Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na+/H+ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1‐dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1‐dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ∼10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady‐state pHi and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process.

Background: The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients.Aim and Methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-alpha, IFN-gamma, and IL-1 beta, and analysing PDZK1 promotor activity, mRNA and protein expression.Results: IL-1 beta was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1 beta-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-beta downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1 beta mediated downregulation of PDZK1, while NF-kappa B, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXR alpha and PPAR alpha was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1 beta -treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPAR alpha-agonist GW7647 stimulated PDZK1mRNA and protein expression and attenuated IL-1 beta -mediated inhibition.Conclusions: The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1 beta -mediated RXR alpha and PPAR alpha repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy.

Background: The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients. Aim and Methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-α, IFN-γ, and IL-1β, and analysing PDZK1 promotor activity, mRNA and protein expression. Results: IL-1β was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1β-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-β downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1β mediated downregulation of PDZK1, while NF-κB, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXRα and PPARα was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1β-treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPARα-agonist GW7647 stimulated PDZK1 mRNA and protein expression and attenuated IL-1β-mediated inhibition. Conclusions: The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1β-mediated RXRα and PPARα repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy.

with LPS in vitro.Overexpression of TGR5 resulted in increased PCNA, CCK8, EdU incorporation and the proportion of cells in S phase, whereas, knockdown of TGR5 caused an opposite effect.Conclusions: Bile acids could promote intestinal epithelial cell proliferation and reduce the mucosal injury by upregulating the expression of TGR5 in obstructive jaundice.

to crypts.Aquaporins are a family of cell plasma membrane channels; AQP3 transports water, and glycerol and putatively also hydrogen peroxide (H 2 O 2 ).AQP3 is expressed on the basolateral membrane of epithelial cells lining the colon, but its exact role in intestinal function in health and disease are not known.To investigate the epithelial specific role of AQP-3 in intestinal crypt formation, we generated small intestinal and colonic enteroids from wild-type and AQP-3 null mice.AQP-3 deficient enteroids exhibited slowed crypt budding and growth on initial isolation.To measure AQP-3 dependent H 2 O 2 transport, enteriods were virally transfected with a ratiometric fluorescent H 2 O 2 sensitive YFP.Enteroids were immobilized on matrigel coated coverslips in a small volume perfusion chamber and live fluorescence imaging carried out.AQP3 deficient enterocytes exhibited slowed membrane hydrogen peroxide transport at high extracellular (>100mM) concentrations.Enteroids grown with hydrogen peroxide on isolation resulted in a change in enteroid morphology.These data suggest that AQP-3 may play a role in crypt formation in response to injury.

Aims: The alarmin HMGB1 is released during cell damage and acts as an early or late stage inflammatory cytokine at multiple levels of target cells. The Na+/H+ exchanger Isoform 3 (NHE3) is the major intestinal salt and fluid absorptive transporter and its function is defective in intestinal inflammation. Aim of the study: We wanted to know if HMGB1 can affect NHE3 mRNA or protein expression as well as its localization in mucosal tissue form patients with ulcerative colitis and in NHE3-expressing Caco2bbe colonic cells. Methods and Results: We studied the mRNA and protein expression and localization of HMGB1 and NHE3 in rectal biopsies from the inflamed and noninflamed mucosa of patients with ulcerative colitis by quantitative qPCR and immunohistochemistry. TNF-α and IFN-γmRNA level was significantly elevated in lesions compared to that in its adjacent normal tissue samples of UC patient's rectal, suggesting a reliable endoscopic sampling of inflamed vs noninflamed tissue. HMGB1 mRNA and protein expression was increased in the inflamed as compared to adjacent normal tissues, and it was mostly distributed in cytoplasm and extracellular region. NHE3 protein was markedly reduced in inflamed compared to adjacent normal tissues, though its mRNA level was not significantly different between inflamed and noninflamed mucosa. When Caco2bbe cells overexpressing human NHE3 (Caco2bbe/ hNHE3) were incubated with high concentration of rh-HMGB1 (10μg/ml), NHE3 mRNA was found to be decreased at 1h (0.385±0.015 vs. 1.011±0.011, p 0.05). NHE3 protein, measured byWestern analysis, was not significantly changed when co-cultured with 10μg/ml rhHMGB1 for 1h or 6h, however, it was dramatically decreased at 24h of co-culture with 10μg/ml rhHMGB1. IFN-gamma levels were markedly increased when Caco2bbe/hNHE3 cells were incubated with 10 μg/ml rhHMGB1 for 1h as compared with normal group (29.855±0.055 vs. 29.525±0.050, p 0.05) and 24h (29.066±0.181 vs. 29.525±0.050, p>0.05) of co-culture with 10μg/ml rhHMGB1 as compared to normal group. This suggests that incubation with rhHMGB1 may down-regulate NHE3 mRNA expression via elevated IFN-gamma level at early stage (1h), resulting in a decrease in NHE3 protein expression at late stage (24h). Conclusion: Decreased NHE3 protein level was accompanied by elevated cytoplasm and extracellular HMGB1 in human ulcerative rectitis tissue. This may result from the regulatory effect of cytokines such as IFNgamma induced by exogenous HMGB1 at late stage of inflammation.

A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.

Hintergrund: Es konnte gezeigt werden, dass der Na+/H+-Austauscher Isoform 1 (NHE1) eine bedeutende Rolle bei der Zellmigration während der Wundheilung und während des invasiven Tumorwachstums zukommt. Das Epithel des Magendarmtrakts zeigt eine sehr schnelle Wundheilung, jedoch sind hier NHE2 und NHE3 stark exprimiert.

Intestinal inflammation results in loss of sodium absorptive function and diarrhea. The present study investigates a potential causal relationship between the inflammation‐induced reduction in expression levels of the PDZ‐adaptor protein PDZK1 and decreased functional activity of its binding partner Na+/H+ exchanger NHE3. Expression of PDZK1 mRNA and protein was found strongly decreased in inflamed intestinal mucosa of murine colitis models, in colonic mucosa of patients with active ulcerative colitis, and in Caco‐2bbe cells treated with Th1 proinflammatory cytokines. Acid‐activated NHE3 transport activity as well as its inhibition by forskolin (FSK) was assessed fluorometrically in microdissected murine and human BCECF‐loaded ileal villi and colonic crypts. The decrease in acid‐activated NHE3 activity and loss of FSK inhibition was similar in PDZK1 +/− enterocytes, in which PDZK1 protein content was reduced by >; 50%, than in inflamed enterocytes, suggesting a causal relationship between PDZK1 downregulation and NHE3 dysfunction, independent of inflammation. Likewise, ShRNA‐mediated PDZK1 knockdown in Caco2bbe resulted in a significant decrease in NHE3 membrane abundance, transport activity and FSK inhibition. We conclude that the marked decrease in the PDZ‐adaptor protein PDZK1 in inflamed murine and human enterocytes is causally related to inflammation‐associated NHE3 dysfunction.