RESUMO
IL-10+ regulatory B (Breg) cells play a vital role in regulating the immune responses in experimental autoimmune encephalomyelitis, colitis, and contact hypersensitivity (CHS). Several stimulants such as lipopolysaccharide (LPS), CD40 ligand, and IL-21 spur the activation and maturation of IL-10+ Breg cells, while the epigenetic mechanism for the IL-10 expression remains largely unknown. It is well accepted that the histone acetylation/ deacetylation is an important mechanism that regulates the expression of IL-10. We found that entinostat, an HDAC inhibitor, stimulated the induction of IL-10+ Breg cells by LPS in vitro and the formation of IL-10+ Breg cells to suppress CHS in vivo. We further demonstrated that entinostat inhibited HDAC1 from binding to the proximal region of the IL-10 expression promoter in splenic B cells, followed by an increase in the binding of NF-κB p65, eventually enhancing the expression of IL-10 in Breg cells. [BMB Reports 2021; 54(10): 534-539].
Assuntos
Linfócitos B Reguladores/metabolismo , Benzamidas/farmacologia , Dermatite de Contato/tratamento farmacológico , Piridinas/farmacologia , Acetilação , Animais , Linfócitos B Reguladores/efeitos dos fármacos , Benzamidas/metabolismo , Células Cultivadas , Colite/metabolismo , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Histona Desacetilase 1/efeitos dos fármacos , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Imunidade/imunologia , Imunidade/fisiologia , Interleucina-10/imunologia , Interleucina-10/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Piridinas/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Mast cells (MCs) play an important role as effector cells that cause allergic responses in allergic diseases. For these reasons, MC is considered an attractive therapeutic target for allergic disease treatment. In this study, we investigated the inhibitory effect of WZ3146, N-[3-[5-chloro-2-[4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]oxyphenyl]prop-2-enamide, and the mechanisms of its actions on the MC activation and IgE-mediated allergic response by using three types of MCs such as rat basophilic leukemia (RBL)-2H3 cells, mouse bone marrow mast cells (BMMCs), and human Laboratory of Allergic Diseases 2 (LAD2) cells. WZ3146 inhibited antigen-stimulated degranulation in a dose-dependent manner (IC50, ~ 0.35⯵M for RBL-2H3 cells; ~ 0.39⯵M for BMMCs; ~ 0.41 for LAD2 cells). WZ3146 also suppressed the production of histamine, tumor necrosis factor (TNF)-α and interleukin (IL)-6, which mediate various allergic responses, in a dose-dependent manner. As the mechanism of WZ3146 to inhibit MCs, it inhibited the activation of spleen tyrosine kinase (Syk) and the downstream signaling proteins of Syk such as linker for activation of T cell (LAT) and phospholipase (PL) Cγ1 in the signaling pathway of FcεRI. In addition, WZ3146 inhibited the activation of Akt, extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). However, WZ3146 did not inhibit degranulation of MCs by thapsigargin or ionomycin, which increase calcium concentration in cytosol. Notably, WZ3146 inhibited the activity of Lyn and Fyn, but not Syk. In an following animal experiment, WZ3146 inhibited IgE-dependent passive cutaneous anaphylaxis (PCA) in a dose-dependent manner (ED50, ~ 20â¯mg/kg). Taken together, in this study we show that the pyrimidine derivative, WZ3146, inhibits the IgE-mediated allergic response by inhibiting Lyn and Fyn Src-family kinases, which are initially activated by antigen stimulation in MCs. Therefore, we propose that WZ3146 could be used as a new therapeutic agent for the treatment of allergic diseases.
Assuntos
Hipersensibilidade/metabolismo , Imunoglobulina E/metabolismo , Mastócitos/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Pirimidinas/farmacologia , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fyn/imunologia , Pirimidinas/química , Ratos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/imunologiaRESUMO
The function of regulatory immune cells in peripheral tissues is crucial to the onset and severity of various diseases. Interleukin-10 (IL-10)-producing regulatory B (IL-10+ Breg) cells are known to suppress various inflammatory diseases. However, evidence for the mechanism by which IL-10+ Breg cells are generated and maintained is still very limited. Here, we found that IL-10+ Breg cells suppress the activation of IL-13-producing type 2 innate lymphoid cells (IL-13+ ILC2s) in an IL-10-dependent manner in mice with oxazolone-induced severe contact hypersensitivity (CHS). Mast cell (MC) IL-5 was important for maintaining the population of IL-10+ Breg cells in peripheral lymphoid tissues. Overall, these results uncover a previously unknown mechanism of MCs as a type of immunoregulatory cell and elucidate the cross-talk among MCs, IL-10+ Breg cells, and IL-13+ ILC2s in CHS.
Assuntos
Linfócitos B Reguladores/imunologia , Linfócitos B Reguladores/metabolismo , Dermatite de Contato/etiologia , Dermatite de Contato/metabolismo , Interleucina-5/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Oxazolona/efeitos adversos , Tolerância Periférica , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Citocinas/metabolismo , Dermatite de Contato/patologia , Modelos Animais de Doenças , Imunofluorescência , Isotipos de Imunoglobulinas/imunologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
Mast cells are the most prominent effector cells of Type 1 hypersensitivity immune responses. CYC116 [4-(2-amino-4-methyl-1,3-thiazol-5-yl)-N-[4-(morpholin-4-yl)phenyl] pyrimidin-2-amine] is under development to be used as an anti-cancer drug, but the inhibitory effects of CYC116 on the activation of mast cells and related allergy diseases have not reported as of yet. In this study, we demonstrated, for the first time, that CYC116 inhibited the degranulation of mast cells by antigen stimulation (IC50, ~1.42 µM). CYC116 also inhibited the secretion of pro-inflammatory cytokines including TNF-α (IC50, ~1.10 µM), and IL-6 (IC50, ~1.24 µM). CYC116 inhibited the mast cell-mediated allergic responses, passive cutaneous anaphylaxis (ED50, ~22.5 mg/kg), and passive systemic anaphylaxis in a dose-dependent manner in laboratory experiments performed on mice. Specifically, CYC116 inhibited the activity of Fyn in mast cells and inhibited the activation of Syk and Syk-dependent signaling proteins including LAT, PLCγ, Akt, and MAP kinases. Our results suggest that CYC116 could be used as an alternative therapeutic medication for mast cell-mediated allergic disorders, such as atopic dermatitis and allergic rhinitis.
RESUMO
Mast cells are critical effector cells in IgE-mediated allergic responses. The aim of this study was to investigate the anti-allergic effects of 3-[(aminocarbonyl)amino]-5-(3-fluorophenyl)-N-(3S)-3-piperidinyl-2-thiophenecarboxamide (AZD7762) in vitro and in vivo. AZD7762 inhibited the antigen-stimulated degranulation from RBL-2H3 (IC50, â¼27.9â¯nM) and BMMCs (IC50, â¼99.3â¯nM) in a dose-dependent manner. AZD7762 also inhibited the production of TNF-α and IL-4. As the mechanism of its action, AZD7762 inhibited the activation of Syk and its downstream signaling proteins, such as Linker of activated T cells (LAT), phospholipase (PL) Cγ1, Akt, and mitogen-activated protein (MAP) kinases (Erk1/2, p38, and JNK) in mast cells. In in vitro protein kinase assay, AZD7762 inhibited the activity of Lyn and Fyn kinases, which are important for the activation of Syk in mast cells. Furthermore, AZD7762 also suppressed the degranulation of LAD2 human mast cells (IC50, â¼49.9â¯nM) and activation of Syk in a dose-dependent manner. As observed in experiments with mast cells in vitro, AZD7762 inhibited antigen-mediated passive cutaneous anaphylaxis in mice (ED50, â¼35.8â¯mg/kg). Altogether, these results suggest that AZD7762 could be used as a new therapeutic agent for mast cell-mediated allergic diseases.
Assuntos
Antialérgicos/farmacologia , Antineoplásicos/farmacologia , Mastócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Tiofenos/farmacologia , Ureia/análogos & derivados , Quinases da Família src/antagonistas & inibidores , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Reposicionamento de Medicamentos/métodos , Humanos , Imunoglobulina E/toxicidade , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Ureia/farmacologia , Quinases da Família src/metabolismoRESUMO
Mast cells are critical cells that prompt various allergic response-inducing factors, contributing to allergic diseases. While used as an antibiotic for livestock, there is no study on the effect of furaltadone on allergic response. This study investigated the effect of furaltadone on mast cells and passive cutaneous anaphylaxis (PCA). Furaltadone inhibited the degranulation of mast cells stimulated by antigen (IC50, ~ 3.9⯵M), and also suppressed the production of tumor necrosis factor (TNF)-α and interleukin (IL)-4 in a concentration dependent manner. In addition, furaltadone inhibited allergic responses in an acute allergy animal model, PCA. Further investigation on the mechanism for these inhibitory effects of furaltadone found that the activities of Lyn/Syk and Syk-dependent downstream proteins such as mitogen-activated protein (MAP) kinases were inhibited by furaltadone in mast cells. Taken together, this study demonstrates that furaltadone inhibits the activation of mast cells by antigen via the suppression of the Lyn/Syk pathway and ameliorates allergic responses in vivo.
Assuntos
Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , Nitrofuranos/farmacologia , Oxazolidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/metabolismo , Quinases da Família src/metabolismo , Animais , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Mastócitos/citologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Anafilaxia Cutânea Passiva/efeitos dos fármacosRESUMO
An allergic reaction occurs when the immune system overreacts to harmless substance called allergen that gains access to the body. Food allergy is a hypersensitive immune reaction to food proteins and the number of patients with food allergy has recently increased. Aloe Vera is used for wellness and medicinal purposes. In particular, Aloe vera has been reported to enhance immunity. However, the effect of Aloe vera on food allergy is not yet known. In this study, we investigated the effects of processed Aloe vera gel (PAG) containing low molecular weight Aloe polysaccharide (AP) on ovalbumin (OVA)-induced food allergy in mice. Allergic symptoms, rectal temperature, and diarrhea were measured in OVA-induced food allergy mice. Other allergic parameters were also analyzed by RT-PCR, ELISA, flow cytometry, and other biochemical methods. As the results, PAG suppressed the decrease of body temperature, diarrhea, and allergic symptoms in OVA-induced food allergy mice. PAG also reduced serum concentrations of type 2â¯helper T cell (Th2) cytokines (Interleukin-(IL)-4, IL-5, and IL-13) as well as histamine, mast cell protease-1 (MCP-1), and immunoglobulin (Ig)E. PAG blocked the degranulation of mast cells and infiltration of eosinophils in intestine. Furthermore, PAG suppressed the population of Th2 cells in spleen and mesenteric lymph nodes. PAG also increased the production of IL-10 and population of type 1 regulatory T (Tr1) cells in mice with food allergy. Taken together, our findings suggest that PAG suppressed Th2 immune responses through, at least partially, stimulating the secretion of IL-10 in food allergy mice.
Assuntos
Hipersensibilidade Alimentar/prevenção & controle , Preparações de Plantas/química , Polissacarídeos/farmacologia , Células Th2/imunologia , Animais , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Hipersensibilidade Alimentar/imunologia , Imunoglobulina E/imunologia , Intestinos/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Polissacarídeos/isolamento & purificação , Baço/imunologiaRESUMO
Regulatory B cells, also well-known as IL-10-producing B cells, play a role in the suppression of inflammatory responses. However, the epigenetic modulation of regulatory B cells is largely unknown. Recent studies showed that the bromodomain and extra-terminal domain (BET) protein inhibitor JQ1 controls the expression of various genes involving cell proliferation and cell cycle. However, the role of BET proteins on development of regulatory B cells is not reported. In this study, JQ1 potently suppressed IL-10 expression and secretion in murine splenic and peritoneal B cells. While bromodomaincontaining protein 4 (BRD4) was associated with NF-κB on IL-10 promoter region by LPS stimulation, JQ1 interfered the interaction of BRD4 with NF-κB on IL-10 promoter. In summary, BRD4 is essential for toll like receptor 4 (TLR4)-mediated IL-10 expression, suggesting JQ1 could be a potential candidate in regulating IL-10-producing regulatory B cells in cancer. [BMB Reports 2017; 50(12): 640-646].
Assuntos
Azepinas/farmacologia , Linfócitos B Reguladores/efeitos dos fármacos , Interleucina-10/biossíntese , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Receptor 4 Toll-Like/metabolismo , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Animais , Azepinas/química , Linfócitos B Reguladores/metabolismo , Proteínas de Ciclo Celular , Relação Dose-Resposta a Droga , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Triazóis/químicaRESUMO
Mast cells trigger IgE-mediated allergic reactions by releasing various allergic mediators. 8-Formyl-7-hydroxy-4-methylcoumarin, also called 4µ8C, was originally known as an inositol-requiring enzyme 1 (IRE1) suppressant, but no study has examined its relationship with mast cells and allergic diseases. Therefore, the purpose of this study was to determine whether 4µ8C is effective in suppressing allergic reactions in mast cells and in IgE-mediated allergic animal model. 4µ8C suppressed the degranulation of IgE-mediated mast cells (IC50=3.2µM) and the production of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in a dose-dependent manner. 4µ8C also suppressed passive cutaneous anaphylaxis (PCA) in mice (ED50=25.1mg/kg). In an experiment on mast cell signaling pathways stimulated by antigen, the phosphorylation and activation of Syk was decreased by 4µ8C, and phosphorylation of downstream signaling molecules, such as linker for activated T cells (LAT), Akt, and the three MAP kinases, ERK, p38, and JNK, were suppressed. Mechanistic studies showed that 4µ8C inhibited the activity of Lyn and Fyn in vitro. Based on the results of those experiments, the suppressor mechanism of allergic reaction by 4µ8C involved reduced activity of Lyn and Fyn, which is pivotal in an IgE-mediated signaling pathway. In summary, for the first time, this study shows that 4µ8C inhibits Lyn and Fyn, thus suppressing allergic reaction by reducing the degranulation and the production of inflammatory cytokines. This suggests that 4µ8C can be used as a new medicinal candidate to control allergic diseases such as seasonal allergies and atopic dermatitis.
Assuntos
Anafilaxia/imunologia , Cumarínicos/farmacologia , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , Quinase Syk/metabolismo , Animais , Degranulação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Interleucina-4/metabolismo , Masculino , Mastócitos/citologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Anafilaxia Cutânea Passiva , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Transdução de Sinais , Quinase Syk/antagonistas & inibidores , Quinase Syk/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The immunoregulatory cytokine Interleukin 10 (IL-10) protein is produced by various cells during the course of inflammatory disorders. Mainly, it downregulates pro-inflammatory cytokines, antigen presentation, and helper T cell activation. In this study, we show that the ratio of IL-10-producing cells was significantly increased in lineage negative (i.e., not T, B, or leukocyte cell lineages) cells than in lineage positive cells in lymphoid and peripheral tissues. We further observed that IL-10-producing innate lymphoid cells (ILCs), here called firstly ILC10, were increased in number in oxazolone-induced contact hypersensitivity (CHS) mice. In detail, IL-10-producing lineage negative cells were elevated in the axillary, inguinal lymph node, and ear tissues of CHS mice. Notably, the cells expressed classical ILC marker proteins such as CD45, CD127, and Sca-1. Altogether, our findings suggest for the first time that ILC10s are present in various physiological settings and could be involved in numerous immune responses as regulatory cells. [BMB Reports 2016; 49(5): 293-296].
Assuntos
Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Imunidade Inata , Interleucina-10/biossíntese , Linfócitos/patologia , Animais , Linhagem da Célula , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
Food allergy is a hypersensitive immune reaction to food proteins. We have previously demonstrated the presence of IL-10-producing CD5(+) B cells and suggested their potential role in regulating cow's milk casein allergy in humans and IgE-mediated anaphylaxis in mice. In this study, we determined whether IL-10-producing CD5(+) regulatory B cells control casein-induced food allergic responses in mice and, if so, the underlying mechanisms. The induction of oral tolerance (OT) by casein suppressed casein-induced allergic responses including the decrease of body temperature, symptom score, diarrhea, recruitment of mast cells and eosinophils into jejunum, and other biological parameters in mice. Notably, the population of IL-10-producing CD5(+) B cells was increased in mesenteric lymph node (MLN), but not in spleen or peritoneal cavity (PeC) in OT mice. The adoptive transfer of CD5(+) B cells from MLN, but not those from spleen and PeC, suppressed the casein-induced allergic responses in an allergen-specific and IL-10-dependent manner. The inhibitory effect of IL-10-producing CD5(+) B cells on casein-induced allergic response was dependent on Foxp3(+) regulatory T cells. Taken together, mesenteric IL-10-producing regulatory B cells control food allergy via Foxp3(+) regulatory T cells and could potentially act as a therapeutic regulator for food allergy.
Assuntos
Alérgenos/imunologia , Linfócitos B Reguladores/imunologia , Linfócitos B Reguladores/metabolismo , Caseínas/imunologia , Interleucina-10/biossíntese , Hipersensibilidade a Leite/imunologia , Hipersensibilidade a Leite/metabolismo , Leite/imunologia , Transferência Adotiva , Animais , Subpopulações de Linfócitos B , Antígenos CD5 , Bovinos , Comunicação Celular , Modelos Animais de Doenças , Feminino , Tolerância Imunológica , Imunoglobulina E/imunologia , Imunomodulação , Linfonodos/imunologia , Linfonodos/metabolismo , Mesentério , Camundongos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40(hi)CD5(+) B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40(hi) is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10(-/-)CD5(+)CD19(+) B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40(hi)CD5(+) Breg cells in mice. However, the population of CD40(hi)CD5(+) B cells was minimal in IL-10(-/-) mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40(hi)CD5(+) B cells and the autocrine effect of IL-10 is critical to the formation of CD40(hi)CD5(+) Breg cells.
Assuntos
Linfócitos B Reguladores/metabolismo , Antígenos CD40/metabolismo , Antígenos CD5/metabolismo , Interleucina-10/metabolismo , Animais , Comunicação Autócrina/efeitos dos fármacos , Linfócitos B Reguladores/imunologia , Diferenciação Celular/efeitos dos fármacos , Interleucina-10/genética , Interleucina-10/farmacologia , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismoRESUMO
We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer's disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Besouros/química , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas de Insetos/farmacologia , Neurônios/citologia , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Proteólise/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meia-Vida , Humanos , Proteínas de Insetos/química , Camundongos , Dados de Sequência Molecular , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ácido Okadáico/farmacologia , Oxidopamina/farmacologia , Peptídeos/químicaRESUMO
Previously, we demonstrated that the erythropoietin receptor (EpoR) is present on fibroblasts, where it regulates focal contact. Here, we assessed whether this action of EpoR is involved in the reduced cell adhesion observed in colonocytes exposed to Clostridium difficile toxin A. EpoR was present and functionally active in cells of the human colonic epithelial cell line HT29 and epithelial cells of human colon tissues. Toxin A significantly decreased activating phosphorylations of EpoR and its downstream signaling molecules JAK-2 (Janus kinase 2) and STAT5 (signal transducer and activator of transcription 5). In vitro kinase assays confirmed that toxin A inhibited JAK 2 kinase activity. Pharmacological inhibition of JAK2 (with AG490) abrogated activating phosphorylations of EpoR and also decreased focal contacts in association with inactivation of paxillin, an essential focal adhesion molecule. In addition, AG490 treatment significantly decreased expression of occludin (a tight junction molecule) and tight junction levels. Taken together, these data suggest that inhibition of JAK2 by toxin A in colonocytes causes inactivation of EpoR, thereby enhancing the inhibition of focal contact formation and loss of tight junctions known to be associated with the enzymatic activity of toxin A.