ABSTRACT
BACKGROUND: Bacterial pneumonia is a major risk factor for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Pseudomonas aeruginosa (PA), an opportunistic pathogen with an increasing resistance acquired against multiple drugs, is one of the main causative agents of ALI and ARDS in diverse clinical settings. Given the anti-inflammatory role of the cannabinoid-2 receptor (CB2R), the effect of CB2R activation in the regulation of PA-induced ALI and inflammation was tested in a mouse model as an alternative to conventional antibiotic therapy. METHODS: In order to activate CB2R, a selective synthetic agonist, JWH133, was administered intraperitoneally (i.p.) to C57BL/6J mice. Furthermore, SR144528 (a selective CB2R antagonist) was administered in combination with JWH133 to test the specificity of the CB2R-mediated effect. PA was administered intratracheally (i.t.) for induction of pneumonia in mice. At 24 h after PA exposure, lung mechanics were measured using the FlexiVent system. The total cell number, protein content, and neutrophil population in the bronchoalveolar lavage fluid (BALF) were determined. The bacterial load in the whole lung was also measured. Lung injury was evaluated by histological examination and PA-induced inflammation was assessed by measuring the levels of BALF cytokines and chemokines. Neutrophil activation (examined by immunofluorescence and immunoblot) and PA-induced inflammatory signaling (analyzed by immunoblot) were also studied. RESULTS: CB2R activation by JWH133 was found to significantly reduce PA-induced ALI and the bacterial burden. CB2R activation also suppressed the PA-induced increase in immune cell infiltration, neutrophil population, and inflammatory cytokines. These effects were abrogated by a CB2R antagonist, SR144528, further confirming the specificity of the CB2R-mediated effects. CB2R-knock out (CB2RKO) mice had a significantly higher level of PA-induced inflammation as compared to that in WT mice. CB2R activation diminished the excess activation of neutrophils, whereas mice lacking CB2R had elevated neutrophil activation. Pharmacological activation of CB2R significantly reduced the PA-induced NF-κB and NLRP3 inflammasome activation, whereas CB2KO mice had elevated NLRP3 inflammasome. CONCLUSION: Our findings indicate that CB2R activation ameliorates PA-induced lung injury and inflammation, thus paving the path for new therapeutic avenues against PA pneumonia.
Subject(s)
Acute Lung Injury , Cannabinoids , Inflammation , Pseudomonas Infections , Receptor, Cannabinoid, CB2 , Animals , Mice , Acute Lung Injury/chemically induced , Acute Lung Injury/genetics , Acute Lung Injury/immunology , Acute Lung Injury/prevention & control , Cannabinoids/pharmacology , Cannabinoids/therapeutic use , Cytokines , Inflammasomes/genetics , Inflammasomes/immunology , Inflammation/genetics , Inflammation/immunology , Inflammation/prevention & control , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Pseudomonas aeruginosa , Receptors, Cannabinoid , Respiratory Distress Syndrome , Receptor, Cannabinoid, CB2/genetics , Receptor, Cannabinoid, CB2/immunology , Pseudomonas Infections/genetics , Pseudomonas Infections/immunology , Pseudomonas Infections/microbiology , Pseudomonas Infections/prevention & control , Disease Models, AnimalABSTRACT
BACKGROUND: Chronic tissue injury was shown to induce progressive scarring in fibrotic diseases such as idiopathic pulmonary fibrosis (IPF), while an array of repair/regeneration and stress responses come to equilibrium to determine the outcome of injury at the organ level. In the lung, type I alveolar epithelial (ATI) cells constitute the epithelial barrier, while type II alveolar epithelial (ATII) cells play a pivotal role in regenerating the injured distal lungs. It had been demonstrated that eukaryotic cells possess repair machinery that can quickly patch the damaged plasma membrane after injury, and our previous studies discovered the membrane-mending role of Tripartite motif containing 72 (TRIM72) that expresses in a limited number of tissues including the lung. Nevertheless, the role of alveolar epithelial cell (AEC) repair in the pathogenesis of IPF has not been examined yet. METHOD: In this study, we tested the specific roles of TRIM72 in the repair of ATII cells and the development of lung fibrosis. The role of membrane repair was accessed by saponin assay on isolated primary ATII cells and rat ATII cell line. The anti-fibrotic potential of TRIM72 was tested with bleomycin-treated transgenic mice. RESULTS: We showed that TRIM72 was upregulated following various injuries and in human IPF lungs. However, TRIM72 expression in ATII cells of the IPF lungs had aberrant subcellular localization. In vitro studies showed that TRIM72 repairs membrane injury of immortalized and primary ATIIs, leading to inhibition of stress-induced p53 activation and reduction in cell apoptosis. In vivo studies demonstrated that TRIM72 protects the integrity of the alveolar epithelial layer and reduces lung fibrosis. CONCLUSION: Our results suggest that TRIM72 protects injured lungs and ameliorates fibrosis through promoting post-injury repair of AECs.
Subject(s)
Alveolar Epithelial Cells/metabolism , Idiopathic Pulmonary Fibrosis/metabolism , Idiopathic Pulmonary Fibrosis/prevention & control , Lung/metabolism , Tripartite Motif Proteins/biosynthesis , Alveolar Epithelial Cells/drug effects , Animals , Bleomycin/toxicity , Female , HEK293 Cells , Humans , Idiopathic Pulmonary Fibrosis/chemically induced , Lung/drug effects , Male , Mice , Mice, 129 Strain , Mice, Knockout , Recombinant Proteins/biosynthesisABSTRACT
Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.
Subject(s)
Macrophages, Alveolar/cytology , Phagocytosis , Pseudomonas aeruginosa/physiology , Animals , Cells, Cultured , Female , Green Fluorescent Proteins/metabolism , Mice, Inbred C57BL , Phagocytosis/physiology , Receptors, Complement/metabolism , Receptors, IgG/metabolismABSTRACT
Studies showed that TRIM72 is essential for repair of alveolar cell membrane disruptions, and exogenous recombinant human TRIM72 protein (rhT72) demonstrated tissue-mending properties in animal models of tissue injury. Here we examine the mechanisms of rhT72-mediated lung cell protection in vitro and test the efficacy of inhaled rhT72 in reducing tissue pathology in a mouse model of ventilator-induced lung injury. In vitro lung cell injury was induced by glass beads and stretching. Ventilator-induced lung injury was modeled by injurious ventilation at 30 ml/kg tidal volume. Affinity-purified rhT72 or control proteins were added into culture medium or applied through nebulization. Cellular uptake and in vivo distribution of rhT72 were detected by imaging and immunostaining. Exogenous rhT72 maintains membrane integrity of alveolar epithelial cells subjected to glass bead injury in a dose-dependent manner. Inhaled rhT72 decreases the number of fatally injured alveolar cells, and ameliorates tissue-damaging indicators and cell injury markers after injurious ventilation. Using in vitro stretching assays, we reveal that rhT72 improves both cellular resilience to membrane wounding and membrane repair after injury. Image analysis detected rhT72 uptake by rat alveolar epithelial cells, which can be inhibited by a cholesterol-disrupting agent. In addition, inhaled rhT72 distributes to the distal lungs, where it colocalizes with phosphatidylserine detection on nonpermeabilized lung slices to label wounded cells. In conclusion, our study showed that inhaled rhT72 accumulates in injured lungs and protects lung tissue from ventilator injury, the mechanisms of which include improving cell resilience to membrane wounding, localizing to injured membrane, and augmenting membrane repair.
Subject(s)
Carrier Proteins/administration & dosage , Pulmonary Alveoli/metabolism , Recombinant Proteins/administration & dosage , Respiration, Artificial/adverse effects , Ventilator-Induced Lung Injury/prevention & control , Wound Healing , Administration, Inhalation , Animals , Cell Membrane/metabolism , Cells, Cultured , Humans , Membrane Proteins , Mice , Pulmonary Alveoli/injuries , Pulmonary Alveoli/pathology , Rats , Ventilator-Induced Lung Injury/etiology , Ventilator-Induced Lung Injury/pathologyABSTRACT
Alcohol exposure can affect brain development, leading to long-lasting behavioral problems, including cognitive impairment, which together is defined as fetal alcohol spectrum disorder (FASD). However, the fundamental mechanisms through which this occurs are largely unknown. In this study, we report that the exposure of postnatal day 7 (P7) mice to ethanol activates caspase-3 via cannabinoid receptor type-1 (CB1R) in neonatal mice and causes a reduction in methylated DNA binding protein (MeCP2) levels. The developmental expression of MeCP2 in mice is closely correlated with synaptogenesis and neuronal maturation. It was shown that ethanol treatment of P7 mice enhanced Mecp2 mRNA levels but reduced protein levels. The genetic deletion of CB1R prevented, and administration of a CB1R antagonist before ethanol treatment of P7 mice inhibited caspase-3 activation. Additionally, it reversed the loss of MeCP2 protein, cAMP response element binding protein (CREB) activation, and activity-regulated cytoskeleton-associated protein (Arc) expression. The inhibition of caspase-3 activity prior to ethanol administration prevented ethanol-induced loss of MeCP2, CREB activation, epigenetic regulation of Arc expression, long-term potentiation (LTP), spatial memory deficits and activity-dependent impairment of several signaling molecules, including MeCP2, in adult mice. Collectively, these results reveal that the ethanol-induced CB1R-mediated activation of caspase-3 degrades the MeCP2 protein in the P7 mouse brain and causes long-lasting neurobehavioral deficits in adult mice. This CB1R-mediated instability of MeCP2 during active synaptic maturation may disrupt synaptic circuit maturation and lead to neurobehavioral abnormalities, as observed in this animal model of FASD.
ABSTRACT
The complement system plays a critical role in immune responses against pathogens. However, its identity and regulation in the lung are not fully understood. This study aimed to explore the role of tripartite motif protein (TRIM) 72 in regulating complement receptor (CR) of the Ig superfamily (CRIg) in alveolar macrophage (AM) and innate immunity of the lung. Imaging, absorbance quantification, and flow cytometry were used to evaluate in vitro and in vivo AM phagocytosis with normal, or altered, TRIM72 expression. Pulldown, coimmunoprecipitation, and gradient binding assays were applied to examine TRIM72 and CRIg interaction. A pneumonia model was established by intratracheal injection of Pseudomonas aeruginosa. Mortality, lung bacterial burden, and cytokine levels in BAL fluid and lung tissues were examined. Our data show that TRIM72 inhibited CR-mediated phagocytosis, and release of TRIM72 inhibition led to increased AM phagocytosis. Biochemical assays identified CRIg as a binding partner of TRIM72, and TRIM72 inhibited formation of the CRIg-phagosome. Genetic ablation of TRIM72 led to improved pathogen clearance, reduced cytokine storm, and improved survival in murine models of severe pneumonia, specificity of which was confirmed by adoptive transfer of wild-type or TRIM72KO AMs to AM-depleted TRIM72KO mice. TRIM72 overexpression promoted bacteria-induced NF-κB activation in murine alveolar macrophage cells. Our data revealed a quiescent, noninflammatory bacterial clearance mechanism in the lung via AM CRIg, which is suppressed by TRIM72. In vivo data suggest that targeted suppression of TRIM72 in AM may be an effective measure to treat fatal pulmonary bacterial infections.
Subject(s)
Carrier Proteins/metabolism , Immunity, Innate/physiology , Lung/immunology , Macrophages, Alveolar/immunology , Receptors, Complement 3b/metabolism , Animals , Carrier Proteins/genetics , Membrane Proteins , Mice, Knockout , NF-kappa B/metabolism , Phagocytosis/physiology , Phagosomes/metabolism , Pneumonia, Bacterial/metabolism , Pneumonia, Bacterial/pathology , Tripartite Motif ProteinsABSTRACT
The present study was undertaken to evaluate the immediate and long-term effects of a single-day exposure to 5-Azacytidine (5-AzaC), a DNA methyltransferase inhibitor, on neurobehavioral abnormalities in mice. Our findings suggest that the 5-AzaC treatment significantly inhibited DNA methylation, impaired extracellular signal-regulated kinase (ERK1/2) activation and reduced expression of the activity-regulated cytoskeleton-associated protein (Arc). These events lead to the activation of caspase-3 (a marker for neurodegeneration) in several brain regions, including the hippocampus and cortex, two brain areas that are essential for memory formation and memory storage, respectively. 5-AzaC treatment of P7 mice induced significant deficits in spatial memory, social recognition, and object memory in adult mice and deficits in long-term potentiation (LTP) in adult hippocampal slices. Together, these data demonstrate that the inhibition of DNA methylation by 5-AzaC treatment in P7 mice causes neurodegeneration and impairs ERK1/2 activation and Arc protein expression in neonatal mice and induces behavioral abnormalities in adult mice. DNA methylation-mediated mechanisms appear to be necessary for the proper maturation of synaptic circuits during development, and disruption of this process by 5-AzaC could lead to abnormal cognitive function.
Subject(s)
Azacitidine/toxicity , Brain Diseases/chemically induced , Enzyme Inhibitors/toxicity , Memory Disorders/etiology , Neurodegenerative Diseases/chemically induced , Animals , Animals, Newborn , Brain/drug effects , Brain/metabolism , DNA Methylation/drug effects , DNA Methylation/genetics , Disease Models, Animal , Female , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Hippocampus/cytology , Hippocampus/drug effects , Long-Term Potentiation/drug effects , Long-Term Potentiation/genetics , Male , Maze Learning/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurodegenerative Diseases/pathology , Phosphopyruvate Hydratase/metabolism , Receptor, Cannabinoid, CB1/deficiency , Receptor, Cannabinoid, CB1/genetics , Signal Transduction/drug effects , Social BehaviorABSTRACT
RNA Polymerase II transcribes beyond what later becomes the 3' end of a mature messenger RNA (mRNA). The formation of most mRNA 3' ends results from pre-mRNA cleavage followed by polyadenylation. In vitro studies have shown that low concentrations of ATP stimulate the 3' cleavage reaction while high concentrations inhibit it, but the origin of these ATP effects is unknown. ATP might enable a cleavage factor kinase or activate a cleavage factor directly. To distinguish between these possibilities, we tested several ATP structural analogs in a pre-mRNA 3' cleavage reaction reconstituted from DEAE-fractionated cleavage factors. We found that adenosine 5'-(ß,γ-methylene)triphosphate (AMP-PCP) is an effective in vitro 3' cleavage inhibitor with an IC50 of â¼300 µM, but that most other ATP analogs, including adenosine 5'-(ß,γ-imido)triphosphate, which cannot serve as a protein kinase substrate, promoted 3' cleavage but less efficiently than ATP. In combination with previous literature data, our results do not support ATP stimulation of 3' cleavage through cleavage factor phosphorylation in vitro. Instead, the more likely mechanism is that ATP stimulates cleavage factor activity through direct cleavage factor binding. The mammalian 3' cleavage factors known to bind ATP include the cleavage factor II (CF IIm) Clp1 subunit, the CF Im25 subunit and poly(A) polymerase alpha (PAP). The yeast homolog of the CF IIm complex also binds ATP through yClp1. To investigate the mammalian complex, we used a cell-line expressing FLAG-tagged Clp1 to co-immunoprecipitate Pcf11 as a function of ATP concentration. FLAG-Clp1 co-precipitated Pcf11 with or without ATP and the complex was not affected by AMP-PCP. Diadenosine tetraphosphate (Ap4A), an ATP analog that binds the Nudix domain of the CF Im25 subunit with higher affinity than ATP, neither stimulated 3' cleavage in place of ATP nor antagonized ATP-stimulated 3' cleavage. The ATP-binding site of PAP was disrupted by site directed mutagenesis but a reconstituted 3' cleavage reaction containing a mutant PAP unable to bind ATP nevertheless underwent ATP-stimulated 3' cleavage. Fluctuating ATP levels might contribute to the regulation of pre-mRNA 3' cleavage, but the three subunits investigated here do not appear to be responsible for the ATP-stimulation of pre-mRNA cleavage.
Subject(s)
Adenosine Triphosphate/metabolism , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional/physiology , mRNA Cleavage and Polyadenylation Factors/metabolism , HeLa Cells , HumansABSTRACT
Alveolar epithelial and endothelial cell injury is a major feature of the acute respiratory distress syndrome, in particular when in conjunction with ventilation therapies. Previously we showed [Kim SC, Kellett T, Wang S, Nishi M, Nagre N, Zhou B, Flodby P, Shilo K, Ghadiali SN, Takeshima H, Hubmayr RD, Zhao X. Am J Physiol Lung Cell Mol Physiol 307: L449-L459, 2014.] that tripartite motif protein 72 (TRIM72) is essential for amending alveolar epithelial cell injury. Here, we posit that TRIM72 improves cellular integrity through its interaction with caveolin 1 (Cav1). Our data show that, in primary type I alveolar epithelial cells, lack of TRIM72 led to significant reduction of Cav1 at the plasma membrane, accompanied by marked attenuation of caveolar endocytosis. Meanwhile, lentivirus-mediated overexpression of TRIM72 selectively increases caveolar endocytosis in rat lung epithelial cells, suggesting a functional association between these two. Further coimmunoprecipitation assays show that deletion of either functional domain of TRIM72, i.e., RING, B-box, coiled-coil, or PRY-SPRY, abolishes the physical interaction between TRIM72 and Cav1, suggesting that all theoretical domains of TRIM72 are required to forge a strong interaction between these two molecules. Moreover, in vivo studies showed that injurious ventilation-induced lung cell death was significantly increased in knockout (KO) TRIM72(KO) and Cav1(KO) lungs compared with wild-type controls and was particularly pronounced in double KO mutants. Apoptosis was accompanied by accentuation of gross lung injury manifestations in the TRIM72(KO) and Cav1(KO) mice. Our data show that TRIM72 directly and indirectly modulates caveolar endocytosis, an essential process involved in repair of lung epithelial cells through removal of plasma membrane wounds. Given TRIM72's role in endomembrane trafficking and cell repair, we consider this molecule an attractive therapeutic target for patients with injured lungs.
Subject(s)
Carrier Proteins/metabolism , Caveolae/metabolism , Endocytosis/physiology , Endothelial Cells/metabolism , Lung/metabolism , Animals , Apoptosis/physiology , Cell Death/physiology , Cell Membrane/metabolism , Cell Movement/physiology , Epithelial Cells/metabolism , Lung/cytology , Membrane Proteins , MiceABSTRACT
The significant consequences of ethanol use during pregnancy are neurobehavioral abnormalities involving hippocampal and neocortex malfunctions that cause learning and memory deficits collectively named fetal alcohol spectrum disorder. However, the molecular mechanisms underlying these abnormalities are still poorly understood and therefore warrant systematic research. Here, we document novel epigenetic abnormalities in the mouse model of fetal alcohol spectrum disorder. Ethanol treatment of P7 mice, which induces activation of caspase 3, impaired DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) levels. Inhibition of caspase 3 activity, before ethanol treatment, rescued DNMT1, DNMT3A proteins as well as DNA methylation levels. Blockade of histone methyltransferase (G9a) activity or cannabinoid receptor type-1 (CB1R), prior to ethanol treatment, which, respectively, inhibits or prevents activation of caspase 3, rescued the DNMT1 and DNMT3A proteins and DNA methylation. No reduction of DNMT1 and DNMT3A proteins and DNA methylation was found in P7 CB1R null mice, which exhibit no ethanol-induced activation of caspase 3. Together, these data demonstrate that ethanol-induced activation of caspase 3 impairs DNA methylation through DNMT1 and DNMT3A in the neonatal mouse brain, and such impairments are absent in CB1R null mice. Epigenetic events mediated by DNA methylation may be one of the essential mechanisms of ethanol teratogenesis. Schematic mechanism of action by which ethanol impairs DNA methylation. Studies have demonstrated that ethanol has the capacity to bring epigenetic changes to contribute to the development of fetal alcohol spectrum disorder (FASD). However, the mechanisms are not well studied. P7 ethanol induces the activation of caspase 3 and impairs DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) proteins (â). The inhibition or genetic ablation of cannabinoid receptor type-1 or inhibition of histone methyltransferase (G9a) by Bix (-----) or inhibition of caspase 3 activation by Q- quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh) () rescue loss of DNMT1, DNMT3A as well as DNA methylation. Hence, the putative DNMT1/DNMT3A/DNA methylation mechanism may have a potential regulatory role in FASD.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/biosynthesis , DNA Methylation/drug effects , DNA Methylation/physiology , Ethanol/toxicity , Receptor, Cannabinoid, CB1/deficiency , Animals , Animals, Newborn , Brain/drug effects , Brain/metabolism , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/antagonists & inhibitors , DNA Methyltransferase 3A , Female , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure.
Subject(s)
Carrier Proteins/metabolism , Cell Membrane/metabolism , Cell Membrane/pathology , Epithelial Cells , Pulmonary Alveoli , Wound Healing , Animals , Carrier Proteins/genetics , Caveolin 1/genetics , Caveolin 1/metabolism , Cell Membrane/genetics , Cells, Cultured , Epithelial Cells/metabolism , Epithelial Cells/pathology , Membrane Proteins , Mice , Mice, Knockout , Pulmonary Alveoli/injuries , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/pathologyABSTRACT
In rodents, many exogenous and endogenous cannabinoids, such as anandamide (AEA) and 2-arachidonyl glycerol (2-AG), have been shown to play an important role in certain hippocampal memory processes. However, the mechanisms by which endogenous AEA regulate this processes are not well understood. Here the effects of AEA on long-term potentiation (LTP), hippocampal-dependent learning and memory tasks, pERK1/2, pCaMKIV, and pCREB signaling events in both cannabinoid receptor type 1 (CB1R) wild-type (WT) and knockout (KO) mice were assessed following administration of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH). Acute administration of URB597 enhanced AEA levels without affecting the levels of 2-AG or CB1R in the hippocampus and neocortex as compared to vehicle. In hippocampal slices, URB597 impaired LTP in CB1R WT but not in KO littermates. URB597 impaired object recognition, spontaneous alternation and spatial memory in the Y-maze test in CB1R WT mice but not in KO mice. Furthermore, URB597 enhanced ERK phosphorylation in WT without affecting total ERK levels in WT or KO mice. URB597 impaired CaMKIV and CREB phosphorylation in WT but not in KO mice. CB1R KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio as compared to WT littermates. Our results indicate that pharmacologically elevated AEA impair LTP, learning and memory and inhibit CaMKIV and CREB phosphorylation, via the activation of CB1Rs. Collectively, these findings also suggest that pharmacological elevation of AEA beyond normal concentrations is also detrimental for the underlying physiological responses.
Subject(s)
Arachidonic Acids/physiology , Endocannabinoids/physiology , Learning/physiology , Long-Term Potentiation/physiology , Memory/physiology , Receptor, Cannabinoid, CB1/physiology , Amidohydrolases/antagonists & inhibitors , Animals , Benzamides/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 4/physiology , Carbamates/pharmacology , Cyclic AMP Response Element-Binding Protein/physiology , Glycerides/physiology , MAP Kinase Signaling System/physiology , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Polyunsaturated Alkamides , Protein Processing, Post-Translational , Receptor, Cannabinoid, CB1/deficiency , Receptor, Cannabinoid, CB1/genetics , Spatial Memory/physiologyABSTRACT
The 3' end formation of mammalian pre-mRNA contributes to gene expression regulation by setting the downstream boundary of the 3' untranslated region, which in many genes carries regulatory sequences. A large number of protein cleavage factors participate in this pre-mRNA processing step, but chemical tools to manipulate this process are lacking. Guided by a hypothesis that a PPM1 family phosphatase negatively regulates the 3' cleavage reaction, we have found a variety of new small molecule activators of the in vitro reconstituted pre-mRNA 3' cleavage reaction. New activators include a cyclic peptide PPM1D inhibitor, a dipeptide with modifications common to histone tails, abscisic acid and an improved l-arginine ß-naphthylamide analog. The minimal concentration required for in vitro cleavage has been improved from 200µM to the 200nM-100µM range. These compounds provide unexpected leads in the search for small molecule tools able to affect pre-mRNA 3' end formation.
Subject(s)
Peptides, Cyclic/pharmacology , RNA 3' End Processing/drug effects , Dose-Response Relationship, Drug , HeLa Cells , Humans , Molecular Structure , Molecular Weight , Structure-Activity RelationshipABSTRACT
BACKGROUND: Ethanol exposure to rodents during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces long-term potentiation and memory deficits. However, the molecular mechanisms underlying these deficits are still poorly understood. METHODS: In the present study, we explored the potential role of epigenetic changes at cannabinoid type 1 (CB1R) exon1 and additional CB1R functions, which could promote memory deficits in animal models of fetal alcohol spectrum disorder. RESULTS: We found that ethanol treatment of P7 mice enhances acetylation of H4 on lysine 8 (H4K8ace) at CB1R exon1, CB1R binding as well as the CB1R agonist-stimulated GTPγS binding in the hippocampus and neocortex, two brain regions that are vulnerable to ethanol at P7 and are important for memory formation and storage, respectively. We also found that ethanol inhibits cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation and activity-regulated cytoskeleton-associated protein (Arc) expression in neonatal and adult mice. The blockade or genetic deletion of CB1Rs prior to ethanol treatment at P7 rescued CREB phosphorylation and Arc expression. CB1R knockout mice exhibited neither ethanol-induced neurodegeneration nor inhibition of CREB phosphorylation or Arc expression. However, both neonatal and adult mice did exhibit enhanced CREB phosphorylation and Arc protein expression. P7 ethanol-treated adult mice exhibited impaired spatial and social recognition memory, which were prevented by the pharmacological blockade or deletion of CB1Rs at P7. CONCLUSIONS: Together, these findings suggest that P7 ethanol treatment induces CB1R expression through epigenetic modification of the CB1R gene, and that the enhanced CB1R function induces pCREB, Arc, spatial, and social memory deficits in adult mice.
Subject(s)
Ethanol/toxicity , Gene Expression Regulation/drug effects , Memory Disorders/chemically induced , Neurodegenerative Diseases/chemically induced , Receptor, Cannabinoid, CB1/drug effects , Receptor, Cannabinoid, CB1/metabolism , AIDS-Related Complex/metabolism , Acetylation/drug effects , Age Factors , Animals , Animals, Newborn/metabolism , Animals, Newborn/psychology , CREB-Binding Protein/metabolism , Central Nervous System Depressants/toxicity , Epigenesis, Genetic/drug effects , Exons/drug effects , Female , Hippocampus/drug effects , Hippocampus/metabolism , Histones/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neocortex/drug effects , Neocortex/metabolism , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/psychology , Phosphorylation/drug effects , Receptor, Cannabinoid, CB1/deficiency , Receptor, Cannabinoid, CB1/genetics , Social Behavior , Up-Regulation/drug effectsABSTRACT
Glycan array analysis of Sclerotium rolfsii lectin (SRL) revealed its exquisite binding specificity to the oncofetal Thomsen-Friedenreich (Galß1-3GalNAcα-O-Ser/Thr, T or TF) antigen and its derivatives. This study shows that SRL strongly inhibits the growth of human colon cancer HT29 and DLD-1 cells by binding to cell surface glycans and induction of apoptosis through both the caspase-8 and -9 mediated signaling. SRL showed no or very weak binding to normal human colon tissues but strong binding to cancerous and metastatic tissues. Intratumor injection of SRL at subtoxic concentrations in NOD-SCID mice bearing HT29 xenografts resulted in total tumor regression in 9 days and no subsequent tumor recurrence. As the increased expression of TF-associated glycans is commonly seen in human cancers, SRL has the potential to be developed as a therapeutic agent for cancer.
Subject(s)
Antigens, Neoplasm/metabolism , Antigens, Tumor-Associated, Carbohydrate/metabolism , Ascomycota/chemistry , Colonic Neoplasms/drug therapy , Lectins/therapeutic use , Animals , Antigens, Neoplasm/immunology , Antigens, Tumor-Associated, Carbohydrate/immunology , Apoptosis/drug effects , Caspase 8/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Humans , Injections , Lectins/isolation & purification , Lectins/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation , Neoplasms, Experimental , Polysaccharides/chemistry , Polysaccharides/immunology , Protein Binding , Signal Transduction/drug effectsABSTRACT
We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate the involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.
Subject(s)
Cytokines/metabolism , Immunomodulation , Lectins/immunology , Leukocyte Common Antigens/immunology , Rhizoctonia/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Cell Proliferation , Cells, Cultured , Humans , Leukocytes, Mononuclear/immunology , Phosphorylation , STAT5 Transcription Factor/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium.
ABSTRACT
BACKGROUND: Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus Rhizoctonia bataticola is highly mitogenic towards human peripheral blood mononuclear cells (PBMC). The lectin has sugar specificity towards N-glycans and binds to glycoproteins containing complex N-glycans (Nagre et al., Glycoconj J. 2010). In this study, we investigated the role of Mitogen Activated Protein Kinase (MAPK) and Signal Transducers and Activators of Transcription (STAT)-5 signaling in RBL-induced proliferation and production of Th1/Th2 cytokines. METHODS: Human PBMC were stimulated with RBL and proliferation was determined by tritiated thymidine incorporation assay, cytokine profiles by ELISA and activation of MAPK and STAT-5 by western blotting. RBL binding was monitored by immunofluorescence staining. Expression of IL-2Rα (CD25) was measured by flow cytometry. RESULTS: The binding and mitogenic activities of RBL were inhibited by glycoproteins- mucin, asialofetuin and fetuin. RBL stimulated expression of IL-2Rα and production of Th1/Th2 cytokines- IL-2, IFN-γ, IL-4 and IL-10. RBL-induced phosphorylation of ERK1/2 and p38 MAPK was detected at 1h and 3h respectively. Significant phosphorylation of STAT-5 (tyr(694)) was observed at 12h. Pharmacological inhibitors of p38 MAPK (SB203580) and JAK/STAT (AG490) but not ERK (PD98059) abrogated proliferation. RBL-induced expression of IL-2Rα and secretion of cytokines were drastically inhibited by SB203580 and AG490. CONCLUSIONS: RBL-induced proliferation and production of Th1/Th2 cytokines are mediated via p38 MAPK and STAT-5 signaling. GENERAL SIGNIFICANCE: RBL, a lectin with complex sugar specificity, is strongly mitogenic to human PBMC and stimulates the production of Th1 and Th2 cytokines. The results identified the signaling mechanism underlying the immunostimulatory activity of RBL.
Subject(s)
Cytokines/metabolism , Fungal Proteins/pharmacology , Lectins/pharmacology , Leukocytes, Mononuclear/drug effects , Rhizoctonia/metabolism , Signal Transduction/drug effects , Blotting, Western , Cell Proliferation/drug effects , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fungal Proteins/metabolism , Humans , Imidazoles/pharmacology , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-2/metabolism , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-4/metabolism , Lectins/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Mitogens/metabolism , Mitogens/pharmacology , Protein Binding , Pyridines/pharmacology , STAT5 Transcription Factor/metabolism , Tyrphostins/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
A lectin with strong mitogenic activity towards human peripheral blood mononuclear cells (PBMCs) and cytotoxic effect on human ovarian cancer cells has been purified from the mycelium of a phytopathogenic fungus, Rhizoctonia bataticola, using ion exchange chromatography and affinity chromatography on asialofetuin-Sepharose. The lectin, termed RBL, is a tetramer of 11-kDa subunits and has unique amino acid sequence at its blocked N-terminus. The purified RBL was blood group nonspecific and its hemagglutination activity was inhibited by mucin (porcine stomach), fetuin (fetal calf serum) and asialofetuin. Glycan array analysis revealed high affinity binding of RBL towards N-glycans and also the glycoproteins containing complex N-glycan chains. Interestingly, the lectin showed high affinity for glycans which are part of ovarian cancer marker CA125, a high molecular weight mucin containing high mannose and complex bisecting type N-linked glycans as well core 1 and 2 type O-glycans. RBL bound to human PBMCs eliciting strong mitogenic response, which could be blocked by mucin, fetuin and asialofetuin demonstrating the carbohydrate-mediated interaction with the cells. Analysis of the kinetics of binding of RBL to PBMCs revealed a delayed mitogenic response indicating a different signaling pathway compared to phytohemagglutinin-L. RBL had a significant cytotoxic effect on human ovarian cancer cell line, PA-1.
Subject(s)
Cell Survival/drug effects , Fungi/chemistry , Lectins/metabolism , Lectins/pharmacology , Mycelium/chemistry , Animals , Cell Line , Cell Line, Tumor , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Female , Flow Cytometry , Glycoproteins/metabolism , Hemagglutination Tests , Humans , Hydrogen-Ion Concentration , Lectins/chemistry , Lectins/isolation & purification , Molecular Weight , Ovarian Neoplasms/metabolism , Polysaccharides/metabolism , Protein Binding , RabbitsABSTRACT
A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80 degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 microg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.