Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha.

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.

Link between inflammation and aquaporin-5 distribution in submandibular gland in Sjögren's syndrome?

OBJECTIVE: To determine whether a link exists between inflammation and aquaporin-5 distribution in submandibular glands from three animal models for Sjögren's syndrome: IQI/JIC, r1ΔT/r2n and non-obese diabetic mice. METHODS: Mice of different ages were used. Inflammatory infiltrates were quantified using the focus score. Acinar aquaporin-5 subcellular distribution was determined by immunohistochemistry and quantified using labelling indices. RESULTS: Minor inflammatory infiltrates were present in r1f/r2n mice. Massive inflammatory infiltrates and acinar destruction were observed in 24-week-old non-obese diabetic mice, 10-and 13-month-old IQI/JIC mice and some r1ΔT/r2n mice. Aquaporin-5 immunoreactivity was primarily apical in submandibular glands from 8- and 24-week-old Balb/C mice, 8-week-old non-obese diabetic mice, 2-, 4- and 7-month-old IQI/JIC mice and r1f/r2n mice. In contrast, decreased apical aquaporin-5 labelling index with concomitant increased apical-basolateral, apical-cytoplasmic and/or apical-basolateral-cytoplasmic aquaporin-5 labelling indices was observed in 24-week-old non-obese diabetic, 10- and 13-month-old IQI/JIC and r1ΔT/r2n mice with a focus score≥1. CONCLUSIONS: Altered aquaporin-5 distribution in submandibular acinar cells from IQI/JIC, non-obese diabetic and r1ΔT/r2n mice with a focus score≥1 appears to be concomitant to the presence of inflammatory infiltrates and acinar destruction.

Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85alpha.

Phosphoinositide 3-kinase (PI3K) activation has been implicated in many cellular responses, including fibroblast growth, transformation, survival, and chemotaxis. Although PI3K is activated by several agents that stimulate T and B cells, the role of PI3K in lymphocyte function is not clear. The mouse gene encoding the PI3K adapter subunit p85alpha and its splice variants p55alpha and p50alpha was disrupted. Most p85alpha-p55alpha-p50alpha-/- mice die within days after birth. Lymphocyte development and function was studied with the use of the RAG2-deficient blastocyst complementation system. Chimeric mice had reduced numbers of peripheral mature B cells and decreased serum immunoglobulin. The B cells that developed had diminished proliferative responses to antibody to immunoglobulin M, antibody to CD40, and lipopolysaccharide stimulation and decreased survival after incubation with interleukin-4. In contrast, T cell development and proliferation was normal. This phenotype is similar to defects observed in mice lacking the tyrosine kinase Btk.

Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase.

The avian sarcoma virus 16 (ASV 16) is a retrovirus that induces hemangiosarcomas in chickens. Analysis of the ASV 16 genome revealed that it encodes an oncogene that is derived from the cellular gene for the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase). The gene is referred to as v-p3k, and like its cellular counterpart c-p3k, it is a potent transforming gene in cultured chicken embryo fibroblasts (CEFs). The products of the viral and cellular p3k genes have PI 3-kinase activity. CEFs transformed with either gene showed elevated levels of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate and activation of Akt kinase.

Cyclosporin A and FK506: molecular mechanisms of immunosuppression and probes for transplantation biology.

The microbial products cyclosporin A (CsA), FK506 and rapamycin are potent immunosuppressive agents. The introduction of CsA in the early 1970's significantly improved the outcome of organ and bone marrow allograft transplantation and advanced therapeutic options in autoimmune diseases. FK506 appears to have a higher therapeutic index than CsA, and has been used with encouraging results in clinical transplantation trials. FK506 and CsA, although structurally unrelated, appear to target similar signal transduction pathways in hematopoietic cells by inhibiting the action of calcineurin, a serine/threonine phosphatase. A structural analog of FK506, rapamycin, inhibits cellular function by a different molecular mechanism. These agents have advanced our understanding of signal transmission pathways in lymphocyte activation.

Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells.

The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.

Disruption of T cell development and repertoire selection by calcineurin inhibition in vivo.

The microbial products FK506 and CsA are potent immunosuppressive agents that prevent early transcriptional events in TcR-mediated activation. Their mode of action is dependent upon the inhibition of calcineurin, a serine/threonine phosphatase positioned within the calcium-dependent signaling pathway. TcR-mediated activation of thymocytes constitutes an important prerequisite for their development and selection to mature T cells. Disruption of the cross-talk between thymic APC and thymocytes results in the loss of normal T cell ontogeny. To study the role of calcineurin in T cell maturation and repertoire selection in vivo, mice were treated with either FK506 or CsA. Administration of either drug inhibited the progression of CD4+CD8+ positive thymocytes to mature single positive T cells. Furthermore, both drugs disrupted the process of negative thymic selection, causing an increased frequency of self-reactive cells among the few positively selected T cells. These effects correlated directly with the degree of inhibition of in vivo calcineurin enzyme activity. Blocking calcineurin activity appears to disrupt positive thymic selection and to prevent the deletion of self-reactive thymocytes.

T cell receptor (beta chain) transgenic mice have selective deficits in gamma delta T cell subpopulations.

TCR-beta (T cell receptor-beta chain) transgenic mice have altered lymphocyte development. TCR-beta transgenic mice are hyporesponsive to alloantigens in vivo and are deficient in gamma delta T cells. In order to begin a study of the relationship between a deficiency of alloreactive gamma delta cells and the defective function of in vivo alloantigen recognition, we analysed the gamma delta T cell development in TCR-beta mice. The presence of the TCR-V beta 8.2 chain transgene is associated with inhibition of gamma chain gene rearrangement. In order to determine how the presence of the TCR-beta transgene affects gamma delta T cell development, gamma delta T cells were studied in the skin, intestine and spleen. TCR-beta mice have dramatically reduced numbers of gamma delta T cells in the spleen and moderately reduced numbers of gamma delta T cells among intestinal intraepithelial lymphocytes. In contrast, these mice have normal numbers of gamma delta dendritic epidermal cells (DEC). These selective deficits could be due to the developmental regulation of transgene transcription during fetal life. We examined transcription of the TCR-beta transgene in the fetal thymus and found that the TCR-beta transgene is first transcribed at high levels on day 16 of fetal life, after DEC have already migrated from the thymus to the epidermis. Furthermore, mRNA from the transgene was detected in DEC, ruling out the formal possibility that DEC bear a gamma delta receptor only because they are incapable of expressing the transgene.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of the investigational mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute lymphoblastic leukemia.

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anticancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here, we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib (DA) in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of DA. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

The role of class I phosphoinositide 3-kinase in T-cell function and autoimmunity.

PI3K (phosphoinositide 3-kinase) regulates diverse cellular responses in the immune system, and members of this enzyme family are considered attractive drug targets for modulating allergy, inflammation and leukaemia. Clearly it is important to understand the function of PI3K in T-lymphocytes, cells that regulate nearly every aspect of immunity. However, the precise role of PI3K in T-cell development and function has been difficult to determine. In this review, I summarize current knowledge of PI3K function in T-cells, focusing on the class I subgroup of PI3K catalytic and regulatory isoforms. I discuss gene disruption studies in mice that reveal redundant or limited roles for individual isoforms, along with evidence for potential autoimmunity when class IA PI3K signalling is reduced.

Lymphocyte cell motility: the twisting, turning tale of phosphoinositide 3-kinase.

The PI3K (phosphoinositide 3-kinase) family of lipid kinases regulate cell motility in diverse organisms and cell types. In mammals, the main PI3K enzyme activated by chemokine receptor signalling is the class IB isoform, p110gamma. Studies of p110gamma-knockout mice have shown an essential function for this isoform in chemotaxis of neutrophils and macrophages both in vitro and in vivo. However, the roles of p110gamma and other PI3K enzymes and regulatory subunits in lymphocyte motility have been more difficult to discern. Recent studies of adoptively transferred, fluorescently labelled lymphocytes have revealed complex and unexpected functions for PI3K in lymphocyte migration in vivo. In this review we highlight cell-type-specific roles for PI3K catalytic and regulatory subunits in the homing and basal motility of lymphocytes in the intact lymph node.

Towards an understanding of isoform specificity in phosphoinositide 3-kinase signalling in lymphocytes.

The PI3K (phosphoinositide 3-kinase) signalling pathway promotes proliferation and transformation in many cell types. This is particularly well illustrated by studies of primary lymphocytes and their leukaemic counterparts. PI3K activation is required for proliferation of T cells and B cells, and certain oncogenes cause leukaemia in part by promoting PI3K signalling. Genetic manipulation of this pathway, together with biochemical studies in primary lymphocytes, has begun to shed light on the molecular mechanisms by which PI3K contributes to the proliferation of normal and transformed lymphocytes. In particular, targeted gene disruption in mice has allowed the identification of specific isoforms of PI3K that are required for distinct cellular responses. Continued investigation of isoform specificity in PI3K signalling, as well as the characterization of critical downstream targets of PI3K signalling, may reveal strategies for the therapeutic control of immune responses and leukaemia.

Structural organization and alternative splicing of the murine phosphoinositide 3-kinase p85 alpha gene.

Phosphoinositide 3-kinase is a lipid and protein kinase composed of a 110-kDa catalytic subunit and an 85-kDa (p85) or 55-kDa (p55) regulatory subunit. In mammals, at least two genes encode catalytic subunits, and at least three genes encode regulatory subunits. Here we report the cloning and structural analysis of the mouse p85 alpha gene. The translated portion of mouse p85 alpha is encoded by 15 exons that span at least 40 kb. We have cloned an alternatively spliced form of p85 alpha from both mouse and rat cDNA libraries. This splice variant encodes a unique 5'-untranslated region, start codon, and 6-amino-acid aminoterminus followed by the carboxyterminal 418 amino acids of p85 alpha. A corresponding exon is present within the p85 alpha genomic locus. In vitro transcription and translation of the splice variant cDNA generate a protein of approximately 45 kDa that is reactive with an anti-p85 alpha antiserum. Northern blot analysis of mouse tissues reveals differential expression of full-length and alternatively spliced p85 alpha, with the splice variant most abundant in the liver.

Immunophilins in protein folding and immunosuppression.

Lymphocyte activation requires the transmission of signals from molecules at the plasma membrane to nuclear signals that regulate gene expression. In recent years, several immunosuppressive compounds have been used as probes to identify important and potentially novel molecules involved in lymphocyte signal transduction processes. The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin have been studied in particular detail. Two distinct classes of immunosuppressant binding proteins have been identified, and collectively termed immunophilins. The cyclophilin family of immunophilins binds CsA, whereas the FK506-binding protein (FKBP) family binds FK506 and rapamycin. This review will discuss both the endogenous functions of immunophilins as well as their roles in mediating immunosuppression.

Phosphoinositide kinases.

Phosphatidylinositol, a component of eukaryotic cell membranes, is unique among phospholipids in that its head group can be phosphorylated at multiple free hydroxyls. Several phosphorylated derivatives of phosphatidylinositol, collectively termed phosphoinositides, have been identified in eukaryotic cells from yeast to mammals. Phosphoinositides are involved in the regulation of diverse cellular processes, including proliferation, survival, cytoskeletal organization, vesicle trafficking, glucose transport, and platelet function. The enzymes that phosphorylate phosphatidylinositol and its derivatives are termed phosphoinositide kinases. Recent advances have challenged previous hypotheses about the substrate selectivity of different phosphoinositide kinase families. Here we re-examine the pathways of phosphoinositide synthesis and the enzymes involved.

Changes in inositol 1,4,5-trisphosphate mass in agonist-stimulated human neutrophils.

Changes in the endogenous synthesis of inositol 1,4,5-trisphosphate (IP3) mass have been quantitated in human peripheral neutrophils stimulated with FMLP, LTB4 and PAF using a recently described, highly specific radioreceptor assay. Each agonist induced a concentration-dependent synthesis of IP3 which was detectable within 10 seconds after stimulation. IP3 production was short-lived, returning to basal levels within 90 seconds. The maximal stimulated level of IP3 in response to FMLP and LTB4 was 30-50 50 pmoles/10(7) neutrophils. PAF was more effective (approximately 100 pmoles IP3/10(7) neutrophils). The response to FMLP was inhibited by pertussis toxin, but was unaffected by cholera toxin. Pretreatment with cytochalasin B did not enhance IP3 synthesis. These findings are generally consistent with previous studies employing [3H]myo-inositol-prelabeled cells, and provide one of the first measurements of IP3 synthesis by mass in agonist-stimulated human neutrophils.

Identification of a physical interaction between calcineurin and nuclear factor of activated T cells (NFATp).

In T lymphocytes, the calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin, plays a pivotal role in transducing membrane-associated signals to the nucleus. One of the putative targets of calcineurin is the pre-existing, cytosolic component of the nuclear factor of activated T cells (NFATp; also referred to as NFAT1), which is one of several transcription factors required for the expression of interleukin 2. Inhibition of calcineurin by the immunosuppressive drugs cyclosporin A and FK506 prevents dephosphorylation of NFATp and its translocation to the nucleus. However, a physical interaction between calcineurin and NFATp has not been demonstrated. Here we demonstrate the binding of NFATp from lysates of T cells to immobilized calcineurin. Stimulation of T cells with calcium ionophore induced a shift in the molecular weight of NFATp that is due to its dephosphorylation. This dephosphorylation was inhibited by treatment of T cells with cyclosporin A or FK506 prior to stimulation. Of note, both the phosphorylated and the dephosphorylated form of NFATp bound to calcineurin. Furthermore, the binding of both forms of NFATp to calcineurin was inhibited by pretreatment of calcineurin with a complex of FK506 and its ligand FKBP12. Taken together these data strongly suggest a direct interaction of calcineurin with NFATp and that this interaction does not depend upon the phosphorylation sites of NFATp affected by activation.

Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A.

The immunosuppressive agents cyclosporin A (CsA) and FK 506 bind to distinct families of intracellular proteins (immunophilins) termed cyclophilins and FK 506-binding proteins (FKBPs). Recently, it has been shown that, in vitro, the complexes of CsA-cyclophilin and FK 506-FKBP-12 bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. We have investigated the effects of drug treatment on phosphatase activity in T lymphocytes. Calcineurin is expressed in T cells, and its activity can be measured in cell lysates. Both CsA and FK 506 specifically inhibit cellular calcineurin at drug concentrations that inhibit interleukin 2 production in activated T cells. Rapamycin, which binds to FKBPs but exhibits different biological activities than FK 506, has no effect on calcineurin activity. Furthermore, excess concentrations of rapamycin prevent the effects of FK 506, apparently by displacing FK 506 from FKBPs. These results show that calcineurin is a target of drug-immunophilin complexes in vivo and establish a physiological role for calcineurin in T-cell activation.

Correlation of calcineurin phosphatase activity and programmed cell death in murine T cell hybridomas.

Ligation of T cell receptor/CD3 complexes induces programmed cell death, or apoptosis, in immature thymocytes and many T cell hybridomas. While it has been demonstrated that T cell receptor-mediated apoptosis requires an increase in intracellular calcium concentration, the specific calcium-dependent signalling events leading to cell death are poorly defined. We have previously shown that T cell receptor/CD3-mediated induction of apoptosis in a murine T cell hybridoma is inhibited by the immunosuppressive drugs cyclosporin A (CsA) and FK506. Recently, it has been determined that these agents inhibit the activity of calcineurin, a calcium- and calmodulin-dependent serine/threonine phosphatase. Using an assay which measures calcineurin activity in cell lysates, we find that calcineurin-dependent dephosphorylation of a phosphopeptide substrate is potently inhibited in hybridomas treated with CsA or FK506. Drug dose-response analyses indicate that the level of cellular calcineurin activity correlates closely with the ability of these cells to undergo apoptosis. Thus, calcineurin appears to be a critical mediator of T cell receptor/CD3 signalling leading to programmed cell death in T cell hybridomas.