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1.
J Physiol ; 593(24): 5269-82, 2015 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-26365358

RESUMEN

KEY POINTS: Interleukin-13 (IL-13) causes intestinal epithelial barrier dysfunction, and is implicated in the pathogenesis of Th2-driven intestinal inflammation (e.g. ulcerative colitis). However, it is unclear whether the epithelial sodium channel (ENaC) - the main limiting factor for sodium absorption in the distal colon - is also influenced by IL-13 and if so, by what mechanism(s). We demonstrate in an intestinal cell model as well as in mouse distal colon that IL-13 causes reduced ENaC activity. We show that IL-13 impairs ENaC-dependent sodium transport by activating the JAK1/2-STAT6 signalling pathway. These results improve our understanding of the mechanisms through which IL-13 functions as a key effector cytokine in ulcerative colitis, thereby contributing to the distinct pathology of this disease. ABSTRACT: Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of ulcerative colitis, possibly by disrupting epithelial integrity. In the distal colon, the epithelial sodium channel (ENaC) is an important factor in the regulation of sodium absorption, and therefore plays a critical role in minimizing intestinal sodium and water losses. In the present study, we investigated whether IL-13 also acts as a potent modulator of epithelial sodium transport via ENaC, and the signalling components involved. The effect of IL-13 on ENaC was examined in HT-29/B6-GR/MR human colon cells, as well as in mouse distal colon, by measuring amiloride-sensitive short-circuit current (ISC ) in Ussing chambers. The expression levels of ENaC subunits and the cellular components that contribute to ENaC activity were analysed by qRT-PCR and promoter gene assay. We show that IL-13, in both the cell model and in native intestinal tissue, impaired epithelial sodium absorption via ENaC (JNa ) as a result of decreased transcription levels of ß- and γ-ENaC subunits and SGK1, a post-translational regulator of ENaC activity, due to impaired promoter activity. The reduction in JNa was prevented by inhibition of JAK1/2-STAT6 signalling. This inhibition also affected the IL-13-induced decrease in p38 MAPK phosphorylation. The contribution of STAT6 to IL-13-mediated ENaC inactivation was confirmed in a STAT6(-/-) mouse model. In conclusion, these results indicate that IL-13, the levels of which are elevated in ulcerative colitis, contributes to impaired ENaC activity via modulation of the STAT6/p38 MAPK pathways.


Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Interleucina-13/farmacología , Mucosa Intestinal/metabolismo , Factor de Transcripción STAT6/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Células HT29 , Humanos , Absorción Intestinal , Mucosa Intestinal/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Sodio/metabolismo
2.
Gastroenterology ; 136(3): 933-42, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19185581

RESUMEN

BACKGROUND & AIMS: The epithelial sodium channel (ENaC) mediates electrogenic sodium absorption in distal colon. In patients with inflammatory bowel disease (IBD), ENaC induction is impaired, mainly through transcriptional suppression by proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha. Glucocorticoid therapy rapidly increases sodium absorption; we investigated the molecular mechanisms underlying the interaction among TNF-alpha, glucocorticoids, and ENaC induction. METHODS: ENaC-mediated sodium transport in glucocorticoid receptor (GR)-expressing HT-29/B6 cells and rat distal colon, under the influence of the synthetic glucocorticoid dexamethasone and TNF-alpha, was quantified in Ussing chambers. ENaC messenger RNA (mRNA) levels were monitored by real-time polymerase chain reaction. GR transactivation and expression were investigated by gene reporter, immunoblot, and confocal immunofluorescence microscopy analyses. The GR mRNA half-life was determined. Signaling pathways were characterized using mitogen-activated protein kinase inhibitors. RESULTS: Dexamethasone not only prevented TNF-alpha-mediated ENaC suppression but caused synergistic induction of ENaC-dependent sodium absorption in HT-29/B6-GR cells and rat distal colon. This synergy resulted from TNF-alpha-mediated increases in GR protein levels because of GR mRNA stabilization and subsequent GR transactivation by dexamethasone. As a consequence, transcription of the ENaC beta- and gamma-subunits was up-regulated, increasing ENaC-dependent sodium absorption. p38 Mitogen-activated protein kinase is required for this synergistic effect: p38 inhibition blocked the increase in GR protein expression and ENaC-dependent sodium absorption. CONCLUSIONS: TNF-alpha and dexamethasone induce ENaC, explaining the rapid and intense proabsorptive effect of glucocorticoid therapies.


Asunto(s)
Dexametasona/farmacología , Canales Epiteliales de Sodio/metabolismo , Proteína Relacionada con TNFR Inducida por Glucocorticoide/farmacología , Mucosa Intestinal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Colon/citología , Colon/efectos de los fármacos , Colon/metabolismo , Sinergismo Farmacológico , Canales Epiteliales de Sodio/genética , Expresión Génica/efectos de los fármacos , Células HT29 , Humanos , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Sodio/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
3.
Gastroenterology ; 134(5): 1436-47, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18355814

RESUMEN

BACKGROUND & AIMS: Crohn's disease (CD) is a chronic inflammatory bowel disease. In this study, we have investigated sodium absorption via epithelial sodium channels (ENaC) in the macroscopically noninflamed colon in active CD. METHODS: Sodium transport via ENaC was investigated in Ussing chambers using biopsy specimens of sigmoid colon from controls and active CD limited to the small intestine. ENaC messenger RNA expression and subcellular localization were studied by real-time polymerase chain reaction and confocal microscopy. Effects of proinflammatory cytokines on ENaC and signaling via mitogen-activated protein kinases were investigated in rat distal colon. Therapeutic inhibition of mitogen-activated protein kinases was studied in CD biopsy specimens. RESULTS: Electrogenic sodium absorption via ENaC was strongly impaired in the macroscopically noninflamed CD colon because of reduced gamma-ENaC transcription, whereas subcellular localization of ENaC was not changed. In contrast to impaired epithelial sodium transport, epithelial barrier function was not altered in noninflamed CD colon, indicating that paracellular leak flux of ions did not contribute to decreased sodium absorption. Exposure of rat distal colon to tumor necrosis factor alpha led to reduced electrogenic sodium absorption because of impaired transcriptional gamma-ENaC induction, which resembled the changes found in CD. Tumor necrosis factor alpha effects were dependent on extracellular signal-regulated kinase 1/2 but not p38 or c-Jun-N-terminal kinase because inhibition of mitogen-activated protein kinase/extracellular regulated kinase (MEK)1/2 but not inhibition of p38 or c-Jun-N-terminal kinase prevented suppression of ENaC. Finally, therapeutic inhibition of MEK1/2 restored electrogenic sodium absorption in CD. CONCLUSIONS: In CD, macroscopically noninflamed colon contributes to diarrhea via impaired ENaC-mediated sodium absorption. Inhibition of extracellular signal-regulated kinase might serve as a potential therapeutic strategy for CD diarrhea.


Asunto(s)
Colon/fisiología , Enfermedad de Crohn/genética , Canales Epiteliales de Sodio/genética , Expresión Génica , Absorción Intestinal/fisiología , ARN/genética , Sodio/metabolismo , Adulto , Animales , Biopsia , Western Blotting , Colon/citología , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Canales Epiteliales de Sodio/biosíntesis , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Transporte Iónico/fisiología , Masculino , Persona de Mediana Edad , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis Espectral , Factor de Necrosis Tumoral alfa/biosíntesis
4.
Biochem Biophys Res Commun ; 382(2): 280-5, 2009 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-19275887

RESUMEN

In the distal colon, the epithelial sodium channel (ENaC) is rate limiting for sodium absorption. Progress in the molecular characterization of ENaC expression and trafficking in response to the mineralocorticoid aldosterone has been hampered, since no epithelial colonic cell line existed expressing functional ENaC stimulated by nanomolar aldosterone via mineralocorticoid receptor (MR). Here, we present a human colonic epithelial cell line inducibly expressing the MR (HT-29/B6-Tet-On-MR) which exhibits aldosterone-dependent ENaC-mediated sodium transport in the presence of the short-chain fatty acid butyrate. Butyrate was necessary for high-level expression of MR which allowed for aldosterone-dependent upregulation of beta- and gamma-ENaC expression. As butyrate alone was not capable of promoting ENaC-mediated sodium transport, aldosterone-induced GILZ (glucocorticoid-induced leucine zipper protein) was identified as a candidate factor increasing apical ENaC levels.


Asunto(s)
Línea Celular , Colon/metabolismo , Canales Epiteliales de Sodio/biosíntesis , Modelos Biológicos , Receptores de Mineralocorticoides/metabolismo , Sodio/metabolismo , Humanos , Mucosa Intestinal/metabolismo
5.
Mol Immunol ; 55(2): 106-9, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23140834

RESUMEN

During innate immune responses the delicate balance of protein synthesis, quality control and degradation is severely challenged by production of radicals and/or the massive synthesis of pathogen proteins. The regulated degradation of ubiquitin-tagged proteins by the ubiquitin proteasome system (UPS) represents one major pathway for the maintenance of cellular proteostasis and regulatory processes under these conditions. In addition, MHC class I antigen presentation is strictly dependent on an appropriate peptide supply by the UPS to efficiently prime CD8(+) T cells and to initiate an adaptive immune response. We here discuss recent efforts in defining the link between innate immune mechanisms like cytokine and ROS production, the induction of an efficient adaptive immune response and the specific involvement of the UPS therein. Cytokines and/or infections induce translation and the production of free radicals, which in turn confer oxidative damage to nascent as well as folded proteins. In parallel, the same signaling cascades are able to accelerate the protein turnover by the concomitantly induced ubiquitin conjugation and degradation of such damaged polypeptides by immunoproteasomes. The ability of immunoproteasomes to efficiently degrade such oxidant-damaged ubiquitylated proteins protects cells from accumulating toxic ubiquitin-rich aggregates. At the same time, this innate immune mechanism facilitates a sufficient peptide supply for MHC class I antigen presentation and connects it to initiation of adaptive immunity.


Asunto(s)
Inmunidad Adaptativa , Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunidad Innata , Ubiquitina-Proteína Ligasas/metabolismo , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo
6.
PLoS One ; 7(11): e49426, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23152905

RESUMEN

In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR), the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR). GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC) was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc)) in Ussing chambers. Endiandrin A (End A) has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF)-α. This was in part due to up-regulation of ß- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK) activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk)-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of ß- and γ-ENaC-subunits along with induction of sgk-1.


Asunto(s)
Colon/metabolismo , Ciclobutanos/farmacología , Canales Epiteliales de Sodio/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Lauraceae/química , Lignanos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Glucocorticoides/agonistas , Animales , Ciclobutanos/química , Dexametasona/farmacología , Activación Enzimática/efectos de los fármacos , Glucocorticoides/farmacología , Células HT29 , Humanos , Inflamación/metabolismo , Inflamación/patología , Absorción Intestinal/efectos de los fármacos , Lignanos/química , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Glucocorticoides/metabolismo , Sodio/metabolismo , Activación Transcripcional/efectos de los fármacos
7.
Eur J Cell Biol ; 90(5): 432-9, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21354648

RESUMEN

The epithelial Na+ channel (ENaC) plays a crucial role in electrogenic Na(+) absorption in the distal colon. ENaC induction via the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) is differentially regulated by modulatory components. As most existing epithelial cell lines including colonic epithelial cell lines miss the co-expression of functional GR and MR, signaling on ENaC is only poorly characterized regarding the interplay of glucocorticoids and mineralocorticoids. In the present study, we show that GR expression and activity are indispensable for MR-dependent induction of ENaC-mediated Na(+) transport. Cooperativity of the two receptors has been studied in the highly differentiated, epithelial colonic cell line HT-29/B6-GR/MR which is equipped with the complete receptor repertoire of both GR and MR due to stable transfection. In contrast to HT-29/B6 cells solely expressing the MR, this cell line displays a physiological response to aldosterone regarding ENaC induction. To achieve this, a pre-incubation step with the GR agonist dexamethasone was required to allow for the subsequent stimulation of ENaC by aldosterone. As a result of cooperative effects between the activated GR and the MR, MR protein levels were elevated and MR-dependent transcription of ENaC subunits ß and γ was increased. As an additional mechanism involved, transcription of SGK-1 (serum- and glucocorticoid-induced kinase 1) and GILZ (glucocorticoid-induced leucin zipper)--both essential for the insertion of ENaC into the apical enterocyte membrane--were also augmented by the activated MR.


Asunto(s)
Aldosterona/metabolismo , Colon/citología , Canales Epiteliales de Sodio/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Línea Celular , Dexametasona/metabolismo , Canales Epiteliales de Sodio/genética , Células HT29 , Humanos , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genética , Sodio/metabolismo
8.
J Biol Chem ; 282(17): 12749-54, 2007 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-17308306

RESUMEN

Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens. The interferon-gamma-inducible proteasome activator PA28 plays an important role in the generation of MHC ligands by proteasomes. Generation of the HLA-A(*)0201 restricted melanoma antigen TRP2(360-368) by the proteasome has been shown to be dependent on the function of PA28 in vitro and in vivo (Sun, Y., Sijts, A. J., Song, M., Janek, K., Nussbaum, A. K., Kral, S., Schirle, M., Stevanovic, S., Paschen, A., Schild, H., Kloetzel, P. M., and Schadendorf, D. (2002) Cancer Res. 62, 2875-2882). Here we analyzed the role of the epitope sequence environment in determining this PA28 dependence. Experiments using the melanoma TRP2(288-296) epitope and the murine cytomegalovirus-derived pp89 epitope precursor peptide for epitope replacement revealed that the TRP2(360-368) flanking sequences can transfer PA28 dependence onto otherwise PA28 independent epitopes. Moreover, the N-terminal flanking sequence is sufficient to establish PA28 dependence of an epitope by allowing PA28-induced coordinated dual cleavages. These results show that N-terminal flanking sequences strongly influence epitope generation efficiency and that PA28 function is particularly relevant for the generation of normally poorly excised peptide products.


Asunto(s)
Antígenos de Neoplasias/química , Epítopos/química , Melanoma/química , Proteínas Musculares , Péptidos/química , Complejo de la Endopetidasa Proteasomal , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/inmunología , Epítopos/inmunología , Antígenos HLA-A/química , Antígenos HLA-A/inmunología , Antígeno HLA-A2 , Humanos , Melanoma/inmunología , Proteínas Musculares/inmunología , Resonancia Magnética Nuclear Biomolecular , Péptidos/inmunología , Complejo de la Endopetidasa Proteasomal/inmunología , Estructura Secundaria de Proteína
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