Insights into the pathophysiology of ulcerative colitis: interleukin‐13 modulates STAT6 and p38 MAPK activity in the colon epithelial sodium channel

Interleukin-13 (IL-13) is a proinflammatory Th2 cytokine identified in the pathogenesis of inflammatory bowel diseases such as ulcerative colitis (UC), and allergic airway diseases like asthma. Interluekin-13 is implicated in epithelial barrier dysfunction by stimulating apoptosis and up-regulating the expression of claudin-2, an epithelial pore-forming protein (Hering et al. 2012). Further, previous research indicates that IL-13 affects ion transport in airway epithelia by regulating the expression of epithelial sodium channel (ENaC) subunits (Anagnostopoulou et al. 2010). ENaC subunits are down-regulated in the colon in UC, and pro-inflammatory cytokines may be the primary regulators (Amasheh et al. 2004). In a recent article in The Journal of Physiology, Dames et al. (2015) set out to determine if IL-13 is involved in regulating ENaC expression on the apical membrane of the distal colon, and if it does, what intercellular signalling is involved in this mechanism. Two different model systems were utilized by Dames et al. (2015); human colorectal cancer cells (HT-29/B6-GR/MR cells) and primary distal colon specimens isolated from both wild type (WT) BALB/c and STAT6−/− mice. The authors used a combination of dexamethasone, butyrate and aldosterone (DBA) to stimulate ENaC dependent sodium absorption (JNa). HT-29/B6-GR/MR cultures treated with IL-13 exhibited a significant reduction in JNa when compared to control cultures after 96 h of treatment (Dames et al. 2015). Distal colon specimens from BALB/c mice treated with IL-13 also had significantly suppressed JNa when compared to control specimens. Dames et al. (2015) then demonstrated that the reduction in JNa was not merely a result of IL-13 treatment-prompted apoptosis, as no difference was identified in caspase-3 (a proteolytic mediator of apoptosis) cleavage in IL-13-treated HT-29/B6-GR/MR cells compared to the control. No significant difference in caspase-3 cleavage was apparent between the treated and control cells, therefore the authors concluded that JNa suppression was not merely due to apoptosis. Dames et al. (2015) used real time-PCR to show that upon stimulation of JNa, HT-29/B6-GR/MR cells treated with IL-13 exhibit significant decreases in mRNA expression of βand γ-ENaC subunits when compared to control cells. These data provide a potential mechanism through which IL-13 treatment may suppress JNa. Luciferase gene reporter assays were also used to examine the influence of IL-13 on γ-ENaC and serum and glucocorticoid-regulated kinase-1 (SGK1; a kinase which triggers apical localization of ENaCs) promoter activity in HT-29/B6-GR/MR cells. Significant reduction in luciferase activity was observed in both γ-ENaC-luciferase and SGK1-luciferase transfected cells treated with IL-13. This suggests that IL-13 may mediate activity of both γ-ENaC and SGK1 promoter activity. Western blot analysis of both phosphorylated and total ERK1/2, JNK and p38 proteins showed that IL-13 treatment increased phosphorylation of only p38 MAPK in HT-29/B6-GR/MR cells after stimulation of JNa. After discerning that IL-13 is involved in inhibition of p38 phosphorylation, Dames et al. (2015) utilized a specific p38 inhibitor (baricitinib) which indeed confirmed that p38 is involved in JNa modulation, even in the absence of IL-13 treatment. Western blot studies revealed that STAT6 is activated by IL-13 treatment. Furthermore, the authors utilized specific inhibitors of STAT6 and JAK1/2 in HT-29/B6-GR/MR cells demonstrating that both of these signalling pathways are involved in JNa suppression caused by IL-13 treatment. To confirm the role of STAT6 in JNa, further experiments using STAT6−/− and WT mice were conducted. Sodium absorption was decreased in STAT6−/− distal colon as compared to distal colon specimens from WT mice, showing that STAT6 signalling is involved in the mechanism through which IL-13 treatment results in JNa repression. After proving that IL-13 treatment inhibits p38 phosphorylation and activates JAK1/2–STAT6, Dames et al. (2015) demonstrated that IL-13 treatmentdependent inhibition of p38 requires STAT6 activation. This was accomplished by monitoring STAT6 phosphorylation levels in the presence of a p38 inhibitor. To further elucidate the signalling cascade through which IL-13 alters JNa, inhibitory antibody experiments were completed to show that IL-13 binds the heterodimeric type-1 receptor (IL-13Rα1/IL-13-4Rα) resulting in downstream activation of the JAK1–2-STAT6 pathway. As cancer cells vary significantly from non-cancerous epithelial cells, extrapolation of findings from colorectal cancer cells, the main model in this study, may not be the best model to study this condition. However, results from ex vivo mouse samples are similar to results observed in HT-29/B6-GR/MR cells, providing some level of confidence for an accurate comparison. The use of DBA to stimulate JNa proved to be extremely effective, but how similar this level of stimulation is with respect to the diseased conditions observed in the colon has not been quantified. However, the signalling pathway identified (IL-13–STAT6–p38) would probably be the same in DBA-stimulated cells, but the observed response may be exaggerated. Previous work demonstrates that IL-13 activates STAT6 resulting in ENaC subunit regulation and thus regulation of JNa in the airway (Anagnostopoulou et al. 2010). However, the present study provides the first evidence demonstrating a mechanism by which increased IL-13 alters sodium absorption in the distal colon, as is seen in individuals with UC. The research presented provides compelling evidence that IL-13 treatment alters JNa by down-regulating transcription of βand γ-ENaC subunits through JAK1/2–STAT6–p38 MAPK signalling pathway modulation in distal colon epithelium. Further, these data add to the general understanding of IL-13 signalling in the colon by demonstrating