Aquaporin-8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis

AIM： To study susceptibility genes which may play a potential role in the pathogenesis and etiology of inflammatory bowel disease （IBD）. METHODS： To identify potential susceptibility genes we performed global gene expression profiling in patients with IBD and control specimens. For determination of an intrinsic gene expression profile in ulcerative colitis （UC） and Crohn＇s disease （CD） compared to normal subjects, mucosal biopsies of non-inflamed regions of the colon and the terminal ileum were subjected to DNA microarray analysis. Real-time RT-PCR and immunohistochemistry were used for verification of selected regulated candidate genes and a genetic analysis was performed. RESULTS： We could show that aquaporin-8 （AQP8） mRNA and protein levels were significantly increased in the colon of UC patients compared to controls. Genetic analysis of the six exons and the promoter region of AQPS, however, revealed no mutations or polymorphisms in IBD patients. CONCLUSION： Our results suggest that upregulation of AQP8 in the colon of UC patients represents a secondary phenomenon which may, due to altered water exchange of the distal intestinal mucosa, disturb the physiologic colonic mucus barrier and thus lead to chronic inflao mmation and ulceration.

Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn's disease and contributes to accelerated epithelial wound healing in vitro

AIM:Eph receptor tyrosine kinases and their membrane bound receptor-like ligands, the ephrins, represent a bi-directional cell-cell contact signaling system that directs epithelial movements in development. The meaning of this system in the adult human gut is unknown. We investigated the Eph/ephrin mRNA expression in the intestinal epithelium of healthy controls and patients with inflammatory bowel disease (IBD). METHODS: mRNA expression profiles of all Eph/ephrin family members in normal small intestine and colon were established by real-time RT-PCR. In addition, differential expression in IBD was investigated by cDNA array technology, and validated by both real-time RT-PCR and immunohistochemistry. Potential effects of enhanced EphB/ephrin-B signaling were analyzed in an in vitro IEC-6 cell scratch wound model. RESULTS: Human adult intestinal mucosa exhibits a complex pattern of Eph receptors and ephrins. Beside the known prominent co-expression of EphA2 and ephrinAl, we found abundantly co-expressed EphB2 and ephrin-B1/2. Interestingly, cDNA array data, validated by real-time PCR and immunohistochemistry, showed upregulation of ephrin-B2 in both perilesional and lesional intestinal epithelial cells of IBD patients, suggesting a role in epithelial homeostasis. Stimulation of ephrin-B signaling in ephrin- B1/2 expressing rat IEC-6-cells with recombinant EphB1-Fc resulted in a significant dose-dependent acceleration of wound closure. Furthermore, fluorescence microscopy showed that EphB1-Fc induced coordinated migration of wound edge cells is associated with enhanced formation of lamellipodial protrusions into the wound, increased actin stress fiber assembly and production of laminin at the wound edge. CONCLUSION: EphB/ephrin-B signaling might represent a novel protective mechanism that promotes intestinal epithelial wound healing, with potential impact on epithelial restitution in IBD.

Serum bile acid profiling reflects enterohepatic detoxification state and intestinal barrier function in inflammatory bowel disease

AIM： To determine free and conjugated serum bile acid （BA） levels in inflammatory bowel disease （IBD） subgroups with defined clinical manifestations. METHODS： Comprehensive serum BA profiling was performed in 358 IBD patients and 310 healthy con- trols by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. RESULTS： Serum levels of hyodeoxycholic acid, the CYP3A4-mediated detoxification product of the second- ary BA lithocholic acid （LCA）, was increased significantly in Crohn＇s disease （CD） and ulcerative colitis （UC）, while most other serum BA species were decreased signifi- cantly. Total BA, total BA conjugate, and total BA glyco- conjugate levels were decreased only in CD, whereas total unconjugated BA levels were decreased only in UC. In UC patients with hepatobiliary manifestations, the conjugated primary BAs glycocholic acid, taurocholic acid, and glycochenodeoxycholic acid were as signifi- cantly increased as the secondary BAs LCA, ursodeoxy- cholic acid, and tauroursodeoxycholic acid compared to UC patients without hepatobiliary manifestations. Finally, we found that in ileocecal resected CD patients, the unconjugated primary BAs, cholic acid and chenode- oxycholic acid, were increased significantly compared to controls and patients without surgical interventions. CONCLUSION： Serum BA profiling in IBD patients that indicates impaired intestinal barrier function and increased detoxification is suitable for advanced diag- nostic characterization and differentiation of IBD sub- groups with defined clinical manifestations.

Lithocholic acid induction of the FGF19 promoter in intestinal cells is mediated by PXR

To study the effect of the toxic secondary bile acid lithocholic acid （LCA） on the expression of fibroblast growth factor 19 （FGF19） in intestinal cells and to characterize the pregnane-X-receptor （PXR） response of the FGF19 promoter region. METHODS： The intestinal cell line LS174T was stimulated with various concentrations of chenodeoxy- cholic acid and lithocholic acid for several time points. FGF19 mRNA levels were determined with quantitative realtime RT-PCR. FGF19 deletion promoter constructs were generated and the LCA response was analzyed in reporter assays. Co-transfections with PXR and RXR were carried out to study FGF19 regulation by these factors, RESULTS： LCA and CDCA strongly up-regulate FGF19 mRNA expression in LS174T cells in a time and dose dependent manner. Using reporter gene assays with several deletion constructs we found that the LCA responsive element in the human FGF19 promoter maps to the proximal regulatory region containing two poten- tial binding sites for PXR. Overexpression of PXR and its dimerization partner retinoid X receptor （RXR） and stimulation with LCA or the potent PXR ligand rifampicin leads to a significant induction of FGF19 promoter activ- ity in intestinal cells. CONCLUSION： LCA induced feedback inhibition of bile acid synthesis in the liver is likely to be regulated by PXR inducing intestinal FGF19 expression.