RESUMEN
Intestinal barrier function is required for the maintenance of mucosal homeostasis. Barrier dysfunction is thought to promote progression of both intestinal and systemic diseases. In many cases, this barrier loss reflects increased permeability of the paracellular tight junction as a consequence of myosin light chain kinase (MLCK) activation and myosin II regulatory light chain (MLC) phosphorylation. Although some details about MLCK activation remain to be defined, it is clear that this triggers perijunctional actomyosin ring (PAMR) contraction that leads to molecular reorganization of tight junction structure and composition, including occludin endocytosis. In disease states, this process can be triggered by pro-inflammatory cytokines including tumor necrosis factor-α (TNF), interleukin-1ß (IL-1ß), and several related molecules. Of these, TNF has been studied in the greatest detail and is known to activate long MLCK transcription, expression, enzymatic activity, and recruitment to the PAMR. Unfortunately, toxicities associated with inhibition of MLCK expression or enzymatic activity make these unsuitable as therapeutic targets. Recent work has, however, identified a small molecule that prevents MLCK1 recruitment to the PAMR without inhibiting enzymatic function. This small molecule, termed Divertin, restores barrier function after TNF-induced barrier loss and prevents disease progression in experimental chronic inflammatory bowel disease.
Asunto(s)
Permeabilidad de la Membrana Celular , Células Epiteliales/metabolismo , Homeostasis , Mucosa Intestinal/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Animales , Humanos , Transducción de SeñalRESUMEN
We engineered and employed a chaperone-like amyloid-binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross-reacted with amyloid beta-peptide (Aß42) protofibrils, but not with Aß40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1-hIAPP complex cross-react with Aß42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation-specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation-sensitive and sequence-independent and can target more than one type of protofibril species.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Amiloide/inmunología , Anticuerpos Monoclonales/inmunología , Fragmentos de Péptidos/inmunología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos/inmunología , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/inmunología , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Ratones , Nucleobindinas/inmunología , Nucleobindinas/metabolismo , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Células Piramidales/inmunología , Células Piramidales/metabolismoRESUMEN
Alzheimer's disease (AD) is the primary cause of age-related dementia. Effective strategies to prevent and treat AD remain elusive despite major efforts to understand its basic biology and clinical pathophysiology. Significant investments in therapeutic drug discovery programs over the past two decades have yielded some important insights but no blockbuster drugs to alter the course of disease. Because significant memory loss and cognitive decline are associated with neuron death and loss of gray matter, especially in the frontal cortex and hippocampus, some focus in drug development has shifted to early prevention of cellular pathology. Although clinical trial design is challenging, due in part to a lack of robust biomarkers with predictive value, some optimism has come from the identification and study of inherited forms of early-onset AD and genetic risk factors that provide insights about molecular pathophysiology and potential drug targets. In addition, better understanding of the Aß amyloid pathway and the tau pathway-leading to amyloid plaques and neurofibrillary tangles, respectively, which are histopathological hallmarks of AD-continues to drive significant drug research and development programs. The main focus of this review is to summarize the most recent basic biology, biochemistry, and pharmacology that serve as a foundation for more than 50 active advanced-phase clinical trials for AD prevention and therapy.
Asunto(s)
Envejecimiento/fisiología , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Inhibidores de la Colinesterasa/uso terapéutico , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Humanos , Inmunoterapia , Memantina/uso terapéutico , Enfermedades Metabólicas , Microglía/fisiología , Chaperonas Moleculares/metabolismo , Ovillos Neurofibrilares/efectos de los fármacos , Ovillos Neurofibrilares/metabolismo , Inflamación Neurogénica/tratamiento farmacológico , Neuroinmunomodulación , Proteínas tau/metabolismoRESUMEN
BACKGROUND & AIMS: Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding. METHODS: We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein-occludin or monomeric red fluorescent protein 1-ZO-1. After injection of high doses of TNF (7.5 µg intraperitoneally), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding. RESULTS: Changes detected included redistribution of the tight junction proteins ZO-1 and occludin to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities. CONCLUSIONS: Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function.
Asunto(s)
Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Citoesqueleto de Actina/metabolismo , Animales , Apoptosis/fisiología , Caspasas/metabolismo , Dinamina II/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteínas Fluorescentes Verdes/genética , Mucosa Intestinal/efectos de los fármacos , Proteínas Luminiscentes/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microtúbulos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Ocludina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte de Proteínas/fisiología , Uniones Estrechas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Proteína de la Zonula Occludens-1 , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo , Proteína Fluorescente RojaRESUMEN
Type 2 diabetes mellitus (T2D) is a major public health concern and is characterized by sustained hyperglycemia due to insulin resistance and destruction of insulin-producing ß cells. One pathological hallmark of T2D is the toxic accumulation of human islet amyloid polypeptide (hIAPP) aggregates. Monomeric hIAPP is a hormone normally co-secreted with insulin. However, increased levels of hIAPP in prediabetic and diabetic patients can lead to the formation of hIAPP protofibrils, which are toxic to ß cells. Current therapies fail to address hIAPP aggregation and current screening modalities do not detect it. Using a stabilizing capping protein, monoclonal antibodies (mAbs) can be developed against a previously nonisolatable form of hIAPP protofibrils, which are protofibril specific and do not engage monomeric hIAPP. Shown here are two candidate mAbs that can detect hIAPP protofibrils in serum and hIAPP deposits in pancreatic islets in a mouse model of rapidly progressing T2D. Treatment of diabetic mice with the mAbs delays disease progression and dramatically increases overall survival. These results demonstrate the potential for using novel hIAPP protofibril-specific mAbs as a diagnostic screening tool for early detection of T2D, as well as therapeutically to preserve ß cell function and target one of the underlying pathological mechanisms of T2D.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Animales , Humanos , Ratones , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/terapia , Insulina , Polipéptido Amiloide de los Islotes PancreáticosRESUMEN
Myosin light-chain kinase-dependent tight junction regulation is a critical event in inflammatory cytokine-induced increases in epithelial paracellular permeability. MLCK is expressed in human intestinal epithelium as two isoforms, long MLCK1 and long MLCK2, and MLCK1 is specifically localized to the tight junction, where it regulates paracellular permeability. The sole difference between these long MLCK splice variants is the presence of an immunoglobulin-like cell-adhesion molecule domain, IgCAM3, in MLCK1. To gain insight into the structure of the IgCAM3 domain, the IgCAM3 domain of MLCK1 has been expressed, purified and crystallized. Preliminary X-ray diffraction data were collected to 2.0â Å resolution and were consistent with the primitive trigonal space group P2(1)2(1)2(1).
Asunto(s)
Quinasa de Cadena Ligera de Miosina/química , Secuencia de Aminoácidos , Fraccionamiento Químico , Cristalización , Cristalografía por Rayos X/métodos , Difusión , Técnica del Anticuerpo Fluorescente , Humanos , Concentración de Iones de Hidrógeno , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiología , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Luminiscencia , Datos de Secuencia Molecular , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Homología de Secuencia de Aminoácido , Solubilidad , Uniones Estrechas/metabolismo , Uniones Estrechas/fisiología , Difracción de Rayos X , Rayos XRESUMEN
Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.
Asunto(s)
Homeostasis , Mucosa Intestinal/metabolismo , Espacio Intracelular/enzimología , Quinasa de Cadena Ligera de Miosina/metabolismo , Actomiosina/metabolismo , Animales , Células CACO-2 , Enfermedad Crónica , Homeostasis/efectos de los fármacos , Humanos , Inflamación/patología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/efectos de los fármacos , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Yeyuno/patología , Ratones , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/química , Fosforilación/efectos de los fármacos , Dominios Proteicos , Bibliotecas de Moléculas Pequeñas/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
During amyloid fibril formation, amyloidogenic polypeptides misfold and self assemble into soluble pre-fibrillar aggregates, i.e., protofibrils, which elongate and mature into insoluble fibrillar aggregates. An emerging class of chaperones, chaperone-like amyloid binding proteins (CLABPs), has been shown to interfere with aggregation of particular misfolded amyloid peptides or proteins. We have discovered that the calcium-binding protein nuclebindin-1 (NUCB1) is a novel CLABP. We show that NUCB1 inhibits aggregation of islet-amyloid polypeptide associated with type 2 diabetes mellitus, a-synuclein associated with Parkinson's disease, transthyretin V30M mutant associated with familial amyloid polyneuropathy, and Aß42 associated with Alzheimer's disease by stabilizing their respective protofibril intermediates. Kinetic studies employing the modeling software AmyloFit show that NUCB1 affects both primary nucleation and secondary nucleation. We hypothesize that NUCB1 binds to the common cross-ß-sheet structure of protofibril aggregates to "cap" and stabilize soluble macromolecular complexes. Transmission electron microscopy and atomic force microscopy were employed to characterize the size, shape and volume distribution of multiple sources of NUCB1-capped protofibrils. Interestingly, NUCB1 prevents Aß42 protofibril toxicity in a cellular assay. NUCB1-stabilized amyloid protofibrils could be used as immunogens to prepare conformation-specific antibodies and as novel tools to develop screens for anti-protofibril diagnostics and therapeutics.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al ADN/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/metabolismo , Prealbúmina/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/química , Péptidos beta-Amiloides/química , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Cinética , Microscopía de Fuerza Atómica , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Nucleobindinas , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Fragmentos de Péptidos/química , Prealbúmina/química , Unión Proteica , Estructura Secundaria de Proteína , alfa-Sinucleína/químicaRESUMEN
There is increasing evidence that hydrogen sulfide (H2S), produced by intestinal sulfate-reducing bacteria (SRB), may be involved in the etiopathogenesis of chronic diseases such as ulcerative colitis and colorectal cancer. The activity of SRB, and thus H2S production, is likely determined by the availability of sulfur-containing compounds in the intestine. However, little is known about the impact of dietary or inorganic sulfate on intestinal sulfate and SRB-derived H2S concentrations. In this study, the effects of short-term (7 day) and long-term (1 year) inorganic sulfate supplementation of the drinking water on gastrointestinal (GI) sulfate and H2S concentrations (and thus activity of resident SRBs), and the density of large intestinal sulfomucin-containing goblet cells, were examined in C3H/HeJBir mice. Additionally, a PCR-denaturing gradient gel electrophoresis (DGGE)-based molecular ecology technique was used to examine the impact of sulfate-amended drinking water on microbial community structure throughout the GI tract. Average H2S concentrations ranged from 0.1 mM (stomach) to 1 mM (cecum). A sulfate reduction assay demonstrated in situ production of H2S throughout the GI tract, confirming the presence of SRB. However, H2S generation and concentrations were greatest in the cecum and colon. Sulfate supplementation of drinking water did not significantly increase intestinal sulfate or H2S concentrations, suggesting that inorganic sulfate is not an important modulator of intestinal H2S concentrations, although it altered the bacterial profiles of the stomach and distal colon of 1-year-old mice. This change in colonic bacterial profiles may reflect a corresponding increase in the density of sulfomucin-containing goblet cells in sulfate-supplemented compared with control mice.
Asunto(s)
Sistema Digestivo/metabolismo , Sulfatos/administración & dosificación , Abastecimiento de Agua , Animales , Cromatografía por Intercambio Iónico , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Sistema Digestivo/microbiología , Sulfuro de Hidrógeno/metabolismo , Ratones , Ratones Endogámicos C3H , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismoRESUMEN
Epithelia form barriers that are essential to life. This is particularly true in the intestine, where the epithelial barrier supports nutrient and water transport while preventing microbial contamination of the interstitial tissues. Along with plasma membranes, the intercellular tight junction is the primary cellular determinant of epithelial barrier function. Disruption of tight junction structure, as a result of specific protein mutations or aberrant regulatory signals, can be both a cause and an effect of disease. Recent advances have provided new insights into the extracellular signals and intracellular mediators of tight junction regulation in disease states as well as into the interactions of intestinal barrier function with mucosal immune cells and luminal microbiota. In this review, we discuss the critical roles of the tight junction in health and explore the contributions of barrier dysfunction to disease pathogenesis.
Asunto(s)
Células Epiteliales/fisiología , Homeostasis/fisiología , Enfermedades Intestinales/fisiopatología , Animales , Membrana Celular/fisiología , Humanos , Mucosa Intestinal/fisiología , Uniones Estrechas/fisiologíaRESUMEN
Epithelial paracellular barrier function, determined primarily by tight junction permeability, is frequently disrupted in disease. In the intestine, barrier loss can be mediated by tumor necrosis factor (alpha) (TNF) signaling and epithelial myosin light chain kinase (MLCK) activation. However, TNF induces only limited alteration of tight junction morphology, and the events that couple structural reorganization to barrier regulation have not been defined. We have used in vivo imaging and transgenic mice expressing fluorescent-tagged occludin and ZO-1 fusion proteins to link occludin endocytosis to TNF-induced tight junction regulation. This endocytosis requires caveolin-1 and is essential for structural and functional tight junction regulation. These data demonstrate that MLCK activation triggers caveolin-1-dependent endocytosis of occludin to effect structural and functional tight junction regulation.
Asunto(s)
Caveolina 1/metabolismo , Endocitosis/fisiología , Proteínas de la Membrana/metabolismo , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Caveolina 1/genética , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Ocludina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transducción de Señal , Proteína de la Zonula Occludens-1RESUMEN
Permeability of the intestinal epithelial barrier is regulated in response to physiological and pathophysiological stimuli. Recent work has characterized a critical role of acute tight junction regulation in diarrhea secondary to T cell activation and cytokine release. The intracellular mediators of the ensuing barrier dysfunction include myosin light chain kinase, which phosphorylates myosin II regulatory light chain and triggers structural tight junction reorganization. While the molecular intermediates in this reorganization are not defined, the new discovery that individual tight junction-associated proteins are highly dynamic at steady state may provide insight into the mechanisms of regulation.
Asunto(s)
Uniones Estrechas/metabolismo , Animales , Permeabilidad de la Membrana Celular , Citocinas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Transducción de SeñalRESUMEN
Villus enterocyte nutrient absorption occurs via precisely orchestrated interactions among multiple transporters. For example, transport by the apical Na(+)-glucose cotransporter, SGLT1, triggers translocation of NHE3, Na(+)-H(+) antiporter isoform 3, to the plasma membrane. This translocation requires activation of p38 mitogen-activated protein kinase (MAPK), Akt2, and ezrin. Akt2 directly phosphorylates ezrin, but the precise role of p38 MAPK in this process remains to be defined. Sequence analysis suggested that p38 MAPK could not directly phosphorylate Akt2. We hypothesized that MAPKAPK-2 might link p38 MAPK and Akt2 activation. MAPKAPK-2 was phosphorylated after initiation of Na(+)-glucose cotransport with kinetics that paralleled activation of p38 MAPK, Akt2, and ezrin. MAPKAPK-2, Akt2, and ezrin phosphorylation were all attenuated by p38 MAPK inhibition but were unaffected by dominant negative ezrin expression. Akt2 inhibition blocked ezrin but not p38 MAPK or MAPKAPK-2 phosphorylation, suggesting that MAPKAPK-2 could be an intermediate in p38 MAPK-dependent Akt2 activation. Consistent with this, MAP-KAPK-2 could phosphorylate an Akt2-derived peptide in vitro. siRNA-mediated MAPKAPK-2 knockdown inhibited phosphorylation of Akt2 and ezrin but not p38 MAPK. MAPKAPK-2 knockdown also blocked NHE3 translocation. Thus, MAP-KAPK-2 controls Akt2 phosphorylation. In so doing, MAP-KAPK-2 links p38 MAPK to Akt2, ezrin, and NHE3 activation after SGLT1-mediated transport.
Asunto(s)
Glucosa/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sodio/metabolismo , Transporte Biológico , Línea Celular Tumoral , Proteínas del Citoesqueleto/metabolismo , Enterocitos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas , Fosforilación , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transportador 1 de Sodio-Glucosa/metabolismo , Intercambiador 3 de Sodio-HidrógenoRESUMEN
Myosin light chain kinase (MLCK) is expressed as long and short isoforms from unique transcriptional start sites within a single gene. Tumor necrosis factor (TNF) augments intestinal epithelial long MLCK expression, which is critical to cytoskeletal regulation. We found that TNF increases long MLCK mRNA transcription, both in human enterocytes in vitro and murine enterocytes in vivo.5'-RACE identified two novel exons, 1A and 1B, which encode alternative long MLCK transcriptional start sites. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis identified two essential Sp1 sites upstream of the exon 1A long MLCK transcriptional start site. Analysis of deletion and truncation mutants showed that a 102-bp region including these Sp1 sites was necessary for basal transcription. A promoter construct including 4-kb upstream of exon 1A was responsive to TNF, AP-1, or NFkappaB, but all except NFkappaB responses were absent in a shorter 2-kb construct, and all responses were absent in a 1-kb construct. Electrophoretic mobility shift assays, ChIP, and site-directed mutagenesis explained these data by identifying three functional AP-1 sites between 2- and 4-kb upstream of exon 1A and two NFkappaB sites between 1- and 2-kb upstream of exon 1A. Analysis of differentiating epithelia showed that only well differentiated enterocytes activated the 4-kb long MLCK promoter in response to TNF, and consensus promoter reporters demonstrated that TNF-induced NFkappaB activation decreased during differentiation while TNF-induced AP-1 activation increased. Thus either AP-1 or NFkappaB can up-regulate long MLCK transcription, but the mechanisms by which TNF up-regulates intestinal epithelial long MLCK transcription from exon 1A are differentiation-dependent.
Asunto(s)
Diferenciación Celular/fisiología , Regulación de la Expresión Génica , Quinasa de Cadena Ligera de Miosina/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal/fisiología , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células CACO-2 , Células Cultivadas , Enterocitos/citología , Enterocitos/fisiología , Exones , Femenino , Humanos , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Quinasa de Cadena Ligera de Miosina/genética , FN-kappa B/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
BACKGROUND & AIMS: Tumor necrosis factor (TNF) plays a critical role in intestinal disease. In intestinal epithelia, TNF causes tight junction disruption and epithelial barrier loss by up-regulating myosin light chain kinase (MLCK) activity and expression. The aim of this study was to determine the signaling pathways by which TNF causes intestinal epithelial barrier loss. METHODS: Caco-2 cells that were either nontransfected or stably transfected with human TNF receptor 1 (TNFR1) or TNFR2 and mouse colonocytes were used for physiologic, morphologic, and biochemical analyses. RESULTS: Colitis induced in vivo by adoptive transfer of CD4(+)CD45RB(hi) T cells was associated with increased epithelial MLCK expression and myosin II regulatory light chain (MLC) phosphorylation as well as morphologic tight junction disruption. In vitro studies showed that TNF caused similar increases in MLCK expression and MLC phosphorylation, as well as barrier dysfunction, in Caco-2 monolayers only after interferon (IFN)-gamma pretreatment. This reductionist model was therefore used to determine the molecular mechanism by which IFN-gamma and TNF synergize to cause intestinal epithelial barrier loss. IFN-gamma priming increased TNFR1 and TNFR2 expression, and blocking antibody studies showed that TNFR2, but not TNFR1, was required for TNF-induced barrier dysfunction. Transgenic TNFR2, but not TNFR1, expression allowed IFN-gamma-independent TNF responses. CONCLUSIONS: IFN-gamma primes intestinal epithelia to respond to TNF by inducing TNFR2 expression, which in turn mediates TNF-induced MLCK-dependent barrier dysfunction. The data further suggest that epithelial TNFR2 blockade may be a novel approach to restore barrier function in intestinal disease.
Asunto(s)
Interferón gamma/metabolismo , Enfermedades Intestinales/metabolismo , Enfermedades Intestinales/fisiopatología , Receptores del Factor de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células CACO-2 , Proteínas de Homeodominio/genética , Humanos , Interferón gamma/farmacología , Enfermedades Intestinales/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Regulación hacia Arriba/fisiologíaRESUMEN
Loss of E2A transcription factor activity or activation of the intracellular form of Notch1 (ICN) leads to the development of leukemia or lymphoma in humans or mice, respectively. Current models propose that ICN functions by suppressing E2A through a pre-T cell receptor (TCR)-dependent mechanism. Here we show that lymphomas arising in E2A(-/-) mice require the activation of Notch1 for their survival and have accumulated mutations in, or near, the Notch1 PEST domain, resulting in increased stability and signaling. In contrast, lymphomas arising in p53(-/-) mice show the activation of Notch1, but no mutations were identified in ICN. The requirement for Notch1 signaling in E2A(-/-) lymphomas cannot be overcome by ectopic expression of pTalpha; however, pTalpha is required for optimal survival and expansion of these cells. Our findings indicate that the activation of Notch1 is an important "second hit" for the transformation of E2A(-/-) T cell lymphomas and that Notch1 promotes survival through pre-TCR-dependent and -independent mechanisms.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Linfoma de Células T/inmunología , Receptor Notch1/genética , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Secuencia de Bases , Northern Blotting , Supervivencia Celular , Transformación Celular Neoplásica , Cartilla de ADN , Humanos , Linfoma de Células T/patología , Ratones , Ratones Noqueados , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
PURPOSE: A peptide inhibitor of myosin light chain kinase (MLCK), termed membrane permeant inhibitor of myosin light chain kinase (PIK), has previously been demonstrated to correct paracellular barrier defects associated with in vitro cell models of infectious and inflammatory intestinal disease. The current study describes a strategy to identify stable analogues of PIK required for future in vivo studies that has resulted in the identification of two promising candidates. METHODS: Because PIK functions at an intracellular site of epithelial cells and is envisaged to be administered orally, hydrolysis patterns were determined for PIK in both extracts of homogenized Caco-2 (a human intestinal epithelial cell line) and in luminal secretions isolated from rat intestine. Based on these hydrolysis patterns, four peptides Ac-RKKYKYRRK-NH(2) (acetylated PIK), rkkykyrrk-NH(2) (D PIK), krrykykkr-NH(2) (Dreverse PIK), and RKKykyRRK-NH(2) (Dpalindrome PIK) were synthesised. Studies were carried out to determine the stability, activity, and selectivity of these PIK analogues. RESULTS: D PIK and Dreverse PIK had much longer half-lives of 3.6 and 13.4 h, respectively, compared to PIK, acetylated (Ac)-PIK, or Dpalindrome PIK. All PIK analogues inhibited MLCK potently, although D PIK was a slightly better inhibitor than the other analogues. Similarly, all PIK analogues enhanced paracellular barrier function in Caco-2 monolayers studied in vitro. No appreciable inhibition of cAMP-dependent protein kinase (PKA) or calcium/calmodulin-dependent protein kinase II (CaMPKII) was detected with any of the analogues. CONCLUSIONS: PIK is quickly degraded within two enzyme-containing preparations that represent different aspects of the intestinal environment. The PIK analogues D PIK and Dreverse PIK demonstrated extended half-lives in these enzyme preparations while retaining the biological activity and specificity of the parent PIK peptide.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/química , Péptido Hidrolasas/química , Tecnología Farmacéutica/métodos , Animales , Células CACO-2 , Permeabilidad de la Membrana Celular/fisiología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Semivida , Humanos , Mucosa Intestinal/metabolismo , Intestinos/química , Intestinos/efectos de los fármacos , Quinasa de Cadena Ligera de Miosina/metabolismo , Oligopéptidos/antagonistas & inhibidores , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptido Hidrolasas/análisis , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Tecnología Farmacéutica/tendenciasRESUMEN
Numerous intestinal diseases are characterized by immune cell activation and compromised epithelial barrier function. We have shown that cytokine treatment of epithelial monolayers increases myosin II regulatory light chain (MLC) phosphorylation and decreases barrier function and that these are both reversed by MLC kinase (MLCK) inhibition. The aim of this study was to determine the mechanisms by which interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha regulate MLC phosphorylation and disrupt epithelial barrier function. We developed a model in which both cytokines were required for barrier dysfunction. Barrier dysfunction was also induced by TNF-alpha addition to IFN-gamma-primed, but not control, Caco-2 monolayers. TNF-alpha treatment of IFN-gamma-primed monolayers caused increases in both MLCK expression and MLC phosphorylation, suggesting that MLCK is a TNF-alpha-inducible protein. These effects of TNF-alpha were not mediated by nuclear factor-kappaB. However, at doses below those needed for nuclear factor-kappaB inhibition, sulfasalazine was able to prevent TNF-alpha-induced barrier dysfunction, MLCK up-regulation, and MLC phosphorylation. Low-dose sulfasalazine also prevented morphologically evident tight junction disruption induced by TNF-alpha. These data show that IFN-gamma can prime intestinal epithelial monolayers to respond to TNF-alpha by disrupting tight junction morphology and barrier function via MLCK up-regulation and MLC phosphorylation. These TNF-alpha-induced events can be prevented by the clinically relevant drug sulfasalazine.
Asunto(s)
Interferón gamma/fisiología , Quinasa de Cadena Ligera de Miosina/biosíntesis , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba , Células CACO-2 , Proliferación Celular , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Interferón gamma/metabolismo , Microscopía Fluorescente , FN-kappa B/metabolismo , Fosforilación , Sulfasalazina/farmacología , Uniones Estrechas , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND & AIMS: Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure. METHODS: Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically. RESULTS: Live-cell imaging of enhanced green fluorescent protein-beta-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds. CONCLUSIONS: These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction.
Asunto(s)
Mucosa Intestinal/fisiopatología , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Cicatrización de Heridas , Citoesqueleto de Actina , Actinas/metabolismo , Células CACO-2 , Colon/metabolismo , Colon/patología , Colon/fisiopatología , Sistemas de Computación , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/fisiopatología , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Cadenas Ligeras de Miosina/metabolismo , Permeabilidad , Fosforilación , Factores de Tiempo , Distribución Tisular , Quinasas Asociadas a rhoRESUMEN
Recent progress in genomics, proteomics, and bioinformatics enables unprecedented opportunities to examine the evolutionary history of molecular, cellular, and developmental pathways through phylogenomics. Accordingly, we have developed a motif analysis tool for phylogenomics (Phylomat, http://alg.ncsa.uiuc.edu/pmat) that scans predicted proteome sets for proteins containing highly conserved amino acid motifs or domains for in silico analysis of the evolutionary history of these motifs/domains. Phylomat enables the user to download results as full protein or extracted motif/domain sequences from each protein. Tables containing the percent distribution of a motif/domain in organisms normalized to proteome size are displayed. Phylomat can also align the set of full protein or extracted motif/domain sequences and predict a neighbor-joining tree from relative sequence similarity. Together, Phylomat serves as a user-friendly data-mining tool for the phylogenomic analysis of conserved sequence motifs/domains in annotated proteomes from the three domains of life.