RESUMEN
Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn's disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.
Asunto(s)
Actinas , Actomiosina , Humanos , Actinas/metabolismo , Actomiosina/metabolismo , Citocinesis , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Miosinas/metabolismo , Uniones Estrechas/metabolismo , Células CACO-2 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Epithelial tight junctions define the paracellular permeability of the intestinal barrier. Molecules can cross the tight junctions via two distinct size-selective and charge-selective paracellular pathways: the pore pathway and the leak pathway. These can be distinguished by their selectivities and differential regulation by immune cells. However, permeability increases measured in most studies are secondary to epithelial damage, which allows non-selective flux via the unrestricted pathway. Restoration of increased unrestricted pathway permeability requires mucosal healing. By contrast, tight junction barrier loss can be reversed by targeted interventions. Specific approaches are needed to restore pore pathway or leak pathway permeability increases. Recent studies have used preclinical disease models to demonstrate the potential of pore pathway or leak pathway barrier restoration in disease. In this Review, we focus on the two paracellular flux pathways that are dependent on the tight junction. We discuss the latest evidence that highlights tight junction components, structures and regulatory mechanisms, their impact on gut health and disease, and opportunities for therapeutic intervention.
Asunto(s)
Membrana Mucosa , Uniones Estrechas , Humanos , Uniones Estrechas/química , Uniones Estrechas/metabolismo , Permeabilidad , Mucosa Intestinal/metabolismoRESUMEN
Environmental enteric dysfunction (EED) is characterized by malabsorption and diarrhea that result in irreversible deficits in physical and intellectual growth. We sought to define the expression of transport and tight junction proteins by quantitative analysis of duodenal biopsies from patients with EED. Biopsies from Pakistani children with confirmed EED diagnoses were compared to those from age-matched North American healthy controls, patients with celiac disease, and patients with nonceliac disease with villous atrophy or intraepithelial lymphocytosis. Expression of brush border digestive and transport proteins and paracellular (tight junction) proteins was assessed by quantitative multiplex immunofluorescence microscopy. EED was characterized by partial villous atrophy and marked intraepithelial lymphocytosis. Epithelial proliferation and enteroendocrine, tuft, and Paneth cell numbers were unchanged, but there was significant goblet cell expansion in EED biopsies. Expression of proteins involved in nutrient and water absorption and that of the basolateral Cl- transport protein NKCC1 were also increased in EED. Finally, the barrier-forming tight junction protein claudin-4 (CLDN4) was significantly upregulated in EED, particularly within villous enterocytes. In contrast, expression of CFTR, CLDN2, CLDN15, JAM-A, occludin, ZO-1, and E-cadherin was unchanged. Upregulation of a barrier-forming tight junction protein and brush border and basolateral membrane proteins that support nutrient and water transport in EED is paradoxical, as their increased expression would be expected to be correlated with increased intestinal barrier function and enhanced absorption, respectively. These data suggest that EED activates adaptive intestinal epithelial responses to enhance nutrient absorption but that these changes are insufficient to restore health.
Asunto(s)
Mucosa Intestinal , Linfocitosis , Niño , Humanos , Mucosa Intestinal/metabolismo , Linfocitosis/metabolismo , Linfocitosis/patología , Uniones Estrechas/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Atrofia/metabolismo , Atrofia/patologíaRESUMEN
OBJECTIVE: Intestinal barrier loss is a Crohn's disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically. DESIGN: Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects. RESULTS: MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects. CONCLUSION: Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.
Asunto(s)
Enfermedad de Crohn , Animales , Humanos , Ratones , Células CACO-2 , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/metabolismo , Mucosa Intestinal/metabolismo , Ratones Noqueados , Quinasa de Cadena Ligera de Miosina/metabolismo , Tacrolimus/farmacología , Proteínas de Unión a Tacrolimus/metabolismo , Uniones Estrechas/fisiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are required to combat invading bacteria, excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen. Thus, neutrophil recruitment needs to be fine-tuned to ensure bacterial killing, while avoiding neutrophil-inflicted tissue damage. We recently showed that the actin-binding protein HS1 promotes neutrophil extravasation; and hypothesized that HS1 is also a critical regulator of sepsis progression. We evaluated the role of HS1 in a model of lethal sepsis induced by cecal-ligation and puncture. We found that septic HS1-deficient mice had a better survival rate compared to WT mice due to absence of lung damage. Lungs of septic HS1-deficient mice showed less inflammation, fibrosis, and vascular congestion. Importantly, systemic CLP-induced neutrophil recruitment was attenuated in the lungs, the peritoneum and the cremaster in the absence of HS1. Lungs of HS1-deficient mice produced significantly more interleukin-10. Compared to WT neutrophils, those HS1-deficient neutrophils that reached the lungs had increased surface levels of Gr-1, ICAM-1, and L-selectin. Interestingly, HS1-deficient neutrophils had similar F-actin content and phagocytic activity, but they failed to polymerize actin and deform in response to CXCL-1 likely explaining the reduced systemic neutrophil recruitment in HS1-deficient mice. Our data show that HS1 deficiency protects against sepsis by attenuating neutrophil recruitment to amounts sufficient to combat bacterial infection, but insufficient to induce tissue damage.
Asunto(s)
Neutrófilos , Sepsis , Animales , Modelos Animales de Enfermedad , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila/fisiología , Neutrófilos/metabolismoRESUMEN
The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.
RESUMEN
The intestinal barrier is an essential component of innate host defense. The single layer of epithelial cells that line the intestine must balance barrier function with both active, transcellular and diffusive, paracellular transport. Tight junctions, which link adjacent cells, form a selectively permeable seal that defines both paracellular transport and barrier properties. Molecules can cross tight junctions by either of two distinct routes, termed pore and the leak pathways, that differ in capacity, charge-selectivity, size-selectivity, and responses to physiological and pathophysiological stimuli. A third intestinal permeability route, the unrestricted pathway, reflects loss of the epithelial barrier, as occurs with mucosal damage, is independent of paracellular and transcellular pathways, and is neither charge- nor size-selective.The most commonly used approach for measuring intestinal permeability in vivo involves gavage of FITC-4 kDa dextran and analysis of the quantity recovered in serum. Unfortunately, this method cannot distinguish between leak and unrestricted pathways, as 4 kDa dextran can cross both. Moreover, 4 kDa dextran is too large to cross the pore pathway and, therefore, provides no information regarding this paracellular flux route. Here we describe a multiplex method that allows simultaneous, independent analysis of each pathway.
Asunto(s)
Uniones Estrechas , Claudinas , Dextranos , Mucosa Intestinal , Intestinos , PermeabilidadRESUMEN
The tight junction protein claudin-2 is upregulated in disease. Although many studies have linked intestinal barrier loss to local and systemic disease, these have relied on macromolecular probes. In vitro analyses show, however, that these probes cannot be accommodated by size- and charge-selective claudin-2 channels. We sought to define the impact of claudin-2 channels on disease. Transgenic claudin-2 overexpression or IL-13-induced claudin-2 upregulation increased intestinal small cation permeability in vivo. IL-13 did not, however, affect permeability in claudin-2-knockout mice. Claudin-2 is therefore necessary and sufficient to effect size- and charge-selective permeability increases in vivo. In chronic disease, T cell transfer colitis severity was augmented or diminished in claudin-2-transgenic or -knockout mice, respectively. We translated the in vitro observation that casein kinase-2 (CK2) inhibition blocks claudin-2 channel function to prevent acute, IL-13-induced, claudin-2-mediated permeability increases in vivo. In chronic immune-mediated colitis, CK2 inhibition attenuated progression in claudin-2-sufficient, but not claudin-2-knockout, mice, i.e., the effect was claudin-2 dependent. Paracellular flux mediated by claudin-2 channels can therefore promote immune-mediated colitis progression. Although the mechanisms by which claudin-2 channels intensify disease remain to be defined, these data suggest that claudin-2 may be an accessible target in immune-mediated disorders, including inflammatory bowel disease.
Asunto(s)
Claudinas/deficiencia , Colitis/etiología , Animales , Claudinas/genética , Claudinas/metabolismo , Colitis/inmunología , Colitis/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucina-13/administración & dosificación , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Regulación hacia ArribaRESUMEN
Neutrophil extravasation is a migratory event in response to inflammation that depends on cytoskeletal dynamics regulated by myosins. Myosin-1e (Myo1e) is a long-tailed class-I myosin that has not yet been studied in the context of neutrophil-endothelial interactions and neutrophil extravasation. Intravital microscopy of TNFα-inflamed cremaster muscles in Myo1e-deficient mice revealed that Myo1e is required for efficient neutrophil extravasation. Specifically, Myo1e deficiency caused increased rolling velocity, decreased firm adhesion, aberrant crawling, and strongly reduced transmigration. Interestingly, we observed a striking discontinuous rolling behavior termed "intermittent rolling," during which Myo1e-deficient neutrophils showed alternating rolling and jumping movements. Surprisingly, chimeric mice revealed that these effects were due to Myo1e deficiency in leukocytes. Vascular permeability was not significantly altered in Myo1e KO mice. Myo1e-deficient neutrophils showed diminished arrest, spreading, uropod formation, and chemotaxis due to defective actin polymerization and integrin activation. In conclusion, Myo1e critically regulates adhesive interactions of neutrophils with the vascular endothelium and neutrophil extravasation. Myo1e may therefore be an interesting target in chronic inflammatory diseases characterized by excessive neutrophil recruitment.
RESUMEN
The actin-related protein complex 2/3 (Arp2/3) generates branched actin networks important for many cellular processes such as motility, vesicular trafficking, cytokinesis, and intercellular junction formation and stabilization. Activation of Arp2/3 requires interaction with actin nucleation-promoting factors (NPFs). Regulation of Arp2/3 activity is achieved by endogenous inhibitory proteins through direct binding to Arp2/3 and competition with NPFs or by binding to Arp2/3-induced actin filaments and disassembly of branched actin networks. Arp2/3 inhibition has recently garnered more attention as it has been associated with attenuation of cancer progression, neurotoxic effects during drug abuse, and pathogen invasion of host cells. In this review, we summarize current knowledge on expression, inhibitory mechanisms and function of endogenous proteins able to inhibit Arp2/3 such as coronins, GMFs, PICK1, gadkin, and arpin. Moreover, we discuss cellular consequences of pharmacological Arp2/3 inhibition.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Citoesqueleto de Actina , Complejo 2-3 Proteico Relacionado con la Actina/antagonistas & inhibidores , Unión Competitiva , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Factor de Maduración de la Glia/química , Factor de Maduración de la Glia/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Tiazolidinas/química , Tiazolidinas/metabolismoRESUMEN
A single layer of polarized epithelial cells lining the colonic mucosa create a semipermeable barrier indispensable for gut homeostasis. The role of intestinal epithelial cell (IEC) polarization in the maintenance of the epithelial homeostasis and in the development of inflammatory bowel diseases is not fully understood. In this review, now we report that IEC polarization plays an essential role in the regulation of IL-6/STAT3 signaling in the colonic mucosa. Our results demonstrate that autocrine STAT3 activation in IECs is mediated by the apical secretion of IL-6 in response to the basolateral stimulation with IFN-γ. This process relies on the presence of functional, IFN-γ-producing CD4+ T cells. In the absence of basolateral IFN-γ, the compartmentalization of the IL-6/STAT3 signaling is disrupted, and STAT3 is activated mainly in macrophages. Thus, in this study, we show that during inflammation, IFN-γ regulates IL-6/STAT3 signaling in IEC in the colonic mucosa.
Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Células CACO-2 , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Inflamación/metabolismo , Interferón gamma/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Inflammatory bowel diseases (IBD) are multifactorial disorders affecting millions of people worldwide with alarmingly increasing incidences every year. Dysfunction of the intestinal epithelial barrier is associated with IBD pathogenesis, and therapies include anti-inflammatory drugs that enhance intestinal barrier function. However, these drugs often have adverse side effects thus warranting the search for alternatives. Compatible solutes such as bacterial ectoines stabilize cell membranes and proteins. AIM: To unravel whether ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) and homoectoine (4,5,6,7-tetrahydro-2-methyl-1H-(1,3)-diazepine-4-carboxylic acid), a synthetic derivative of ectoine, have beneficial effects during dextran sulfate sodium (DSS)-induced colitis in mice. METHODS/RESULTS: We found that the disease activity index was significantly reduced by both ectoines. DSS-induced edema formation, epithelial permeability, leukocyte recruitment and tissue damage were reduced by ectoine and homoectoine, with the latter having stronger effects. Interestingly, the claudin switch usually observed during colitis (decreased expression of claudin-1 and increased expression of the leaky claudin-2) was completely prevented by homoectoine, whereas ectoine only reduced claudin-2 expression. Concomitantly, only homoectoine ameliorated the drop in transepithelial electrical resistance induced by IFN-γ and TNF-α in Caco-2 cells. Both ectoines inhibited loss of ZO-1 and occludin and prevented IFN-γ/TNF-α-induced increased paracellular flux of 4 kDa FITC-dextran in vitro. Moreover, both ectoines reduced expression of pro-inflammatory cytokines and oxidative stress during colitis. CONCLUSION: While both ectoine and homoectoine have protective effects on the epithelial barrier during inflammation, only homoectoine completely prevented the inflammatory claudin switch in tight junctions. Thus, homoectoine may serve as diet supplement in IBD patients to reach or extend remission.
Asunto(s)
Aminoácidos Diaminos/farmacología , Claudina-1/efectos de los fármacos , Claudina-2/efectos de los fármacos , Colitis/patología , Epitelio/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Animales , Células CACO-2 , Claudina-1/genética , Claudina-1/metabolismo , Claudina-2/genética , Claudina-2/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Edema , Impedancia Eléctrica , Humanos , Técnicas In Vitro , Interferón gamma/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Estrés Oxidativo/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Changes in vascular permeability occur during inflammation and the actin cytoskeleton plays a crucial role in regulating endothelial cell contacts and permeability. We demonstrated recently that the actin-binding protein cortactin regulates vascular permeability via Rap1. However, it is unknown if the actin cytoskeleton contributes to increased vascular permeability without cortactin. As we consistently observed more actin fibres in cortactin-depleted endothelial cells, we hypothesised that cortactin depletion results in increased stress fibre contractility and endothelial barrier destabilisation. Analysing the contractile machinery, we found increased ROCK1 protein levels in cortactin-depleted endothelium. Concomitantly, myosin light chain phosphorylation was increased while cofilin, mDia and ERM were unaffected. Secretion of the barrier-stabilising hormone adrenomedullin, which activates Rap1 and counteracts actomyosin contractility, was reduced in plasma from cortactin-deficient mice and in supernatants of cortactin-depleted endothelium. Importantly, adrenomedullin administration and ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling actomyosin contractility with consequences for endothelial barrier integrity.
Asunto(s)
Adrenomedulina/metabolismo , Permeabilidad Capilar/fisiología , Cortactina/deficiencia , Células Endoteliales/fisiología , Actomiosina/fisiología , Animales , Proteínas Contráctiles/biosíntesis , Proteínas Contráctiles/genética , Cortactina/antagonistas & inhibidores , Cortactina/genética , Cortactina/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Pulmón/citología , Masculino , Ratones , Interferencia de ARN , ARN Interferente Pequeño/genética , Complejo Shelterina , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/fisiologíaRESUMEN
The correct regulation of tissue barriers is of utmost importance for health. Barrier dysfunction accompanies inflammatory disorders and, if not controlled properly, can contribute to the development of chronic diseases. Tissue barriers are formed by monolayers of epithelial cells that separate organs from their environment, and endothelial cells that cover the vasculature, thus separating the blood stream from underlying tissues. Cells within the monolayers are connected by intercellular junctions that are linked by adaptor molecules to the cytoskeleton, and the regulation of these interactions is critical for the maintenance of tissue barriers. Many endogenous and exogenous molecules are known to regulate barrier functions in both ways. Proinflammatory cytokines weaken the barrier, whereas anti-inflammatory mediators stabilize barriers. Adrenomedullin (ADM) and intermedin (IMD) are endogenous peptide hormones of the same family that are produced and secreted by many cell types during physiologic and pathologic conditions. They activate certain G-protein-coupled receptor complexes to regulate many cellular processes such as cytokine production, actin dynamics and junction stability. In this review, we summarize current knowledge about the barrier-stabilizing effects of ADM and IMD in health and disease.