Fine tuning T lymphocytes: A role for the lipid phosphatase SHIP-1

The phosphoinositide 3-kinase signaling pathway regulates a range of T lymphocyte cellular functions including growth, proliferation, cytokine secretion and survival. Aberrant regulation of phosphoinositide 3-kinase-dependent signaling in T lymphocytes has been implicated in inflammatory and autoimmune diseases. In common with much of the immune system, several mechanisms exist to ensure the pathway is tightly regulated to elicit appropriate responses. One level of control involves the Src homology 2 domain-containing inositol-5-phosphatase-1 (SHIP-1) that modulates phosphoinositide 3-kinase signaling by degrading the key signaling lipid PI(3,4,5)P₃ to PI(3,4)P₂, but also serves as a key scaffolding molecule in the formation of multi-protein complexes. Here we discuss the role of SHIP-1 in regulating T lymphocyte and immune function, as well as its potential as a therapeutic target.     

Involvement of the phosphoinositide 3-kinase/Akt signaling pathway in the resistance to therapeutic treatments of human leukemias

A major factor undermining successful cancer treatment is the occurrence of resistance to conventional treatments such as chemotherapy and ionizing radiation. Evidence accumulated over the recent years has indicated the phosphoinositide 3-kinase/Akt signal transduction pathway as one of the major factors implicated in cancer resistance to conventional therapies. Indeed, the phosphoinositide 3-kinase/Akt axis regulates the expression and/or function of many anti-apoptotic proteins which strongly contributes to cancer cell survival. As a result, small molecules designed to specifically target key components of this signaling network are now being developed for clinical use as single therapeutic agents and/or in combination with other forms of therapy to overcome resistance. Initially, the phosphoinositide 3-kinase/Akt signal transduction pathway has been mainly investigated in solid tumors. Recently, however, this network has also been recognized as an important therapeutic target in human leukemias. Specific inhibition of this signalling pathway may be a valid approach to treat these diseases and increase the efficacy of standard types of therapy.     

Phosphatidylinositol 3-kinase

Currently, a central question in biology is how signals from the cell surface modulate intracellular processes. In recent years phosphoinositides have been shown to play a key role in signal transduction. Two phosphoinositide pathways have been characterized, to date. In the canonical phosphoinositide turnover pathway, activation of phosphatidylinositol-specific phospholipase C results in the hydrolysis of phosphatidylinositol 4,5-bisphospate and the generation of two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. The 3-phosphoinositide pathway involves protein-tyrosine kinase-mediated recruitment and activation of phosphatidylinositol 3-kinase, resulting in the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. The 3-phosphoinositides are not substrates of any known phospholipase C, are not components of the canonical phosphoinositide turnover pathway, and may themselves act as intracellular mediators. The 3-phosphoinositide pathway has been implicated in growth factor-dependent mitogenesis, membrane ruffling and glucose uptake. Furthermore the homology of the yeast vps34 with the mammalian phosphatidylinositol 3-kinase has suggested a role for this pathway in vesicular trafficking. In this review the different mechanisms employed by protein-tyrosine kinases to activate phosphatidylinositol 3-kinase, and its involvement in the signaling cascade initiated by tyrosine phosphorylation, are examined.     

OX40 complexes with phosphoinositide 3-kinase and protein kinase B (PKB) to augment TCR-dependent PKB signaling

T lymphocyte activation requires signal 1 from the TCR and signal 2 from costimulatory receptors. For long-lasting immunity, growth and survival signals imparted through the Akt/protein kinase B (PKB) pathway in activated or effector T cells are important, and these can be strongly influenced by signaling from OX40 (CD134), a member of the TNFR superfamily. In the absence of OX40, T cells do not expand efficiently to Ag, and memory formation is impaired. How most costimulatory receptors integrate their signals with those from Ag through the TCR is not clear, including whether OX40 directly recruits PKB or molecules that regulate PKB. We show that OX40 after ligation by OX40L assembled a signaling complex that contained the adapter TNFR-associated factor 2 as well as PKB and its upstream activator phosphoinositide 3-kinase (PI3K). Recruitment of PKB and PI3K were dependent on TNFR-associated factor 2 and on translocation of OX40 into detergent-insoluble membrane lipid microdomains but independent of TCR engagement. However, OX40 only resulted in strong phosphorylation and functional activation of the PI3K-PKB pathway when Ag was recognized. Therefore, OX40 primarily functions to augment PKB signaling in T cells by enhancing the amount of PI3K and PKB available to the TCR. This highlights a quantitative role of this TNFR family second signal to supplement signal 1.     

Lpr T cell hyporesponsiveness to mitogens linked to deficient receptor-stimulated phosphoinositide hydrolysis

The T lymphocytes that expand with age in the peripheral lymphoid organs of autoimmune disease-prone mice homozygous for the lpr mutation display deficient activation and proliferation in response to mitogenic lectins or antigen. In the present study, an attempt was made to correlate the deficient agonist-induced proliferation of these lpr T cells with early transmembrane signaling events mediated by receptor-coupled phosphoinositide hydrolysis. lpr T cells were capable of binding the agonistic lectin, phytohemagglutinin, in a normal manner. In addition, they expressed on their surface the antigen-specific T cell receptor-CD3 complex, which is required for T cell activation, albeit at a lower density than that found on congenic +/+ T cells. Furthermore, lpr T cells contained normal levels of the Ca2+- and phospholipid-dependent enzyme, protein kinase C, and the enzyme was translocated from the cytosol to the particulate fraction upon phorbol ester treatment. On the other hand, the lpr T cells displayed a markedly deficient agonist-induced phosphoinositide hydrolysis in comparison with their congenic +/+ counterparts, as indicated by the minimal accumulation of the phosphoinositide-derived second messengers, inositol phosphates and diacylglycerol. The defective step(s) in transmembrane signaling was bypassed by a combination of phorbol ester plus Ca2+ ionophore, which reconstituted proliferative responses of lpr T cells to normal levels, suggesting that: (a) the phosphoinositide signaling pathway plays an obligatory role in T cell activation; and (b) signaling events subsequent to phosphoinositide hydrolysis are, for the most part, intact in lpr T cells. The deficient step(s) in lpr T cell activation precedes, therefore, the generation of phosphoinositide-derived second messengers and could be due to defective function of the T cell receptor-CD3 complex, GTP-binding proteins, and/or phosphoinositide-specific phosphodiesterase. It remains to be determined whether the deficient signaling event(s) in lpr T cells is a direct pathologic consequence of the lpr gene, or rather, reflects the immature status of a normally minor thymic subset that is aberrantly exported and expanded in lpr mice.     

The long isoform of cellular FLIP is essential for T lymphocyte proliferation through an NF-kappaB-independent pathway

Although the long isoform of cellular FLIP (c-FLIP(L)) has been implicated in TCR-mediated signaling, its role in T cell proliferation remains controversial. Some studies have demonstrated that overexpression of c-FLIP(L) promotes T cell proliferation and NF-kappaB activation, whereas others have reported that c-FLIP(L) overexpression has no effect or even inhibits T cell proliferation. To establish the role of c-FLIP(L) in T lymphocyte proliferation, we have generated a conditional knockout mouse strain specifically lacking c-FLIP(L) in T lymphocytes. c-FLIP(L)(-/-) mice exhibit severely impaired effector T cell development after Listeria monocytogenes infection in vivo and c-FLIP(L)-deficient T cells display defective TCR-mediated proliferation in vitro. However, c-FLIP(L)(-/-) T cells exhibit normal NF-kappaB activity upon TCR stimulation. These results demonstrate that c-FLIP(L) is essential for T lymphocyte proliferation through an NF-kappaB-independent pathway.     

Recent advances in the protein kinase B signaling pathway

The phosphoinositide 3' kinase signaling pathway is activated in response to a plethora of growth factors and cytokines, and initiates a cascade of signaling events primarily via the induction of specific protein-serine/threonine kinases. Interest in the pathway has been driven by its frequent aberrant activation in disease and its impact on cell fate decisions owing to roles in survival signaling and metabolic control. There have been recent advances in our understanding of the primary components of this pathway, namely phosphoinositide-dependent kinase-1, protein kinase B and glycogen synthase kinase-3, including insights into their mechanisms of regulation, substrate proteins and cellular functions.     

PI3K signaling controls cell fate at many points in B lymphocyte development and activation

Many receptors on diverse cell types activate phosphoinositide 3-kinase (PI3K). The lipid products of PI3K, termed 3-phosphoinositides, regulate numerous cellular processes by recruiting specific proteins to membrane signaling complexes. In the B lymphocyte lineage, PI3K activation is a critical control point at various stages of development, proliferation and differentiation. PI3K signaling is promoted by stimulatory receptors such as surface immunoglobulin, CD40, Toll-like receptors and cytokine receptors, and opposed by the inhibitory receptor FcgammaRIIB1. Genetic dissection of the PI3K pathway in mice has indicated that certain B cell functions are regulated by a limited set of PI3K isoforms and downstream effectors. Here we review our current understanding of how signals are relayed to and from PI3K in B cells.     

How a RING Finger Protein and a Steroid Hormone Control Autophagy?

Previous work in our laboratory has indicated that the steroid hormone ecdysone triggers programmed autophagy in the fat body of Drosophila larvae by downregulating the class I phosphoinositide 3-kinase (PI3K) pathway. We recently found evidence that Deep orange (Dor), a Drosophila RING finger protein implicated in late-endosomal trafficking, controls ecdysone signaling as well as autolysosome fusion, thus exerting a dual regulation of autophagy. We found that dor mutants are defective in programmed autophagy. The mutant larvae showed impaired upregulation of ecdysone signaling during development, accompanied by a failure to downregulate the PI3K pathway. Downregulation of the PI3K pathway could be restored by feeding the dor mutants with ecdysone. Even though ecdysone signaling and autophagy were impaired in the dor mutants, we detected an accumulation of autophagosomes in dor mutant fat bodies. This could probably be attributed to the failure of autophagosomes to fuse with lysosomes. In this Addendum we review these findings and provide some speculations about how Dor may control both ecdysone signalling and autolysosomal fusion.

Addendum to:

A Dual Function for Deep Orange in Programmed Autophagy in the Drosophila melanogaster Fat Body

K. Lindmo, A. Simonsen, A. Brech, K. Finley, T.E. Rusten and H. Stenmark

Exp Cell Res 2006; Epub ahead of print     

How a RING finger protein and a steroid hormone control autophagy

Previous work in our laboratory has indicated that the steroid hormone ecdysone triggers programmed autophagy in the fat body of Drosophila larvae by downregulating the class I phosphoinositide 3-kinase (PI3K) pathway. We recently found evidence that Deep orange (Dor), a Drosophila RING finger protein implicated in late-endosomal trafficking, controls ecdysone signaling as well as autolysosome fusion, thus exerting a dual regulation of autophagy. We found that dor mutants are defective in programmed autophagy. The mutant larvae showed impaired upregulation of ecdysone signaling during development, accompanied by a failure to downregulate the PI3K pathway. Downregulation of the PI3K pathway could be restored by feeding the dor mutants with ecdysone. Even though ecdysone signaling and autophagy were impaired in the dor mutants, we detected an accumulation of autophagosomes in dor mutant fat bodies. This could probably be attributed to the failure of autophagosomes to fuse with lysosomes. In this Addendum we review these findings and provide some speculations about how Dor may control both ecdysone signalling and autolysosomal fusion.     

The effects of polyunsaturated fatty acids and antioxidant vitamins on atrial oxidative stress,nitrotyrosine residues,and connexins following extracorporeal circulation in patients undergoing cardiac surgery

Obesity-induced low-grade inflammation (metaflammation) impairs insulin receptor signaling. This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, inhibitor of NF-kB kinase complex β (IKKβ), AMP-activated protein kinase, protein kinase C, Rho-associated coiled-coil containing protein kinase, and RNA-activated protein kinase. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in type 2 diabetes mellitus (T2-DM). Identifying the specific protein kinases involved in obesity-induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity-induced T2-DM.     

Inhibition of T cell antigen receptor-dependent phosphorylation of CD4 in human immunodeficiency virus type 1 infected cells.

Inhibitory effects of human immunodeficiency virus (HIV) on T lymphocyte function have been linked to perturbation of signaling through the T cell antigen receptor-CD3 complex. Comparative biochemical analyses of signaling responses were performed in T cells that were either uninfected or chronically infected with the HIV-1/IIIB strain. Stimulation with antibodies to CD3 triggered both Ca2+ accumulation and phosphoinositide hydrolysis responses that were equivalent in uninfected and infected cells. Treatment with anti-CD3 or with phorbol diester also stimulated serine phosphorylation of CD4 molecules in uninfected T cells. However, phosphorylation of CD4 was not observed after anti-CD3 treatment in HIV-infected T cells despite normal phosphorylation responses to phorbol diester. Identical results were obtained using a T cell line that was infected with an env (gp160/120-) HIV-1 defective variant. These studies indicate that infection with HIV-1 inhibits the activation of protein kinase associated with the T cell receptor-CD3 complex by a mechanism which is independent of viral env protein components.     

Dexamethasone suppresses osteogenesis of osteoblast via the PI3K/Akt signaling pathway in vitro and in vivo

The hypofunction of osteoblasts induced by glucocorticoids (GCs) has been identified as a major contributing factor for GC-induced osteoporosis (GIO). However, the biological mechanism underlying the effect of GC in osteoblasts are not fully elucidated. Recent studies implicated an important role of phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway in the regulation of bone growth. We propose that the PI3K/Akt signaling may be implicated in the process of GC-induced osteogenic inhibition in osteoblasts. In this study, primary osteoblasts were used in vitro and in rats in vivo to evaluate the biological significance of the PI3K/Akt pathway in GC-induced bone loss. In vivo, dexamethasone (Dex)-treated rats had low bone mineral density and decreased expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and phosphorylated Akt (p-Akt) in bone tissue. In vitro study shows that Dex over the dose of 10^–8 M remarkably inhibited cellular osteogenesis, as represented by decreased cell viability, lessened ALP activity, and suppressed osteogenic protein expressions including ALP and OCN. Meanwhile, a dramatic downregulation in the PI3K/Akt pathway phosphorylation was also observed in Dex-treated osteoblasts. These changes were marked rescued by treatment with a PI3K agonist 740Y-P. Moreover, downregulation of ALP and OCN expressions by LY294002 can mimic the suppressive effects of Dex. These data together reveal that the suppressed PI3K/Akt pathway is involved in the regulatory action of Dex on osteogenesis.     

Alzheimer's presenilin-1 mutation potentiates inositol 1,4,5-trisphosphate-mediated calcium signaling in Xenopus oocytes

Perturbations in intracellular Ca2+ signaling may represent one mechanism underlying Alzheimer's disease (AD). The presenilin-1 gene (PS1), associated with the majority of early onset familial AD cases, has been implicated in this signaling pathway. Here we used the Xenopus oocyte expression system to investigate in greater detail the role of PS1 in intracellular Ca2+ signaling pathways. Treatment of cells expressing wild-type PS1 with a cell surface receptor agonist to stimulate the phosphoinositide second messenger pathway evoked Ca2+-activated Cl- currents that were significantly potentiated relative to controls. To determine which elements of the signal transduction pathway are responsible for the potentiation, we used photolysis of caged inositol 1,4,5-trisphosphate (IP3) and fluorescent Ca2+ imaging to demonstrate that PS1 potentiates IP3-mediated release of Ca2+ from internal stores. We show that an AD-linked mutation produces a potentiation in Ca2+ signaling that is significantly greater than that observed for wild-type PS1 and that cannot be attributed to differences in protein expression levels. Our findings support a role for PS1 in modulating IP3-mediated Ca2+ liberation and suggest that one pathophysiological mechanism by which PS1 mutations contribute to AD neurodegeneration may involve perturbations of this function.     

Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.     

The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway

The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic pathway in the human colon cancer HT-29 cells. Here we demonstrate that the wild-type PTEN is expressed in this cell line. Its overexpression directed by an inducible promoter counteracts the interleukin-13 down-regulation of macroautophagy. This effect was dependent upon the phosphoinositide phosphatase activity of PTEN as determined by using the mutant G129E, which has only protein phosphatase activity. The role of Akt/PKB in the signaling control of interleukin-13-dependent macroautophagy was investigated by expressing a constitutively active form of the kinase ((Myr)PKB). Under these conditions a dramatic inhibition of macroautophagy was observed. By contrast a high rate of autophagy was observed in cells expressing a dominant negative form of PKB. These data demonstrate that the signaling control of macroautophagy overlaps with the well known PI 3-kinase/PKB survival pathway and that the loss of PTEN function in cancer cells inhibits a major catabolic pathway.     

Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ inter- acted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.     

多糖调控T/B淋巴细胞免疫应答机制的研究进展

淋巴细胞是机体适应性免疫系统的重要组成,多糖对其刺激作用在生物医药领域受到广泛的关注。目前,大部分的相关研究仅限于多糖对淋巴细胞增殖及细胞因子或抗体表达水平的调控,系统的分子机制解析少见报道。综合多糖对淋巴细胞的免疫调节作用发现:活性多糖可同时刺激T/B细胞、也可选择性刺激T细胞或选择性刺激B细胞;多糖刺激T细胞免疫应答的信号通道主要为TCR/CD3→PTK→PI3-K→PKC/PLCγ→Ca2+→calcineurin→NFAT和TCR/CD3→PTK→MAPKs→AP-1;而刺激B细胞的信号通道主要包括TLR2/4→TRAF6→IKKc→NF-κB、TLR2/4→PTK→MAPKs→AP-1和IgM/CD79→PTK→MAPKs→AP-1。同时,归纳多糖刺激淋巴细胞活性的构效关系,旨在为相关领域的研究提供参考。