Abstracts

Day 2: Wednesday, June 27, 2012

Session III: Young Investigator Presentations

Inactivation of p110d PI 3-kinase unlocks anti-cancer immune responses
Khaled Ali, Centre for Cell Signalling, Barts Cancer Institute, Queen Mary University of London

The PI3K pathway is frequently activated in tumor cells and is an important therapeutic target in cancer. Mammals have eight isoforms of PI3K, with the p110d isoform of PI3K being highly expressed in leukocytes in which it modulates immune functions. Broad-spectrum PI3K inhibition in cancer is generally expected to suppress immune surveillance. Here, we document that p110d inactivation in mice protects against solid tumor growth and metastasis, by decreasing cancer-induced immunosuppression by regulatory T cells and polymorphonuclear myeloid-derived suppressor cells, allowing an effective CD8⁺ T cell-dependent anti-tumor response. Thus, p110d inhibition is not immunosuppressive in cancer and is a druggable target to stimulate anti-tumor immune responses.
 

Coauthors: Dalya R. Soond², Roberto Pineiro¹, Wayne Pearce¹, Cheryl Scudamore³, Heather Maecker⁴, Lori Friedman⁴, Alexander Rudensky⁵, Martin Turner², Klaus Okkenhaug², Bart Vanhaesebroeck^1
 
1Centre for Cell Signalling, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK
²Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge CB22 3AT, UK
³Department of Pathology and Infectious Diseases, Royal Veterinary College, Hawkshead Lane, North Mymms Hatfield, Hertfordshire AL9 7TA, UK
⁴Cancer Signalling and Translational Oncology, Genentech Inc, South San Francisco, CA, USA
⁵Memorial Sloan-Kettering Cancer Centre, New York
 

PI3K-AKT-MTORC1-S6K1/2 Axis Controls TH17 Differentiation
Shigenori Nagai, PhD, Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan & Core Research for Evolutional Science and Technology, JST, Tokyo, Japan

The PI3K-Akt-mTORC1 axis contributes to the activation, survival, and proliferation of CD4⁺ Tcells upon stimulation through TCR and CD28. In this work, we demonstrate that the suppression of this axis by deletion of p85α or PI3K/mTORC1 inhibitors as well as T cell-specific deletion of raptor, an essential component of mTORC1, impairs Th17 differentiation in vitro and in vivo in a S6K1/2-dependent fashion. Inhibition of PI3K-Akt-mTORC1-S6K1 axis impairs the downregulation of Gfi1, a negative regulator of Th17 differentiation. Furthermore, we demonstrate that S6K2, a nuclear counterpart of S6K1, is induced by the PI3K-Akt-mTORC1 axis, binds RORγ, and carries RORγ to the nucleus. These results point toward a pivotal role of PI3K-Akt-mTORC1-S6K1/2 axis in Th17 differentiation.
 

Coauthors: Yutaka Kurebayashi, MD¹, Ai Ikejiri, PhD¹, Masashi Ohtani, PhD³, Kenji Ichiyama, PhD^{1, 2}, Yukiko Baba, MSc^{1, 2}, Taketo Yamada, MD, PhD⁴, Shohei Egami, MD¹, Takayuki Hoshii, PhD⁵, Atsushi Hirao, MD, PhD⁵, Satoshi Matsuda, PhD³, Shigeo Koyasu, PhD^1
 
1Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan
²Core Research for Evolutional Science and Technology, JST, Tokyo, Japan
³Department of Cell Signaling, Institute of Biomedical Science, Kansai Medical University, Osaka, Japan
⁴Department of Pathology, Keio University School of Medicine, Tokyo, Japan
⁵Division of Molecular Genetics, Cancer Research Institute, Kanazawa University, Ishikawa, Japan
⁶Research Center for Science Systems, Japan Society for the Promotion of Science, Tokyo, Japan
⁷RIKEN Center for Allergy and Immunology, Kanagawa, Japan
 

Osteolineage expression of SHIP1 is required for BM microenvironment signaling
Iyer, S., Dept. of Microbiology & Immunology, SUNY Upstate Medical University

Germline SHIP^-/- mice exhibit profound osteoporosis that was proposed to result from hyper-resorptive action by SHIP1-deficient osteoclasts (OC) (Takeshita et al., Nature Med, 2002). A subsequent study confirmed that SHIP^-/- OC are hyper-resorptive ex vivo and attributed this behavior to a failure of SHIP1 to impede PI3K recruitment to the cytoplasmic tail of DAP12 associated with key OC activating receptors (Peng et al., Science Signaling, 2010). To ablate SHIP1 expression specifically in osteoblasts, we used mice expressing Cre-recombinase driven by the osteoblast specific Osterix promoter (Osx^Cre). Here we show that osteolineage expression of SHIP1 is required for development of osteoblasts such that normal body growth, bone formation and mineralization are impaired in mice. Additionally, in the absence of osteolineage expression of SHIP1 the development of mesenchymal stem/progenitor cells is biased toward adipogenesis such that marrow fat content is also increased. The loss of SHIP1 expression by osteolineage cells causes stress in the BM microenvironment and leads to increased peripheralization of hematopoietic stem/progenitors to the spleen. Surprisingly, we find that mice with myeloid-restricted ablation of SHIP1, including OC, show no reduction in bone mass, length, mineralization or body size. Hence, diminished bone mass, growth and density in the SHIP1-deficient host results from SHIP-deficiency in osteoblasts. Intriguingly, mice with a SHIP-deficient osteolineage compartment also exhibit decreased OC numbers. Consistent with this decrease in bone resorptive cells, mice with a SHIP-deficient osteolineage compartment do not exhibit age-associated bone loss. These findings demonstrate a novel role for SHIP1 in fate determination of mesenchymal stem/progenitor populations, bone growth and OB support of HSC and suggest that reductions in SHIP1 expression or its signaling might be used to abrogate age-related loss of bone density.
 

Coauthors: Margulies, B.S.,² and Kerr, W.G.^1,3,4
 
1Dept. of Microbiology & Immunology, SUNY Upstate Medical University
²Dept. of Orthopedic Surgery, SUNY Upstate Medical University
³Dept. of Pediatrics, SUNY Upstate Medical University, Syracuse, NY, USA
⁴Dept. of Chemistry, Syracuse University, Syracuse, NY 13210
 

Class III PI3K VPS34 Plays an Essential Role in Autophagy, Endocytosis, and Heart and Liver Function
Nadia Jaber, Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY

Mammalian Vps34 is the sole member of the class III phosphatidylinositol 3-kinases, which mediates its function in membrane trafficking pathways by phosphorylating PtdIns to generate PtdIns(3)P. Vps34 is suggested to have a central role in endocytosis, autophagy and recently, mTOR signaling. Interestingly, a conditional deletion of Vps34 in sensory neurons reported a disruption in the endocytic pathway, but not the autophagic pathway. This prompted us to generate a conditional deletion of Vps34 in mouse heart and liver, and to fully characterize the effect on autophagy. Deletion of Vps34 in mouse embryonic fibroblasts via adenoviral Cre infection results in the diminishment of localized PtdIns(3)P production, as well as blocked autophagosome formation and flux. Vps34-null cells accumulate large, translucent vacuoles which appear to have characteristics of late endosomes. Early endosome function of transferring recycling is preserved, whereas late endocytic/lysosomal degradation of EGFR is blocked. In addition, amino acid-stimulated, but not serum-, glucose- or insulinstimulated mTOR activation is suppressed in the absence of Vps34. Starvationinduced autophagosome formation is blocked in both Vps34-null heart and liver. Liver-specific Albumin-Cre;Vps34f/f mice developed hepatomegaly and hepatic steatosis, as well as impaired protein turnover. Ablation of Vps34 in the heart of muscle creatine kinase-Cre;Vps34f/f mice led to cardiomegaly and decreased contractility. Taken together, our results indicate that Vps34 plays an essential role in regulating autophagy, endocytic trafficking, mTOR signaling and organ function.
 

Coauthors: Zhixun Dou¹, Juei-Suei Chen¹, Joseph Catanzaro¹, Ya-Ping Jiang², MD, Elzbieta Selinger², Xiaosen Ouyang³, PhD, Richard Lin², MD, PhD, Jianhua Zhang³, PhD, and Wei-Xing Zong¹, PhD
 
¹Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY ²Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY ³Department of Pathology, University of Alabama, Birmingham, AL
 

Session IV: Modulation of Inositol Phospholipid Signaling in Immune Function

PI3K p110delta in T cell Immunity and Inflammation
Klaus Okkenhaug, PhD, Babraham Institute, United Kingdom

PI3Ks regulate key T cell functions and can be activated by diverse families of transmembrane receptors. Although all class I PI3K isoforms are expressed in T cells, p110delta is predominantly activated by tyrosine-kinase linked antigen, costimulatory and cytokine receptors, whereas p110gamma is activated by primarily by chemokine receptors. Inhibition of PI3K in T cells profoundly impairs their ability to produce cytokines and to support other cells of the immune system upon antigenic challenge. PI3Ks also control the trafficking of T cells between different lymphoid compartment and to sites of inflammation. Our results also show that p110delta-deficient T cells show defects in their capacity to interact productively with antigen presenting cells which we propose has a substantial impact on the capacity of p110delta-deficient T cells to fully integrate the plethora of signals provided by antigen presenting cells during the first 24 hours of T cell activation. However, regulatory T cell function is also compromised by p110delta inhibition. Therefore, the net effect of p110delta-inhibitors on a given immune response is unpredictable and in some cases leads to enhance immunity. I will discuss recent results from experiments testing the effect of p110delta inhibition on immune responses to pathogens and tumours.
 

mTOR Kinase Inhibitors Modulate Lymphocyte Function via the AKT-FOXO Axis
David Fruman, PhD, University of California, Irvine

The mechanistic target of rapamycin (mTOR) is a kinase that functions in two cellular complexes, mTORC1 and mTORC2. Compared to rapamycin, ATP-competitive mTOR kinase inhibitors have greater anti-tumor activity in several mouse models of solid and hematological cancers. Previous studies attributed the improved anti-cancer activity to more complete inhibition of mTORC1. Here we report that inhibition of mTORC2 contributes to the pro-apoptotic effects of mTOR kinase inhibitors in pre-B cell leukemias. mTORC2 inhibition leads to nuclear entry of the AKT/SGK substrate FOXO1 and activates transcription of FOXO target genes. In sensitive cell lines, mTOR kinase inhibitors induce expression of pro-apoptotic FOXO targets such as TRAIL. In resistant cell lines, these pro-apoptotic genes are silenced epigenetically but their expression can be induced by combined treatment with mTOR kinase inhibitors and histone deacetylase inhibitors (HDACi). This drug combination causes a synergistic increase in apoptosis in cell lines and primary patient-derived leukemia cells. We also present evidence that treatment of activated normal B cells with mTOR kinase inhibitors alters differentiation through the AKT-FOXO axis. In splenic B cells stimulated with polyclonal mitogens, mTOR kinase inhibition augments class-switch recombination in a FOXO-dependent manner, while suppressing plasma cell differentiation. Transient treatment of mice with the mTOR kinase inhibitor AZD8055 augments high affinity antibody production in response to a protein antigen. In summary, the ability of mTOR kinase inhibitors to augment FOXO function is a key factor in the anti-leukemic efficacy of this class of compounds and also modulates the differentiation of antigen-stimulated mature B cells.
 

Coauthors: Jose J. Limon¹, Brandon Beagle¹, Matthew R. Janes^1,2, Yi Liu², and Christian Romme^1,2
1University of California, Irvine, Irvine, CA
²Intellikine, Inc., La Jolla, CA

Role of SHIP1 in Inflammation at Mucosal Surfaces
William G. Kerr, PhD, Murphy Family Professor of Children's Oncology Research

T cells are proposed to play a critical role in immune surveillance and host protection at mucosal surfaces. However, genetic mutations that impair T cell function do not routinely lead to infection at these sites due to compensatory protection mediated by innate immune effectors such as neutrophils and macrophages. SHIP deficient mice are known to succumb to profound inflammatory disease proposed to be mediated by macrophages and neutrophils that occurs in two distinct mucosal immune sites, lung and small intestine. Here I will discuss how SHIP1-deficiency leads to inflammatory disease in these mucosal sites and how this is prevented when SHIP deficient mice are selectively reconstituted with SHIP-competent T cells. Moreover, we find that persistence of mature CD4⁺ and CD8⁺ T cells in the periphery is dependent upon SHIP and its enzymatic activity. Thus, by promoting protective T cell immunity, while also restraining myeloid immune effectors, SHIP1 maintains the hierarchy of adaptive and innate immune functions necessary for mucosal barrier function. These findings reveal a previously unappreciated role for SHIP1 in mature T cell biology that have important implications for therapeutic use of T cells and in inflammatory diseases of the lung and intestine.
 

Control of T Cell Homeostasis and Responses by PTEN
Laurence Turka, MD, Beth Israel Deaconess Medical Center

Multiple T cell surface receptors, most notably the T cell receptor for antigen and the interleukin-2 receptor, signal at least in part via PI-3 kinase (PI-3K). The tumor suppressor gene PTEN is a key regulator of PI-3K in multiple cell types, and our lab has focused on its roles in T cell development and the control of T cell responses. Using mice with deletion of Pten targeted to the T cell compartment, we have shown distinct context dependent roles for PTEN at differing stages of T cell maturity. During thymic maturation, mice which lack PTEN uniformly develop T cell lymphoma, characterized by a 14:15 chromosomal translocation involving the oncogene c-myc and the TCRα locus. These tumors do not appear to exhibit or depend on Notch activation. In contrast, if translocations are prevented via the use of a recombination-deficient background, Pten-targeted mice develop Notch-dependent lymphomas. Tumor development in Pten-targeted mice always occurs during thymic development, as demonstrated by the ability of early thymectomy to prevent malignancy, and the fact that adoptively transferred mature T cells never undergo transformation in their hosts. In these circumstances however, thymectomized Pten-targeted mice develop progressive autoimmunity, with production of anti-dsDNA antibodies and T cell infiltration of visceral organs. These data, as well as more recent studies using site-directed Pten knock-in mutants, will be discussed.
 

Session V: Modulation of Inositol Phospholipid Signaling in Inflammatory Disease

Targeting PI3k-gamma & PI3k-delta in Inflammation
Vito J. Palombella, PhD, Infinity Pharmaceuticals

The phosphoinositide-3-kinase (PI3K) family of enzymes is involved in multiple cellular functions such as cell proliferation and survival, metabolism, cell differentiation, and cellular trafficking. Specifically, the PI3K-δ and PI3K-γ isoforms are necessary for adaptive and innate immunity, and the role of these enzymes in various immune cells hassupported the development of small molecule inhibitors of either or both PI3K isoforms for the treatment of immune mediated diseases. Isoform-specific PI3K-δ and PI3K-γ inhibitors are expected to have therapeutic effects without interfering with general PI3K signaling critical to the normal function of other cellular systems. IPI-145 is a potent, novel, synthetic small molecule designed to reversibly inhibit the enzymatic activity of both PI3K-δ and PI3K-γ isoforms, and is being developed as an orally administered therapeutic forhematologic malignancies and inflammation. IPI-145 is competitive with ATP and is highly selective over other protein kinases.Inhibition of both PI3K-&delta and PI3K-γ presents a unique therapeutic opportunity for the treatment of a variety of complex immune-mediated inflammatory diseases.
 

AQX-1125: A Novel Allosteric Activator of SHIP1. From Preclinical Studies to Proof-of-Concept Clinical Studies
David Chernoff, MD, Aquinox, Inc.

The SH2-containing inositol-5'-phosphatase 1 (SHIP1) is selectively expressed in cells of hematopoietic lineage and metabolizes PI(3,4,5)P3 to PI(3,4)P2. SHIP1-deficient mice exhibit progressive pulmonary inflammation and fibrosis. AQX-1125, a novel small-molecule SHIP1 activator, increased the catalytic activity of human recombinant SHIP1. This effect depends on the C2 region of the enzyme. Consistent with its effects on phosphoinositide metabolism, AQX-1125 inhibited Akt phosphorylation in SHIP1-proficient, but not in SHIP1-deficient monocytes, inhibited the activation of murine mast cells from wild-type, but not of SHIP1^-/- mice, and reduced cytokine production in murine splenocytes. The SHIP1 activator exerted a pleiotropic inhibitory effect on human leukocyte chemotaxis (IC₅₀<1 µM) and it exerted anti-inflammatory effects in rodent models of bacterial lipopolysaccharide (LPS)-induced lung inflammation; the efficacy was absent in SHIP1^-/- mice. Pharmacological SHIP1 activation was also efficacious in rodent models of ovalbumin-induced airway inflammation, a glucocorticoid-resistant model of cigarette smoke-induced lung inflammation and in a bleomycin model of pulmonary fibrosis. AQX-1125 exhibited >80% oral bioavailability and >5h terminal half-life in preclinical models, and showed good dose-proportionality and a 20h terminal half-life in humans. The translational value of the concept of pharmacological SHIP1 activation is highlighted by the fact that AQX-1125 attenuated neutrophil counts in induced sputum of healthy human volunteers following LPS inhalation. Thus, pharmacological SHIP1 activation is a novel approach for the experimental therapy of asthma, chronic obstructive pulmonary disease, pulmonary fibrosis and other inflammatory diseases.
 

Coauthors: Lloyd F. Mackenzie¹, Grant R. Stenton¹, Patrick Tam¹, Jennifer L. Cross¹, Curtis Harwig¹, Jeffrey Raymond¹, Judy Toews¹, Joyce Wu¹, Nancy Ogden¹, Stephen P. Smith², Vanessa Brown³, J. Stuart Elborn³, Emanuela Esposito⁴, Salvatore Cuzzocrea⁴, Thomas MacRury⁴, and Csaba Szabo^4
1Aquinox Pharmaceuticals Inc., Richmond, BC, Canada
²Celerion, Belfast, Northern Ireland, UK
³Respiratory Medicine Group, Centre for Infection and Immunity, Queen's University, Belfast, Northern Ireland, UK
⁴Department of Pharmacology, University of Messina, Messina, Italy

PI3Kdelta Inhibition as a Future Paradigm for the Inhaled Therapy of Respiratory Diseases
Augustin Amour, PhD, GlaxoSmithKline

Chronic lung inflammation and airway obstruction are the main features of the two most common respiratory diseases, asthma and COPD. In the majority of cases, combinations of inhaled bronchodilators and steroids are effective in providing symptomatic treatment, but a proportion of patients remain poorly controlled by the current standard of care. PI3Kdelta inhibition has the potential to suppress the key inflammatory cell types activated in asthma and COPD and to broaden the effectiveness of steroids. It could therefore provide a future alternative treatment alongside the existing respiratory standard of care. Here, we present pre-clinical data of a potent and selective inhaled inhibitor of PI3Kdelta. We observed that it prevented inflammatory cell recruitment and release of pro-inflammatory mediators in response to disease relevant triggers (e.g. allergen, cigarette smoke and viruses). In addition, it improved steroid sensitivity under conditions where glucocorticoid receptor function is muted. Finally, in our first clinical study, this inhibitor was well tolerated and its exposure provided high lung to plasma ratios supporting the profile for a safe and potentially effective inhaled drug.
 

PI3K Gamma Inactivation in Inflammation and Autoimmune Disease
Wai-Ping Fung-Leung, PhD, Johnson & Johnson Pharmaceuticals

Phosphoinositide 3-kinase gamma (PI3Kγ) is a lipid kinase expressed in leukocytes and activated by G-protein coupled-receptors. PI3Kγ plays an important role in cell migration and is also involved in other cellular responses such as neutrophil oxidative burst, T cell activation and mast degranulation. The function of PI3Kγ depends on its kinase activity and therefore small molecules inhibiting its kinase activity could be promising medicines for treatment of inflammation and autoimmune diseases. Inactivation of PI3Kγ with genetic and pharmacological approaches has been taken to validate its role in development of autoimmune diseases and our findings will be discussed in the presentation.
 

PI3Kγ Signaling — Gateway to Inflammation and Obesity
Matthias P. Wymann, Department of Biomedicine, University of Basel

Phosphoinositide 3-kinasePI3Kγ (PI3Kγ) plays a central role in inflammation [1], allergy [2], cardiovascular [3; 4] and metabolic disease [5]. Structural investigations have provided a basis for the development of strategies to target PI3Kγ in chronic inflammatory conditions such as rheumatoid arthritis [6], atherosclerosis [7] and allergic responses [2; 8]. Novel insights in localized, PI3Kγ-mediated PtdIns(3,4,5)P₃ production [8] and a PI3Kγγ-adapter-specifc role of Ras [9], are currently exploited to modulate cell-specific PI3Kγ activation in inflammatory, proliferative and metabolic disease. PI3Kγγ in- and outputs can be controlled by upstream kinases such as protein kinase A (PKA; [5]) and protein kinase C (PKC; Walser et al.), and regulate PI3Kγγsignaling in a cell-specific fashion. Obesity is accompanied by chronic, low-grade inflammation: as PI3Kγγ plays a major role in leukocyte recruitment, targeting of PI3Kγγ has been considered as a strategy to attenuate progression of obesity to insulin resistance and type 2 diabetes. Indeed, PI3Kγ null mice are protected from high fat diet-induced obesity, metabolic inflammation, fatty liver and insulin resistance. The lean phenotype of the PI3Kγ null mice has been linked to an increased thermogenesis and energy expenditure. Surprisingly, the increase in fat mass and metabolic aberrations were not linked to PI3Kγ activity in the hematopoietic compartment. Thermogenesis and oxygen consumption are modulated by PI3Kγ lipid kinase-dependent and lipid kinase-independent signaling mechanisms [5]. Altogether, PI3Kγ emerges as a signaling node integrating upstream signals from G protein-coupled receptors, but can be switched to process alternative inputs. PI3Kγ signaling has now developed a signature controlling metabolic and inflammatory stress, and can provide entry points for therapeutic strategies in metabolic disease, inflammation and cardiovascular disease. For reviews see: [10-12].
 
References
1. Hirsch, E. et al. (2000). Science 287, 1049-1053
2. Laffargue, M. et al. (2002). Immunity 16, 441-451
3. Patrucco, E. et al. (2004). Cell 118, 375-387
4. Perino, A. et al. (2011). Mol Cell 42, 84-95
5. Becattini, B. et al. (2011). Proc Natl Acad Sci USA 108, E854-63
6. Camps, M. et al. (2005). Nat Med 11, 936-943
7. Fougerat, A. et al. (2008). Circulation 117, 1310-1317
8. Bohnacker, T. et al. (2009). Sci Signal 2, ra27
9. Kurig, B. et al. (2009). Proc Natl Acad Sci USA 106, 20312-20317
10. Marone, R. et al. (2008). Biochim Biophys Acta 1784, 159-185
11. Wymann, M. P., and Schneiter, R. (2008). Nat Rev Mol Cell Biol 9, 162-176
12. Wymann, M. P. (2012). PI3Ks-Drug Targets in Inflammation and Cancer. In Balla, T., M. P. Wymann, & J. D. York (Eds.), Phosphoinositides I: Enzymes of Synthesis and Degradation, Vol 58 (111-181). Springer Netherlands.
 

Session VI: Inositol Phospholipid Signaling and Metabolic Disease

PI3Kgamma Signaling — Gateway to Inflammation and Obesity
Matthias P. Wymann, PhD, University of Basel, Switzerland

The Role of Lipid Kinases/Phosphatase Signaling in the Hypothalamus and Metabolic Disease
Jens Claus Brüning, MD, University of Cologne, Germany

Insulin and leptin act in the CNS to control energy homeostasis and peripheral glucose metabolism. Although both hormones act through different classes of receptors, they both activate PI3-kinase signaling in neurons. PI3-kinase activation on one hand controls expression ad processing of key neuropeptides, which control food intake and the regulation of hepatic gluconeogenesis, such as proopiomelanocortin, but also also regulates neuronal excitability via control of ATP-dependent potassium channels. The presentation will focus on the molecular mechanisms of how PI3-kianse signaling regulates feeding and glucose metabolism via its action in distince hypothalamic neurocircuits as well as on the molecular basis by which these signaling pathway are altered upon obesity development.
 

Phospholipase D Signaling in Diabetes and Cardiovascular Disease
Michael A. Frohman, MD, PhD, Stony Brook University

Phospholipase D (PLD), which generates the signaling lipid phosphatidic acid, and PI4P5-Kinase (PIP5K), which generates PI4,5P₂, have long been proposed to facilitate regulated exocytosis in specialized secretory cells such as pancreatic β-cells. Studies on mice lacking PLD and PIP5K isoforms though have revealed unexpected findings, due in part, seemingly, to the complex compensatory responses activated in response to loss of the signaling sources. I will review our recent findings that PI4P5-Kinase α and PLD2 undertake regulatory roles to inhibit glucose-stimulated insulin secretion rather than to facilitate it, and discuss underlying mechanisms we have identified, which include effects on the actin cytoskeleton, cis-Golgi structure, and calcium signaling. I will also discuss a new role for PLD2 in the control of blood pressure that has been uncovered by genetic linkage studies and may ensue from PLD2 regulation of signaling pathways previously reported.
 

Inpp4b, A Modulator of Osteoclast Differentiation
Jean Vacher, PhD, Clinical Research Institute of Montreal, Canada

Osteoclasts derive from the hematopoietic monocyte/macrophage lineage and are responsible for bone resorption. Defective differentiation or loss of osteoclast activity leads to malignant osteopetrosis, a genetic disease characterized by abnormal increase in bone mass and severe reduction in bone marrow compartment. We are studying the grey-lethal (gl) mouse mutant that displays a severe recessive osteopetrotic phenotype closely related to the human infantile malignant osteopetrosis. We identified a gl target gene associated with osteopetrosis in gl/gl mice, the murine Inositol Polyphosphate 4-phosphatase type II (Inpp4b). Inpp4b expression is normally detected during osteoclast differentiation and in mature cells. To gain insight into Inpp4b function in osteoclast, ex vivo overexpression of Inpp4b in RAW 264.7 was generated and upon RANKL stimulation caused major decrease in osteoclast-like cells (OCLs) differentiation. In contrast, overexpression of a mutated Inpp4b in the phosphatase function highly stimulated OCL differentiation. Importantly, we showed that Inpp4b regulation of osteoclast differentiation occurs via modulation of NFATc1 nuclear translocation and specific target genes transcription. To define the role of Inpp4b in vivo on bone mass regulation, we produced a conditional null allele in the mouse. Ablation of Inpp4b resulted in severe decrease of bone mineral density and led to osteoporosis with increased osteoclast population and resorption. We then interrogated for a role of Inpp4b in human bone mineral density by analysis of genetic SNPs. These results detected an Inpp4b variant associated with bone mineral density variability in two cohorts of premenopausal women. Together our ex vivo and in vivo studies defined Inpp4b as a major modulator of the osteoclast lineage and a novel potential risk factor for human osteoporosis.