The role of SHIP in growth factor induced signalling

The recently cloned, hemopoietic-specific, src homology 2 (SH2)-containing inositol phosphatase, SHIP, is rapidly gaining prominence as a potential regulator of all phosphatidylinositol (PI)-3 kinase mediated events since it has been shown both in vitro and in vivo to hydrolyze the 5′ phosphate from phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P₃). Thus SHIP, and its more widely expressed counterpart, SHIP2, could play a central role in determining PI-3,4,5-P₃ and PI-3,4-P₂ levels in many cell types. To explore the in vivo function of SHIP further we recently generated a SHIP knock out mouse and in this review we discuss experiments carried out with bone marrow derived mast cells (BMMCs) from these animals.     

Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling

The inositol phosphatase SHIP binds to the FcgammaRIIB1 receptor and plays a critical role in FcgammaRIIB1-mediated inhibition of B-cell proliferation and immunoglobulin synthesis. The molecular details of SHIP function are not fully understood. While point mutations of the signature motifs in the inositol phosphatase domain abolish SHIP's ability to inhibit calcium flux in B cells, little is known about the function of the evolutionarily conserved, putative noncatalytic regions of SHIP in vivo. In this study, through a systematic mutagenesis approach, we identified the inositol phosphatase domain of SHIP between amino acids 400 and 866. Through reconstitution of a SHIP-deficient B-cell line with wild-type and mutant forms of SHIP, we demonstrate that the catalytic domain alone is not sufficient to mediate FcgammaRIIB1/SHIP-dependent inhibition of B-cell receptor signaling. Expression of a truncation mutant of SHIP that has intact phosphatase activity but lacks the last 190 amino acids showed that the noncatalytic region in the C terminus is essential for inhibitory signaling. Mutation of two tyrosines within this C-terminal region, previously identified as important in binding to Shc, showed a reduced inhibition of calcium flux. However, studies with an Shc-deficient B-cell line indicated that Shc-SHIP complex formation is not required and that other proteins that bind these tyrosines may be important in FcgammaRIIB1/SHIP-mediated calcium inhibition. Interestingly, membrane targeting of SHIP lacking the C terminus is able to restore this inhibition, suggesting a role for the C terminus in localization or stabilization of SHIP interaction at the membrane. Taken together, these data suggest that the noncatalytic carboxyl-terminal 190 amino acids of SHIP play a critical role in SHIP function in B cells and may play a similar role in several other receptor systems where SHIP functions as a negative regulator.     

The role of glypicans in Hedgehog signaling

Glypicans (GPCs) are a family of proteoglycans that are bound to the cell surface by a glycosylphosphatidylinositol anchor. Six glypicans have been found in the mammalian genome (GPC1 to GPC6). GPCs regulate several signaling pathways, including the pathway triggered by Hedgehogs (Hhs). This regulation, which could be stimulatory or inhibitory, occurs at the signal reception level. In addition, GPCs have been shown to be involved in the formation of Hh gradients in the imaginal wing disks in Drosophila. In this review we will discuss the role of various glypicans in specific developmental events in the embryo that are regulated by Hh signaling. In addition, we will discuss the mechanism by which loss-of-function GPC3 mutations alter Hh signaling in the Simpson–Golabi–Behmel overgrowth syndrome, and the molecular basis of the GPC5-induced stimulation of Hh signaling and tumor progression in rhabdomyosarcomas.     

食欲素信号系统在成瘾中的作用

成瘾是对成瘾物质的强迫性、持续性需求并缺乏控制能力的行为,它会导致大脑中枢奖赏回路的改变。下丘脑是调控自然奖赏的重要脑区,它能特异性地表达一种被称为食欲素(orexins/hypocretins)的神经肽。食欲素通过作用于食欲素受体调控睡眠、觉醒状态,同时,食欲素受体在药物成瘾和奖赏相关的行为中也有重要作用,投射到不同脑区的食欲素对不同药物导致的成瘾调节作用也不同,调控食欲素信号系统,将可能成为治疗成瘾的重要方法。本文重点总结了食欲素信号系统在不同药物成瘾过程中的作用的最新研究进展。     

The role of lymphotoxin receptor signaling in diseases

LT, LIGHT, and TNF are core family members of the TNFR superfamily of cytokines. LT and LIGHT, produced primarily by lymphocytes, interact with LTbetaR expressed by stromal and epithelial cells. Extensive studies over the last decade have revealed a critical role of LT-LTbetaR interactions for organogenesis and maintenance of the secondary lymphoid organs and in the generation of an efficient humoral immune response to various pathogens. LTbetaR's function beyond the lymphoid organs shows valuable potential yet remains largely undefined. Recent studies indicate that LTbetaR signaling is required for liver regeneration, hepatitis, and hepatic lipid metabolism. The balance of beneficial and detrimental effects of LTbetaR is critical for understanding the mechanisms of autoimmune disease and liver function and may open a new avenue for therapeutic intervention. This review will discuss recent advances in understanding LTbetaR's role in various human and murine disease models while focusing on its regulation of and implications in various liver related diseases.     

The role of TRADD in death receptor signaling

TRADD (TNFR1-associated death domain protein) was initially identified as an adaptor molecule that transduces the signal downstream of the TNFR1 (tumor necrosis factor receptor 1). TNFR1 belongs to the so-called death receptor (DR) family of receptors that depending on the context can induce either apoptosis or proliferation, as well as NF-κB and MAP kinase activation. The receptors of this group contain death domain (DD) that is necessary for the induction of apoptosis. This review summarizes the recent advances in the field of DR signaling and in particular the role of TRADD.     

The role of TRADD in death receptor signaling

TRADD (TNFR1-associated death domain protein) was initially identified as an adaptor molecule that transduces the signal downstream of the TNFR1 (tumor necrosis factor receptor 1). TNFR1 belongs to the so-called death receptor (DR) family of receptors that depending on the context can induce either apoptosis or proliferation, as well as NF-κB and MAP kinase activation. The receptors of this group contain death domain (DD) that is necessary for the induction of apoptosis. This review summarizes the recent advances in the field of DR signaling and in particular the role of TRADD.     

Role of the SHP-2 tyrosine phosphatase in cytokine-induced signaling and cellular response

Cytokines and growth factors are important extracellular regulatory proteins. They exert their biological functions through binding to their cognate receptors on the cell surface and triggering intracellular signaling cascades. However, the intracellular signaling mechanisms of cytokines and growth factors are not well understood. Accumulating evidence has shown that protein phosphorylation and dephosphorylation carried out by protein kinases and protein phosphatases are fundamental biochemical events in intracellular signal transduction. SHP-2, a Src homology (SH) 2 domain-containing protein tyrosine phosphatase (PTP), is widely involved in a variety of signaling pathways triggered by cytokines and growth factors, including the MAP kinase, Jak-Stat, and PI3 kinase pathways. Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signals relayed from the cell surface to the nucleus, and is a critical intracellular regulator in cytokine and growth factor-induced cell survival, proliferation, and differentiation.     

The role of cathepsin X in cell signaling

Cathepsin X is a lysosomal cysteine protease, found predominantly in cells of monocyte/macrophage lineage. It acts as a monocarboxypepidase and has a strict positional and narrower substrate specificity relative to the other human cathepsins. In our recent studies we identified—β₂ subunit of integrin receptors and α and γ enolase as possible substrates for cathepsin X carboxypeptidase activity. In both cases cathepsin X is capable to cleave regulatory motifs at C-terminus affecting the function of targeted molecules. We demonstrated that via activation of β₂ integrin receptor Mac-1 (CD11b/CD18) active cathepsin X enhances adhesion of monocytes/macrophages to fibrinogen and regulates the phagocytosis. By activation of Mac-1 receptor cathepsin X may regulate also the maturation of dendritic cells, a process, which is crucial in the initiation of adaptive immunity. Cathepsin X activates also the other β₂ integrin receptor, LFA-1 (CD11a/CD18) which is involved in the proliferation of T lymphocytes. By modulating the activity of LFA-1 cathepsin X causes cytoskeletal rearrangements and morphological changes of T lymphocytes enhancing ameboid-like migration in 2-D and 3-D barriers and increasing homotypic aggregation. The cleavage of C-terminal amino acids of α and γ enolase by cathepsin X abolishes their neurotrophic activity affecting neuronal cell survival and neuritogenesis.Key words: cathepsin X, integrin, enolase, T lymphocyte, macrophage, dendritic cell, adhesion, migration, neuritogenesisProteases comprise a group of enzymes that catalyse the cleavage of a peptide bond in a polypeptide chain by nucleophilic attack on the carbonyl carbon. The proteases are either exopeptidases cleaving one or a few amino acids at the N- or C-terminus of polypeptide chain or endopeptidases that cleave the peptide bond internally. According to the catalytic mechanism the endopeptidases are divided into aspartic, cysteine, serine, threonine and metallo endoproteases—see MEROPS database.¹ To date, 561 genes encoding for proteases have been identified in human genome. Among them 148 genes encode for cysteine proteases including a group of eleven lysosomal cysteine proteases (members of C1 family) also called cathepsins. They exhibit different expression patterns, levels and specificities, all of which contribute to their differential physiological roles. Some of them, like cathepsins B, H, L and C are ubiquitously present in tissues, whereas others (cathepsins S, V, X, O, K, F and W) are expressed by specific cell types. Cysteine cathepsins were long believed to be responsible for the terminal protein degradation in the lysosomes, however, this view has changed dramatically when they have been found to be involved in a number of important cellular processes and pathologies.^2,3In contrast to other cathepsins, cathepsin X was discovered only recently. Its gene,^4,5 structure^6,7 and activity properties^8,9 show several unique features that distinguish it clearly from other human cysteine proteases. It has a very short pro-region⁷ and a three residue insertion motif which forms a characteristic “mini loop.”⁶ Cathepsin X exhibits carboxypeptidase activity⁶ and, in contrast to cathepsin B, the other carboxypeptidase, it does not act as an endopeptidase. Contrary to the first reports,⁴ cathepsin X is not widely expressed in cells and tissues, but is restricted to the cells of the immune system, predominantly monocytes, macrophages and dendritic cells.¹⁰ Higher levels of cathepsin X were also found in tumor and immune cells of prostate¹¹ and gastric¹² carcinomas and in macrophages of gastric mucosa, especially after infection by Helicobacter pylori.¹³ Recently it was shown that cathepsin X is abundantly expressed in mouse brain cells, in particular glial cells. Its upregulation was also detected in the brains of patients with Alzheimer disease.¹⁴The involvement of cathepsin X in signal transduction is implied by the integrin-binding motifs, present in its pro-form (RGD: Arg-Gly-Asp) and mature form (ECD: Glu-Cys-Asp).^4,5 Moreover, cathepsin X binds cell surface heparan sulfate proteoglycans¹⁵ which are also involved in integrin regulation. A strong co-localization of pro-cathepsin X with β₃ integrin subunit was demonstrated in our study in pro-monocytic U-937 cells.¹⁶ Further, it was reported that the pro-form of cathepsin X interacts with α_vβ₃ integrin through the RGD motif in lamellipodia of human umbilical vein endothelial cells (HUVECs).¹⁷ However, we showed that the active form of cathepsin X co-localized predominantly with β₂ integrin subunit in various cells of monocytes/macrophage lineage. Active cathepsin X was shown to regulate β₂ integrin-dependent adhesion, phagocytosis and T lymphocyte activation by interaction with macrophage antigen-1 (CD11b/CD18, Mac-1). We showed that inhibitors and monoclonal antibodies, capable to impair cathepsin X enzymatic activity, reduced the binding of differentiated U-937 cells to fibrinogen and polystyrene surface in a dose dependent manner. The co-localization of active cathepsin X with β₂ integrin chain was particularly enhanced in interactions of monocyte/macrophages with endothelial and tumor cells.Besides in monocytes and macrophages the active cathepsin X plays a role in β₂ integrin activation also in dendritic cells (DC), which are crucial for effective antigen presentation and initiation of T cell dependent immune response. Maturation of dendritic cells is accompanied by a range of morphological and cytoskeleton structure changes. In response to maturation stimuli in vitro, DCs rapidly adhere, develop polarity and assemble actin rich structures at the leading edge, known as podosomes.¹⁸ The adhesion of immature DCs to the extracellular matrix, is accompanied by recruitment of Mac-1 integrin receptor, which can be activated by cathepsin X. We have shown that, during maturation, cathepsin X translocates to the plasma membrane of maturing DCs, enabling Mac-1 activation and, consequently, cell adhesion.¹⁹ In mature DCs cathepsin X redistributes from the membrane to the perinuclear region, which coincides with the de-adhesion of DCs, formation of cell clusters and acquisition of the mature phenotype. Again, the inhibition of cathepsin X activity during DC differentiation and maturation reduced the capacity of DCs to stimulate T lymphocytes.β₂ integrin receptors are important also in T lymphocyte functions, such as migration and invasion across the endothelium and tissues. Lymphocyte function-associated antigen-1 (CD11a/CD18, LFA-1), the predominant β₂ integrin receptor in T lymphocytes enables cell-cell interactions and homotypic aggregation via LFA-1-ICAM-1 (intracellular adhesion molecule-1) interactions. LFA-1 can act also as a true signaling receptor, causing F-actin reorganization that leads to cytoskeletal changes of the cell²⁰ and a switch from a spherical to a polarized shape.²¹ Although the concentration of cathepsin X in T lymphocytes is lower compared to monocytes and macrophages, we showed that it interacts with LFA-1 promoting cytoskeleton-dependent morphological changes and migration across 2D and 3D models of ICAM-1 and Matrigel.^22,23 Its co-localization with LFA-1 was particularly evident at the trailing edge protrusion, the uropod, which plays an important role in T lymphocyte migration and cell-cell interactions (Fig. 1). Uropodal active cathepsin X cleaves C-terminal amino acids of β chain in LFA-1 promoting its high affinity conformation and the binding of the cytoskeletal protein talin. This interaction stabilizes the uropod and promotes its elongation (Jevnikar, et al. submitted).Open in a separate windowFigure 1Activation of LFA-1 integrin receptor by cathepsin X at the uropod of T lymphocyte promotes cytoskeleton-dependent morphological changes and cell migration.³⁰We demonstrated that uropods of cathepsin X upregulated T lymphocytes elongate to extreme length and form cell-to-cell connections, the nanotubes (Obermajer, et al. in press). Membrane (or tunneling) nanotubes were recently found as a new principle of cell-to-cell communication enabling transmission of complex and specific messages to distant cells through a physically connected network. Calcium fluxes, vesicles and cell-surface components can all traffic between cells connected by nanotubes. In immune system nanotubes integrate communities of cells for a better coordination of their action in various stages of immune response. We showed that nanotubes of cathepsin X upregulated T lymphocytes could readily transfer cellular organelles such as mitochondria and lysosomes and proposed that nanotube mediated transfer makes possible T lymphocyte activation without the need for direct contact with antigen presenting cells.The exact mechanism of cathepsin X translocation towards plasma membrane and degradation of C-terminal amino acids of β chain remains unclear. In lysosomes cathepsin X can be found as a pro- and active form. After cell activation cathepsin X containing vesicles translocate towards the plasma membrane,¹⁶ as observed also for some other lysosomal proteases.²⁴ During this process it is possible that pro-cathepsin X is activated by the other cysteine protease cathepsin L, as shown already in vitro.⁸ Both proteases were strongly co-localized with β₂ integrin chain at plasma membrane of activated monocytes/macrophages and at uropodes of T lymphocytes. Simultaneous co-localization with the lysosomal markers demonstrates that at least the initial translocation of cathepsin X towards cytoplasmic tail of β₂ integrin chain is vesicular. The interaction of cathepsin X with β₂ integrin subunit was confirmed by immunoprecipitation and FRET.²² According to in vitro experiments we propose that cathepsin X cleaves sequentially C-terminal aminoacids F⁷⁶⁶, A⁷⁶⁷, E⁷⁶⁸ and S⁷⁶⁹ of β₂ integrin subunit (Fig. 2) until reaching proline in penultimate position, confirming previous observation that the proline in S2 position leads to resistance to cathepsin X proteolysis.²⁵ Also, our results are in agreement with the previously mentioned monocarboxypeptidase activity of cathepsin X.^26,27 Since the signaling to and from the integrins is mainly regulated by the short cytoplasmic tail of β₂ subunit,²⁸ cathepsin X mediated β₂ integrin truncation leads to regulation of the receptor signaling. The interaction of cytoplasmic tail with different cytoskeletal and regulatory proteins, such as talin, filamin, radixin and α-actinin is crucial for signal transduction and modulation of cytoskeleton.²⁹Open in a separate windowFigure 2Cathepsin X activates LFA-1 by sequential cleavage of C-terminal amino acids of β₂ integrin subunit.Besides β₂ integrin chain we recently identified isozymes α and γ enolases as another molecular target for cathepsin X carboxypeptidase activity (Obermajer, et al. submitted). We demonstrated that cathepsin X sequentially cleaves C-terminal amino acids of both isozymes, abolishing their neurotrophic activity. On this way the neuronal cell survival and neuritogenesis can be regulated. Inhibition of cathepsin X activity increases the generation of plasmin, essential for neuronal differentiation and changes the length distribution of neurites, especially in the early phase of neurite outgrowth. Moreover, cathepsin X inhibition increases neuronal survival and reduces serum deprivation induced apoptosis, particularly in the absence of nerve growth factor.     

The role of cathepsin X in cell signaling

Cathepsin X is a lysosomal cysteine protease, found predominantly in cells of monocyte/macrophage lineage. It acts as a monocarboxypepidase and has a strict positional and narrower substrate specificity relative to the other human cathepsins. In our recent studies we identified ? β2 subunit of integrin receptors and α and γ enolase as possible substrates for cathepsin X carboxypeptidase activity. In both cases cathepsin X is capable to cleave regulatory motifs at C-terminus affecting the function of targeted molecules. We demonstrated that via activation of β2 integrin receptor Mac-1 (CD11b/CD18) active cathepsin X enhances adhesion of monocytes/macrophages to fibrinogen and regulates the phagocytosis. By activation of Mac-1 receptor cathepsin X may regulate also the maturation of dendritic cells, a process, which is crucial in the initiation of adaptive immunity. Cathepsin X activates also the other β2 integrin receptor, LFA-1 (CD11a/CD18) which is involved in the proliferation of T lymphocytes. By modulating the activity of LFA-1 cathepsin X causes cytoskeletal rearrangements and morphological changes of T lymphocytes enhancing ameboid-like migration in 2-D and 3-D barriers and increasing homotypic aggregation. The cleavage of C-terminal amino acids of α and γ enolase by cathepsin X abolishes their neurotrophic activity affecting neuronal cell survival and neuritogenesis.     

A role for SHIP in stem cell biology and transplantation

Inositol phospholipid signaling pathways have begun to emerge as important players in stem cell biology and bone marrow transplantation [1-4]. The SH2-containing Inositol Phosphatase (SHIP) is among the enzymes that can modify endogenous mammalian phosphoinositides. SHIP encodes an isoform specific to pluripotent stem (PS) cells [5,6] plays a role in hematopoietic stem (HS) cell biology [7,8] and allogeneic bone marrow (BM) transplantation [1,2,9,10]. Here I discuss our current understanding of the cell and molecular pathways that SHIP regulates that influence PS/HS cell biology and BM transplantation. Genetic models of SHIP-deficiency indicate this enzyme is a potential molecular target to enhance both autologous and allogeneic BM transplantation. Thus, strategies to reversibly target SHIP expression and their potential application to stem cell therapies and allogeneic BMT are also discussed.     

Heterologous regulation of chemokine receptor signaling by the lipid phosphatase SHIP in lymphocytes

The SH2 domain-containing inositol polyphosphate 5-phosphatase (SHIP) is known to play an important role in the negative regulation by FcgammaRIIB of PI3K-dependent signaling cascades activated by the B cell antigen receptor (BCR) as well as several tyrosine-kinase coupled cytokine receptors. However, to date the role of SHIP in the regulation of PI3K-dependent signals elicited by G-protein-coupled receptors (GPCR) such as chemokine receptors has not been investigated. In this study, we report that ligation of the G-protein-coupled chemokine receptor CXCR4 by SDF-1/CXCL12 has no effect on the tyrosine phosphorylation of SHIP in the murine B cell lymphoma A20. However, co-ligation of the B cell antigen receptor and FcgammaRIIB inhibits the PI3K-dependent phosphorylation of PKB and ERK1/2 in response to CXCL12. We have also utilised a constitutively active membrane-localised SHIP mutant expressed in the Jurkat leukaemic T cell line (which do not normally express SHIP), in order to investigate the effect of this mutant on CXCL12 stimulated PI3K-dependent signaling events. Experiments have revealed that CXCL12-mediated PKB phosphorylation, chemotaxis and lipid accumulation are inhibited in the presence of this SHIP mutant. Thus, it appears that heterologous activation of SHIP by non-G-protein-coupled receptor-mediated routes can impinge on PI3K-dependent signaling pathways activated by independently ligated G-protein-coupled chemokine receptors.     

The role of pseudophosphatases as signaling regulators

Pseudophosphatases are atypical members of the protein tyrosine phosphatase superfamily. Mutations within their catalytic signature motif render them catalytically inactive. Despite this lack of catalytic function, pseudophosphatases have been implicated in various diseases such as Charcot Marie-Tooth disorder, cancer, metabolic disorder, and obesity. Moreover, they have roles in various signaling networks such as spermatogenesis, apoptosis, stress response, tumorigenesis, and neurite differentiation. This review highlights the roles of pseudophosphatases as essential regulators in signaling cascades, providing insight into the function of these catalytically inactive enzymes.     

Death receptors bind SHP-1 and block cytokine-induced anti-apoptotic signaling in neutrophils.

Death domain-containing receptors of the tumor necrosis factor (TNF)/nerve growth factor (NGF) family can induce apoptosis upon activation in many cellular systems. We show here that a conserved phosphotyrosine-containing motif within the death domain of these receptors can mediate inhibitory functions. The Src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), SHP-2 and SH2-containing inositol phosphatase (SHIP) bound to this motif in a caspase-independent but cell-dependent manner. We also found that stimulation of death receptors disrupted anti-apoptosis pathways initiated (at least under certain conditions) by survival factors in neutrophils. In these cells, activation of the tyrosine kinase Lyn, an important anti-apoptotic event, was prevented as a consequence of death-receptor stimulation, most likely through association of the receptor with activated SHP-1. Thus, we provide molecular and functional evidence for negative signaling by death receptors.     

Caspase-activated ROCK-1 allows erythroblast terminal maturation independently of cytokine-induced Rho signaling

Stem cell factor (SCF) and erythropoietin are strictly required for preventing apoptosis and stimulating proliferation, allowing the differentiation of erythroid precursors from colony-forming unit-E to the polychromatophilic stage. In contrast, terminal maturation to generate reticulocytes occurs independently of cytokine signaling by a mechanism not fully understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size, chromatin condensation in the nucleus and disappearance of organelles, which requires transient caspase activation. These events are followed by nucleus extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Here, we show that in early step, SCF stimulates the Rho/ROCK pathway until the basophilic stage. Thereafter, ROCK-1 is activated independently of Rho signaling by caspase-3-mediated cleavage, allowing terminal maturation at least in part through phosphorylation of the light chain of myosin II. Therefore, in this differentiation system, final maturation occurs independently of SCF signaling through caspase-induced ROCK-1 kinase activation.     

The role of heme oxygenase signaling in various disorders

Modern methods of cell and molecular biology, augmented by molecular technology, have great potential for helping to unravel the complex mechanisms of various diseases. They also have the potential to help us try to dissect the events which follow the altered physiological conditions. Thus, there is every reason to believe that some of the potential mechanisms will be translated sooner or later into the clinic. Heme oxygenase (HO)-related mechanisms play an important role in several aspects of different diseases. In the past several years, significant progress has been made in our understanding of the function and regulation of HO. The objective of this article is to review current knowledge relating to the importance of HO mechanism in various diseases including myocardial ischemia/reperfusion, hypertension, cardiomyopathy, organ transplantation, endotoxemia, lung diseases, and immunosuppression. The morbidity and mortality of these diseases remain high even with optimal medical management. Furthermore, in this review, we consider various factors influencing the HO system and finally assess current pharmacological approaches to their control.