Glycogen synthase kinase-3β is a prosurvival signal for the maintenance of human mast cell homeostasis

Homeostasis of mature tissue-resident mast cells is dependent on the relative activation of pro- and antiapoptotic regulators. In this study, we investigated the role of glycogen synthase kinase 3β (GSK3β) in the survival of neoplastic and nonneoplastic human mast cells. GSK3β was observed to be phosphorylated at the Y(216) activating residue under resting conditions in both the neoplastic HMC1.2 cell line and in peripheral blood-derived primary human mast cells (HuMCs), suggesting constitutive activation of GSK3β in these cells. Lentiviral-transduced short hairpin RNA knockdown of GSK3β in both the HMC1.2 cells and HuMCs resulted in a significant reduction in cell survival as determined with the MTT assay. The decrease in stem cell factor (SCF)-mediated survival in the GSK3β knockdown HuMCs was reflected by enhancement of SCF withdrawal-induced apoptosis, as determined by Annexin V staining and caspase cleavage, and this was associated with a pronounced reduction in SCF-mediated phosphorylation of Src homology 2 domain-containing phosphatase 2 and ERK1/2 and reduced expression of the antiapoptotic proteins Bcl-xl and Bcl-2. These data show that GSK3β is an essential antiapoptotic factor in both neopastic and nontransformed primary human mast cells through the regulation of SCF-mediated Src homology 2 domain-containing phosphatase 2 and ERK activation. Our data suggest that targeting of GSK3β with small m.w. inhibitors such as CHIR 99021 may thus provide a mechanism for limiting mast cell survival and subsequently decreasing the intensity of the allergic inflammatory response.     

Docosahexaenoic acid and palmitic acid reciprocally modulate monocyte activation in part through endoplasmic reticulum stress

Palmitic acid (C16:0) and TLR2 ligand induce, but docosahexaenoic acid (DHA) inhibits monocyte activation. C16:0 and TLR2 or TLR4 ligand induce certain ER stress markers; thus, we determined whether ER stress induced by these agonists is sufficient to induce monocyte activation, and whether the ER stress is inhibited by DHA which is known to inhibit C16:0- or ligand-induced TLR activation. Monocyte activation and ER stress were assessed by TLR/inflammasome-induced IL-1β production, and phosphorylation of IRE-1 and eIF2 and expression of CHOP, respectively in THP-1 cells. TLR2 ligand Pam3CSK4 induced phosphorylation of eIF2, but not phosphorylation of IRE-1 and CHOP expression. LPS also induced phosphorylation of both IRE-1 and eIF2 but not CHOP expression suggesting that TLR2 or TLR4 ligand, or C16:0 induces different ER stress responses. C16:0-, Pam3CSK4-, or LPS-induced IL-1β production was inhibited by 4-phenylbutyric acid, an inhibitor of ER stress suggesting that IL-1β production induced by these agonists is partly mediated through ER stress. Among two ER stress-inducing molecules, thapsigargin but not tunicamycin led to the expression of pro-IL-1β and secretion of IL-1β. Thus, not all types of ER stress are sufficient to induce inflammasome-mediated IL-1β secretion in monocytes. Although both C16:0 and thapsigargin-induced IL-1β secretion was inhibited by DHA, only C16:0-mediated ER stress was responsive to DHA. These findings suggest that the anti-inflammatory effects of DHA are at least in part mediated through modulating ER homeostasis and that the propensity of ER stress can be differentially modulated by the types of dietary fat we consume.     

Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells

Mast cells play a critical role in host defense against bacterial infection. Murine mast cells produce cytokines in response to bacterial peptidoglycan and LPS via Toll-like receptor (TLR) TLR2- and TLR4-dependent mechanisms. The expression of TLRs by human mast cells and responses to known TLR activators was examined. Human mast cells expressed mRNA for TLR1, TLR2, and TLR6 but not TLR4. Bacterial peptidoglycan and yeast zymosan were potent inducers of GM-CSF and IL-1beta and also induced substantial short-term cysteinyl leukotriene generation. In contrast, a synthetic triacylated lipopeptide induced short-term degranulation but failed to induce cysteinyl leukotriene production. The TLR4 activator Escherichia coli LPS did not induce a GM-CSF, IL-1beta leukotriene, or degranulation response. These data demonstrate highly selective production of different classes of mast cell mediators in response to distinct TLR activators of potential importance to the host response to bacterial or fungal pathogens.     

Cell death of prostate cancer cells by specific amino acid restriction depends on alterations of glucose metabolism

Selective amino acid restriction targets mitochondria resulting in DU145 and PC3 prostate cancer cell death. This study shows that restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln), or methionine (Met) differentially modulates glucose metabolism, glycogen synthase kinase 3β (GSK3β), p53, and pyruvate dehydrogenase (PDH) in these two cell lines. In DU145 cells, Gln and Met restriction increase glucose consumption, but Tyr/Phe restriction does not. Addition of glucose to culture media diminishes cell death induced by Tyr/Phe‐restriction. Addition of pyruvate reduces cell death due to Tyr/Phe and Gln restriction. Tyr/Phe, Gln and Met restriction increase phosphorylation of GSK3β‐Ser⁹, phosphorylation of p53‐Ser¹⁵ and reduce the mitochondrial localization of PDH. Addition of glucose or pyruvate to cultures significantly reverses the alterations in GSK3β, p53 and PDH induced by amino acid restriction. In p53‐null PC3 cells, Tyr/Phe, Gln and Met restriction decreases glucose consumption, reduces phosphorylation of Akt‐Ser⁴⁷³, and increases phosphorylation of GSK3β‐Ser⁹. Addition of pyruvate or glucose reduces death of Met‐restricted cells. Addition of glucose increases phosphorylation of Akt‐Ser⁴⁷³ in amino acid‐restricted cells reduces phosphorylation of GSK3β‐Ser⁹ in Tyr/Phe and Gln restricted cells and increases phosphorylation of GSK3β‐Ser⁹ in Met restricted cells. Addition of pyruvate reduces phosphorylation of GSK3β‐Ser⁹ in all amino acid‐restricted cells. In summary, cell death induced by specific amino acid restriction is dependent on or closely related to the modulation of glucose metabolism. GSK3β (DU145 and PC3) and p53 (DU145) are crucial switches connecting metabolism and these signaling molecules to cell survival during amino acid restriction. J. Cell. Physiol. 224: 491–500, 2010. © 2010 Wiley‐Liss, Inc.     

Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells

BackgroundWhile Syk has been shown to associate with TLR4, the immune consequences of Syk–TLR interactions and related molecular mechanisms are unclear.MethodsGain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting.ResultsSyk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr–Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K–Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses.ConclusionsSyk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K–Akt and NF-κB signaling pathways.General significanceSyk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.     

Differential Effects of the Toll-Like Receptor 2 Agonists,PGN and Pam3CSK4 on Anti-IgE Induced Human Mast Cell Activation

Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.     

Mast Cells Mobilize Myeloid-Derived Suppressor Cells and Treg Cells in Tumor Microenvironment via IL-17 Pathway in Murine Hepatocarcinoma Model

Tumor immunosuppression is commonly braided with chronic inflammation during tumor development. However, the relationship between immunosuppression and inflammation in tumor microenvironment is still unclear. We have demonstrated that mast cells are accumulated and exacerbate the inflammation and immunosuppression in tumor microenvironment via SCF/c-kit signaling pathway. Here, we further elucidate the underlying mechanism, which involves both myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our data showed that mast cells mobilized the infiltration of MDSCs to tumor and induced the production of IL-17 by MDSCs; MDSCs-derived IL-17 indirectly attracted Treg cells, enhanced their suppressor function, and induced the IL-9 production by Treg cells; in turn, IL-9 strengthened the survival and protumor effect of mast cells in tumor microenvironment. Our findings disclose a closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment, which provides a new insight into the paralleled developments of inflammation and immunosuppression in tumor microenvironment. Based on these findings, we propose that targeting tumor inflammation might be a potential strategy to reverse the immunosuppression of tumor microenvironment, thus facilitating cancer immunotherapy.     

TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells

Recent studies have revealed that murine bone marrow-derived cultured mast cells (BMMC), which are phenotypically immature mast cells, express functional TLR2 and TLR4 that recognize distinct pathogen-associated molecules. However, it remains relatively uncertain whether mast cells express other TLR. We recently established a method to obtain large numbers of murine fetal skin-derived cultured mast cells (FSMC); these cells exhibit important features of connective tissue type mast cells. Working with FSMC and BMMC, the TLR mRNA expression profiles were compared between both cell types. Although TLR2 and TLR4 mRNA were detected in both cells at comparable levels, TLR3, TLR7, and TLR9 mRNA were expressed by FSMC at higher levels than by BMMC, suggesting distinct TLR expression profiles among different mast cell populations. With respect to their functional aspects, FSMC, but not BMMC, dose dependently produced proinflammatory cytokines (TNF-alpha and IL-6) and chemokines (RANTES, MIP-1alpha, and MIP-2) in response to poly(I:C), R-848, and CpG oligodeoxynucleotide, which are TLR3, TLR7, and TLR9 activators, respectively. Interestingly, these TLR activators failed to induce degranulation and IL-13 production by both mast cells, although peptidoglycan and LPS (TLR2 and TLR4 activators, respectively) induced IL-13 production by both cells. Mast cells, thus, may have potential to recruit other immune cells to the infected sites by responding to various bacterial and viral components through TLR signaling pathways, presumably being involved in initiating innate immunity and subsequently linking innate and acquired immune responses.     

Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed α, β, and γ subunits of high-affinity immunoglobulin E (IgE) receptor (FcεRI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, and cyclooxygenase 2, and production of prostaglandin D(2) and leukotriene C(4) in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-α expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on FcεRI- and TLR-mediated effector functions of mast cells.     

Citreorosein inhibits degranulation and leukotriene C4 generation through suppression of Syk pathway in mast cells

The aim of this study was to evaluate whether citreorosein (CIT), a naturally occurring anthraquinone isolated from Polygoni cuspidati (P. cuspidati) radix, modulates degranulation and 5-lipoxygenase (5-LO)-dependent leukotriene C(4) (LTC(4)) generation in mast cells. Cit suppresses both degranulation and the generation of LTC(4) in a dose-dependent manner in stem cell factor (SCF)-mediated mouse bone marrow-derived mast cells (BMMCs). With regard to its molecular mechanism of action, we investigated the effects of CIT on intracellular signaling and mast cell activation employing BMMCs. Binding of SCF to c-Kit on mast cell membranes induced increases in intrinsic tyrosine kinase Syk activity and activation of multiple downstream events including phosphorylation of phospholipase Cγ (PLCγ), mobilization of intracellular Ca(2+), phosphatidylinositol 3-kinase (PI3K), Akt, MAP kinases (MAPKs), translocation of phospho-phospholipase A(2) (PLA(2)) and 5-LO. The results from the biochemical analysis demonstrate that CIT attenuates degranulation and LTC(4) generation through the suppression of multiple step signaling and would be beneficial for the prevention of allergic inflammation.     

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6-bis(3-chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT-116, SW480, and Caco-2 cells, but not in primary human dermal fibroblast cells. MCPP-induced concentration-dependent apoptotic cell death in colon cancer cells was measured by fluorescence-activated cell sorter (FACS) analysis. Treatment of HCT-116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein (GRP)-78, phosphorylation of eukaryotic initiation factor-2α (eIF-2α), suggesting the induction of ER stress. MCPP also increased GSK3α/β(Tyr270/216) phosphorylation and reduced GSK3α/β(Ser21/9) phosphorylation time-dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP-mediated cell apoptosis. Treatment of MCPP also increased caspase-7, caspase-9, and caspase-3 activity. The inhibition of caspase activity by z-DEVE-FMK or z-VAD-FMK significantly reduced MCPP-induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP-induced GRP and CHOP up-regulation, and pro-caspase-3 and pro-caspase-9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/β activation in mediating the MCPP-induced human colon cancer cell apoptosis.     

Stimulation of mouse connective tissue-type mast cells by hemopoietic stem cell factor, a ligand for the c-kit receptor.

The proliferative capacity of mouse connective tissue-type mast cells (CTMC) was analyzed by using a newly discovered c-kit ligand, termed stem cell factor (SCF). More than 90% of CTMC in the peritoneal cavity responded to recombinant rat SCF (rrSCF) and were able to give rise to pure mast cell colonies in methylcellulose culture. Serial observation (mapping) of growth of individual CTMC in culture containing rrSCF confirmed their striking proliferative ability. No serum but accessory cells (non-CTMC cells) in the peritoneal population were required for the clonal growth of CTMC induced by rrSCF in our methylcellulose culture of whole peritoneal cells. The rrSCF-induced mast cell colony formation from peritoneal CTMC was completely inhibited by the addition of anti-c-kit antibody, which can block the binding of SCF to c-kit, to the culture. When IL-3 was combined with rrSCF, mast cell colonies dramatically increased in size. Mapping studies revealed that the combination of the two factors augmented the proliferative rate of CTMC. Approximately 60% of the constituent cells of the mast cell colonies which were formed from peritoneal CTMC in the culture containing rrSCF alone were stained with berberine sulfate, which is a characteristic of CTMC. However, most mast cells which were induced by rrSCF+IL-3 from peritoneal CTMC contained berberine(-)-safranin(-)-Alcian blue(+) granules. Although IL-4 exhibited little synergism with rrSCF in the induction of CTMC proliferation, the addition of IL-4 to the culture containing rrSCF+IL-3 resulted in an increase in mast cells which retained CTMC characteristics.     

Silencing of H4R inhibits the production of IL-1β through SAPK/JNK signaling in human mast cells

Context: Mast cell (MC) activation through H4R releases various inflammatory mediators which are associated with allergic asthma.

Objectives: To investigate the siRNA-mediated gene silencing effect of H4R on human mast cells (HMCs) functions and the activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) signaling pathways for the release of ineterleukin-1β (IL-1β) in HMCs.

Materials and methods: H4R expression was analyzed by RT-PCR and western blotting in human mast cell line-1 (HMC-1) cells and H4RsiRNA transfected cells. The effect of H4RsiRNA and H4R-antagonist on H4R mediated MC functions such as intracellular Ca²⁺ release, degranulation, IL-6 and IL-1β release, and the activation SAPK/JNK signaling pathways were studied. HMC-1 cells were stimulated with 10?μM of histamine (His) and 4-methylhistamine (4-MH) and pretreated individually with H4R-antagonist JNJ7777120 (JNJ), histamine H1 receptor (H1R)-antagonist mepyramine, and signaling molecule inhibitors SP600125 (SP) and Bay117082.

Results: We found that the HMC-1 cells expressed H4R and H4RsiRNA treatment down regulated the H4R expression in HMC-1 cells. Both His and 4-MH induced the intracellular Ca²⁺ release and degranulation whereas; H4R siRNA and JNJ inhibited the effect. Furthermore, the activation of H4R caused the phosphorylation of SAPK/JNK pathways. H4R gene silencing and pretreatment with SP and JNJ decreased His and 4-MH induced phosphorylation of SAPK/JNK. We found that the activation of H4R caused the release of IL-1β (124.22?pg/ml) and IL-6 (122.50?pg/ml) on HMC-1 cells. Whereas, SAPK/JNK inhibitor (68.36?pg/ml) inhibited the H4R mediated IL-1β release.

Conclusions: Taken together, the silencing of H4R inhibited the H4R mediated MC functions and SAPK/JNK phosphorylation. Furthermore, the H4R activation utilized SAPK/JNK signaling pathway for IL-1β release in HMC-1 cells.     

Cleavage of GSK‐3β by calpain counteracts the inhibitory effect of Ser9 phosphorylation on GSK‐3β activity induced by H2O2

Glycogen synthase kinase‐3 beta (GSK‐3β) dysfunction may play an essential role in the pathogenesis of psychiatric, metabolic, neurodegenerative diseases, in which oxidative stress exists concurrently. Some studies have shown that GSK‐3β activity is up‐regulated under oxidative stress. This study evaluated how oxidative stress regulates GSK‐3β activity in human embryonic kidney 293 (HEK293)/Tau cells treated with hydrogen peroxide (H₂O₂). Here, we show that H₂O₂ induced an obvious increase of GSK‐3β activity. Surprisingly, H₂O₂ dramatically increased phosphorylation of GSK‐3β at Ser9, an inactive form of GSK‐3β,while there were no changes of phosphorylation of GSK‐3β at Tyr216. Moreover, H₂O₂ led to a transient [Ca²⁺]_i elevation, and simultaneously increased the truncation of GSK‐3β into two fragments of 40 kDa and 30 kDa, whereas inhibition of calpain decreased the truncation and recovered the activity of GSK‐3β. Furthermore, tau was hyperphosphorylated at Ser396, Ser404, and Thr231, three most common GSK‐3β targeted sites after 100 μM H₂O₂ administration in HEK293/Tau cells, whereas inhibition of calpain blocked the tau phosphorylation. In addition, we found that there were no obvious changes of Cyclin‐dependent kinase 5 (CDK5) expression (responsible for tau phosphorylation) and of p35 cleavage, the regulatory subunit of CDK5 in H₂O₂‐treated HEK293/Tau cells. In conclusion, Ca²⁺‐dependent calpain activation leads to GSK‐3β truncation, which counteracts the inhibitory effect of Ser9 phosphorylation, up‐regulates GSK‐3β activity, and phosphorylates tau in H₂O₂‐treated HEK293/Tau cells.     

Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism

PGE(2) is an endogenously synthesized inflammatory mediator that is over-produced in chronic inflammatory disorders such as allergic asthma. In this study, we investigated the regulatory effects of PGE(2) on mast cell degranulation and the production of cytokines relevant to allergic disease. Murine bone marrow-derived mast cells (BMMC) were treated with PGE(2) alone or in the context of IgE-mediated activation. PGE(2) treatment alone specifically enhanced IL-6 production, and neither induced nor inhibited degranulation and the release of other mast cell cytokines, including IL-4, IL-10, IFN-gamma, and GM-CSF. IgE/Ag-mediated activation of BMMC induced the secretion of IL-4, IL-6, and GM-CSF, and concurrent PGE(2) stimulation synergistically increased mast cell degranulation and IL-6 and GM-CSF, but not IL-4, production. A similar potentiation of degranulation and IL-6 production by PGE(2), in the context of IgE-directed activation, was observed in the well-established IL-3-dependent murine mast cell line, MC/9. RT-PCR analysis of unstimulated MC/9 cells revealed the expression of EP(1), EP(3), and EP(4) PGE receptor subtypes, including a novel splice variant of the EP(1) receptor. Pharmacological studies using PGE receptor subtype-selective analogs showed that the potentiation of IgE/Ag-induced degranulation and IL-6 production by PGE(2) is mediated through EP(1) and/or EP(3) receptors. Our results suggest that PGE(2) may profoundly alter the nature of the mast cell degranulation and cytokine responses at sites of allergic inflammation through an EP(1)/EP(3)-dependent mechanism.