Regulation Of Mast Cell Function Research Articles

Differential regulation of Nr4a subfamily nuclear receptors following mast cell activation

The biological function of the Nr4a subfamily of nuclear receptors is only partially understood. Here we show for the fist time that mast cell (MC) activation processes involve the regulation of Nr4a factors. Exposure of murine bone marrow-derived MCs (BMMCs) to live bacteria causes a robust and selective upregulation of all Nr4a members (Nr4a1–Nr4a3). In response to purified LPS, strong upregulation of Nr4a3, but not of Nr4a1 or Nr4a2 was seen. Nr4a3 expression was also induced after the activation of BMMCs by IgE receptor cross-linking. Moreover, Nr4a expression was induced in activated human MCs. As shown by Western blot analysis, Nr4a phosphorylation was induced by IgE receptor cross-linking and calcium ionophore stimulation of BMMCs and LAD2 cells, respectively. By using various inhibitors of signaling pathways, Nr4a3 induction in BMMCs was shown to be strongly dependent on Gö6976-sensitive kinases and partially dependent on the nuclear factor of activated T-cells (NFAT) pathway, while nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) inhibition failed to inhibit Nr4a3 expression in BMMCs. Together, these data reveal selective induction of Nr4a family members in activated MCs and implicate Nr4a family nuclear receptors in the regulation of MC function.

3K1558 マスト細胞ミトコンドリアのカルシウムイメージング(細胞生物的課題4,第49回年会講演予稿集)

3K1558 マスト細胞ミトコンドリアのカルシウムイメージング(細胞生物的課題4,第49回年会講演予稿集)

Analysis of Tie2 function in mast cells

BackgroundMast cells are most widely acknowledged as a centralmediator of allergic reactions. Recent literature has alsoimplicated mast cells in a variety of biological andpathological conditions, spurring an interest in thegenetic regulation of mast cell function and develop-ment. In a survey of global gene expression, we identi-fied higher Tie2 mRNA expression in bone marrowderived mast cells (BMMC) in relation to a Lin

Protein Tyrosine Nitration of Aldolase in Mast Cells: A Plausible Pathway in Nitric Oxide-Mediated Regulation of Mast Cell Function

NO is a short-lived free radical that plays a critical role in the regulation of cellular signaling. Mast cell (MC)-derived NO and exogenous NO regulate MC activities, including the inhibition of MC degranulation. At a molecular level, NO acts to modify protein structure and function through several mechanisms, including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MCs, we investigated protein tyrosine nitration in human MC lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione. Using two-dimensional gel Western blot analysis with an anti-nitrotyrosine Ab, together with mass spectrometry, we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MCs. The nitration of aldolase A was associated with a reduction in the maximum velocity of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance analysis showed that despite these changes in the activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that S-nitrosoglutathione treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose 1,6-bisphosphate. Interestingly, fructose 1,6-bisphosphate inhibited IgE-mediated MC degranulation in LAD2 cells. Thus, for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target. This posttranslational nitration of aldolase A may be an important pathway that regulates MC phenotype and function.

Differential Role of Class 1A PI 3-Kinase Regulatory Subunits in Mast Cell Growth, Maturation and Survival.

Abstract 77Stem cell factor (SCF) mediated c-Kit receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from c-Kit are poorly understood. Mast cells express multiple regulatory subunits of class 1A PI 3-kinase including p85α, p85β, p50α, and p55α. While it is known that PI 3-kinase plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including maturation, growth, and survival are not known. Using mice deficient in the expression of p85α or p85β or combination of both p85α/p55α/p50α as well as all four subunits we have examined the role of these subunits in mast cell functions. We show that loss of p85α subunit alone results in impaired bone marrow derived mast cell (BMMC) maturation, growth, and survival compared to wild-type (WT) controls, in spite of the continuous expression of p85β, p55α, and p50α subunits in these cells. Restoring the expression of p85α in p85α deficient mast cells restores the maturation and growth defects. To assess the contribution of p50α and p55α subunits, we generated mice using the Cre lox system that were deficient in the expression of all three subunits (i.e. p85α/p55α/p50α). Deficiency of p85α/p55α/p50α subunits in bone marrow cells completely blocked mast cell maturation and growth, suggesting an essential role for the smaller subunits p50 and p55 in addition to the full length form of p85. Curiously, over-expression of p50α in p85α deficient BMMCs only marginally rescued mast cell maturation and growth, suggesting that the full length form of p85α functions with specificity in regulating mast cell functions. Since the major difference between the shorter isoforms and the full length form of p85α is the absence of the amino terminal SH3 and BH domains, we generated two mutants of p85α lacking either the SH3 or the BH domain and expressed them in p85α−/− BMMCs. While both these mutants completely restored the maturation defect associated with p85α deficiency and showed normal binding to the c-Kit receptor upon SCF stimulation as well as to the p110 catalytic subunits; none of these mutants completely rescued SCF induced proliferation (50% and 70% respectively, n=3, p<0.004). Biochemically, lack of SCF induced growth rescue in p85α−/− BMMCs expressing p85αΔSH3 and p85αΔBH mutants was associated with a lack of rescue in the activation of Akt and Erk, but complete rescue in the activation of JNK (n=3). Consistently, while transplantation of p85α deficient bone marrow cells transduced with p85α into mast cell deficient Wsh mice resulted in complete restoration of gastrointestinal mast cells as well as mast cells in the stomach and spleen, p85αΔSH3 and p85αΔBH mutants restored mast cells only partially. These results indicate that other domains (SH3 and BH) of p85α are required for mast cell growth. In contrast to p85α, deficiency of p85β alone resulted in increased BMMC maturation, growth and survival compared to controls (1.2 fold, n=3, p<0.003). Consistently, over-expression of p85β in WT bone marrow cells resulted in a profound reduction in the maturation of mast cells as well as proliferation. We studied whether reduced maturation and proliferation due to the loss or over-expression of p85β was a result of altered c-Kit receptor internalization and degradation. Our results revealed significantly more c-Kit receptor internalization and degradation in p85β expressing cells compared to p85α expressing cells (2 fold, n=5, p<0.001). Since Cbl family of ubiquitin ligases are involved in the down-regulation of tyrosine kinase receptors, we analyzed whether c-Cbl is involved in p85β mediated c-Kit receptor internalization and degradation. Phosphorylation of c-Cbl and ubiquitination of c-Kit receptor was more in p85β expressing cells compared to p85 expressing cells (n=3). In conclusion, while the current dogma in the field of PI3Kinase signaling suggests that all regulatory subunits of PI3Kinase function in a similar manner; we provide genetic and biochemical evidence to suggest that p85 regulatory subunits differentially regulate growth and maturation of mast cells. Disclosures:Munugalavadla:Genentech: Employment, Patents & Royalties.

Galectin-9 Is a High Affinity IgE-binding Lectin with Anti-allergic Effect by Blocking IgE-Antigen Complex Formation

Galectin (Gal)-9 was first described as an eosinophil chemoattractant. With the progress in research, Gal-9 has come to be known as a versatile immunomodulator that is involved in various aspects of immune regulations, and the entire picture of the function still remains elusive. To uncover as-yet unknown activity of Gal-9, we have been examining the effect of the protein in various disease animal models. Here we show that Gal-9 attenuated asthmatic reaction in guinea pigs and suppressed passive-cutaneous anaphylaxis in mice. These results indicate the mast cell stabilizing effect of Gal-9. In vitro studies of mast cell degranulation involving RBL-2H3 cells demonstrated that Gal-9 suppressed degranulation from the cells stimulated by IgE plus antigen and that the inhibitory effect was completely abrogated in the presence of lactose, indicating lectin activity of Gal-9 is critical. We found that Gal-9 strongly and specifically bound IgE, which is a heavily glycosylated immunoglobulin, and that the interaction prevented IgE-antigen complex formation, clarifying the mode of action of the anti-degranulation effect. Gal-9 is expressed by several mast cells including mouse mast cell line MC/9. The fact that immunological stimuli of MC/9 cells augmented Gal-9 secretion from the cells implies that Gal-9 is an autocrine regulator of mast cell function to suppress excessive degranulation. Collectively, these findings shed light on a novel function of Gal-9 in mast cells and suggest a beneficial utility of Gal-9 for the treatment of allergic disorders including asthma.

Inhibition of IgE-induced mast cell activation by ethyl tertiary-butyl ether, a bioethanol-derived fuel oxygenate

Abstract Objectives The effect of ethyl tertiary-butyl ether (ETBE), which is widely used as a fuel oxygenate commonly produced from bioethanol, on immunoglobulin (Ig)E-dependent mast cell activation was investigated. Methods The rat mast cell line RBL2H3 sensitised with monoclonal anti-ovalbumin IgE was challenged with ovalbumin in the presence or absence of ETBE, tert-butanol (TBA), which is the main metabolite of ETBE in humans, and ethanol. Degranulation of RBL2H3 was examined by the release of β-hexosaminidase. To understand the mechanisms responsible for regulating mast cell function, the effects of ETBE, TBA and ethanol on the levels of intracellular calcium, phosphorylation of Akt (as a marker of phosphatidylinositol 3-kinase) and global tyrosine phosphorylation were also measured as indicators of mast cell activation. Key findings In the presence of ETBE, TBA or ethanol, IgE-induced release of β-hexosaminidase was decreased. These compounds also attenuated the IgE-mediated increase in the levels of intracellular Ca2+, phosphorylation of Akt and global tyrosine phosphorylation in RBL2H3 cells. Conclusions ETBE, TBA and ethanol inhibited mast cell degranulation by inhibiting the increase in intracellular calcium ion concentration and activation of phosphatidylinositol 3-kinase and protein tyrosine kinase activation, suggesting that exposure to ETBE might affect immune responses, particularly in allergic diseases.

Inhibition of IgE-induced mast cell activation by ethyl tertiary-butyl ether, a bioethanol-derived fuel oxygenate

The effect of ethyl tertiary-butyl ether (ETBE), which is widely used as a fuel oxygenate commonly produced from bioethanol, on immunoglobulin (Ig)E-dependent mast cell activation was investigated. The rat mast cell line RBL2H3 sensitised with monoclonal anti-ovalbumin IgE was challenged with ovalbumin in the presence or absence of ETBE, tert-butanol (TBA), which is the main metabolite of ETBE in humans, and ethanol. Degranulation of RBL2H3 was examined by the release of beta-hexosaminidase. To understand the mechanisms responsible for regulating mast cell function, the effects of ETBE, TBA and ethanol on the levels of intracellular calcium, phosphorylation of Akt (as a marker of phosphatidylinositol 3-kinase) and global tyrosine phosphorylation were also measured as indicators of mast cell activation. In the presence of ETBE, TBA or ethanol, IgE-induced release of beta-hexosaminidase was decreased. These compounds also attenuated the IgE-mediated increase in the levels of intracellular Ca(2+), phosphorylation of Akt and global tyrosine phosphorylation in RBL2H3 cells. ETBE, TBA and ethanol inhibited mast cell degranulation by inhibiting the increase in intracellular calcium ion concentration and activation of phosphatidylinositol 3-kinase and protein tyrosine kinase activation, suggesting that exposure to ETBE might affect immune responses, particularly in allergic diseases.

Comparison of inhibitory activities of zinc oxide ultrafine and fine particulates on IgE-induced mast cell activation

The effects of ultrafine and fine particles of zinc oxide (ZnO) on IgE-dependent mast cell activation were investigated. The rat mast cell line RBL2H3 sensitized with monoclonal anti-ovalbumin (OVA) IgE was challenged with OVA in the presence or absence of ZnO particles and zinc sulfate (ZnSO4). Degranulation of RBL2H3 was examined by the release of β-hexosaminidase. To understand the mechanisms responsible for regulating mast cell functions, the effects of ZnO particles on the levels of intracellular Zn2+, Ca2+, phosphorylated-Akt, and global tyrosine phosphorylation were also measured. IgE-induced release of b-hexosaminidase was obviously attenuated by ultrafine ZnO particles and ZnSO4, whereas it was very weakly inhibited by fine ZnO particles. The intracellular Zn2+ concentration was higher in the cells incubated with ultrafine ZnO particles than in those with fine ZnO particles. Consistent with inhibitory effect on release of b-hexosaminidase, ultrafine ZnO particles and ZnSO4, but not fine ZnO particle, strongly attenuated the IgE-mediated increase of phosphorylated-Akt and tyrosine phosphorylations of 100 and 70 kDa proteins in RBL2H3 cells. These findings indicate that ultrafine ZnO particles, with a small diameter and a large total surface area/mass, could release Zn2+ easily and increase intracellular Zn2+ concentration efficiently, thus decreasing FceRI-mediated mast cell degranulation through inhibitions of PI3K and protein tyrosine kinase activation. Exposure to ZnO particles might affect immune responses, especially in allergic diseases.

Nitration of aldolase: a critical post-translational modification in nitric oxide-mediated regulation of mast cell function (80.6)

Abstract Nitric oxide (NO) is a short-lived free radical that regulates mast cell (MC) activities including inhibition of MC degranulation. To elucidate the molecular mechanisms underlying the effects of NO in MC, we investigated protein tyrosine nitration in human mast cell lines treated with the NO donor S-Nitrosoglutathione. Using 2D gel western blot analysis with an anti-nitrotyrosine antibody together with mass spectroscopy, we identified aldolase A, an enzyme of the glycolytic pathway as a target for tyrosine nitration in MC. Aldolase A nitration was associated with reduction in the Km and Vmax of aldolase in HMC-1 and LAD-2. Nuclear Magnetic Resonance analysis showed that despite these changes in activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was increased. Elevated levels of lactate and pyruvate suggested that NO treatment enhanced glycolysis in MC. Reduced MC aldolase activity was associated with increased intracellular levels of its substrate, fructose 1,6 bisphosphate (FBP). Interestingly, FBP inhibited IgE-mediated MC degranulation in LAD-2 cells. Thus for the first time we report evidence of protein tyrosine nitration in two human MC lines and identify aldolase A as a prominent target that may regulate MC phenotype and function. Funded by: Canadian Institutes of Health Research, The Lung Association, Alberta &amp; NWT and 75th Anniversary Award, University of Alberta.

Antagonistic Signaling by IL-4 and TGFb Regulates Mast Cell Function (80.5)

Abstract Mast cells, like all cells of the immune system, are regulated by crosstalk from multiple cytokines, which collectively form a signaling network. Cytokine networking is an important concept for understanding how the immune system is controlled. We hypothesized that the initial presence of pro-inflammatory cytokines in the absence of anti-inflammatory cytokines could desensitize mast cells from anti-inflammatory signals, rendering them more likely to be activated in allergic reactions. Conversely, we hypothesized that the initial presence of anti-inflammatory cytokines in the absence of pro-inflammatory cytokines could shield mast cells from activation by pro-inflammatory cytokines in allergic type responses. In this study, we show that IL-4 and TGFβ1 act as an antagonistic pair in controlling the mast cell response. Mast cell pre-culture with IL-4 decreased TGFβR1 and TGFβR2 RNA and protein levels through a STAT6-dependent mechanism. Further, IL-4 diminished TGFβ1 signaling, blocking SMAD2 phosphorylation. IL-4 also blocked TGFβ1-mediated mast cell death and suppressed TGFβ1-mediated mast cell migration. Mast cell pre-culture with TGFβ1 mirrored this antagonistic effect. TGFβ1 pre-culture reduced IL-4R protein levels, suppressed Il-4-mediated Stat6 phosphorylation, and diminished mast cell migration toward IL-4. When both are present simultaneously, TGFβ1 appears to have a dominating inhibitory effect over IL-4 by blocking proliferation in vitro and recruitment in vivo. These data are important in understanding how cytokine networks can impact mast-cell mediated diseases like asthma. Supported by NIH grants 1R01 AI59638 and U19A1077435.

Activation and Function of the mTORC1 Pathway in Mast Cells

Little is known about the signals downstream of PI3K which regulate mast cell homeostasis and function following FcepsilonRI aggregation and Kit ligation. In this study, we investigated the role of the mammalian target of rapamycin complex 1 (mTORC1) pathway in these responses. In human and mouse mast cells, stimulation via FcepsilonRI or Kit resulted in a marked PI3K-dependent activation of the mTORC1 pathway, as revealed by the wortmannin-sensitive sequential phosphorylation of tuberin, mTOR, p70S6 kinase (p70S6K), and 4E-BP1. In contrast, in human tumor mast cells, the mTORC1 pathway was constitutively activated and this was associated with markedly elevated levels of mTORC1 pathway components. Rapamycin, a specific inhibitor of mTORC1, selectively and completely blocked the FcepsilonRI- and Kit-induced mTORC1-dependent p70S6K phosphorylation and partially blocked the 4E-BP1 phosphorylation. In parallel, although rapamycin had no effect on FcepsilonRI-mediated degranulation or Kit-mediated cell adhesion, it inhibited cytokine production, and kit-mediated chemotaxis and cell survival. Furthermore, Rapamycin also blocked the constitutive activation of the mTORC1 pathway and inhibited cell survival of tumor mast cells. These data provide evidence that mTORC1 is a point of divergency for the PI3K-regulated downstream events of FcepsilonRI and Kit for the selective regulation of mast cell functions. Specifically, the mTORC1 pathway may play a critical role in normal and dysregulated control of mast cell homeostasis.

Regulation of Mast Cell Function by Regulatory T Cells

Mast cell activation is associated with atopic and inflammatory diseases. Despite this, the natural controls of mast cell homeostasis are poorly understood. We have found that IL‐10 and TGFb1 suppress mast cell function and have investigated how these cytokines might be available to mast cells during the inflammatory response. Regulatory T cells (Treg) are a sub‐class of CD4+ T cells that can produce both IL‐10 and TGFb1. We hypothesized that these cells could function in mast cell homeostasis. In this study, we demonstrate that mast cells can recruit Treg. Furthermore, Treg, but not CD4+/CD25− T cells, suppress mast cell FceRI expression. Despite the inhibitory effects of IL‐10 and TGFb1, Treg effects were independent of IL‐10 and TGF‐b1, and required cell contact. Surprisingly, co‐culture with either Treg or CD25− T cells enhanced IgE‐mediated cytokine production by mast cells. These data are the first demonstration that mast cells can recruit Treg and that these interactions may alter the mast cell response. Supported by NIH grant 1R01 AI59638.

IL-10 Suppresses Mast Cell IgE Receptor Expression and Signaling In Vitro and In Vivo

Mast cells are known for their roles in allergy, asthma, systemic anaphylaxis, and inflammatory disease. IL-10 can regulate inflammatory responses and may serve as a natural regulator of mast cell function. We examined the effects of IL-10 on in vitro-cultured mouse and human mast cells, and evaluated the effects of IL-10 on FcepsilonRI in vivo using mouse models. IgE receptor signaling events were also assessed in the presence or absence of IL-10. IL-10 inhibited mouse mast cell FcepsilonRI expression in vitro through a Stat3-dependent process. This down-regulation was consistent in mice tested in vivo, and also on cultured human mast cells. IL-10 diminished expression of the signaling molecules Syk, Fyn, Akt, and Stat5, which could explain its ability to inhibit IgE-mediated activation. Studies of passive systemic anaphylaxis in IL-10-transgenic mice showed that IL-10 overexpression reduced the IgE-mediated anaphylactic response. These data suggest an important regulatory role for IL-10 in dampening mast cell FcepsilonRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.

The Early Growth Response Factor-1 Is Involved in Stem Cell Factor (SCF)-induced Interleukin 13 Production by Mast Cells, but Is Dispensable for SCF-dependent Mast Cell Growth

The stem cell factor (SCF) plays a central role in the regulation of mast cell function and growth. However, roles of transcription factors involved in these processes remain incompletely defined. The early growth response factor-1 (Egr-1) is a member of the zinc finger transcription factor family. A role for Egr-1 in SCF-induced mast cell activation and growth was investigated in mouse bone marrow-derived mast cells (BMMC). The stimulation of BMMC with SCF induced a strong expression of Egr-1 mRNA. SCF-induced Egr-1 nuclear translocation and DNA binding were demonstrated by electrophoretic mobility shift assay (EMSA) and immunofluorescence assay. SCF-induced IL-13 expression was significantly reduced at both mRNA and protein levels in Egr-1-deficient BMMC. In addition, the synergy between IgE and SCF on IL-13 and IL-4 production was reduced in Egr-1-deficient mast cells. Interestingly, Egr-1 deficiency had little effect on SCF-induced mast cell growth. SCF-induced Egr activation likely requires tyrosine phosphorylation because a tyrosine kinase inhibitor PP2 blocked SCF-induced nuclear protein binding to Egr probe as determined by EMSA. Thus, Egr-1 is required for SCF-induced IL-13 expression, but not mast cell growth. Egr-1 represents a novel mechanism for SCF-induced mast cell activation.

Mast Cell Homeostasis: A Fundamental Aspect of Allergic Disease

Mast cells are well known for their role in allergic disease and have recently been implicated in inflammatory disorders, including autoimmune arthritis, multiple sclerosis, and atherosclerosis. Although aberrant mast cell activation is the focus of many studies, much less is known about normal mast cell homeostasis. Because loss of the normal constraints on mast cell activation, proliferation, and survival may be central to disease etiology, understanding these issues warrants attention. This review summarizes the knowledge of mast cell homeostasis controlled by IgE and the regulatory cytokines IL-4, IL-10, and TGF-beta1. Because each of these proteins plays an important role in immune responses tied to mast cell-associated disease, this group represents a potential set of factors altered in atopic or autoimmune patients. It is interesting to note, for example, that polymorphisms within each of these factors or their receptors are linked to allergic disease. By first understanding how cytokines and IgE regulate mast cell function and survival, we may then predict how these factors may function in disease onset and progression.

An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response

Cross-linking of the FcɛRI activates the phosphatidyl inositol 3 kinase (PI3K) and mitogen-activated protein kinase pathways. Previous studies demonstrate that Ras guanyl nucleotide-releasing protein (RasGRP)1 is essential in T cell receptor–mediated Ras-Erk activation. Here, we report that RasGRP1 plays an important role in FcɛRI-mediated PI3K activation and mast cell function. RasGRP1-deficient mice failed to mount anaphylactic allergic reactions. RasGRP1−/− mast cells had markedly reduced degranulation and cytokine production. Although FcɛRI-mediated Erk activation was normal, PI3K activation was diminished. Consequently, activation of Akt, PIP3-dependent kinase, and protein kinase C δ was defective. Expression of a constitutively active form of N-Ras could rescue the degranulation defect and Akt activation. We further demonstrated that RasGRP1−/− mast cells were defective in granule translocation, microtubule formation, and RhoA activation. Our results identified RasGRP1 as an essential regulator of mast cell function.

Negative Regulation of FcϵRI-mediated Signaling and Mast Cell Function by the Adaptor Protein LAX

LAX is a transmembrane adaptor protein that is expressed in both T and B cells. Upon stimulation via the antigen receptors, it is tyrosine-phosphorylated and binds Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. Disruption of the Lax gene causes hyperresponsiveness in T and B lymphocytes. Here, we showed that LAX was also expressed in mast cells. Upon engagement of the Fc epsilonRI, LAX was also phosphorylated and interacted with Grb2 and p85. LAX-deficient mast cells were hyperresponsive to stimulation via the Fc epsilonRI, as evidenced by enhanced degranulation, p38 MAPK, Akt, and phosphatidylinositol 3-kinase activation. This hyperresponsiveness was likely a consequence of reduced LAB expression after sensitization of mast cells with anti-dinitrophenyl IgE. In addition, Fc epsilonRI-mediated cytokine production and cell survival were also enhanced. These data suggested that LAX negatively regulates mast cell function.

FcɛRI control of Ras via inositol (1,4,5) trisphosphate 3-kinase and inositol tetrakisphosphate

The inositol (1,4,5) trisphosphate 3-kinase (ITP3K) phosphorylates Ins (1,4,5) P3 to produce Ins (1,3,4,5) P4. The ITP3K substrate, InsP3, and its product, InsP4, both have the potential to regulate mast cell function. Here, we explore the effects of dominant inhibition of ITP3K upon secretory responses and Ras GTPase activation following antigenic cross-linking of the mast cell immunoreceptor, FcɛRI. Inhibition of ITP3K potentiates both calcium release from intracellular stores and calcium-dependent secretory responses in mast cells. Moreover, mast cells with dominantly inhibited ITP3K display constitutive activation of Ras and certain Ras effector pathways. We propose three mechanisms by which ITP3K inhibition could influence Ras activation. The protection of InsP3 that results from ITP3K inhibition may lead to enhanced activation of calcium-sensitive Ras-GAPs or -GRFs. Similarly, the deficit in InsP4 may change the behavior of the InsP4 receptor, the GAP1 IP4BP. Our data are inconsistent with calcium-sensitive Ras-GAP activation being the primary consequence of ITP3K inhibition in mast cells. Rather, we observe potentiation of Ras responses in mast cells transfected with dominant negative GAP1 IP4BP. Moreover, shRNA-mediated knockdown of GAP1 IP4BP potentiates FcɛRI-mediated Ras activation, indicating that this InsP4-binding GAP protein may be used by the FcɛRI immunoreceptor to regulate Ras.

TGF-β1 Inhibits Mast Cell FcεRI Expression

Mast cell activation through the high affinity IgE receptor (FcepsilonRI) is a critical component of atopic inflammation. The cytokine TGF-beta1 has been shown to inhibit IgE-dependent mast cell activation, possibly serving to dampen mast cell-mediated inflammatory responses. We present proof that TGF-beta1 inhibits mast cell FcepsilonRI expression through a reversible pathway that diminishes protein, but not mRNA, expression of the FcepsilonRI subunit proteins alpha, beta, and gamma. The stability of the expressed proteins and the assembled cell surface complex was unaltered by TGF-beta1 treatment. However, TGF-beta1 decreased the rate of FcepsilonRI beta-chain synthesis, arguing that this inhibitory cytokine exerts its effects at the level of mRNA translation. TGF-beta1 consistently diminished FcepsilonRI expression on cultured human or mouse mast cells as well as freshly isolated peritoneal mast cells. The related cytokines, TGF-beta2 and TGF-beta3, had similar effects. We propose that TGF-beta1 acts as a negative regulator of mast cell function, in part by decreasing FcepsilonRI expression.