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BACKGROUND: MARCKS (myristoylated alanine-rich C kinase substrates) serves as a substrate for protein kinase C, residing in the plasma membrane while acts as an actin filament crosslinking protein. This investigation aims to elucidate phosphorylated MARCKS (p-MARCKS) levels and activity in allergic asthma patients and explore the therapeutic potential of peptide inhibitors targeting p-MARCKS in an acute mouse model of allergic asthma. METHODS: Immunohistochemistry and histology staining were employed on lung tissue slides to evaluate p-MARCKS expression and allergic asthma symptoms. Airway resistance was measured using invasive whole-body plethysmography. Flow cytometry detected lung dendritic cell migration, and migration/maturation assays were conducted on isolated murine bone marrow-derived dendritic cells (BM-DCs). RESULTS: Elevated p-MARCKS expression was observed in both human asthmatic tissues and animal models immunized with ovalbumin or Alternaria alternata. Remarkably, asthmatic individuals showed elevated high p-MARCKS expression in lung tissues. Intraperitoneal injection of the peptide MPS, targeting the MARCKS phosphorylation site domain, before allergen challenged, effectively suppressed MARCKS phosphorylation in murine lung tissues. MPS inhibited both in vivo and in vitro migration and maturation of dendritic cells (BM-DCs) and reduced Th2-related lymphocyte activation in bronchoalveolar lavage fluid (BALF). MPS pretreatment additionally suppressed all symptoms associated with allergic airway asthma, including a reduction in inflammatory cell influx, airway mucous cell metaplasia, and airway hyperreactivity. CONCLUSION: These findings suggest that phosphorylated MARCKS occurs in asthmatic lung tissue, and the inhibition of MARCKS phosphorylation by the MPS peptide reduces dendritic cell migration and Th2-related lymphocytes in the lungs in a murine model of acute asthma.
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Ferroptosis is an iron-dependent programmed cell death associated with severe kidney diseases, linked to decreased glutathione peroxidase 4 (GPX4). However, the spatial distribution of renal GPX4-mediated ferroptosis and the molecular events causing GPX4 reduction during ischemia-reperfusion (I/R) remain largely unknown. Using spatial transcriptomics, we identify that GPX4 is situated at the interface of the inner cortex and outer medulla, a hyperactive ferroptosis site post-I/R injury. We further discover OTU deubiquitinase 5 (OTUD5) as a GPX4-binding protein that confers ferroptosis resistance by stabilizing GPX4. During I/R, ferroptosis is induced by mTORC1-mediated autophagy, causing OTUD5 degradation and subsequent GPX4 decay. Functionally, OTUD5 deletion intensifies renal tubular cell ferroptosis and exacerbates acute kidney injury, while AAV-mediated OTUD5 delivery mitigates ferroptosis and promotes renal function recovery from I/R injury. Overall, this study highlights a new autophagy-dependent ferroptosis module: hypoxia/ischemia-induced OTUD5 autophagy triggers GPX4 degradation, offering a potential therapeutic avenue for I/R-related kidney diseases.
Assuntos
Injúria Renal Aguda , Ferroptose , Traumatismo por Reperfusão , Humanos , Rim , Autofagia , IsquemiaRESUMO
The myristoylated alanine-rich C-kinase substrate (MARCKS) is a membrane-associated protein kinase C (PKC) substrate ubiquitously expressed in eukaryotic cells. MARCKS plays important roles in multiple cellular processes, including cell adhesion and motility, mucin secretion, exocytosis, and inflammatory response. Aberrant MARCKS signaling has been observed in the development and progression of multiple cancer types. In addition, MARCKS facilitates cancer metastasis through modulating cancer cell migration and invasion. Moreover, MARCKS contributes to treatment resistance, likely by promoting cancer stem cell renewal as well as immunosuppression. In this review, we describe MARCKS protein structure, cellular localization, and biological functions. We then discuss the role of MARCKS in cancer metastasis as well as its mechanisms of action in solid tumors. Finally, we review recent advances in targeting MARCKS as a new therapeutic strategy in cancer management.
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Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.-Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.
Assuntos
Fibroblastos/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Fosfatidilinositóis/metabolismo , Fibrose Pulmonar/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Proliferação de Células , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Substrato Quinase C Rico em Alanina Miristoilada/genética , Fosfatidilinositóis/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose Pulmonar/induzido quimicamenteRESUMO
BACKGROUND AND PURPOSE: Myristoylated alanine-rich C kinase substrate (MARCKS), a PKC substrate, facilitates mucus production and neutrophil migration. However, the effects of therapeutic procedures targeting the phosphorylation site of MARCKS on steroid-resistant asthma and the mechanisms underlying such effects have not yet been investigated. We designed a peptide that targets the MARCKS phosphorylation site (MPS peptide) and assessed its therapeutic potential against steroid-resistant asthma. EXPERIMENTAL APPROACH: Mice were sensitized with ovalbumin (OVA), alum, and challenged with aerosolized OVA five times a week for 1 month. The mice were intratracheally administered MPS peptides three times a week, 1 hr before OVA challenge. Asthma symptoms and cell profiles in the bronchoalveolar lavage were assessed, and key proteins were analysed using Western blotting. KEY RESULTS: Phosphorylated (p)-MARCKS was highly expressed in inflammatory and bronchial epithelial cells in OVA-immunized mice. MPS peptide reduced eosinophils, neutrophils, mucus production, collagen deposition, and airway hyper-responsiveness. Dexamethasone (Dexa) did not alleviate steroid-resistant asthma symptoms. MPS peptide caused a decrease in p-MARCKS, nitrotyrosine and the expression of oxidative stress enzymes, NADPH oxidase dual oxidase 1 and inducible NOS, in lung tissues. Compared to Dexa, MPS peptides inhibited C5a production and attenuated IL-17A and KC production in the airway more effectively, thus suppressing asthma symptoms. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that targeting MARCKS phosphorylation through MPS treatment may inhibit neutrophilic inflammation and relieve asthma symptoms, thereby highlighting its potential as a therapeutic agent for steroid-resistant asthma.
Assuntos
Asma/tratamento farmacológico , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Peptídeos/uso terapêutico , Corticosteroides/uso terapêutico , Alérgenos , Animais , Asma/metabolismo , Asma/patologia , Modelos Animais de Doenças , Resistência a Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos BALB C , Ovalbumina , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacosRESUMO
Cholera toxin (CT) is a bacterial component that increases intracellular cAMP levels in host cells and suppresses T-cell activation. Recently, CT was reported to induce T helper type 17-skewing dendritic cells and activate interleukin-17A (IL-17A) production in CD4+ T cells through a cAMP-dependent pathway. However, the underlying mechanism by which cAMP regulates IL-17A production in T cells is not completely defined. In this study, we took advantage of a small molecule protein kinase A (PKA) inhibitor (H89) and different cAMP analogues: a PKA-specific activator (N6-benzoyl-adenosine-cAMP), an exchange protein activated by cAMP-specific activator (Rp-8-chlorophenylthio-2'-O-methyl cAMP), and a PKA inhibitor (Rp-8-bromo-cAMP), to elucidate the signalling cascade of cAMP in IL-17A regulation in T cells. We found that CT induced IL-17A production and IL-17A promoter activity in activated CD4+ T cells through a cAMP/PKA pathway. Moreover, this regulation was via cAMP-response element binding protein (CREB) -mediated transcriptional activation by using the transfection of an IL-17A promoter-luciferase reporter construct and CREB small interfering RNA in Jurkat cells. Also, we showed that CREB bound to the CRE motif located at -183 of the IL-17A promoter in vitro. Most interestingly, not only in CD4+ T cells, CT also enhanced cAMP/PKA-dependent IL-17A production and CREB phosphorylation in CD8+ T cells. In conclusion, our data suggest that CT induces an IL-17A-dominated immune microenvironment through the cAMP/PKA/CREB signalling pathway. Our study also highlights the potentials of CT and cAMP in modulating T helper type 17 responses in vivo.
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Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Toxina da Cólera/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Interleucina-17/biossíntese , Interleucina-17/genética , Sítios de Ligação , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais , Ativação TranscricionalRESUMO
Cigarette smoke has been shown to trigger aberrant signaling pathways and pathophysiological processes; however, the regulatory mechanisms underlying smoke-induced gene expression remain to be established. Herein, we observed that two smoke-responsive genes, HO-1 and CYP1A1, are robustly induced upon smoke by different mechanisms in human bronchial epithelia. CYP1A1 is mediated by aryl hydrocarbon receptor signaling, while induction of HO-1 is regulated by oxidative stress, and suppressed by N-acetylcysteine treatment. In light of a pivotal role of NRF2 and BACH1 in response to oxidative stress and regulation of HO-1, we examined if smoke-induced HO-1 expression is modulated through the NRF2/BACH1 axis. We demonstrated that smoke causes significant nuclear translocation of NRF2, but only a slight decrease in nuclear BACH1. Knockdown of NRF2 attenuated smoke-induced HO-1 expression while down-regulation of BACH1 had stimulatory effects on both basal and smoke-induced HO-1 with trivial influence on NRF2 nuclear translocation. Chromatin immunoprecipitation assays showed that smoke augments promoter-specific DNA binding of NRF2 but suppresses BACH1 binding to the HO-1 promoter ARE sites, two of which at -1.0 kb and -2.6 kb are newly identified. These results suggest that the regulation of NRF2 activator and BACH1 repressor binding to the ARE sites are critical for smoke-mediated HO-1 induction.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Heme Oxigenase-1/genética , Fator 2 Relacionado a NF-E2/genética , Fumar/genética , Brônquios/metabolismo , Brônquios/patologia , Linhagem Celular , Núcleo Celular/genética , Citocromo P-450 CYP1A1/genética , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica/genética , Humanos , Estresse Oxidativo/genética , Regiões Promotoras Genéticas , Mapas de Interação de Proteínas/genética , Transdução de Sinais/genética , Fumar/patologiaRESUMO
In the context of the human airway, interleukin-17A (IL-17A) signaling is associated with severe inflammation, as well as protection against pathogenic infection, particularly at mucosal surfaces such as the airway. The intracellular molecule Act1 has been demonstrated to be an essential mediator of IL-17A signaling. In the cytoplasm, it serves as an adaptor protein, binding to both the intracellular domain of the IL-17 receptor as well as members of the canonical nuclear factor kappa B (NF-κB) pathway. It also has enzymatic activity, and serves as an E3 ubiquitin ligase. In the context of airway epithelial cells, we demonstrate for the first time that Act1 is also present in the nucleus, especially after IL-17A stimulation. Ectopic Act1 expression can also increase the nuclear localization of Act1. Act1 can up-regulate the expression and promoter activity of a subset of IL-17A target genes in the absence of IL-17A signaling in a manner that is dependent on its N- and C-terminal domains, but is NF-κB independent. Finally, we show that nuclear Act1 can bind to both distal and proximal promoter regions of DEFB4, one of the IL-17A responsive genes. This transcriptional regulatory activity represents a novel function for Act1. Taken together, this is the first report to describe a non-adaptor function of Act1 by directly binding to the promoter region of IL-17A responsive genes and directly regulate their transcription.
Assuntos
Elementos de Resposta/fisiologia , Transdução de Sinais/fisiologia , Transcrição Gênica/fisiologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Regulação para Cima/fisiologia , Células A549 , Proteínas Adaptadoras de Transdução de Sinal , Humanos , Interleucina-17/biossíntese , Interleucina-17/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Domínios Proteicos , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , beta-Defensinas/biossíntese , beta-Defensinas/genéticaRESUMO
The root of Polygonum multiflorum (also called He-Shou-Wu in Chinese) is a common herb and medicinal food in Asia used for its anti-aging properties. Our study investigated the therapeutic potential of an extract of the root of Polygonum multiflorum (PME) in allergic asthma by using a mouse model. Feeding of 0.5 and 1 mg/mouse PME inhibited ovalbumin (OVA)-induced allergic asthma symptoms, including airway inflammation, mucus production, and airway hyper-responsiveness (AHR), in a dose-dependent manner. To discern PME's mechanism of action, we examined the profile and cytokine production of inflammatory cells in bronchial alveolar lavage fluid (BALF). We found that eosinophils, the main inflammatory cell infiltrate in the lung of OVA-immunized mice, significantly decreased after PME treatment. Th2 cytokine levels, including interleukin (IL)-4, IL-5, IL-13, eotaxin, and the proinflammatory cytokine tumor necrosis factor (TNF)-[Formula: see text], decreased in PME-treated mice. Elevated mRNA expression of Th2 transcription factor GATA-3 in the lung tissue was also inhibited after oral feeding of PME in OVA-immunized mice. Thus, we conclude that PME produces anti-asthma activity through the inhibition of Th2 cell activation.
Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Fallopia multiflora/química , Fitoterapia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Administração Oral , Animais , Asma/metabolismo , Asma/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Fator de Transcrição GATA3/metabolismo , Mediadores da Inflamação/metabolismo , Pulmão/metabolismo , Camundongos Endogâmicos BALB C , Muco/metabolismo , Ovalbumina , Raízes de PlantasRESUMO
Epithelial-mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3ß inactivation, cytoplasmic accumulation, and nuclear translocation of ß-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and ß-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3ß/ß-catenin signaling pathway.
Assuntos
Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Proteína HMGB1/fisiologia , Antígenos de Neoplasias/metabolismo , Linhagem Celular , Movimento Celular , Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Mucosa Respiratória/citologia , Transdução de Sinais , beta Catenina/metabolismoRESUMO
Numerous studies have shown that TH17 cells and their signature cytokine IL-17A are critical to host defense against various bacterial and fungal infections. The protective responses mediated by TH17 cells and IL-17A include the recruitment of neutrophils, release of antimicrobial peptides and chemokines, and enhanced tight junction of epithelial cells. Due to the importance of TH17 cells in infections, efforts have been made to develop TH17-based vaccines. The goal of vaccination is to establish a protective immunological memory. Most currently approved vaccines are antibody-based and have limited protection against stereotypically different strains. Studies show that T-cell-based vaccines may overcome this limitation and protect hosts against infection of different strains. Two main strategies are used to develop TH17 vaccines: identification of TH17-specific antigens and TH17-skewing adjuvants. Studies have revealed that cholera toxin (CT) induces a potent Th17 response following vaccination. Antigen vaccination along with CT induces a robust TH17 response, which is sometimes accompanied by TH1 responses. Due to the toxicity of CT, it is hard to apply CT in a clinical setting. Thus, understanding how CT modulates TH17 responses may lead to the development of successful TH17-based vaccines.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Toxina da Cólera/imunologia , Células Th17/citologia , Células Th17/imunologia , Vacinas/imunologia , Animais , Diferenciação Celular , Toxina da Cólera/administração & dosagem , AMP Cíclico/metabolismo , Células Dendríticas/imunologia , Humanos , Infecções/imunologia , Interleucina-17/imunologia , Vacinas/administração & dosagemRESUMO
Accumulating evidence has suggested that myristoylated alanine-rich C-kinase substrate (MARCKS) is critical for regulating multiple pathophysiological processes. However, the molecular mechanism underlying increased phosphorylation of MARCKS at Ser159/163 (phospho-MARCKS) and its functional consequence in neoplastic disease remain to be established. Herein, we investigated how phospho-MARCKS is regulated in breast carcinoma, and its role in the context of chemotherapy. In a screen of patients with breast tumors, we find that the abundance of phospho-MARCKS, not MARCKS protein per se, increased in breast cancers and positively correlated with tumor grade and metastatic status. Among chemotherapeutic agents, mitotic inhibitors, including paclitaxel, vincristine or eribulin, notably promoted phospho-MARCKS accumulation in multiple breast cancer cells. We further show that phospho-MARCKS acted upstream of Src activation upon paclitaxel exposure. Reduction of phospho-MARCKS by knockdown of MARCKS or pharmacological agents increased paclitaxel sensitivity. Particularly, a known phospho-MARCKS inhibitor, MANS peptide, was demonstrated to increase paclitaxel efficacy and attenuate angiogenesis/metastasis of xenografted breast cancer cells by decreasing abundance of phospho-MARCKS and messages of inflammatory mediators. Our data suggest that unresponsiveness of breast cancer to paclitaxel treatment is, at least in part, mediated by phospho-MARCKS and also provide an alternative therapeutic strategy against breast cancer by improving taxanes sensitivity.
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Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Paclitaxel/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células MCF-7 , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Substrato Quinase C Rico em Alanina Miristoilada , Neovascularização Patológica/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Fosforilação , Interferência de RNA , RNA Interferente PequenoRESUMO
Pneumonia remains one of the leading causes of death in both adults and children worldwide. Despite the adoption of a wide variety of therapeutics, the mortality from community-acquired pneumonia has remained relatively constant. Although viral and fungal acute airway infections can result in pneumonia, bacteria are the most common cause of community-acquired pneumonia, with Streptococcus pneumoniae isolated in nearly 50% of cases. Pneumolysin is a cholesterol-dependent cytolysin or pore-forming toxin produced by Streptococcus pneumonia and has been shown to play a critical role in bacterial pathogenesis. Airway epithelium is the initial site of many bacterial contacts and its barrier and mucosal immunity functions are central to infectious lung diseases. In our studies, we have shown that the prior exposure to statins confers significant resistance of airway epithelial cells to the cytotoxicity of pneumolysin. We decided to take this study one step further, assessing changes in both the transcriptome and lipidome of human airway epithelial cells exposed to toxin, statin or both. Our current work provides the first global view in human airway epithelial cells of both the transcriptome and the lipid interactions that result in cellular protection from pneumolysin.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Metabolismo dos Lipídeos , Metaboloma , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Estreptolisinas/toxicidade , Transcriptoma , Proteínas de Bactérias/toxicidade , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Análise por Conglomerados , Biologia Computacional , Células Epiteliais , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Lipídeos de Membrana/metabolismo , Substâncias Protetoras/farmacologia , Transdução de SinaisRESUMO
Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.
Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Proteínas Hemolisinas/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Imunidade Inata/efeitos dos fármacos , Sinvastatina/farmacologia , Estreptolisinas/antagonistas & inibidores , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Linhagem Celular Transformada , Células Epiteliais/imunologia , Células Epiteliais/patologia , Proteínas Hemolisinas/toxicidade , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/imunologia , Injeções Intraperitoneais , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pravastatina/imunologia , Pravastatina/farmacologia , Cultura Primária de Células , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Sinvastatina/imunologia , Staphylococcus aureus/química , Streptococcus pneumoniae/química , Estreptolisinas/toxicidadeRESUMO
Combination treatment for non-small cell lung cancer (NSCLC) is becoming more popular due to the anticipation that it may be more effective than single drug treatment. In addition, there are efforts to genetically screen patients for specific mutations in light of attempting to administer specific anticancer agents that are most effective. In this study, we evaluate the anticancer and anti-angiogenic effects of low dose erlotinib-cisplatin combination in NSCLC in vitro and in vivo. In NSCLC cells harboring epidermal growth factor receptor (EGFR) mutations, combination erlotinib-cisplatin treatment led to synergistic cell death, but there was minimal efficacy in NSCLC cells with wild-type EGFR. In xenograft models, combination treatment also demonstrated greater inhibition of tumor growth compared to individual treatment. The anti-tumor effect observed was secondary to the targeting of angiogenesis, evidenced by decreased vascular endothelial growth factor (VEGF) levels and decreased levels of CD31 and microvessel density. Combination treatment targets angiogenesis through down-regulation of the c-MYC/hypoxia inducible factor 1-alpha (HIF-1α) pathway. In fact, cell lines with EGFR exon 19 deletions expressed high basal levels of c-MYC and HIF-1α and correlate with robust responses to combination treatment. These results suggest that low dose erlotinib-cisplatin combination exhibits its anti-tumor activity by targeting angiogenesis through the modulation of the c-MYC/HIF-1α/VEGF pathway in NSCLC with EGFR exon 19 deletions. These findings may have significant clinical implications in patients with tumors harboring EGFR exon 19 deletions as they may be particularly sensitive to this regimen.
Assuntos
Adenocarcinoma/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Receptores ErbB/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/patologia , Neovascularização Patológica/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cloridrato de Erlotinib , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Mutação , Quinazolinas/administração & dosagemRESUMO
Club (Clara) Cell Secretory Protein (CCSP, or CC16) is produced mainly by non-ciliated airway epithelial cells including bronchiolar club cells and the change of its expression has been shown to associate with the progress and severity of Chronic Obstructive Pulmonary Disease (COPD). In an animal model, the lack of CC16 renders the animal susceptible to the tumorigenic effect of a major CS carcinogen. A recent population-based Tucson Epidemiological Study of Airway Obstructive Diseases (TESAOD) has indicated that the low serum CC16 concentration is closely linked with the smoke-related mortality, particularly that driven by the lung cancer. However, the study of CC16 expression in well-defined smoke exposure models has been lacking, and there is no experimental support for the potential causal link between CC16 and CS-induced pathophysiological changes in the lung. In the present study, we have found that airway CC16 expression was significantly repressed in COPD patients, in monkey CS exposure model, and in CS-induced mouse model of COPD. Additionally, the lack of CC16 exacerbated airway inflammation and alveolar loss in the mouse model. Therefore, CC16 may play an important protective role in CS-related diseases.
Assuntos
Pulmão/metabolismo , Fumar/metabolismo , Uteroglobina/metabolismo , Animais , Regulação para Baixo , Expressão Gênica , Pulmão/imunologia , Macaca mulatta , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumaça , Fumar/efeitos adversos , Nicotiana , Uteroglobina/genéticaRESUMO
RATIONALE: Phosphorylation of myristoylated alanine-rich C kinase substrate (phospho-MARCKS) at the phosphorylation site domain (PSD) is crucial for mucus granule secretion and cell motility, but little is known concerning its function in lung cancer. OBJECTIVES: We aimed to determine if MARCKS PSD activity can serve as a therapeutic target and to elucidate the molecular basis of this potential. METHODS: The clinical relevance of phospho-MARCKS was first confirmed. Next, we used genetic approaches to verify the functionality and molecular mechanism of phospho-MARCKS. Finally, cancer cells were pharmacologically inhibited for MARCKS activity and subjected to functional bioassays. MEASUREMENTS AND MAIN RESULTS: We demonstrated that higher phospho-MARCKS levels were correlated with shorter overall survival of lung cancer patients. Using shRNA silencing and ectopic expression of wild-type and PSD-mutated (S159/163A) MARCKS, we showed that elevated phospho-MARCKS promoted cancer growth and erlotinib resistance. Further studies demonstrated an interaction of phosphoinositide 3-kinase with MARCKS, but not with phospho-MARCKS. Interestingly, phospho-MARCKS acted in parallel with increased phosphatidylinositol (3,4,5)-triphosphate pools and AKT activation in cells. Through treatment with a 25-mer peptide targeting the MARCKS PSD motif (MPS peptide), we were able to suppress tumor growth and metastasis in vivo, and reduced levels of phospho-MARCKS, phosphatidylinositol (3,4,5)-triphosphate, and AKT activity. This peptide also enhanced the sensitivity of lung cancer cells to erlotinib treatment, especially those with sustained activation of phosphoinositide 3-kinase/AKT signaling. CONCLUSIONS: These results suggest a key role for MARCKS PSD in cancer disease and provide a unique strategy for inhibiting the activity of MARCKS PSD as a treatment for lung cancer.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Modelos Animais de Doenças , Cloridrato de Erlotinib , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Substrato Quinase C Rico em Alanina Miristoilada , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Transdução de Sinais/fisiologiaRESUMO
Airway epithelial cells are the first line of defense against viral infections and are instrumental in coordinating the inflammatory response. In this study, we demonstrate the synergistic stimulation of CXCL10 mRNA and protein, a key chemokine responsible for the early immune response to viral infection, following treatment of airway epithelial cells with IFN γ and influenza virus. The synergism also occurred when the cells were treated with IFN γ and a viral replication mimicker (dsRNA) both in vitro and in vivo. Despite the requirement of type I interferon (IFNAR) signaling in dsRNA-induced CXCL10, the synergism was independent of the IFNAR pathway since it wasn't affected by the addition of a neutralizing IFNAR antibody or the complete lack of IFNAR expression. Furthermore, the same synergistic effect was also observed when a CXCL10 promoter reporter was examined. Although the responsive promoter region contains both ISRE and NFκB sites, western blot analysis indicated that the combined treatment of IFN γ and dsRNA significantly augmented NFκB but not STAT1 activation as compared to the single treatment. Therefore, we conclude that IFN γ and dsRNA act in concert to potentiate CXCL10 expression in airway epithelial cells via an NFκB-dependent but IFNAR-STAT independent pathway and it is at least partly regulated at the transcriptional level.