RESUMO
Thrombospondin-1 (TSP1) is a matricellular protein associated with the regulation of cell migration through direct binding interactions with integrin proteins and by associating with other receptors known to regulate integrin function, including CD47 and CD36. We previously demonstrated that deletion of an epithelial TSP1 receptor CD47 attenuates epithelial wound repair following intestinal mucosal injury. However, the mechanisms by which TSP1 contributes to intestinal mucosal repair remains poorly understood. Our results show upregulated TSP1 expression in colonic mucosal wounds and impaired intestinal mucosal wound healing in vivo upon intestinal epithelial specific loss of TSP1 (VillinCre/+Thbs1f/f or Thbs1ΔIEC). We report that exposure to exogenous TSP1 enhanced migration of IECs in a CD47- and TGFß1-dependent manner, and that deficiency of TSP1 in primary murine colonic epithelial cells resulted in impaired wound healing. Mechanistically, TSP1 modulated epithelial actin cytoskeletal dynamics by suppression of RhoA activity, activation of Rac1, and changes in F-actin bundling. Overall, TSP1 was found to regulate intestinal mucosal wound healing via CD47 and TGFß1, coordinate integrin-containing cell-matrix adhesion dynamics and remodel the actin cytoskeleton in migrating epithelial cells to enhance cell motility and promote wound repair.
RESUMO
The metal ion transporter SLC39A8 is associated with physiological traits and diseases, including blood manganese (Mn) levels and inflammatory bowel diseases (IBD). The mechanisms by which SLC39A8 controls Mn homeostasis and epithelial integrity remain elusive. Here, we generate Slc39a8 intestinal epithelial cell-specific-knockout (Slc39a8-IEC KO) mice, which display markedly decreased Mn levels in blood and most organs. Radiotracer studies reveal impaired intestinal absorption of dietary Mn in Slc39a8-IEC KO mice. SLC39A8 is localized to the apical membrane and mediates 54Mn uptake in intestinal organoid monolayer cultures. Unbiased transcriptomic analysis identifies alkaline ceramidase 1 (ACER1), a key enzyme in sphingolipid metabolism, as a potential therapeutic target for SLC39A8-associated IBDs. Importantly, treatment with an ACER1 inhibitor attenuates colitis in Slc39a8-IEC KO mice by remedying barrier dysfunction. Our results highlight the essential roles of SLC39A8 in intestinal Mn absorption and epithelial integrity and offer a therapeutic target for IBD associated with impaired Mn homeostasis.
Assuntos
Ceramidase Alcalina , Proteínas de Transporte de Cátions , Doenças Inflamatórias Intestinais , Mucosa Intestinal , Manganês , Camundongos Knockout , Animais , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Manganês/metabolismo , Camundongos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ceramidase Alcalina/metabolismo , Ceramidase Alcalina/genética , Humanos , Camundongos Endogâmicos C57BL , Homeostase , Masculino , Colite/metabolismo , Colite/genética , Colite/patologia , Absorção Intestinal , Células Epiteliais/metabolismoRESUMO
Claudin family tight junction proteins form charge- and size-selective paracellular channels that regulate epithelial barrier function. In the gastrointestinal tract, barrier heterogeneity is attributed to differential claudin expression. Here, we show that claudin-23 (CLDN23) is enriched in luminal intestinal epithelial cells where it strengthens the epithelial barrier. Complementary approaches reveal that CLDN23 regulates paracellular ion and macromolecule permeability by associating with CLDN3 and CLDN4 and regulating their distribution in tight junctions. Computational modeling suggests that CLDN23 forms heteromeric and heterotypic complexes with CLDN3 and CLDN4 that have unique pore architecture and overall net charge. These computational simulation analyses further suggest that pore properties are interaction-dependent, since differently organized complexes with the same claudin stoichiometry form pores with unique architecture. Our findings provide insight into tight junction organization and propose a model whereby different claudins combine to form multiple distinct complexes that modify epithelial barrier function by altering tight junction structure.
Assuntos
Claudinas , Junções Íntimas , Junções Íntimas/metabolismo , Claudinas/genética , Claudinas/química , Simulação por Computador , Células Epiteliais/metabolismoRESUMO
BACKGROUND: Incidences of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, are escalating worldwide and can be considered a global public health problem. Given that the gold standard approach to IBD therapeutics focuses on reducing the severity of symptoms, there is an urgent unmet need to develop alternative therapies that halt not only inflammatory processes but also promote mucosal repair. Previous studies have identified increased stem cell factor (SCF) expression in inflamed intestinal mucosal tissues. However, the role that SCF plays in mediating intestinal inflammation and repair has not been explored. METHODS: Changes in the expression of SCF were evaluated in the colonic tissue of healthy mice and during dextran sodium sulfate (DSS)-induced colitis. Furthermore, mucosal wound healing and colitis severity were analyzed in mice subjected to either mechanical biopsy or DSS treatment, respectively, following intestinal epithelial cell-specific deletion of SCF or anti-SCF antibody administration. RESULTS: We report robust expression of SCF by intestinal epithelial cells during intestinal homeostasis with a switch to immune cell-produced SCF during colitis. Data from mice with intestinal epithelial cell-specific deletion of SCF highlight the importance of immune cell-produced SCF in driving the pathogenesis of colitis. Importantly, antibody-mediated neutralization of total SCF or the specific SCF248 isoform decreased immune cell infiltration and enhanced mucosal wound repair following biopsy-induced colonic injury or DSS-induced colitis. CONCLUSIONS: These data demonstrate that SCF functions as a pro-inflammatory mediator in mucosal tissues and that specific neutralization of SCF248 could be a viable therapeutic option to reduce intestinal inflammation and promote mucosal wound repair in individuals with IBD.
Our investigation demonstrates that blocking cleavable SCF248 isoform by administration of specific stem cell factor antibodies enhances healing of the intestinal mucosa and restores critical barrier function, suggesting an alternative therapeutic option to treat individuals with active IBD.
Assuntos
Colite Ulcerativa , Colite , Doenças Inflamatórias Intestinais , Animais , Camundongos , Colite/tratamento farmacológico , Colite/patologia , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/patologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Fator de Células-Tronco/antagonistas & inibidores , Fator de Células-Tronco/metabolismoRESUMO
Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.
Assuntos
Inflamação/patologia , Projetos de Pesquisa , Animais , Colite/induzido quimicamente , Colite/patologia , Análise de Dados , Sulfato de Dextrana , Modelos Animais de Doenças , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Índice de Gravidade de DoençaRESUMO
The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal-epithelial-specific Dsc2 knockdown (KD) (Dsc2ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell-cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.
Assuntos
Adesão Celular/fisiologia , Desmocolinas/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Desmocolinas/genética , Desmocolinas/fisiologia , Desmogleína 2/metabolismo , Caderinas de Desmossomos/metabolismo , Caderinas de Desmossomos/fisiologia , Desmossomos/metabolismo , Humanos , Junções Intercelulares/metabolismo , Mucosa Intestinal , Masculino , Camundongos , Camundongos KnockoutRESUMO
Food-triggered anaphylaxis can encompass a variety of systemic and intestinal symptoms. Murine-based and clinical studies have revealed a role for histamine and H1R and H2R-pathway in the systemic response; however, the molecular processes that regulate the gastrointestinal (GI) response are not as well defined. In the present study, by utilizing an IgE-mast cell (MC)-dependent experimental model of oral antigen-induced anaphylaxis, we define the intestinal epithelial response during a food-induced anaphylactic reaction. We show that oral allergen-challenge stimulates a rapid dysregulation of intestinal epithelial transcellular and paracellular transport that was associated with the development of secretory diarrhea. Allergen-challenge induced (1) a rapid intestinal epithelial Cftr-dependent Cl- secretory response and (2) paracellular macromolecular leak that was associated with modification in epithelial intercellular junction proteins claudin-1, 2, 3 and 5, E-cadherin and desmosomal cadherins. OVA-induced Cftr-dependent Cl- secretion and junctional protein degradation was rapid occurring and was sustained for 72 h following allergen-challenge. Blockade of both the proteolytic activity and Cl- secretory response was required to alleviate intestinal symptoms of food-induced anaphylaxis. Collectively, these data suggest that the GI symptom of food-induced anaphylactic reaction, secretory diarrhea, is a consequence of CFTR-dependent Cl- secretion and proteolytic activity.
Assuntos
Anafilaxia/etiologia , Anafilaxia/metabolismo , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Hipersensibilidade Alimentar/etiologia , Hipersensibilidade Alimentar/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Alérgenos/imunologia , Anafilaxia/patologia , Animais , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Hipersensibilidade Alimentar/patologia , Imunoglobulina E/imunologia , Transporte de Íons , Mastócitos/imunologia , Mastócitos/metabolismo , CamundongosRESUMO
The intestinal mucosa is lined by a single layer of epithelial cells that forms a tight barrier, separating luminal antigens and microbes from underlying tissue compartments. Mucosal damage results in a compromised epithelial barrier that can lead to excessive immune responses as observed in inflammatory bowel disease. Efficient wound repair is critical to reestablish the mucosal barrier and homeostasis. Intestinal epithelial cells (IEC) exclusively express the desmosomal cadherins, Desmoglein-2 and Desmocollin-2 (Dsc2) that contribute to mucosal homeostasis by strengthening intercellular adhesion between cells. Despite this important property, specific contributions of desmosomal cadherins to intestinal mucosal repair after injury remain poorly investigated in vivo. Here we show that mice with inducible conditional knockdown (KD) of Dsc2 in IEC (Villin-CreERT2; Dsc2 fl/fl) exhibited impaired mucosal repair after biopsy-induced colonic wounding and recovery from dextran sulfate sodium-induced colitis. In vitro analyses using human intestinal cell lines after KD of Dsc2 revealed delayed epithelial cell migration and repair after scratch-wound healing assay that was associated with reduced cell-matrix traction forces, decreased levels of integrin ß1 and ß4, and altered activity of the small GTPase Rap1. Taken together, these results demonstrate that epithelial Dsc2 is a key contributor to intestinal mucosal wound healing in vivo.
Assuntos
Movimento Celular , Desmocolinas/metabolismo , Integrinas/metabolismo , Mucosa Intestinal/patologia , Cicatrização , Animais , Adesão Celular , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Deleção de Genes , Humanos , Inflamação/patologia , Camundongos Endogâmicos C57BL , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Epitélio/metabolismo , Mucosa/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Cicatrização , Proteína ADAM10/metabolismo , Animais , Biomarcadores , Epitélio/patologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Mucosa/patologia , NF-kappa B/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
OBJECTIVE: This work aimed to determine if cataractous changes associated with EMT occurring in the K14E6 mice lenses are associated with TGF-ß and Wnt/ß-catenin signaling activation. MATERIALS AND METHODS: Cataracts of K14E6 mice were analysed histologically; and components of TGF-ß and Wnt/ß-catenin signaling were evaluated by Western blot, RT-qPCR, in situ RT-PCR, IHC, or IF technics. Metalloproteinases involved in EMT were also assayed using zymography. The endogenous stabilisation of Smad7 protein was also assessed using an HDAC inhibitor. RESULTS: The K14E6 mice, which displayed binocular cataracts in 100% of the animals, exhibited loss of tissue organisation, cortical liquefaction, and an increase in the number of hyperproliferative-nucleated cells with mesenchymal-like characteristics in the lenses. Changes in lenses' cell morphology were due to actin filaments reorganisation, activation of TGF-ß and Wnt/ß-catenin pathways, and the accumulation of MTA1 protein. Finally, the stabilisation of Smad7 protein diminishes cell proliferation, as well as MTA1 protein levels. CONCLUSION: The HPV16-E6 oncoprotein induces EMT in transgenic mice cataracts. The molecular mechanism may involve TGF-ß and Wnt/ß-catenin pathways, suggesting that the K14E6 transgenic mouse could be a useful model for the study or treatment of EMT-induced cataracts.
Assuntos
Catarata/metabolismo , Transição Epitelial-Mesenquimal , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/biossíntese , Proteínas Repressoras/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt , Animais , Catarata/genética , Catarata/patologia , Modelos Animais de Doenças , Papillomavirus Humano 16/genética , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multicellular three-dimensional structures capable of giving rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumour-derived extracellular matrix (ECM), which severely limits the use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs, such that three-dimensional structures are never embedded in tumour-derived ECM. We also demonstrate that the hydrogel serves as an injection vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.
Assuntos
Colo/efeitos dos fármacos , Hidrogéis/farmacologia , Intestinos/efeitos dos fármacos , Organoides/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Humanos , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Células-Tronco Pluripotentes/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas de Sinalização Intercelular CCN/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antígenos CD11/metabolismo , Proliferação de Células , Colo/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Inflamação , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , CicatrizaçãoRESUMO
The intestinal epithelium forms a highly dynamic and selective barrier that controls absorption of fluid and solutes while restricting pathogen access to underlying tissues. Barrier properties are achieved by intercellular junctions that include an apical tight junction (TJ) and subjacent adherens junctions and desmosomes. The TJ tetraspan claudin proteins form pores between epithelial cells to control paracellular fluid and ion movement. In addition to regulation of barrier function, claudin family members control epithelial homeostasis and are expressed in a spatiotemporal manner in the intestinal crypt-luminal axis. This delicate balance of physiologic differential claudin protein expression is altered during mucosal inflammation. Inflammatory mediators influence transcriptional regulation, as well as endocytic trafficking, targeting, and retention of claudins in the TJ. Increased expression of intestinal epithelial claudin-1, -2, and -18 with downregulation of claudin-3, -4, -5, -7, -8, and -12 has been observed in intestinal inflammatory disorders. Such changes in claudin proteins modify the epithelial barrier function in addition to influencing epithelial and mucosal homeostasis. An improved understanding of the regulatory mechanisms that control epithelial claudin proteins will provide strategies to strengthen the epithelial barrier function and restore mucosal homeostasis in inflammatory disorders.
Assuntos
Claudinas/metabolismo , Homeostase , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Animais , Claudina-1/genética , Claudina-1/metabolismo , Claudina-3/genética , Claudina-3/metabolismo , Claudinas/genética , Humanos , Inflamação/genética , Modelos Biológicos , Junções Íntimas/metabolismoRESUMO
Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2-dependent downregulation of claudin-2 (CLDN-2) and upregulation of claudin-4 (CLDN-4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN-2 downregulation and CLDN-4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN-2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF-induced changes on CLDN-2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus, a process that depends on Src activation. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF-induced downregulation of CLDN-2, but they are also implicated in the EGF-induced CLDN-4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN-2 and -4 proteins and mRNAs through Src and STAT3 activity.
Assuntos
Claudina-2/biossíntese , Claudina-4/biossíntese , Fator de Crescimento Epidérmico/fisiologia , Fator de Transcrição STAT3/metabolismo , Quinases da Família src/metabolismo , Animais , Butadienos/farmacologia , Claudina-2/genética , Claudina-4/genética , Cães , Regulação para Baixo , Impedância Elétrica , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Indóis/farmacologia , Células Madin Darby de Rim Canino , Maleimidas/farmacologia , Nitrilas/farmacologia , Fosforilação , Biossíntese de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/biossíntese , Fator de Transcrição STAT3/biossíntese , Junções Íntimas/fisiologia , Transcrição Gênica , Regulação para Cima , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/biossínteseRESUMO
In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.