chemokines. These data resolve the complexities of chemokine‐induced neutrophilia and PI3K signaling and define the γ isoform as a promising target for new therapeutics to treat airway inflammatory diseases.

Long‐term p110α PI3K inactivation exerts a beneficial effect on metabolism

The insulin/insulin‐like growth factor‐1 signalling (IIS) pathway regulates cellular and organismal metabolism and controls the rate of aging. Gain‐of‐function mutations in p110α, the principal mammalian IIS‐responsive isoform of PI 3‐kinase (PI3K), promote cancer. In contrast, loss‐of‐function mutations in p110α impair insulin signalling and cause insulin resistance, inducing a pre‐diabetic state. It remains unknown if long‐term p110α inactivation induces further metabolic deterioration over time, leading to overt unsustainable pathology. Surprisingly, we find that chronic p110α partial inactivation in mice protects from age‐related reduction in insulin sensitivity, glucose tolerance and fat accumulation, and extends the lifespan of male mice. This beneficial effect of p110α inactivation derives in part from a suppressed down‐regulation of insulin receptor substrate (IRS) protein levels induced by age‐related hyperinsulinemia, and correlates with enhanced insulin‐induced Akt signalling in aged p110α‐deficient mice. This temporal metabolic plasticity upon p110α inactivation indicates that prolonged PI3K inhibition, as intended in human cancer treatment, might not negatively impact on organismal metabolism.

Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans

Mutant Pik3ca gives rise to venous malformations. PI3K-ing the best treatment Venous malformations are a type of congenital vascular anomalies composed of dilated blood vessels, which can cause a variety of complications such as pain, disfigurement, and bleeding. The available treatments for these malformations are invasive and not particularly effective. Now, Castel et al. and Castillo et al. have both identified mutations in the phosphatidylinositol 3-kinase (PI3K) pathway as a cause of venous malformations, studied these in numerous mouse models, and demonstrated that they can be effectively treated by inhibiting PI3K activity, paving the way for future clinical trials. Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3caH1047R, a constitutively active mutant of the p110α isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3caH1047R resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.