RESUMO
Development of pneumonia is the most lethal consequence of influenza, increasing mortality more than 50-fold compared with uncomplicated infection. The spread of viral infection from conducting airways to the alveolar epithelium is therefore a pivotal event in influenza pathogenesis. We found that mitogenic stimulation with keratinocyte growth factor (KGF) markedly accelerated mortality after infectious challenge with influenza A virus (IAV). Coadministration of KGF with IAV markedly accelerated the spread of viral infection from the airways to alveoli compared with challenge with IAV alone, based on spatial and temporal analyses of viral nucleoprotein staining of lung tissue sections and dissociated lung cells. To better define the temporal relationship between KGF administration and susceptibility to IAV infection in vivo, we administered KGF 120, 48, 24, and 0 h before intrapulmonary IAV challenge and assessed the percentages of proliferating and IAV-infected, alveolar type II (AECII) cells in dispersed lung cell populations. Peak AECII infectivity coincided with the timing of KGF administration that also induced peak AECII proliferation. AECII from mice that were given intrapulmonary KGF before isolation and then infected with IAV ex vivo exhibited the same temporal pattern of proliferation and infectious susceptibility. KGF-induced increases in mortality, AECII proliferation, and enhanced IAV susceptibility were all reversed by pretreatment of the animals with the mTOR inhibitor rapamycin before mitogenic stimulation. Taken together, these data suggest mTOR signaling-dependent, mitogenic conditioning of AECII is a determinant of host susceptibility to infection with IAV.
Assuntos
Células Epiteliais Alveolares/metabolismo , Proliferação de Células/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos/farmacologia , Vírus da Influenza A/metabolismo , Mitógenos/farmacologia , Infecções por Orthomyxoviridae/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Suscetibilidade a Doenças/induzido quimicamente , Feminino , Camundongos , Camundongos Endogâmicos DBA , Infecções por Orthomyxoviridae/patologiaRESUMO
Lung surfactant proteins (SPs) play critical roles in surfactant function and innate immunity. SP-A and SP-D, members of the collectin family of C-type lectins, exhibit distinct ligand specificities, effects on surfactant structure, and host defense functions despite extensive structural homology. SP-A binds to dipalmitoylphosphatidylcholine (DPPC), the major surfactant lipid component, but not phosphatidylinositol (PI), whereas SP-D shows the opposite preference. Additionally, SP-A and SP-D recognize widely divergent pathogen-associated molecular patterns. Previous studies suggested that a ligand-induced surface loop conformational change unique to SP-A contributes to lipid binding affinity. To test this hypothesis and define the structural features of SP-A and SP-D that determine their ligand binding specificities, a structure-guided approach was used to introduce key features of SP-D into SP-A. A quadruple mutant (E171D/P175E/R197N/K203D) that introduced an SP-D-like loop-stabilizing calcium binding site into the carbohydrate recognition domain was found to interconvert SP-A ligand binding preferences to an SP-D phenotype, exchanging DPPC for PI specificity, and resulting in the loss of lipid A binding and the acquisition of more avid mannan binding properties. Mutants with constituent single or triple mutations showed alterations in their lipid and sugar binding properties that were intermediate between those of SP-A and SP-D. Structures of mutant complexes with inositol or methyl-mannose revealed an attenuation of the ligand-induced conformational change relative to wild-type SP-A. These studies suggest that flexibility in a key surface loop supports the distinctive lipid binding functions of SP-A, thus contributing to its multiple functions in surfactant structure and regulation, and host defense.
Assuntos
Modelos Moleculares , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Cinética , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Ligantes , Lipídeo A/química , Lipídeo A/metabolismo , Lipossomos , Mutagênese Sítio-Dirigida , Mutação , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Redobramento de Proteína , Estabilidade Proteica , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.
Assuntos
Cristalografia por Raios X/métodos , Proteolipídeos/metabolismo , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animais , Sítios de Ligação , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Conformação Proteica , Proteína A Associada a Surfactante Pulmonar/genética , RatosRESUMO
Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of toxic and infectious insults. In prior studies we found that recombinant human KGF accelerates clearance of bacteria from the murine lung by augmenting the function of alveolar macrophages (AM). In this study we tested the hypothesis that endogenous KGF plays a role in the maintenance of innate pulmonary defense against gram-negative bacterial infections. KGF-deficient mice exhibited delayed clearance of Escherichia coli from the lungs, attenuated phagocytosis by AM, and decreased antimicrobial activity in bronchoalveolar lavage (BAL) fluid, due in part to reductions in levels of surfactant protein A, surfactant protein D, and lysozyme. These immune deficits were accompanied by lower alveolar type II epithelial cell counts and reduced alveolar type II epithelial cell expression of collectin and lysozyme genes on a per cell basis. No significant between-group differences were detected in selected inflammatory cytokines or BAL inflammatory cell populations at baseline or after bacterial challenge in the wild-type and KGF-deficient mice. A single intranasal dose of recombinant human KGF reversed defects in bacterial clearance, AM function, and BAL fluid antimicrobial activity. We conclude that KGF supports alveolar innate immune defense through maintenance of alveolar antimicrobial protein levels and functions of AM. Together these data demonstrate a role for endogenous KGF in maintenance of normal pulmonary innate immune function.
Assuntos
Infecções por Escherichia coli/imunologia , Fator 7 de Crescimento de Fibroblastos/fisiologia , Imunidade Inata , Macrófagos Alveolares/imunologia , Pneumonia Bacteriana/imunologia , Animais , Células Cultivadas , Colectinas/genética , Colectinas/metabolismo , Infecções por Escherichia coli/metabolismo , Feminino , Expressão Gênica , Humanos , Macrófagos Alveolares/microbiologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muramidase/genética , Muramidase/metabolismo , Pneumonia Bacteriana/metabolismo , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/microbiologiaRESUMO
Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease affecting almost exclusively female-sexed individuals. The cysts represent regions of lung destruction caused by smooth muscle tumors containing mutations in one of the two tuberous sclerosis (TSC) genes. mTORC1 inhibition slows but does not stop LAM advancement. Furthermore, monitoring disease progression is hindered by insufficient biomarkers. Therefore, new treatment options and biomarkers are needed. LAM cells express melanocytic markers, including glycoprotein non-metastatic melanoma protein B (GPNMB). The function of GPNMB in LAM is currently unknown; however, GPNMB's unique cell surface expression on tumor versus benign cells makes GPNMB a potential therapeutic target, and persistent release of its extracellular ectodomain suggests potential as a serum biomarker. Here, we establish that GPNMB expression is dependent on mTORC1 signaling, and that GPNMB regulates TSC2-null tumor cell invasion in vitro. Further, we demonstrate that GPNMB enhances TSC2-null xenograft tumor growth in vivo, and that ectodomain release is required for this xenograft growth. We also show that GPNMB's ectodomain is released from the cell surface of TSC2-null cells by proteases ADAM10 and 17, and we identify the protease target sequence on GPNMB. Finally, we demonstrate that GPNMB's ectodomain is present at higher levels in LAM patient serum compared to healthy controls and that ectodomain levels decrease with mTORC1 inhibition, making it a potential LAM biomarker.
Assuntos
Biomarcadores Tumorais , Linfangioleiomiomatose , Glicoproteínas de Membrana , Linfangioleiomiomatose/metabolismo , Linfangioleiomiomatose/patologia , Linfangioleiomiomatose/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Animais , Biomarcadores Tumorais/metabolismo , Feminino , Camundongos , Linhagem Celular Tumoral , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proliferação de CélulasRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Ficus hirta Vahl., a traditional Chinese medicine commonly used in the Lingnan region, has been extensively used for liver disease treatment in China. Its notable antioxidant and anti-inflammatory properties have been reported in previous studies. However, its potential effect and underlying mechanism on liver fibrosis remains unclear. AIM OF STUDY: This study was aimed to investigate the effect and its underlying mechanism of Ficus hirta Vahl on liver fibrosis in vitro and in vivo. MATERIALS AND METHODS: The main components of Ficus hirta Vahl in blood were investigated by using UPLC-Q/TOF-MS/MS. Two animal models of liver fibrosis, the CCl4 and MCD induced mice, were used to assess the efficacy of Ficus hirta Vahl on liver fibrosis. Metabolomics was used to detect the level of metabolites in the serum of liver fibrosis mice after Ficus hirta Vahl treatment. Furthermore, the mechanism was validated in vitro using the human liver stellate cell line LX-2. The binding affinities of the active ingredients of Ficus hirta Vahl to the main targets of liver fibrosis were also determined. Finally, we identified the key active ingredients responsible for the treatment of liver fibrosis in vivo. RESULTS: Fibrosis and inflammatory markers were significant down-regulation in both CCl4 and MCD induced liver fibrosis mice after Ficus hirta Vahl administration in a dose-dependent manner. We found that Ficus hirta Vahl may primarily exert its effect on liver fibrosis through the glutathione metabolic pathway. Importantly, the glutathione metabolic pathway is closely associated with ferroptosis, and our subsequent in vitro experiments provided evidence supporting this association. Ficus hirta Vahl was found to modulate the GSH/GPX4 pathway, ultimately leading to the amelioration of liver fibrosis. Moreover, using serum pharmacochemistry and molecular docking, we successfully identified apigenin as a probable efficacious monomer for the management of liver fibrosis and subsequently validated its efficacy in mice with CCl4-induced hepatic fibrosis. CONCLUSION: Ficus hirta Vahl triggered the ferroptosis of hepatic stellate cell by regulating the GSH/GPX4 pathway, thereby alleviating liver fibrosis in mice. Moreover, apigenin is a key compound in Ficus hirta Vahl responsible for the effective treatment of liver fibrosis.
Assuntos
Ferroptose , Ficus , Glutationa , Células Estreladas do Fígado , Cirrose Hepática , Animais , Ficus/química , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Ferroptose/efeitos dos fármacos , Masculino , Humanos , Camundongos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Glutationa/metabolismo , Linhagem Celular , Tetracloreto de Carbono , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Extratos Vegetais/farmacologiaRESUMO
The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored mechanisms of silica-induced pulmonary fibrosis in human lung samples collected from patients with occupational exposure to silica and in a longitudinal mouse model of silicosis using multiple modalities including whole-lung single-cell RNA sequencing and histological, biochemical, and physiologic assessments. In addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor κΒ ligand (RANKL) in pulmonary lymphocytes, and alveolar type II cells. Anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated pulmonary fibrosis. We conclude that silica induces differentiation of pulmonary osteoclast-like cells leading to progressive lung injury, likely due to sustained elaboration of bone-resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.
Assuntos
Diferenciação Celular , Osteoclastos , Fibrose Pulmonar , Dióxido de Silício , Silicose , Dióxido de Silício/toxicidade , Animais , Humanos , Osteoclastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismo , Camundongos , Silicose/patologia , Silicose/metabolismo , Silicose/etiologia , Diferenciação Celular/efeitos dos fármacos , Ligante RANK/metabolismo , Modelos Animais de Doenças , Masculino , Pulmão/patologia , Pulmão/metabolismo , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Macrófagos Alveolares/efeitos dos fármacos , FemininoRESUMO
Cutaneous melanoma is one of the most prevalent tumors, and it is still a huge challenge in the current clinical treatment. Isoliquiritigenin (ISL), which is isolated from Glycyrrhiza uralensis Fisch., has been reported for its anti-tumor effect. However, the underlying mechanism and targets of ISL are still not be revealed clearly. In this study, differentiallyexpressedproteins were identified bylabel-free quantitative mass spectrometry. Two isoforms of the histone variant H2A.Z, including H2A.Z.1 and H2A.Z.2, were significantly down regulated after administration of ISL in melanoma. H2A.Z.1 was highly expressed in melanoma and correlated with poor prognosis of melanoma. The expression of H2A.Z was inhibited by ISL in a concentration-dependent manner. Overexpression of H2A.Z.1 in melanoma cell lines partly restored the repressed cell proliferation and cell cycle by ISL. Moreover, E2F1 was identified as one downstream target of H2A.Z.1, which was also highly expressed in melanoma and correlated with poor prognosis of melanoma. Furthermore, in vivo assays validated the inhibitory role of ISL in melanoma proliferation and the expression of H2A.Z.1 and E2F1.Aboveall,it is indicated that ISL inhibit melanoma proliferation via targeting H2A.Z.1-E2F1 pathway. These findings explain the anti-tumor mechanism of ISL and provide potential therapeutic targets for melanoma.
Assuntos
Chalconas , Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/metabolismo , Histonas , Neoplasias Cutâneas/tratamento farmacológico , Linhagem Celular Tumoral , Chalconas/farmacologia , Chalconas/uso terapêutico , Fator de Transcrição E2F1 , Melanoma Maligno CutâneoRESUMO
The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored the mechanisms of silica-induced pulmonary fibrosis in a mouse model using multiple modalities including whole-lung single-nucleus RNA sequencing. These analyses revealed that in addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor-κB ligand (RANKL) in pulmonary lymphocytes and alveolar type II cells. Furthermore, anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated silica-induced pulmonary fibrosis. We conclude that silica induces osteoclast-like differentiation of distinct recruited and tissue resident monocyte populations, leading to progressive lung injury, likely due to sustained elaboration of bone resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.
RESUMO
Pulmonary alveolar microlithiasis is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a pulmonary alveolar microlithiasis lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes and other proteins suggested a role for osteoclast-like cells in the host response to microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activation in a receptor activator of nuclear factor-κB ligand and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.
Assuntos
Pneumopatias , Osteogênese , Humanos , Homeostase , PulmãoRESUMO
Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of insults. In this study, we tested the hypothesis that KGF augments pulmonary host defense. We found that a single dose of intrapulmonary KGF enhanced the clearance of Escherichia coli or Pseudomonas aeruginosa instilled into the lungs 24 h later. KGF augmented the recruitment, phagocytic activity, and oxidant responses of alveolar macrophages, including lipopolysaccharide-stimulated nitric oxide release and zymosan-induced superoxide production. Less robust alveolar macrophage recruitment and activation was observed in mice treated with intraperitoneal KGF. KGF treatment was associated with increased levels of MIP1γ, LIX, VCAM, IGFBP-6, and GM-CSF in the bronchoalveolar lavage fluid. Of these, only GM-CSF recapitulated in vitro the macrophage activation phenotype seen in the KGF-treated animals. The KGF-stimulated increase in GM-CSF levels in lung tissue and alveolar lining fluid arose from the epithelium, peaked within 1 h, and was associated with STAT5 phosphorylation in alveolar macrophages, consistent with epithelium-driven paracrine activation of macrophage signaling through the KGF receptor/GM-CSF/GM-CSF receptor/JAK-STAT axis. Enhanced bacterial clearance did not occur in response to KGF administration in GM-CSF(-/-) mice, or in mice treated with a neutralizing antibody to GM-CSF. We conclude that KGF enhances alveolar host defense through GM-CSF-stimulated macrophage activation. KGF administration may constitute a promising therapeutic strategy to augment innate immune defenses in refractory pulmonary infections.
Assuntos
Fator 7 de Crescimento de Fibroblastos/farmacologia , Imunidade Inata , Pulmão/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Comunicação Parácrina/imunologia , Animais , Epitélio/imunologia , Fator 7 de Crescimento de Fibroblastos/administração & dosagem , Fator 7 de Crescimento de Fibroblastos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos KnockoutRESUMO
Surfactant protein A (SP-A), a C-type lectin, plays an important role in innate lung host defense against inhaled pathogens. Crystallographic SP-A·ligand complexes have not been reported to date, limiting available molecular information about SP-A interactions with microbial surface components. This study describes crystal structures of calcium-dependent complexes of the C-terminal neck and carbohydrate recognition domain of SP-A with d-mannose, D-α-methylmannose, and glycerol, which represent subdomains of glycans on pathogen surfaces. Comparison of these complexes with the unliganded SP-A neck and carbohydrate recognition domain revealed an unexpected ligand-associated conformational change in the loop region surrounding the lectin site, one not previously reported for the lectin homologs SP-D and mannan-binding lectin. The net result of the conformational change is that the SP-A lectin site and the surrounding loop region become more compact. The Glu-202 side chain of unliganded SP-A extends out into the solvent and away from the calcium ion; however, in the complexes, the Glu-202 side chain translocates 12.8 Å to bind the calcium. The availability of Glu-202, together with positional changes involving water molecules, creates a more favorable hydrogen bonding environment for carbohydrate ligands. The Lys-203 side chain reorients as well, extending outward into the solvent in the complexes, thereby opening up a small cation-friendly cavity occupied by a sodium ion. Binding of this cation brings the large loop, which forms one wall of the lectin site, and the adjacent small loop closer together. The ability to undergo conformational changes may help SP-A adapt to different ligand classes, including microbial glycolipids and surfactant lipids.
Assuntos
Metabolismo dos Carboidratos , Carboidratos/farmacologia , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/metabolismo , Animais , Cristalografia por Raios X , Glicerol/metabolismo , Glicerol/farmacologia , Lectinas/química , Lectinas/metabolismo , Ligantes , Manose/metabolismo , Manose/farmacologia , Metilmanosídeos/metabolismo , Metilmanosídeos/farmacologia , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína/efeitos dos fármacos , RatosRESUMO
Biohydrogen using migrated ammonia as nitrogen source, and biogas upgrading with hydrogen collected at biocathode in an integrated bioelectrochemical system (BES) were investigated, during the anaerobic digestion of Taihu blue algae. Under an applied voltage of 0.4 V, biohydrogen (202.87 mL) reached 2.34 and 2.90 times than groups with applied voltage of 0 V and 0.8 V, respectively. Moreover, biohydrogen of the group with 1000 mg/L initial ammonia addition (524.16 mL) reached 1.53 times than that the of the control. With 0.25 bar of H2 injected at the beginning (R1), highest methane production (286.62) mL and content (75.73%) were obtained. Comparing to other groups, not only microbial genus responsible for both aceticlastic and hydrogenotrophic methanogens of the group R1 were apparently enriched, but key enzymes related to methane production also acquired better abundances. Therefore, it's promising to conduct the ammonia alleviating, hydrogen producing and biogas upgrading simultaneously using BES.
Assuntos
Amônia , Biocombustíveis , Anaerobiose , Reatores Biológicos , Hidrogênio , Metano , NitrogênioRESUMO
Long noncoding RNAs (lncRNAs) play very important roles in cell differentiation. Our recent study has demonstrated that a novel lncRNA named lnc-OAD modulated 3T3-L1 adipocyte differentiation. In the present study, we examined the roles of lnc-OAD in bone morphogenetic protein 2- (BMP-2-) induced osteoblast differentiation. Lnc-OAD expression was increased during BMP-2-induced osteoblast differentiation in C3H10T1/2 mesenchymal stem cells and MC3T3-E1 preosteoblast cells. Knockdown of lnc-OAD expression by specific siRNA remarkably decreased early osteoblast differentiation. In addition, stable knockdown of lnc-OAD by lentivirus vector also significantly inhibited late osteoblast differentiation and matrix mineralization in vitro. Conversely, stably overexpressed lnc-OAD with lentiviral vector accelerated osteoblast differentiation. Mechanistically, knockdown of lnc-OAD reduced significantly the phosphorylation of AKT and the expression of Osterix induced by BMP-2, while overexpression of lnc-OAD enhanced the phosphorylation of AKT and the expression of Osterix. Taken together, our study suggests that lnc-OAD plays an important role in modulating BMP-2-induced osteoblast differentiation via, at least in part, regulating the AKT-Osterix signaling axis.
Assuntos
Proteína Morfogenética Óssea 2/biossíntese , Diferenciação Celular , Osteoblastos/metabolismo , RNA Longo não Codificante/biossíntese , Transdução de Sinais , Células 3T3-L1 , Animais , Proteína Morfogenética Óssea 2/genética , Camundongos , Osteoblastos/citologia , RNA Longo não Codificante/genéticaRESUMO
The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A-null mice and was increased in SP-D-overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D-mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/farmacologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/microbiologia , Divisão Celular/efeitos dos fármacos , Colectinas/farmacologia , Dactinomicina/farmacocinética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/metabolismo , Corantes Fluorescentes/farmacocinética , Predisposição Genética para Doença , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Testes de Sensibilidade Microbiana , Proteína A Associada a Surfactante Pulmonar/deficiência , Proteína A Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/biossíntese , Proteína D Associada a Surfactante Pulmonar/genética , Ratos , Ratos Sprague-Dawley , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/metabolismoRESUMO
We have reported that Gram-negative organisms decorated with rough lipopolysaccharide (LPS) are particularly susceptible to the direct antimicrobial actions of the pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D). In this study, we examined the lipid and LPS components required for the permeabilizing effects of the collectins on model bacterial membranes. Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), with or without rough Escherichia coli LPS (J5), smooth E. coli LPS (B5), or cholesterol, were loaded with self-quenching probes and exposed to native or oxidatively modified SP-A. Fluorescence that resulted from permeabilization of liposomes and diffusion of dyes was assessed by microscopy or fluorimetry. Human SP-A and melittin increased the permeability of J5 LPS/POPE liposomes, but not B5 LPS/POPE liposomes or control (POPE only) liposomes. At a human SP-A concentration of 100 microg/mL, the permeability of the J5 LPS/POPE membranes increased 4.4-fold (p < 0.02) compared to the control with no added SP-A. Rat SP-A and SP-D also permeabilized the J5-containing liposomes. Incorporation of cholesterol into J5 LPS/POPE liposomes at a POPE:cholesterol molar ratio of 1:0.15 blocked human SP-A or melittin-induced permeability (p < 0.05) compared to cholesterol-free liposomes. Exposure of human SP-A to surfactant lipid peroxidation blocked the permeabilizing activity of the protein. We conclude that SP-A permeabilizes phospholipid membranes in an LPS-dependent and rough LPS-specific manner, that the effect is neither SP-A- nor species-specific, and that oxidative damage to SP-A abolishes its membrane destabilizing properties. Incorporation of cholesterol into the membrane enhances resistance to permeabilization by SP-A, most likely by increasing the packing density and membrane rigidity.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Colectinas/farmacologia , Lipopolissacarídeos/metabolismo , Pulmão/metabolismo , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Colesterol/metabolismo , Colesterol/farmacologia , Colectinas/metabolismo , Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Lipossomos/metabolismo , Modelos Biológicos , Antígenos O/metabolismo , Antígenos O/farmacologia , Fosfatidiletanolaminas/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/farmacologia , RatosRESUMO
Surfactant protein B (SP-B) is secreted into the airspaces with surfactant phospholipids where it reduces surface tension and prevents alveolar collapse at end expiration. SP-B is a member of the saposin-like family of proteins, several of which have antimicrobial properties. SP-B lyses negatively charged liposomes and was previously reported to inhibit the growth of Escherichia coli in vitro; however, a separate study indicated that elevated levels of SP-B in the airspaces of transgenic mice did not confer resistance to infection. The goal of this study was to assess the antimicrobial properties of native SP-B and synthetic peptides derived from the native peptide. Native SP-B aggregated and killed clinical isolates of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and group B streptococcus by increasing membrane permeability; however, SP-B also lysed RBC, indicating that the membranolytic activity was not selective for bacteria. Both the antimicrobial and hemolytic activities of native SP-B were inhibited by surfactant phospholipids, suggesting that endogenous SP-B may not play a significant role in alveolar host defense. Synthetic peptides derived from native SP-B were effective at killing both Gram-positive and Gram-negative bacteria at low peptide concentrations (0.15-5.0 microM). The SP-B derivatives selectively lysed bacterial membranes and were more resistant to inhibition by phospholipids; furthermore, helix 1 (residues 7-22) retained significant antimicrobial activity in the presence of native surfactant. These results suggest that the role of endogenous SP-B in host defense may be limited; however, synthetic peptides derived from SP-B may be useful in the treatment of bacterial pneumonias.
Assuntos
Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Peptídeos/farmacologia , Proteína B Associada a Surfactante Pulmonar/farmacologia , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeos/genética , Proteína B Associada a Surfactante Pulmonar/genéticaRESUMO
We have reported that surfactant protein A kills some Gram-negative organisms by increasing membrane permeability. In this study, we investigated the physiologic importance of this activity and the effect of oxidative stress on the antimicrobial functions of SP-A in vitro and in vivo. Concentrated bronchoalveolar lavage fluids from SP-A+/+ mice increased the permeability of the Escherichia coli K12 cell membrane to a greater extent than lavage from SP-A-/- animals. Similarly, calcium-dependent surfactant-binding proteins of SP-A+/+ mice increased membrane permeability more than those from SP-A-/- mice and produced greater zonal killing of agar-embedded bacteria in a radial diffusion assay. Exposure of human SP-A to copper-initiated surfactant phospholipid peroxidation or to free radicals generated by human neutrophils in vitro increased the level of SP-A-associated carbonyl moieties and blocked the permeabilizing function of the protein. We also found that exposure of mice to 90% O2 for 4 days, sufficient to lead to consumption of glutathione, oxidation of protein thiols, and accumulation of airspace protein-associated carbonyl moieties, blocked the permeabilizing activity of lavage fluid from SP-A+/+ mice. We conclude that SP-A is a major microbial permeablizing factor in lavage fluid and that oxidative stress inhibits the antibacterial activity of SP-A by a mechanism that includes oxidative modification and functional inactivation of the protein.
Assuntos
Infecções por Escherichia coli/metabolismo , Pneumonia/metabolismo , Proteinose Alveolar Pulmonar/metabolismo , Alvéolos Pulmonares/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Cálcio/metabolismo , Colectinas/metabolismo , Humanos , Hiperóxia/metabolismo , Peroxidação de Lipídeos , Camundongos , Camundongos Mutantes , Oxirredução , Permeabilidade , Alvéolos Pulmonares/microbiologia , Proteína A Associada a Surfactante Pulmonar/genéticaRESUMO
Surfactant proteins A (SP-A) and D (SP-D) play an important role in the innate immune defenses of the respiratory tract. SP-A binds to the lipid A region of lipopolysaccharide (LPS), and SP-D binds to the core oligosaccharide region. Both proteins induce aggregation, act as opsonins for neutrophils and macrophages, and have direct antimicrobial activity. Bordetella pertussis LPS has a branched core structure and a nonrepeating terminal trisaccharide. Bordetella bronchiseptica LPS has the same structure, but lipid A is palmitoylated and there is a repeating O-antigen polysaccharide. The ability of SP-A and SP-D to agglutinate and permeabilize wild-type and LPS mutants of B. pertussis and B. bronchiseptica was examined. Previously, wild-type B. pertussis was shown to resist the effects of SP-A; however, LPS mutants lacking the terminal trisaccharide were susceptible to SP-A. In this study, SP-A was found to aggregate and permeabilize a B. bronchiseptica mutant lacking the terminal trisaccharide, while wild-type B. bronchiseptica and mutants lacking only the palmitoyl transferase or O antigen were resistant to SP-A. Wild-type B. pertussis and B. bronchiseptica were both resistant to SP-D; however, LPS mutants of either strain lacking the terminal trisaccharide were aggregated and permeabilized by SP-D. We conclude that the terminal trisaccharide protects Bordetella species from the bactericidal functions of SP-A and SP-D. The O antigen and palmitoylated lipid A of B. bronchiseptica play no role in this resistance.