Ran Zhuang

miR-195 competes with HuR to modulate stim1 mRNA stability and regulate cell migration

Stromal interaction molecule 1 (Stim1) functions as a sensor of Ca2+ within stores and plays an essential role in the activation of store-operated Ca2+ entry (SOCE). Although lowering Stim1 levels reduces store-operated Ca2+ entry and inhibits intestinal epithelial repair after wounding, the mechanisms that control Stim1 expression remain unknown. Here, we show that cellular Stim1 abundance is controlled posttranscriptionally via factors that associate with 3′-untranslated region (3′-UTR) of stim1 mRNA. MicroRNA-195 (miR-195) and the RNA-binding protein HuR competed for association with the stim1 3′-UTR and regulated stim1 mRNA decay in opposite directions. Interaction of miR-195 with the stim1 3′-UTR destabilized stim1 mRNA, whereas the stability of stim1 mRNA increased with HuR association. Interestingly, ectopic miR-195 overexpression enhanced stim1 mRNA association with argonaute-containing complexes and increased the colocalization of tagged stim1 RNA with processing bodies (P-bodies); the translocation of stim1 mRNA was abolished by HuR overexpression. Moreover, decreased levels of Stim1 by miR-195 overexpression inhibited cell migration over the denuded area after wounding but was rescued by increasing HuR levels. In sum, Stim1 expression is controlled by two factors competing for influence on stim1 mRNA stability: the mRNA-stabilizing protein HuR and the decay-promoting miR-195.

Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by differentially modulating STIM1 and STIM2

Early epithelial restitution occurs as a consequence of intestinal epithelial cell (IEC) migration after wounding, and its defective regulation is implicated in various critical pathological conditions. Polyamines stimulate intestinal epithelial restitution, but their exact mechanism remains unclear. Canonical transient receptor potential-1 (TRPC1)-mediated Ca(2+) signaling is crucial for stimulation of IEC migration after wounding, and induced translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane activates TRPC1-mediated Ca(2+) influx and thus enhanced restitution. Here, we show that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca(2+) signaling by altering the ratio of STIM1 to STIM2. Increasing cellular polyamines by ectopic overexpression of the ornithine decarboxylase (ODC) gene stimulated STIM1 but inhibited STIM2 expression, whereas depletion of cellular polyamines by inhibiting ODC activity decreased STIM1 but increased STIM2 levels. Induced STIM1/TRPC1 association by increasing polyamines enhanced Ca(2+) influx and stimulated epithelial restitution, while decreased formation of the STIM1/TRPC1 complex by polyamine depletion decreased Ca(2+) influx and repressed cell migration. Induced STIM1/STIM2 heteromers by polyamine depletion or STIM2 overexpression suppressed STIM1 membrane translocation and inhibited Ca(2+) influx and epithelial restitution. These results indicate that polyamines differentially modulate cellular STIM1 and STIM2 levels in IECs, in turn controlling TRPC1-mediated Ca(2+) signaling and influencing cell migration after wounding.

Activation of Wnt3a signaling stimulates intestinal epithelial repair by promoting c-Myc-regulated gene expression

In response to mucosal injury, epithelial cells modify the patterns of expressed genes to repair damaged tissue rapidly. Our previous studies have demonstrated that the transcription factor c-Myc is necessary for stimulation of epithelial cell renewal during mucosal healing, but the up-stream signaling initiating c-Myc gene expression after injury remains unknown. Wnts are cysteine-rich glycoproteins that act as short-range ligands to locally activate receptor-mediated signaling pathways and correlate with the increased expression of the c-Myc gene. The current study tested the hypothesis that Wnt3a signaling is implicated in intestinal epithelial repair after wounding by stimulating c-Myc expression. Elevated Wnt3a signaling in intestinal epithelial cells (IEC-6 line) by coculturing with stable Wnt3a-transfected fibroblasts or ectopic overexpression of the Wnt3a gene enhanced intestinal epithelial repair after wounding. This stimulatory effect on epithelial repair was prevented by silencing the Wnt coreceptor LRP6 or by c-Myc silencing. Activation of the Wnt3a signaling pathway increased β-catenin nuclear translocation by decreasing its phosphorylation and stimulated c-Myc expression during epithelial repair after wounding. In stable Wnt3a-transfected IEC-6 cells, increased levels of c-Myc were associated with an increase in expression of c-Myc-regulated genes cyclcin D1 and cyclin E, whereas c-Myc silencing inhibited expression of cyclin D1 and cyclin E and delayed epithelial repair. These results indicate that elevated Wnt3a signaling in intestinal epithelial cells after wounding stimulates epithelial repair by promoting c-Myc-regulated gene expression.

Src-mediated caveolin-1 phosphorylation regulates intestinal epithelial restitution by altering Ca2+ influx after wounding

Early mucosal restitution occurs as a consequence of intestinal epithelial cell (IEC) migration to reseal superficial wounds, but its exact mechanism remains largely unknown. Caveolin-1 (Cav1), a major component associated with caveolar lipid rafts in the plasma membrane, is implicated in many aspects of cellular functions. This study determined if c-Src kinase (Src)-induced Cav1 phosphorylation promotes intestinal epithelial restitution after wounding by activating Cav1-mediated Ca(2+) signaling. Src directly interacted with Cav1, formed Cav1-Src complexes, and phosphorylated Cav1 in IECs. Inhibition of Src activity by its chemical inhibitor PP2 or suppression of the functional caveolin scaffolding domain by caveolin-scaffolding domain peptides prevented Cav1-Src interaction, reduced Cav1 phosphorylation, decreased Ca(2+) influx, and inhibited cell migration after wounding. Disruption of caveolar lipid raft microdomains by methyl-β-cyclodextrin reduced Cav1-mediated Ca(2+) influx and repressed epithelial restitution. Moreover, Src silencing prevented subcellular redistribution of phosphorylated Cav1 in migrating IECs. These results indicate that Src-induced Cav1 phosphorylation stimulates epithelial restitution by increasing Cav1-mediated Ca(2+) signaling after wounding, thus contributing to the maintenance of gut mucosal integrity under various pathological conditions.

HuR Enhances Early Restitution of the Intestinal Epithelium by Increasing Cdc42 Translation

ABSTRACT The mammalian intestinal mucosa exhibits a spectrum of responses after acute injury and repairs itself rapidly to restore the epithelial integrity. The RNA-binding protein HuR regulates the stability and translation of target mRNAs and is involved in many aspects of gut epithelium homeostasis, but its exact role in the regulation of mucosal repair after injury remains unknown. We show here that HuR is essential for early intestinal epithelial restitution by increasing the expression of cell division control protein 42 (Cdc42) at the posttranscriptional level. HuR bound to the Cdc42 mRNA via its 3′ untranslated region, and this association specifically enhanced Cdc42 translation without an effect on the Cdc42 mRNA level. Intestinal epithelium-specific HuR knockout not only decreased Cdc42 levels in mucosal tissues, but it also inhibited repair of damaged mucosa induced by mesenteric ischemia/reperfusion in the small intestine and by dextran sulfate sodium in the colon. Furthermore, Cdc42 silencing prevented HuR-mediated stimulation of cell migration over the wounded area by altering the subcellular distribution of F-actin. These results indicate that HuR promotes early intestinal mucosal repair after injury by increasing Cdc42 translation and demonstrate the importance of HuR deficiency in the pathogenesis of delayed mucosal healing in certain pathological conditions.

292 Deletion of RNA-Binding Protein HuR in Intestinal Epithelial Cells Delays Mucosal Repair After Ischemia/Reperfusion-Induced Injury

Acute gut mucosal injury occurs commonly during critical pathological conditions, leading to mucosal hemorrhage, epithelial barrier dysfunction, and the translocation of luminal toxic substances and bacteria to the blood stream, but the exact mechanism underlying mucosal injury/repair are still obscure. RNA-binding proteins (RBPs) posttranscriptionally regulate gene expression and are implicated in many aspects of pathophysiology. HuR is among the most prominent sequence-specific RBP and has emerged as a master regulator of the maintenance of the homeostasis of gut mucosa. Our previous studies show that HuR regulates intestinal epithelial cell proliferation and apoptosis, but the exact role of HuR in gut mucosal repair after injury remains unknown. This study tested the hypothesis that HuR plays a role in intestinal mucosal repair after ischemia/reperfusion (I/R)-induced Injury.Methods: Studies were conducted in our recently generating intestinal epithelial tissue-specific HuR knockout (IE-HuR-/-) mice and wild-type littermates. Mucosal injury was induced by exposed to 30min of mesenteric ischemia, followed by 2-h reperfusion. Barrier function was detected by paracellular tracer flux assay using FITC-dextran. Results: The mucosa of the animals subjected to mesenteric I/R displayed signs of remarkable damage. Macroscopically, the intestines of the animals exposed to I/R exhibited swollen and edematous with areas of red streaks in both littermates and IE-HuR-/animals. Microscopic analysis showed that there were severed damages in the small intestinal mucosa as indicated by sloughed cells, denuded villi with dilated capillaries, and frank hemorrhage. Although there were no significant differences in the injury scores between littermates and IE-HuR-/mice when measured immediately after I/R, HuR deletion delayed the process of early mucosal repair after I/Rinduced injury. The mucosal surface remained discontinuous, showing sloughed cells and debris in IE-HuR-/mice (injury score: 3.3 ± 0.12) 6 h after I/R, whereas the mucosa in littermates was almost completely recovered (0.2 ± 0.03). Both littermate and IE-HuR-/mice exhibited significant gut barrier dysfunction after I/R-induced injury, but increased levels of gut permeability in IE-HuR-/mice were much greater than those observed in littermates. The recovery of gut barrier function after I/R was also inhibited by HuR deletion. Moreover, expression of the Wnt co-receptor LRP6 and ARP2 in the intestinal mucosa of HuR-/mice decreased dramatically, which are necessary for normal mucosal repair after injury. Conclusions: These results indicate that 1) HuR is crucial for maintenance of normal gut mucosal epithelial integrity and 2) HuR deletion in intestinal epithelial cells delays mucosal repair and represses the recovery of gut barrier function after I/R-induced mucosal injury.

Tu1733 MicroRNA-322/503 Repress Translation of Smurf1 and Smurf2 to Regulate TGFβ/SMAD Signaling and Gut Epithelial Homeostasis

Background and aims: The intestinal epithelium is a vigorously self-renewing tissue. Stem cells and transient-amplifying cells are located at the base of the crypts and give rise to terminally differentiated cells which migrate up the villous tip. A tightly regulated balance between proliferation and cell death is of utmost importance. Survivin is a unique member of the inhibitor of apoptosis (IAP) family with bifunctional properties involved in controlling apoptosis and cell division, respectively. To elucidate the contribution of survivin in the intestine we analysed mice with a specific deletion of survivin in intestinal epithelial cells (IECs). Methods: Survivinfl mice were crossbred to Villin-Cre or Villin-CreERT2 mice, resulting in conditional knockout mice (SurvivinΔIEC) or inducible knockout mice (SurviviniΔIEC). SurviviniΔIEC and control mice were histologically investigated by immuno¬histochemistry. The gene and protein expression pattern of IECs of Survivini ΔIEC mice was analysed by quantitative PCR and Western-Blot. To analyse the timepoint of the survivin deletion and intestinal epithelial cell death we focused on the in-vitro cultivation of organoids from SurviviniΔIEC mice. Results: We initially intended to generate mice with a specific deletion of the survivin gene in IECs but mice were embryonic lethal. Therefore, we created mice with an inducible deletion of the survivin gene in IECs, triggered by a daily injection of tamoxifen. Immunohistochemical staining of control gut tissue revealed a localisation of survivin in the crypt compartment. Detailed analysis showed specific presence of survivin in transient amplifying cells and stem cells. The specific deletion of survivin in IECs leads to a fatal destruction of the intestinal epithelium. IECs of Survivini ΔIEC mice showed abnormally enlarged nuclei, suggesting a defect in cell cycle progression. Furthermore, we observed accumulations of encapsulated debri and a massive dislocation of the crypt compartment. Western-blot analysis revealed elevated protein levels of activated caspase-3 and caspase-8, indicating dysregulated cell death of survivin depleted IECs. Quantitative PCR showed reduced gene expression levels of stem cell genes like lgr5 and olfm4, as well as for the proliferation marker ki67. Conclusions: The dramatic phenotype of Survivini ΔIEC mice demonstrated the absolute necessity of survivin in the intestinal epithelium. The lack of survivin results in a complete loss of the crypt-villous structure and the prominent formation of enlarged nuclei, arrested in cell cycle. The loss of stem cells as well as transient amplifying cells demonstrates the important contribution of survivin for the intestinal stem cell niche and the preservation of epithelial cells in the gut.

985 Induced β-Pix Signaling by Polyamines Activates RAC1 and Stimulates Epithelial Restitution After Wounding

BACKGROUND/AIM: Though antisecretory drugs such as histamine H2-receptor antagonists (H2-RAs) and proton pump inhibitors (PPIs) are commonly used for the treatment of upper gastrointestinal mucosal lesions induced by NSAIDs, it has recently been reported that these drugs exacerbated small intestinal lesions induced by NSAIDs in rats. However, there are few effective agents for the treatment of this complication. We examined the effect of various mucosal protective agents (MPAs) and mucin (MUC) on NSAID-induced intestinal lesions and the exacerbation of intestinal lesions caused by antisecretory drugs. Effect of drugs on intestinal motility was also examined. METHODS: Male Wistar rats (180-220 g, 7 rats/ group) were treated with diclofenac Na (DIC) with or without ranitidine (RAN) or omeprazole (OPZ) concomitantly. RAN or OPZ was administered p.o. 30 min before DIC. Each of MPAs (misoprostol, MIS; irsogladine, IRS; rebamipide, REB) and MUC was administered p.o. 30 min before RAN or OPZ. Intestinal lesions were examined 24 h after DIC administration. Intestinal motility was measured by using a balloon method under urethane anesthesia. RESULTS: 1) DIC (10 mg/kg, p.o.) produced many lesions in the middle and lower parts of the small intestine. The lesion index (LI, total length of individual lesions) in the vehicletreated group was 100.6±10.8 mm. Both MPAs (MIS: 0.03-0.3 mg/kg, IRS: 1-10 mg/kg, and REB: 30-300mg/kg) andMUC (30-300mg/kg) dose-dependently inhibited the formation of lesions. 2) Lower dose of DIC (3 mg/kg) or RAN (30 mg/kg) alone did not cause significant lesions in the small intestine, but marked lesions were observed following co-administration of RAN and DIC; the LI was 92.0±12.0 mm (P,0.001). The increase of lesions was prevented by the MPAs and MUC dose-dependently. 3) DIC (6 mg/kg) caused mild lesions in the small intestine, the LI in the vehicle-treated group was 38.3±4.5 mm. OPZ (100 mg/kg) did not cause lesions in the small intestine, but significantly increased the lesions caused by DIC; the LI was 78.5±10.9 mm (P,0.001). The increase of lesions was inhibited by the MPAs and MUC dose-dependently. 4) DIC (3-30 mg/kg, s.c.) increased the ileal motility dose-dependently. The increase of motility was inhibited by s.c. administration of MIS (0.1 mg/kg) and atropine (10 mg/kg), but was not affected by IRS (10 mg/kg) and REB (300 mg/kg). CONCLUSIONS: 1) Both RAN andOPZ enhanced the intestinal lesions caused by low (clinical) doses of DIC, supporting the previous findings that antisecretory drugs exacerbated intestinal lesions induced by NSAIDs; 2) MPAs as well as MUC prevented the formation of intestinal lesions induced by DIC alone and inhibited the exacerbation of intestinal lesions by RAN and OPZ, suggesting the usefulness of MPAs for the treatment of NSAID-induced small intestinal lesions with or without antisecretory drugs.