RESUMEN

BACKGROUND & AIMS: CXADR-like membrane protein (CLMP) is structurally related to coxsackie and adenovirus receptor. Pathogenic variants in CLMP gene have been associated with congenital short bowel syndrome, implying a role for CLMP in intestinal development. However, the contribution of CLMP to regulating gut development and homeostasis is unknown. METHODS: In this study, we investigated CLMP function in the colonic epithelium using complementary in vivo and in vitro approaches, including mice with inducible intestinal epithelial cell (IEC)-specific deletion of CLMP (ClmpΔIEC), intestinal organoids, IECs with overexpression, or loss of CLMP and RNA sequencing data from individuals with colorectal cancer. RESULTS: Loss of CLMP enhanced IEC proliferation and, conversely, CLMP overexpression reduced proliferation. Xenograft experiments revealed increased tumor growth in mice implanted with CLMP-deficient colonic tumor cells, and poor engraftment was observed with CLMP-overexpressing cells. ClmpΔIEC mice showed exacerbated tumor burden in an azoxymethane and dextran sulfate sodium-induced colonic tumorigenesis model, and CLMP expression was reduced in human colorectal cancer samples. Mechanistic studies revealed that CLMP-dependent regulation of IEC proliferation is linked to signaling through mTOR-Akt-ß-catenin pathways. CONCLUSIONS: These results reveal novel insights into CLMP function in the colonic epithelium, highlighting an important role in regulating IEC proliferation, suggesting tumor suppressive function in colon cancer.

Asunto(s)

Colitis , Neoplasias del Colon , Animales , Humanos , Ratones , Proliferación Celular , Colitis/inducido químicamente , Colitis/metabolismo , Neoplasias del Colon/patología , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Células Epiteliales/patología , Mucosa Intestinal/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo

RESUMEN

Intrinsic activity of aldehyde dehydrogenase (ALDH)2, a cardiac mitochondrial enzyme, is vital in detoxifying 4-hydroxy-2-nonenal (4HNE) like cellular reactive carbonyl species (RCS) and thereby conferring cardiac protection against pathological stress. It was also known that a single point mutation (E487K) in ALDH2 (prevalent in East Asians) known as ALDH2*2 reduces its activity intrinsically and was associated with increased cardiovascular diseases. We and others have shown that ALDH2 activity is reduced in several pathologies in WT animals as well. Thus, exogenous augmentation of ALDH2 activity is a good strategy to protect the myocardium from pathologies. In this study, we will test the efficacy of intracardiac injections of the ALDH2 gene in mice. We injected both wild type (WT) and ALDH2*2 knock-in mutant mice with ALDH2 constructs, AAv9-cTNT-hALDH2-HA tag-P2A-eGFP or their control constructs, AAv9-cTNT-eGFP. We found that intracardiac ALDH2 gene transfer increased myocardial levels of ALDH2 compared to GFP alone after 1 and 3 weeks. When we subjected the hearts of these mice to 30 min global ischemia and 90 min reperfusion (I-R) using the Langendorff perfusion system, we found reduced infarct size in the hearts of mice with ALDH2 gene vs GFP alone. A single time injection has shown increased myocardial ALDH2 activity for at least 3 weeks and reduced myocardial 4HNE adducts and infarct size along with increased contractile function of the hearts while subjected to I-R. Thus, ALDH2 overexpression protected the myocardium from I-R injury by reducing 4HNE protein adducts implicating increased 4HNE detoxification by ALDH2. In conclusion, intracardiac ALDH2 gene transfer is an effective strategy to protect the myocardium from pathological insults.

Asunto(s)

Miocardio , Mutación Puntual , Ratones , Animales , Miocardio/metabolismo , Aldehído Deshidrogenasa Mitocondrial/genética , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Mitocondrias/metabolismo , Infarto/metabolismo

RESUMEN

PCR is a powerful tool for generating specific fragments of DNA that can be used to create gene variations or tagged expression constructs. Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we developed an optimized protocol. To accomplish this, three significant changes were made; 1) touchdown PCR cycling parameters were used to eliminate the need for optimizing PCR cycling conditions, 2) the high-fidelity, high-processivity Q5 DNA polymerase was used to improve full-length amplification quality, and 3) a reduced amount of primer in the final PCR amplification step decreased non-specific amplimers. This modified protocol results in consistent generation of gene fusion products, with little to no background and enhanced efficiency of the transgene construction process.

RESUMEN

Adenovirus-associated virus is a powerful vector system for transducing cells in vivo. It is widely used in animal systems due to high transduction efficiency of non-dividing cells with more than a dozen serotypes that have preferential tissue tropism. The viral genome remains episomal in the nucleus but maintains sustained expression in terminally differentiated cells for several weeks to months. Despite the popularity of recombinant AAV (rAAV) vectors, quality control testing of the virus after production is largely limited to physical characteristics such as viral genomes/ml determinations and silver staining acrylamide gels to determine purity. Functional testing, in vivo, is not practical due to high cost and restricted access of animal care and long duration of the assay (2-3 weeks). Some functional testing can be accomplished in cultured cells such as HEK293 cells, but HEK293 cells limit the types of rAAV constructs that can be tested. Many rAAV constructs are designed to study neurons in the brain with neural-specific promoters and many are floxed with loxp sites to be "activated" only in Cre-expressing neurons in transgenic animals. To develop a reporter cell line for rapid rAAV quality control assessment of these neural-specific, floxed rAAV constructs, we used the lentiviral system to stably express Cre recombinase in the SH-SY5Y neuroblastoma cell line. •A simple and economic method to evaluate recombinant AAV in vitro.•Allows functional validation of rAAV across a wide range of serotypes and promoters.•Allows functional validation of Cre-dependent rAAV constructs.

RESUMEN

Pathobiology of several chronic inflammatory disorders, including ulcerative colitis and Crohn's disease is related to intermittent, spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). In this report, we show that TNFα promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor, Src and Rac1 signaling to promote wound closure. Consistent with these findings, delayed colonic mucosal repair was observed after administration of a neutralizing TNFα antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa, the pro-inflammatory milieu containing TNFα and PAF sets the stage for reparative events mediated by PAFR signaling.

Asunto(s)

Epitelio/metabolismo , Membrana Mucosa/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Cicatrización de Heridas , Proteína ADAM10/metabolismo , Animales , Biomarcadores , Epitelio/patología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Membrana Mucosa/patología , FN-kappa B/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Especies Reactivas de Oxígeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Proteína de Unión al GTP rac1/metabolismo

RESUMEN

Junctional adhesion molecule-A (JAM-A) is a transmembrane glycoprotein expressed on leukocytes, endothelia, and epithelia that regulates biological processes including barrier function and immune responses. While JAM-A has been reported to facilitate tissue infiltration of leukocytes under inflammatory conditions, the contributions of leukocyte-expressed JAM-A in vivo remain unresolved. We investigated the role of leukocyte-expressed JAM-A in acute peritonitis induced by zymosan, lipopolysaccharide (LPS), or TNFα using mice with selective loss of JAM-A in myelomonocytic cells (LysM-Cre;Jam-afl/fl). Surprisingly, in LysM-Cre;Jam-afl/fl mice, loss of JAM-A did not affect neutrophil (PMN) recruitment into the peritoneum in response to zymosan, LPS, or TNFα although it was significantly reduced in Jam-aKO mice. In parallel, Jam-aKO peritoneal macrophages exhibited diminished CXCL1 chemokine production and decreased activation of NF-kB, whereas those from LysM-Cre;Jam-afl/fl mice were unaffected. Using Villin-Cre;Jam-afl/fl mice, targeted loss of JAM-A on intestinal epithelial cells resulted in increased intestinal permeability along with reduced peritoneal PMN migration as well as lower levels of CXCL1 and active NF-kB similar to that observed in Jam-aKO animals. Interestingly, in germ-free Villin-Cre;Jam-afl/fl mice, PMN recruitment was unaffected suggesting dependence on gut microbiota. Such observations highlight the functional link between a leaky gut and regulation of innate immune responses.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Neutrófilos/inmunología , Peritonitis/inmunología , Receptores de Superficie Celular/metabolismo , Uniones Estrechas/patología , Animales , Moléculas de Adhesión Celular/genética , Células Cultivadas , Quimiocina CXCL1/metabolismo , Modelos Animales de Enfermedad , Microbioma Gastrointestinal , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/metabolismo , Infiltración Neutrófila , Peritonitis/inducido químicamente , Permeabilidad , Receptores de Superficie Celular/genética , Zimosan

RESUMEN

Junctional adhesion molecule-A (JAM-A), an epithelial tight junction protein, plays an important role in regulating intestinal permeability through association with a scaffold signaling complex containing ZO-2, Afadin, and the small GTPase Rap2. Under inflammatory conditions, we report that the cytoplasmic tail of JAM-A is tyrosine phosphorylated (p-Y280) in association with loss of barrier function. While barely detectable Y280 phosphorylation was observed in confluent monolayers of human intestinal epithelial cells under basal conditions, exposure to cytokines TNFα, IFNγ, IL-22, or IL-17A, resulted in compromised barrier function in parallel with increased p-Y280. Phosphorylation was Src kinase dependent, and we identified Yes-1 and PTPN13 as a major kinase and phosphatase for p-JAM-A Y280, respectively. Moreover, cytokines IL-22 or IL-17A induced increased activity of Yes-1. Furthermore, the Src kinase inhibitor PP2 rescued cytokine-induced epithelial barrier defects and inhibited phosphorylation of JAM-A Y280 in vitro. Phosphorylation of JAM-A Y280 and increased permeability correlated with reduced JAM-A association with active Rap2. Finally, we observed increased phosphorylation of Y280 in colonic epithelium of individuals with ulcerative colitis and in mice with experimentally induced colitis. These findings support a novel mechanism by which tyrosine phosphorylation of JAM-A Y280 regulates epithelial barrier function during inflammation.

Asunto(s)

Células Epiteliales/metabolismo , Inflamación/patología , Intestinos/patología , Molécula A de Adhesión de Unión/metabolismo , Fosfotirosina/metabolismo , Secuencia de Aminoácidos , Animales , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Citocinas/farmacología , Sulfato de Dextran , Células HEK293 , Humanos , Intestinos/química , Ratones Endogámicos C57BL , Modelos Biológicos , Fosforilación/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 13/metabolismo , Proteínas Proto-Oncogénicas c-yes/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteínas de Unión al GTP rap/metabolismo

RESUMEN

Desmosomal cadherins mediate intercellular adhesion and have also been shown to regulate homeostatic signaling in epithelial cells. We have previously reported that select pro-inflammatory cytokines induce Dsg2 ectodomain cleavage and shedding from intestinal epithelial cells (IECs). Dsg2 extracellular cleaved fragments (Dsg2 ECF) function to induce paracrine pro-proliferative signaling in epithelial cells. In this study, we show that exposure of IECs to pro-inflammatory cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) resulted in Dsg2 intracellular cleavage and generation of a ~55 kDa fragment (Dsg2 ICF). Dsg2 intracellular cleavage is mediated by caspase-8 and occurs prior to Dsg2 extracellular cleavage and the execution of apoptosis. Expression of exogenous Dsg2 ICF in model IECs resulted in increased sensitivity to apoptotic stimuli and apoptosis execution. Additionally, expression of the Dsg2 ICF repressed the anti-apoptotic Bcl-2 family member proteins Bcl-XL and Mcl1. Taken together, our findings identify a novel mechanism by which pro-inflammatory mediators induce modification of Dsg2 to activate apoptosis and eliminate damaged cells, while also promoting release of Dsg2 ECF that promotes proliferation of neighboring cells and epithelial barrier recovery.

Asunto(s)

Apoptosis , Desmogleína 2/metabolismo , Células Epiteliales/metabolismo , Interferón gamma/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Caspasa 8/metabolismo , Desmogleína 2/genética , Células Epiteliales/citología , Humanos , Interferón gamma/genética , Procesamiento Proteico-Postraduccional , Factor de Necrosis Tumoral alfa/genética

RESUMEN

In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.

Asunto(s)

Proteínas CCN de Señalización Intercelular/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Antígenos CD11/metabolismo , Proliferación Celular , Colon/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Inflamación , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Cicatrización de Heridas

RESUMEN

The intestinal epithelium is a dynamic barrier that maintains the distinct environments of intestinal tissue and lumen. Epithelial barrier function is defined principally by tight junctions, which, in turn, depend on the regulated expression of claudin family proteins. Claudins are expressed differentially during intestinal epithelial cell (IEC) differentiation. However, regulatory mechanisms governing claudin expression during epithelial differentiation are incompletely understood. We investigated the molecular mechanisms regulating claudin-7 during IEC differentiation. Claudin-7 expression is increased as epithelial cells differentiate along the intestinal crypt-luminal axis. By using model IECs we observed increased claudin-7 mRNA and nascent heteronuclear RNA levels during differentiation. A screen for potential regulators of the CLDN7 gene during IEC differentiation was performed using a transcription factor/DNA binding array, CLDN7 luciferase reporters, and in silico promoter analysis. We identified hepatocyte nuclear factor 4α as a regulatory factor that bound endogenous CLDN7 promoter in differentiating IECs and stimulated CLDN7 promoter activity. These findings support a role of hepatocyte nuclear factor 4α in controlling claudin-7 expression during IEC differentiation.

Asunto(s)

Diferenciación Celular/genética , Claudinas/metabolismo , Células Epiteliales/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Mucosa Intestinal/metabolismo , Células CACO-2 , Claudinas/genética , Células Epiteliales/citología , Regulación de la Expresión Génica , Células HT29 , Factor Nuclear 4 del Hepatocito/genética , Humanos , Mucosa Intestinal/citología , Regiones Promotoras Genéticas

RESUMEN

Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD.

Asunto(s)

Anexina A1/fisiología , Exosomas/fisiología , Mucosa Intestinal/fisiopatología , Animales , Anexina A1/administración & dosificación , Antiinflamatorios/administración & dosificación , Línea Celular , Colitis/sangre , Colitis/fisiopatología , Humanos , Mucosa Intestinal/efectos de los fármacos , Ratones Noqueados , Nanopartículas , Péptidos/administración & dosificación , Cicatrización de Heridas

RESUMEN

The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. ß-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/ß-catenin signaling. Here we show that inhibition of Akt/ß-catenin-mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated ß-catenin 552 (pß-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits ß-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes ß-catenin transactivation through Akt-dependent C-terminal phosphorylation of ß-catenin to promote its association with 14.3.3ζ. Augmented ß-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/ß-catenin from the nucleus, thereby inhibiting ß-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.

Asunto(s)

Proteínas 14-3-3/metabolismo , Interferón gamma/farmacología , Mucosa Intestinal/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , beta Catenina/antagonistas & inhibidores , Animales , Células CHO , Línea Celular , Proliferación Celular , Cricetulus , Activación Enzimática , Inflamación , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal

RESUMEN

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.

Asunto(s)

Claudina-4/metabolismo , Claudinas/metabolismo , Interferón gamma/fisiología , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Animales , Células CHO , Células CACO-2 , Cricetinae , Cricetulus , Células HeLa , Humanos , Ratones , Multimerización de Proteína

RESUMEN

BACKGROUND: Allergic fungal rhinosinusitis (AFRS) is a disease demonstrating substantial eosinophilic inflammation and characteristic radiographic bony erosion/expansion. Periostin is an extracellular matrix protein associated with eosinophil accumulation in eosinophilic esophagitis, allergic asthma mucus production, and chronic rhinosinusitis (CRS) polyp formation. Receptor activator of nuclear factor κ-B ligand (RANKL) is an osteoclast activator present in osteoporosis and periodontal disease. We sought to evaluate periostin and RANKL expression in AFRS and correlate these levels with radiographic scales of disease severity. METHODS: Thirty sinus tissue specimens were intraoperatively collected from 3 patient groups: AFRS; CRS without nasal polyps (CRSsNP); and controls (n = 10 per group). Specimens were analyzed by semiquantitative reverse-transcription polymerase chain reaction (sq-RT-PCR) and immunofluorescence (IF) labeling/confocal microscopy for the presence of both periostin and RANKL. Immunofluorescence staining intensity was quantified by pixel density analysis. Preoperative computed tomography (CT) scans from each patient were scored using both the Lund-Mackay and CT bone erosion scoring systems. RESULTS: Periostin was significantly elevated in AFRS sinus tissue compared to CRSsNP and controls, as demonstrated by IF (p < 0.001) and PCR (p = 0.011). RANKL was not detected in sinus tissue by IF or PCR. Periostin levels positively correlated with radiographic indices of disease severity for both soft tissue and bone, using Lund-Mackay (r = 0.926 [PCR] and r = 0.581 [IF]) and CT bone erosion (r = 0.672 [PCR] and r = 0.616 [IF]) scoring systems, respectively. CONCLUSION: Periostin is increased in AFRS tissue compared to CRSsNP and controls. Periostin levels positively correlate with radiologic disease severity scores. The increased levels of periostin in AFRS are possibly tied to its intense eosinophilic inflammatory etiology.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Micosis/metabolismo , Ligando RANK/metabolismo , Rinitis Alérgica/metabolismo , Sinusitis/metabolismo , Adulto , Anciano , Moléculas de Adhesión Celular/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Micosis/diagnóstico por imagen , Pólipos Nasales/diagnóstico por imagen , Pólipos Nasales/metabolismo , Ligando RANK/genética , ARN Mensajero/metabolismo , Rinitis Alérgica/diagnóstico por imagen , Índice de Severidad de la Enfermedad , Sinusitis/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Adulto Joven

RESUMEN

Regional expression of Wingless/Int (Wnt) genes plays a central role in regulating intestinal development and homeostasis. However, our knowledge of such regional Wnt proteins in the colon remains limited. To understand further the effect of Wnt signaling components in controlling intestinal epithelial homeostasis, we investigated whether the physiological heterogeneity of the proximal and distal colon can be explained by differential Wnt signaling. With the use of a Wnt signaling-specific PCR array, expression of 84 Wnt-mediated signal transduction genes was analyzed, and a differential signature of Wnt-related genes in the proximal versus distal murine colon was identified. Several Wnt agonists (Wnt5a, Wnt8b, and Wnt11), the Wnt receptor frizzled family receptor 3, and the Wnt inhibitory factor 1 were differentially expressed along the colon length. These Wnt signatures were associated with differential epithelial cell proliferation and migration in the proximal versus distal colon. Furthermore, reduced Wnt/ß-catenin activity and decreased Wnt5a and Wnt11 expression were observed in mice lacking commensal bacteria, an effect that was reversed by conventionalization of germ-free mice. Interestingly, myeloid differentiation primary response gene 88 knockout mice showed decreased Wnt5a levels, indicating a role for Toll-like receptor signaling in regulating Wnt5a expression. Our results suggest that the morphological and physiological heterogeneity within the colon is in part facilitated by the differential expression of Wnt signaling components and influenced by colonization with bacteria.

Asunto(s)

Bacterias/metabolismo , Colon/microbiología , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Proliferación Celular , Colon/anatomía & histología , Colon/metabolismo , Regulación Bacteriana de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microbiota , Organismos Libres de Patógenos Específicos , Proteínas Wnt/genética , Proteína Wnt-5a

RESUMEN

The gastrointestinal epithelium functions as an important barrier that separates luminal contents from the underlying tissue compartment and is vital in maintaining mucosal homeostasis. Mucosal wounds in inflammatory disorders compromise the critical epithelial barrier. In response to injury, intestinal epithelial cells (IECs) rapidly migrate to reseal wounds. We have previously observed that a membrane-associated, actin binding protein, annexin A2 (AnxA2), is up-regulated in migrating IECs and plays an important role in promoting wound closure. To identify the mechanisms by which AnxA2 promotes IEC movement and wound closure, we used a loss of function approach. AnxA2-specific shRNA was utilized to generate IECs with stable down-regulation of AnxA2. Loss of AnxA2 inhibited IEC migration while promoting enhanced cell-matrix adhesion. These functional effects were associated with increased levels of ß1 integrin protein, which is reported to play an important role in mediating the cell-matrix adhesive properties of epithelial cells. Because cell migration requires dynamic turnover of integrin-based adhesions, we tested whether AnxA2 modulates internalization of cell surface ß1 integrin required for forward cell movement. Indeed, pulse-chase biotinylation experiments in IECs lacking AnxA2 demonstrated a significant increase in cell surface ß1 integrin that was accompanied by decreased ß1 integrin internalization and degradation. These findings support an important role of AnxA2 in controlling dynamics of ß1 integrin at the cell surface that in turn is required for the active turnover of cell-matrix associations, cell migration, and wound closure.

Asunto(s)

Anexina A2/metabolismo , Movimiento Celular/fisiología , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Integrina beta1/metabolismo , Mucosa Intestinal/metabolismo , Anexina A2/genética , Células CACO-2 , Adhesión Celular/fisiología , Matriz Extracelular/genética , Humanos , Integrina beta1/genética , Transporte de Proteínas/fisiología , Proteolisis , Cicatrización de Heridas/fisiología

RESUMEN

N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1(-/-IEC) and AnxA1(-/-) mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.

Asunto(s)

Anexina A1/metabolismo , Colitis Ulcerosa/metabolismo , Mucosa Intestinal/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas/metabolismo , Transducción de Señal , Cicatrización de Heridas , Animales , Anexina A1/genética , Línea Celular , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Femenino , Regulación de la Expresión Génica/genética , Humanos , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , NADPH Oxidasas/genética , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Péptidos/genética , Péptidos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 12/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 12/metabolismo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

Asunto(s)

Uniones Adherentes/metabolismo , Cadherinas/biosíntesis , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas de la Membrana/biosíntesis , Animales , Línea Celular , Claudina-1 , Perros , Epitelio/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Ocludina , Permeabilidad

RESUMEN

Symbionts often exhibit significant reductions in genome complexity while pathogens often exhibit increased complexity through acquisition and diversification of virulence determinants. A few organisms have evolved complex life cycles in which they interact as symbionts with one host and pathogens with another. How the predicted and opposing influences of symbiosis and pathogenesis affect genome evolution in such instances, however, is unclear. The Polydnaviridae is a family of double-stranded (ds) DNA viruses associated with parasitoid wasps that parasitize other insects. Polydnaviruses (PDVs) only replicate in wasps but infect and cause severe disease in parasitized hosts. This disease is essential for survival of the parasitoid's offspring. Thus, a true mutualism exists between PDVs and wasps as viral transmission depends on parasitoid survival and parasitoid survival depends on viral infection of the wasp's host. To investigate how life cycle and ancestry affect PDVs, we compared the genomes of Campoletis sonorensis ichnovirus (CsIV) and Microplitis demolitor bracovirus (MdBV). CsIV and MdBV have no direct common ancestor, yet their encapsidated genomes share several features including segmentation, diversification of virulence genes into families, and the absence of genes required for replication. In contrast, CsIV and MdBV share few genes expressed in parasitized hosts. We conclude that the similar organizational features of PDV genomes reflect their shared life cycle but that PDVs associated with ichneumonid and braconid wasps have likely evolved different strategies to cause disease in the wasp's host and promote parasitoid survival.

Asunto(s)

Genoma Viral , Polydnaviridae/genética , Polydnaviridae/patogenicidad , Animales , ADN Viral/genética , Lepidópteros/parasitología , Datos de Secuencia Molecular , Filogenia , Polydnaviridae/clasificación , Polydnaviridae/fisiología , Secuencias Repetitivas de Ácidos Nucleicos , Especificidad de la Especie , Simbiosis/genética , Virulencia/genética , Replicación Viral/genética , Avispas/virología

RESUMEN

The covalent attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues of target proteins, a process termed sumoylation, is a recently discovered protein modification that plays an important role in regulating many diverse cellular processes. For this reason there is significant interest in identifying new sumoylated proteins and the lysine residue(s) within these target proteins where SUMO attachment occurs. Such knowledge will allow determination of the functional consequences of sumoylation through mutation of the relevant sequences. This unit describes two different experimental approaches for ascertaining specific protein sumoylation: the first is based on immunoprecipitation of the protein of interest followed by SUMO immunoblotting. The second involves incubation of the protein (either an in vitro translation product or a purified recombinant protein) in a reconstituted in vitro sumoylation enzymatic reaction followed by visualization of sumoylated protein as a higher than normal molecular-weight band in SDS-PAGE.

Asunto(s)

Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Electroforesis en Gel de Poliacrilamida , Inmunoprecipitación