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Key pointsr 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).r We demonstrate in an intestinal cell model as well as in mouse distal colon that IL-13 causes reduced ENaC activity.r We show that IL-13 impairs ENaC-dependent sodium transport by activating the JAK1/2-STAT6 signalling pathway.r 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 (I SC ) 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 (J Na ) as a result of decreased transcription levels of β-and γ-ENaC subunits and SGK1, a post-translational regulator of ENaC activity, due to impaired promoter activity. The reduction in J Na 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.Please note that Figures 1, 3, 4, 5 and 6 that appear in this final copy-edited version have been updated since the paper was published online as an Accepted Article. The new figures have been supplied by the authors and have been approved by the Editor. The conclusions of the study remain the same. Abbreviations DBA, dexamethasone plus butyrate and aldosterone; ENaC, epithelial sodium channel; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HRE, hormone response element; IL-13, interleukin-13; I SC , amiloride sensitive short-circuit current; J Na , epithelial sodium absorption via the ENaC; MAPK, mitoge...

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Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens. The interferon-␥-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.The generation of major histocompatibility complex (MHC) 2 class I ligands and the recognition of MHC class I-ligand complexes by CD8ϩ T cells is an effective tool for the elimination of infected or disordered cells from organisms. The ubiquitin-proteasome system represents the major source for MHC class I-presented peptides exposed to CD8ϩ T cells (1). Ubiquitin-proteasome system-mediated peptide generation is influenced by structural changes in the 20S catalytic core (20S proteasome), which occur by exchange of the standard catalytic subunits by immunosubunits. The ATP-dependent 26S proteasome, composed of a 20S core and a 19S regulatory complex, recognizes substrates by a multi-ubiquitin signal. In contrast, 20S proteasomes can accept larger non-ubiquitinated peptides as substrates for ATP-independent degradation (3). However, 20S proteasomes exist in a latent state within the cells. In consequence, proteasome activity and function is regulated in part by modulating substrate entry by the PA28 complex, which attaches ATP-independent to the outer ␣-rings of the 20S proteasome. Detailed kinetic analysis using fluorogenic peptide substrates has shown that activation of the 20S proteasome by PA28 occurs by facilitating either substrate entry and/or product exit, with no effects on the active sites (3). In support, structural analyses have demonstrated that attachment of the activator to the 20S core leads to the opening of the central gate of the ␣-ring (4).The expression of PA28 is constitutively enhanced in cells with specialized antigen-presenting function and is induced by stimulation of cells with interferon-␥. The presentation of a number of viral MHC class I epitopes is enhanced in the presence...

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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 (Isc) 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 β- 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 β- and γ-ENaC-subunits along with induction of sgk-1.

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