RESUMO
The mechanisms by which cytokine signals prevent the activation and mitochondrial targeting of the proapoptotic protein Bax are unclear. Here we show, using primary human eosinophils, that in the absence of the prosurvival cytokines granulocyte-macrophage colony-stimulating factor and interleukin 5, Bax spontaneously underwent activation and initiated mitochondrial disruption. Inhibition of Bax resulted in less eosinophil apoptosis, even in the absence of cytokines. Granulocyte-macrophage colony-stimulating factor induced activation of the kinase Erk1/2, which phosphorylated Thr167 of Bax; this facilitated new interaction of Bax with the prolyl isomerase Pin1. Blockade of Pin1 led to cleavage and mitochondrial translocation of Bax and caspase activation, regardless of the presence of cytokines. Our findings indicate that Pin1 is a key mediator of prosurvival signaling and is a regulator of Bax function.
Assuntos
Eosinófilos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Interleucina-5/imunologia , Peptidilprolil Isomerase/imunologia , Proteína X Associada a bcl-2/imunologia , Morte Celular , Sobrevivência Celular , Células Cultivadas , Eosinófilos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-5/metabolismo , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/metabolismo , Fosforilação , Transporte Proteico , Proteína X Associada a bcl-2/metabolismoRESUMO
Eosinophils (Eos) are prominent inflammatory cells found in the sputum, airways, and airway walls during and after exacerbations of allergic asthma. These cells are potent secretors of a wide array of cytotoxic granule proteins, cytokines, and lipid mediators involved in the initiation and maintenance of the Th2-type inflammatory reaction. Even though respiratory viral and bacterial infections are known risk factors contributing to severity and prognosis, the induction of anti-microbial Th1 type responses can offset allergic Th2 responses. Emerging evidence suggests that the prolyl isomerase Pin1 plays important roles in both Th1 and Th2 immunity in the lung and especially during allergic disease. In this review, we summarize recent findings related to the role of Pin1 in the regulation of Eos differentiation, survival, migration, cytokine expression, and antiviral immunity in asthma.
Assuntos
Eosinófilos/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Infecções Respiratórias/imunologia , Células Th2/imunologia , Viroses/imunologia , Animais , Diferenciação Celular , Sobrevivência Celular , Citocinas/metabolismo , Eosinófilos/imunologia , Regulação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos , Peptidilprolil Isomerase de Interação com NIMA/genética , Vesículas Secretórias/metabolismo , Equilíbrio Th1-Th2 , Receptor 7 Toll-Like/metabolismoRESUMO
The response of eosinophils (Eos) to respiratory virus has emerged as an important link between pulmonary infection and allergic asthmatic exacerbations. Eos activate innate immune responses through TLR signaling. In this study, using mouse and human Eos and mice lacking the prolyl isomerase Pin1 selectively in Eos, we show that Pin1 is indispensable for eosinophilopoiesis in the bone marrow (BM) and mature cell function in the presence of TLR7 activation. Unbiased in vivo analysis of mouse models of allergic airway inflammation revealed that TLR7 activation in knockout mice resulted in systemic loss of Eos, reduced IFN production, and an inability to clear respiratory viruses. Consistent with this finding, BM mouse Eos progenitors lacking Pin1 showed markedly reduced cell proliferation and survival after TLR7 activation. Mechanistically, unlike wild-type cells, Pin1 null mouse Eos were defective in the activation of the endoplasmic reticulum stress-induced unfolded protein response. We observed significant reductions in the expression of unfolded protein response components and target genes, aberrant TLR7 cleavage and trafficking, and reduced granule protein production in knockout Eos. Our data strongly suggest that Pin1 is required for BM Eos generation and function during concurrent allergen challenge and viral infection.
Assuntos
Eosinófilos/fisiologia , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Hipersensibilidade Respiratória/imunologia , Infecções por Respirovirus/imunologia , Vírus Sendai/fisiologia , Estresse Fisiológico/imunologia , Receptor 7 Toll-Like/metabolismo , Animais , Asma/imunologia , Diferenciação Celular , Células Cultivadas , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptidilprolil Isomerase de Interação com NIMA/genética , Transdução de Sinais , Resposta a Proteínas não DobradasRESUMO
RATIONALE: Leukocyte recruitment to sites of allergic inflammation depends on the local production of priming cytokines, chemokines, and potentially other mediators. Previously, we showed that eosinophils (Eos) express numerous orphan G-protein-coupled receptors, including Epstein-Barr virus-induced gene 2 (EBI2). Despite its contribution to inflammatory diseases, the role of EBI2 in pulmonary eosinophilia is unknown. OBJECTIVES: To determine whether oxysterol ligands for EBI2 are increased in asthma exacerbation, and if or how they promote Eos pulmonary migration. METHODS: EBI2 ligands and pulmonary eosinophilia were measured in the bronchoalveolar lavage fluid from patients with mild asthma 48 hours after acute allergen challenge. In vitro, the ability of EBI2 ligands alone or in combination with IL-5 priming to induce the migration of human blood Eos was assessed. MEASUREMENTS AND MAIN RESULTS: EBI2 was constitutively and stably expressed in peripheral blood Eos. Eos treated with the EBI2 ligands showed significantly increased transwell migration that was enhanced by priming with physiologic doses of IL-5. Migration was suppressed by inhibitors of the prolyl isomerase Pin1 or extracellular signal-regulated kinases (ERK) 1/2 or by pertussis toxin. EBI2 signaling activated Pin1 isomerase activity through a cascade that was sensitive to ERK inhibitors and pertussis toxin. The concentration of EBI2 ligands was significantly increased in the bronchoalveolar lavage fluid 48 hours after segmental allergen challenge and strongly correlated with the increased numbers of Eos, lymphocytes, and neutrophils in the airways. CONCLUSIONS: Oxysterols are increased in inflamed airways after allergen challenge and, through G-protein subunit α, ERK, and Pin1 signaling, likely participate in the regulation of Eos migration into the lung in people with asthma.
Assuntos
Alérgenos/imunologia , Asma/imunologia , Leucócitos/imunologia , Pulmão/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Movimento Celular , Eosinófilos/imunologia , Humanos , Contagem de Leucócitos , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
IL-5 is a major therapeutic target to reduce eosinophilia. However, all of the eosinophil-activating cytokines, such as IL-5, IL-3, and GM-CSF, are typically present in atopic diseases, including allergic asthma. As a result of the functional redundancy of these three cytokines on eosinophils and the loss of IL-5R on airway eosinophils, it is important to take IL-3 and GM-CSF into account to efficiently reduce tissue eosinophil functions. Moreover, these three cytokines signal through a common ß-chain receptor but yet differentially affect protein production in eosinophils. Notably, the increased ability of IL-3 to induce the production of proteins, such as semaphorin-7A, without affecting mRNA levels suggests a unique influence of IL-3 on translation. The purpose of this study was to identify the mechanisms by which IL-3 distinctively affects eosinophil function compared with IL-5 and GM-CSF, with a focus on protein translation. Peripheral blood eosinophils were used to study intracellular signaling and protein translation in cells activated with IL-3, GM-CSF, or IL-5. We establish that, unlike GM-CSF or IL-5, IL-3 triggers prolonged signaling through activation of ribosomal protein S6 (RPS6) and the upstream kinase 90-kDa ribosomal S6 kinase (p90S6K). Blockade of p90S6K activation inhibited phosphorylation of RPS6 and IL-3-enhanced semaphorin-7A translation. Furthermore, in an allergen-challenged environment, in vivo phosphorylation of RPS6 and p90S6K was enhanced in human airway compared with circulating eosinophils. Our findings provide new insights into the mechanisms underlying differential activation of eosinophils by IL-3, GM-CSF, and IL-5. These observations identify IL-3 and its downstream intracellular signals as novel targets that should be considered to modulate eosinophil functions.
Assuntos
Eosinófilos/fisiologia , Interleucina-3/imunologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteína S6 Ribossômica/metabolismo , Asma/imunologia , Células Cultivadas , Ativação Enzimática , Eosinofilia/imunologia , Eosinófilos/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Hipersensibilidade/imunologia , Interleucina-5/imunologia , Subunidade alfa de Receptor de Interleucina-5/metabolismo , Fosforilação , RNA Mensageiro/biossíntese , Proteína S6 Ribossômica/genética , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Semaforinas/biossíntese , Semaforinas/genética , Transdução de Sinais/genéticaRESUMO
Eosinophils (Eos) are potent inflammatory cells and abundantly present in the sputum and lung of patients with allergic asthma. During both transit to and residence in the lung, Eos contact prosurvival cytokines, particularly IL-3, IL-5 and GM-CSF, that attenuate cell death. Cytokine signaling modulates the expression and function of a number of intracellular pro- and anti-apoptotic molecules. Both intrinsic mitochondrial and extrinsic receptor-mediated pathways are affected. This article discusses the fundamental role of the extracellular and intracellular molecules that initiate and control survival decisions by human Eos and highlights the role of the cis-trans isomerase, Pin1 in controlling these processes.
Assuntos
Apoptose , Eosinófilos/fisiologia , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Sobrevivência Celular , Citocinas/fisiologia , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/fisiologia , Transdução de SinaisRESUMO
Interstitial pulmonary fibrosis is caused by the excess production of extracellular matrix (ECM) by Fb in response to TGF-ß1. Here, we show that the peptidyl-prolyl isomerase Pin1 modulates the production of many pro- and antifibrogenic cytokines and ECM. After acute, bleomycin injury, Pin1(-/-) mice showed reduced, pulmonary expression of collagens, tissue inhibitors of metalloproteinases, and fibrogenic cytokines but increased matrix metalloproteinases, compared with WT mice, despite similar levels of inflammation. In primary fibroblasts, Pin1 was required for TGF-ß-induced phosphorylation, nuclear translocation, and transcriptional activity of Smad3. In Pin1(-/-) cells, inhibitory Smad6 was found in the cytoplasm rather than nucleus. Smad6 knockdown in Pin1(-/-) fibroblasts restored TGF-ß-induced Smad3 activation, translocation, and target gene expression. Therefore, Pin1 is essential for normal Smad6 function and ECM production in response to injury or TGF-ß and thus may be an attractive therapeutic target to prevent excess scarring in diverse lung diseases.
Assuntos
Peptidilprolil Isomerase/metabolismo , Fibrose Pulmonar/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Proteína Smad6/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Bleomicina , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Mutação , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Interferência de RNA , Proteína Smad3/genética , Proteína Smad6/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND: Current treatments for Alzheimer's disease (AD) have largely failed to yield significant therapeutic benefits. Novel approaches are desperately needed to help address this immense public health issue. Data suggests that early intervention at the first stages of mild cognitive impairment may have a greater chance for success. The calcineurin (CN)-Pin1 signaling cascade can be selectively targeted with tacrolimus (FK506), a highly specific, FDA-approved CN inhibitor used safely for > 20 years in solid organ transplant recipients. AD prevalence was significantly reduced in solid organ recipients treated with FK506. METHODS: Time release pellets were used to deliver constant FK506 dosage to APP/PS1 mice without deleterious manipulation or handling. Immunofluorescence, histology, molecular biology, and behavior were used to evaluate changes in AD pathology. RESULTS: FK506 can be safely and consistently delivered into juvenile APP/PS1 mice via time-release pellets to levels roughly seen in transplant patients, leading to the normalization of CN activity and reduction or elimination of AD pathologies including synapse loss, neuroinflammation, and cognitive impairment. Pin1 activity and function were rescued despite the continuing presence of high levels of transgenic Aß42. Indicators of neuroinflammation including Iba1 positivity and IL-6 production were also reduced to normal levels. Peripheral blood mononuclear cells (PBMC) obtained during treatment or splenocytes isolated at euthanasia activated normally after mitogens. CONCLUSIONS: Low-dose, constant FK506 can normalize CNS CN and Pin1 activity, suppress neuroinflammation, and attenuate AD-associated pathology without blocking peripheral IL-2 responses making repurposed FK506 a viable option for early, therapeutic intervention in AD.
Assuntos
Doença de Alzheimer , Animais , Humanos , Camundongos , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Calcineurina/metabolismo , Modelos Animais de Doenças , Interleucina-2/imunologia , Interleucina-2/metabolismo , Leucócitos Mononucleares/patologia , Camundongos Transgênicos , Doenças Neuroinflamatórias , Fenótipo , Presenilina-1/genética , Linfócitos T/patologia , Tacrolimo/farmacologia , Tacrolimo/uso terapêuticoRESUMO
BACKGROUND: High temperatures will occur more frequently with global warming, with potential impacts on the efficacy of biological control agents. Heat shock proteins (HSPs) are induced by high temperature, but their possible roles in pest resistance to entomopathogens remain unexplored. We investigated the effects of high temperature (35 °C) on Helicoverpa armigera resistance to H. armigera nucleopolyhedrovirus (HearNPV) and the putative roles of HSPs in this process. RESULTS: Even short periods (24 h) of high temperature (35 °C) reduced mortality in HearNPV-infected H. armigera larvae. Sustained 35 °C exposure significantly shortened developmental time, and increased fresh weight and locomotor activity in infected larvae. Moreover, high temperature inhibited virus replication and thickened the epidermis of H. armigera, resulting in reduced spread of infection from cadavers. Real-time polymerase chain reaction (PCR) analysis showed that expression of 11 HSP genes was altered by the 35 °C treatment, and that mostly small heat shock protein (sHSP) genes were up-regulated, the same sHSPs were induced when larvae were infected with HearNPV. Finally, RNA interference (RNAi) suppression of these sHSPs showed that only Hsp24.91 and Hsp21.8 diminished H. armigera defensive responses to HearNPV infection. CONCLUSION: Even short periods of exposure to high temperature can significantly reduce susceptibility of H. armigera larvae to HearNPV by stimulating the production of sHSPs which enhance immune responses, with important implications for the use of entomopathogens as biological control agents under global warming scenarios. © 2022 Society of Chemical Industry.
Assuntos
Mariposas , Nucleopoliedrovírus , Animais , Agentes de Controle Biológico , Proteínas de Choque Térmico/genética , Larva , Mariposas/genética , Nucleopoliedrovírus/fisiologia , TemperaturaRESUMO
BACKGROUND: The cotton bollworm, Helicoverpa armigera, is a worldwide polyphagous pest, causing huge economic losses in vegetable, cotton and corn crops, among others. Owing to long-term exposure to Bacillus thuringiensis (Bt) toxins, evolution of resistance has been detected in this pest. As a conservative and effective neurotransmitter, dopamine (DA) has an important role in insect growth and development. In this study, we investigated the regulatory functions of DA and its associated non-coding RNA in metamorphosis in H. armigera. RESULTS: Expression profiles indicated that DA and DA pathway genes were highly expressed during larval-pupal metamorphosis in H. armigera. RNA interference and pharmacological experiments confirmed that tyrosine hydroxylase (TH), dopa decarboxylase, vesicular amine transporter and DA receptor 2 are critical genes related to the development of H. armigera from larvae to pupae. We also found that miR-14 and miR-2766 targeted the 3' untranslated region to post-transcriptionally regulate HaTH function. Application of miR-2766 and miR-14 antagomirs significantly increased levels of HaTH transcripts and proteins, while injection of miR-2766 and miR-14 agomirs not only suppressed messenger RNA and protein levels of HaTH, but also resulted in defective pupation in H. armigera. CONCLUSION: These results suggest that DA deficiency inhibits larval-pupal metamorphosis in H. armigera. Potentially, DA pathway genes and their microRNAs could be used as a novel target for H. armigera management. © 2022 Society of Chemical Industry.
Assuntos
MicroRNAs , Mariposas , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Larva , MicroRNAs/genética , MicroRNAs/metabolismo , Pupa/genética , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)-induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2-induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts.
Assuntos
Proteínas Morfogenéticas Ósseas/biossíntese , Calcificação Fisiológica/fisiologia , Regulação para Baixo/fisiologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Transativadores/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Calcificação Fisiológica/genética , Células Cultivadas , Regulação para Baixo/genética , Feminino , Fêmur/fisiologia , Camundongos , Camundongos Knockout , Osteogênese/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Transcrição Sp7 , Transativadores/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
Eosinophilic inflammation is a cornerstone of chronic asthma that often culminates in subepithelial fibrosis with variable airway obstruction. Pulmonary eosinophils (Eos) are a predominant source of TGF-beta1, which drives fibroblast proliferation and extracellular matrix deposition. We investigated the regulation of TGF-beta1 and show here that the peptidyl-prolyl isomerase (PPIase) Pin1 promoted the stability of TGF-beta1 mRNA in human Eos. In addition, Pin1 regulated cytokine production by both in vitro and in vivo activated human Eos. We found that Pin1 interacted with both PKC-alpha and protein phosphatase 2A, which together control Pin1 isomerase activity. Pharmacologic blockade of Pin1 in a rat asthma model selectively reduced eosinophilic pulmonary inflammation, TGF-beta1 and collagen expression, and airway remodeling. Furthermore, chronically challenged Pin1(-/-) mice showed reduced peribronchiolar collagen deposition compared with wild-type controls. These data suggest that pharmacologic suppression of Pin1 may be a novel therapeutic option to prevent airway fibrosis in individuals with chronic asthma.
Assuntos
Asma/imunologia , Eosinófilos/imunologia , Peptidilprolil Isomerase/metabolismo , Fibrose Pulmonar/imunologia , Hipersensibilidade Respiratória/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Alérgenos/imunologia , Animais , Antígenos de Superfície/metabolismo , Asma/genética , Asma/patologia , Brônquios/química , Colágeno/análise , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Camundongos , Camundongos Mutantes , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/antagonistas & inibidores , Peptidilprolil Isomerase/genética , Proteína Quinase C-alfa/metabolismo , Proteína Fosfatase 2/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/patologia , Fator de Crescimento Transformador beta1/genéticaRESUMO
Driven by global climate change and urbanization, urban heat island (UHI) and urban storm flood (USF) have become the most frequent and influential hazards in recent decades. Land-use optimization can effectively cope with these hazards. However, the trade-offs between multi-hazard mitigation and economic development impose many limitations in practice. Furthermore, current region-based optimization methods no longer meet the precise management demand, and both subdivision and spatial heterogeneity identification have the potential for wider applicability. Hence, a systematic integration of climate adaptation and urban construction through land-use planning is urgently required. This paper proposes a new land-use decision approach for improving climate adaptability of urbanization. This approach involves multi-objective optimization, spatial subdivision, and urbanization stage identification, which enable the simultaneous achievement of environmental and economic benefits. Taking Xiamen as case study, the results showed that excessive pursuit of land economic output (LEO) limits the chance of mitigating UHI and USF. Improving the LEO per unit area of construction land could disrupt the link between land exploitation and the increasing side effects of climate hazards. Future urbanization hotspots in Xiamen will likely emerge at the urban fringe in Tong'an District and Xiang'an District. Within each developing unit, the upper limit of construction land was 81.06 hm2 and the green space was recommended to be 7.29-21.94 hm2. Construction land and bare land contributed most to UHI and USF, while forest and grassland were highly efficient in heat and runoff mitigation. The developed approach proved to be effective and practicable, especially for reducing the impacts of extreme UHI and USF.
RESUMO
Eosinophils become polarized in response to cytokines such IL-5 or eotaxin prior to directional migration. Polarization is preceded by F-actin assembly, but the mechanisms that regulate these events and how the shape change influences cell migration from the peripheral blood into the lung remain unclear. In this study, we show that the prolyl isomerase, Pin1, is required for IL-5-induced Eos polarization and migration. Co-immunoprecipitation and immunofluorescence analysis revealed that Pin1 directly interacts with members of Rho GTPase family. Mouse eosinophils lacking Pin1 or human cells treated with Pin1 inhibitors showed significantly reduced IL-5-induced GTPase activity and cofilin phosphorylation, resulting in reduced F-actin polymerization, cell polarization, and directional migration to chemokines. Our result suggests that Pin1 regulates cytoskeletal re-organization, eosinophil morphology, and cell migration through the modulation of Rho GTPase activity. Targeting Pin1 along with GTPases could provide a new approach to reduce pulmonary Eos accumulation during asthmatic exacerbations.
Assuntos
Movimento Celular , Polaridade Celular , Eosinófilos/citologia , Eosinófilos/metabolismo , Interleucina-5/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Humanos , Camundongos Endogâmicos C57BL , Polimerização , Ligação Proteica , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Insect metamorphosis is a complex process involving many metabolic pathways, such as juvenile hormones and molting hormones, bioamines, microRNAs (miRNAs), etc. However, relatively little is known about the biogenic amines and their miRNAs to regulate cotton bollworm metamorphosis. Here we show that one miRNA, miR-277 regulates larval-pupal and pupal-adult metamorphosis of cotton bollworm by targeting the 3'UTR of Dopa decarboxylase (DDC), a synthetic catalytic enzyme of dopamine. Injection of miR-277 agomir inhibited the expression of DDC at the mRNA and protein levels, leading to defects in the pupation and emergence of H. armigera that was consistent with the phenotype obtained by injection of DDC double-stranded RNA (dsRNA). Injection of miR-277 antagomir induced the mRNA and protein expression of DDC and rescued the phenotype of pupation failure caused by DDC gene silencing. Unexpectedly, miR-277 antagomir can also cause failure of emergence of H. armigera and both agomir and antagomir of miR-277 injection could cause abnormal phenotypes in wing veins. This study reveals that elaborate regulation of miRNA and its target gene expression is prerequisite for insect development, which provides a new insight to study the developmental mechanisms of insect wing veins.
Assuntos
Dopa Descarboxilase/metabolismo , Metamorfose Biológica/genética , MicroRNAs/metabolismo , Mariposas/genética , Animais , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismoRESUMO
BACKGROUND: Infiltration, accumulation, and degranulation of eosinophils in the lung are hallmarks of active allergic asthma. The pulmonary response to inhaled allergen triggers the secretion of eosinophil chemoattractants and antiapoptotic cytokines, including GM-CSF, IL-3, IL-4, IL-5, and eotaxin, among others. We recently showed that in vitro Pin1 regulated eosinophil production of and response to GM-CSF. OBJECTIVE: We sought to determine the effect of Pin1 inhibition on pulmonary eosinophilia after allergen challenge. METHODS: The Pin1 inhibitor juglone (5-hydroxy-1,4-naphthoquinone) was administered to allergen-sensitized and allergen-challenged Brown Norway rats. Bronchoalveolar lavage fluid and lungs were assessed for inflammation, cytokine expression, and Pin1 activity. RESULTS: Juglone-treated rats showed a dramatic reduction (approximately 75%) in bronchoalveolar lavage fluid and pulmonary eosinophilia but no change in lymphocyte, monocyte/macrophage, or neutrophil numbers. GM-CSF and IL-5 expression were also significantly reduced, whereas Pin1-independent cytokines, such as eotaxin or IL-4, as well as housekeeping mRNAs and proteins, including actin, were unaffected by juglone. The eosinophils present in the lung in juglone-treated rats showed significantly greater apoptosis. CONCLUSION: These data suggest that in vivo Pin1 blockade attenuates GM-CSF and IL-5 production and can selectively reduce eosinophilic allergic inflammation. CLINICAL IMPLICATIONS: Eosinophils can be selectively reduced by Pin1 blockade, despite allergen challenge.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Eosinófilos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Interleucina-5/antagonistas & inibidores , Eosinofilia Pulmonar/imunologia , Hipersensibilidade Respiratória/imunologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Apoptose , Eosinófilos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-5/genética , Interleucina-5/metabolismo , Camundongos , Camundongos Mutantes , Naftoquinonas/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Glutaredoxins play crucial roles in maintaining intracellular redox homeostasis via scavenging of excess reactive oxygen species. In this study, a glutaredoxin domain-containing cysteine-rich gene from Helicoverpa armigera, named HaGdccr, was characterized. Sequence analysis revealed that it contains a glutaredoxin domain and a conserved cysteine and shares high sequence identity with other insect genes. HaGdccr mRNA expression was highest in molting larvae of the 3rd instar and was mainly detected in the central nervous system of larvae and the wings of adults. Quantitative real-time PCR results revealed that the expression of HaGdccr was suppressed at 1 and 6â¯h and increased at 24â¯h after the larvae were treated with 4⯰C and hydrogen peroxide. When the larvae were exposed to 20⯰C, HaGdccr decreased at 1â¯h and was induced at 12 and 24â¯h. HaGdccr transcription level was downregulated at 2 and 12â¯h and upregulated at 24â¯h after the adults were exposed to 0⯰C. However, transcript levels were increased by high temperature in both larvae and adults. After knockdown of HaGdccr by RNA interference, the expression of antioxidant genes, including thioredoxin-like (Trx-like), catalase (CAT), glutathione-S-transferase (GST), thioredoxin reductase (TrxR), and thioredoxin (Trx), was increased, whereas that of thioredoxin peroxidase (Tpx) was decreased. In addition, we found that HaGdccr knockdown enhanced the enzymatic activity of superoxide dismutase and the contents of hydrogen peroxide and ascorbate. Taken together, these results indicate that HaGdccr may play significant roles in protecting organisms against oxidative damage.
Assuntos
Antioxidantes/metabolismo , Glutarredoxinas/genética , Mariposas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Glutarredoxinas/metabolismo , Peróxido de Hidrogênio , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/metabolismo , Masculino , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Análise de Sequência de DNARESUMO
Thioredoxins (Trxs), a member of the thioredoxin system, play crucial roles in maintaining intracellular redox homeostasis and protecting organisms against oxidative stress. In this study, we cloned and characterized two genes, GmTrx2 and GmTrx-like1, from Grapholita molesta. Sequence analysis showed that GmTrx2 and GmTrx-like1 had highly conserved active sites CGPC and CXXC motif, respectively, and shared high sequence identity with selected insect species. The quantitative real-time polymerase chain reaction results revealed that GmTrx2 was mainly detected at first instar, whereas GmTrx-like1 was highly concentrated at prepupa day. The transcripts of GmTrx2 and GmTrx-like1 were both highly expressed in the head and salivary glands. The expression levels of GmTrx2 and GmTrx-like1 were induced by low or high temperature, E. coli, M. anisopliae, H2O2, and pesticides (emamectin benzoate). We further detected interference efficiency of GmTrx2 and GmTrx-like1 in G. molesta larvae and found that peroxidase capacity, hydrogen peroxide content, and ascorbate content all increased after knockdown of GmTrx2 or GmTrx-like1. Furthermore, the hydrogen peroxide concentration was increased by emamectin benzoate and the sensitivity for larvae to emamectin benzoate was improved after GmTrx2 or GmTrx-like1 was silenced. Our results indicated that GmTrx2 and GmTrx-like1 played vital roles in protecting G. molesta against oxidative damage and also provided the theoretical basis for understanding the antioxidant defense mechanisms of the Trx system in insects.
RESUMO
Phototaxis in nocturnal moths is widely utilized to control pest populations in practical production. However, as an elusive behavior, phototactic behavior is still not well understood. Determination of whether the opsin gene plays a key role in phototaxis is an interesting topic. This study was conducted to analyze expression levels and biological importance of three opsin genes (Se-uv, Se-bl, and Se-lw) and phototactic behavior of Spodoptera exigua. The three opsin genes exhibited higher expression levels during daytime, excluding Se-bl in females, whose expression tended to increase at night. And cycling of opsin gene levels tended to be upregulated at night, although the magnitude of increase in females was lower than that in males exposed to constant darkness. The results of western blotting were consistent with those of qRT-PCR. Furthermore, opsin gene expression was not influenced by light exposure during the scotophase, excluding Se-uv in males, and tended to be downregulated by starvation in females and copulation in both female and male moths. To determine the relationship between opsin gene expression and phototactic behavior, Se-lw was knocked down by RNA interference. Moths with one opsin gene knocked down showed enhanced expression of the other two opsin genes, which may play important roles in compensation in vision. The Se-lw-knockdown moths exhibited reduced phototactic efficiency to green light, suggesting that Se-LW contributes to phototaxis, and increases phototactic efï¬ciency to green light. Our finding provides a sound theoretical basis for further investigation of visual expression pattern and phototactic mechanisms in nocturnal moths.