Small noncoding vault RNA2-1 disrupts gut epithelial barrier function via interaction with HuR.

Vault RNAs (vtRNAs) are small noncoding RNAs and highly expressed in many eukaryotes. Here, we identified vtRNA2-1 as a novel regulator of the intestinal barrier via interaction with RNA-binding protein HuR. Intestinal mucosal tissues from patients with inflammatory bowel diseases and from mice with colitis or sepsis express increased levels of vtRNAs relative to controls. Ectopically expressed vtRNA2-1 decreases the levels of intercellular junction (IJ) proteins claudin 1, occludin, and E-cadherin and causes intestinal epithelial barrier dysfunction in vitro, whereas vtRNA2-1 silencing promotes barrier function. Increased vtRNA2-1 also decreases IJs in intestinal organoid, inhibits epithelial renewal, and causes Paneth cell defects ex vivo. Elevating the levels of tissue vtRNA2-1 in the intestinal mucosa increases the vulnerability of the gut barrier to septic stress in mice. vtRNA2-1 interacts with HuR and prevents HuR binding to claudin 1 and occludin mRNAs, thus decreasing their translation. These results indicate that vtRNA2-1 impairs intestinal barrier function by repressing HuR-facilitated translation of claudin 1 and occludin.

Long noncoding RNA uc.230/CUG-binding protein 1 axis sustains intestinal epithelial homeostasis and response to tissue injury.

Intestinal epithelial integrity is commonly disrupted in patients with critical disorders, but the exact underlying mechanisms are unclear. Long noncoding RNAs transcribed from ultraconserved regions (T-UCRs) control different cell functions and are involved in pathologies. Here, we investigated the role of T-UCRs in intestinal epithelial homeostasis and identified T-UCR uc.230 as a major regulator of epithelial renewal, apoptosis, and barrier function. Compared with controls, intestinal mucosal tissues from patients with ulcerative colitis and from mice with colitis or fasted for 48 hours had increased levels of uc.230. Silencing uc.230 inhibited the growth of intestinal epithelial cells (IECs) and organoids and caused epithelial barrier dysfunction. Silencing uc.230 also increased IEC vulnerability to apoptosis, whereas increasing uc.230 levels protected IECs against cell death. In mice with colitis, reduced uc.230 levels enhanced mucosal inflammatory injury and delayed recovery. Mechanistic studies revealed that uc.230 increased CUG-binding protein 1 (CUGBP1) by acting as a natural decoy RNA for miR-503, which interacts with Cugbp1 mRNA and represses its translation. These findings indicate that uc.230 sustains intestinal mucosal homeostasis by promoting epithelial renewal and barrier function and that it protects IECs against apoptosis by serving as a natural sponge for miR-503, thereby preserving CUGBP1 expression.

Circular RNA CircHIPK3 Promotes Homeostasis of the Intestinal Epithelium by Reducing MicroRNA 29b Function.

BACKGROUND & AIMS: Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that form covalently closed circles. Although circRNAs influence many biological processes, little is known about their role in intestinal epithelium homeostasis. We surveyed circRNAs required to maintain intestinal epithelial integrity and identified circular homeodomain-interacting protein kinase 3 (circHIPK3) as a major regulator of intestinal epithelial repair after acute injury. METHODS: Intestinal mucosal tissues were collected from mice exposed to cecal ligation and puncture for 48 hours and patients with inflammatory bowel diseases and sepsis. We isolated primary enterocytes from the small intestine of mice and derived intestinal organoids. The levels of circHIPK3 were silenced in intestinal epithelial cells (IECs) by transfection with small interfering RNAs targeting the circularization junction of circHIPK3 or elevated using a plasmid vector that overexpressed circHIPK3. Intestinal epithelial repair was examined in an in vitro injury model by removing part of the monolayer. The association of circHIPK3 with microRNA 29b (miR-29b) was determined by biotinylated RNA pull-down assays. RESULTS: Genome-wide profile analyses identified ∼300 circRNAs, including circHIPK3, differentially expressed in the intestinal mucosa of mice after cecal ligation and puncture relative to sham mice. Intestinal mucosa from patients with inflammatory bowel diseases and sepsis had reduced levels of circHIPK3. Increasing the levels of circHIPK3 enhanced intestinal epithelium repair after wounding, whereas circHIPK3 silencing repressed epithelial recovery. CircHIPK3 silencing also inhibited growth of IECs and intestinal organoids, and circHIPK3 overexpression promoted intestinal epithelium renewal in mice. Mechanistic studies revealed that circHIPK3 directly bound to miR-29b and inhibited miR-29 activity, thus increasing expression of Rac1, Cdc42, and cyclin B1 in IECs after wounding. CONCLUSIONS: In studies of mice, IECs, and human tissues, our results indicate that circHIPK3 improves repair of the intestinal epithelium at least in part by reducing miR-29b availability.

MicroRNA-195 regulates Tuft cell function in the intestinal epithelium by altering translation of DCLK1.

Intestinal Tuft cells sense luminal contents to influence the mucosal immune response against eukaryotic infection. Paneth cells secrete antimicrobial proteins as part of the mucosal protective barrier. Defects in Tuft and Paneth cells occur commonly in various gut mucosal disorders. MicroRNA-195 (miR-195) regulates the stability and translation of target mRNAs and is involved in many aspects of cell processes and pathologies. Here, we reported the posttranscriptional mechanisms by which miR-195 regulates Tuft and Paneth cell function in the small intestinal epithelium. Mucosal tissues from intestinal epithelial tissue-specific miR-195 transgenic (miR195-Tg) mice had reduced numbers of double cortin-like kinase 1 (DCLK1)-positive (Tuft) and lysozyme-positive (Paneth) cells, compared with tissues from control mice, but there were no effects on Goblet cells and enterocytes. Intestinal organoids expressing higher miR-195 levels from miR195-Tg mice also exhibited fewer Tuft and Paneth cells. Transgenic expression of miR-195 in mice failed to alter growth of the small intestinal mucosa but increased vulnerability of the gut barrier in response to lipopolysaccharide (LPS). Studies aimed at investigating the mechanism underlying regulation of Tuft cells revealed that miR-195 directly interacted with the Dclk1 mRNA via its 3'-untranslated region and inhibited DCLK1 translation. Interestingly, the RNA-binding protein HuR competed with miR-195 for binding Dclk1 mRNA and increased DCLK1 expression. These results indicate that miR-195 suppresses the function of Tuft and Paneth cells in the small intestinal epithelium and further demonstrate that increased miR-195 disrupts Tuft cell function by inhibiting DCLK1 translation via interaction with HuR.

Long Noncoding RNA H19 Impairs the Intestinal Barrier by Suppressing Autophagy and Lowering Paneth and Goblet Cell Function.

BACKGROUND & AIMS: The protective intestinal mucosal barrier consists of multiple elements including mucus and epithelial layers and immune defense; nonetheless, barrier dysfunction is common in various disorders. The imprinted and developmentally regulated long noncoding RNA H19 is involved in many cell processes and diseases. Here, we investigated the role of H19 in regulating Paneth and goblet cells and autophagy, and its impact on intestinal barrier dysfunction induced by septic stress. METHODS: Studies were conducted in H19-deficient (H19-/-) mice, mucosal tissues from patients with sepsis, primary enterocytes, and Caco-2 cells. Septic stress was induced by cecal ligation and puncture (CLP), and gut permeability was detected by tracer fluorescein isothiocyanate-dextran assays. The function of Paneth and goblet cells was examined by immunostaining for lysozyme and mucin 2, respectively, and autophagy was examined by microtubule-associated proteins 1A/1B light chain 3 II immunostaining and Western blot analysis. Intestinal organoids were isolated from H19-/- and control littermate mice and treated with lipopolysaccharide (LPS). RESULTS: Intestinal mucosal tissues in mice 24 hours after exposure to CLP and in patients with sepsis showed high H19 levels, associated with intestinal barrier dysfunction. Targeted deletion of the H19 gene in mice enhanced the function of Paneth and goblet cells and promoted autophagy in the small intestinal mucosa. Knockout of H19 protected Paneth and goblet cells against septic stress, preserved autophagy activation, and promoted gut barrier function after exposure to CLP. Compared with organoids from control littermate mice, intestinal organoids isolated from H19-/- mice had increased numbers of lysozyme- and mucin 2-positive cells and showed increased tolerance to LPS. Conversely, ectopic overexpression of H19 in cultured intestinal epithelial cells prevented rapamycin-induced autophagy and abolished the rapamycin-induced protection of the epithelial barrier against LPS. CONCLUSIONS: In investigations of mice, human tissues, primary organoids, and intestinal epithelial cells, we found that increased H19 inhibited the function of Paneth and goblet cells and suppressed autophagy, thus potentially contributing to barrier dysfunction in intestinal pathologies.

Cooperative Repression of Insulin-Like Growth Factor Type 2 Receptor Translation by MicroRNA 195 and RNA-Binding Protein CUGBP1.

Insulin-like growth factor type 2 (IGF2) receptor (IGF2R) recognizes mannose 6-phosphate-containing molecules and IGF2 and plays an important role in many pathophysiological processes, including gut mucosal adaptation. However, the mechanisms that control cellular IGF2R abundance are poorly known. MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) critically regulate gene expression programs in mammalian cells by modulating the stability and translation of target mRNAs. Here we report that miRNA 195 (miR-195) and RBP CUG-binding protein 1 (CUGBP1) jointly regulate IGF2R expression at the posttranscriptional level in intestinal epithelial cells. Both miR-195 and CUGBP1 interacted with the 3' untranslated region (3'-UTR) of Igf2r mRNA, and the association of CUGBP1 with Igf2r mRNA enhanced miR-195 binding to Igf2r mRNA. Ectopically expressed CUGBP1 and miR-195 repressed IGF2R translation cooperatively without altering the stability of Igf2r mRNA. Importantly, the miR-195- and CUGBP1-repressed levels of cellular IGF2R led to a disruption in the structure of the trans-Golgi network. These findings indicate that IGF2R expression is controlled posttranscriptionally by two factors that associate with Igf2r mRNA and suggest that miR-195 and CUGBP1 dampen IGF signaling by inhibiting IGF2R translation.

CUGBP1 and HuR regulate E-cadherin translation by altering recruitment of E-cadherin mRNA to processing bodies and modulate epithelial barrier function.

The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.

Competitive binding of CUGBP1 and HuR to occludin mRNA controls its translation and modulates epithelial barrier function.

RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HuR are highly expressed in epithelial tissues and modulate the stability and translation of target mRNAs. Here we present evidence that CUGBP1 and HuR jointly regulate the translation of occludin and play a crucial role in the maintenance of tight junction (TJ) integrity in the intestinal epithelial cell monolayer. CUGBP1 and HuR competed for association with the same occludin 3'-untranslated region element and regulated occludin translation competitively and in opposite directions. CUGBP1 overexpression decreased HuR binding to occludin mRNA, repressed occludin translation, and compromised the TJ barrier function, whereas HuR overexpression inhibited CUGBP1 association with occludin mRNA and promoted occludin translation, thereby enhancing the barrier integrity. Repression of occludin translation by CUGBP1 was due to the colocalization of CUGBP1 and tagged occludin RNA in processing bodies (P-bodies), and this colocalization was prevented by HuR overexpression. These findings indicate that CUGBP1 represses occludin translation by increasing occludin mRNA recruitment to P-bodies, whereas HuR promotes occludin translation by blocking occludin mRNA translocation to P-bodies via the displacement of CUGBP1.

Chk2-dependent HuR phosphorylation regulates occludin mRNA translation and epithelial barrier function.

Occludin is a transmembrane tight junction (TJ) protein that plays an important role in TJ assembly and regulation of the epithelial barrier function, but the mechanisms underlying its post-transcriptional regulation are unknown. The RNA-binding protein HuR modulates the stability and translation of many target mRNAs. Here, we investigated the role of HuR in the regulation of occludin expression and therefore in the intestinal epithelial barrier function. HuR bound the 3'-untranslated region of the occludin mRNA and enhanced occludin translation. HuR association with the occludin mRNA depended on Chk2-dependent HuR phosphorylation. Reduced HuR phosphorylation by Chk2 silencing or by reduction of Chk2 through polyamine depletion decreased HuR-binding to the occludin mRNA and repressed occludin translation, whereas Chk2 overexpression enhanced (HuR/occludin mRNA) association and stimulated occludin expression. In mice exposed to septic stress induced by cecal ligation and puncture, Chk2 levels in the intestinal mucosa decreased, associated with an inhibition of occludin expression and gut barrier dysfunction. These results indicate that HuR regulates occludin mRNA translation through Chk2-dependent HuR phosphorylation and that this influence is crucial for maintenance of the epithelial barrier integrity in the intestinal tract.

Polyamines regulate the stability of JunD mRNA by modulating the competitive binding of its 3' untranslated region to HuR and AUF1.

Polyamines critically regulate all mammalian cell growth and proliferation by mechanisms such as the repression of growth-inhibitory proteins, including JunD. Decreasing the levels of cellular polyamines stabilizes JunD mRNA without affecting its transcription, but the exact mechanism whereby polyamines regulate JunD mRNA degradation has not been elucidated. RNA-binding proteins HuR and AUF1 associate with labile mRNAs bearing AU-rich elements located in the 3' untranslated regions (3'-UTRs) and modulate their stability. Here, we show that JunD mRNA is a target of HuR and AUF1 and that polyamines modulate JunD mRNA degradation by altering the competitive binding of HuR and AUF1 to the JunD 3'-UTR. The depletion of cellular polyamines enhanced HuR binding to JunD mRNA and decreased the levels of JunD transcript associated with AUF1, thus stabilizing JunD mRNA. The silencing of HuR increased AUF1 binding to the JunD mRNA, decreased the abundance of HuR-JunD mRNA complexes, rendered the JunD mRNA unstable, and prevented increases in JunD mRNA and protein in polyamine-deficient cells. Conversely, increasing the cellular polyamines repressed JunD mRNA interaction with HuR and enhanced its association with AUF1, resulting in an inhibition of JunD expression. These results indicate that polyamines modulate the stability of JunD mRNA in intestinal epithelial cells through HuR and AUF1 and provide new insight into the molecular functions of cellular polyamines.

STIM1 translocation to the plasma membrane enhances intestinal epithelial restitution by inducing TRPC1-mediated Ca2+ signaling after wounding.

Early epithelial restitution is an important repair modality in the gut mucosa and occurs as a consequence of epithelial cell migration. Canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca2+ channel (SOCs) in intestinal epithelial cells (IECs) and regulates intestinal restitution, but the exact upstream signals initiating TRPC1 activation after mucosal injury remain elusive. Stromal interaction molecule 1 (STIM1) is a single membrane-spanning protein and is recently identified as essential components of SOC activation. The current study was performed to determine whether STIM1 plays a role in the regulation of intestinal epithelial restitution by activating TRPC1 channels. STIM1 translocation to the plasma membrane increased after wounding, which was followed by an increase in IEC migration to reseal wounds. Increased STIM1 levels at the plasma membrane by overexpressing EF-hand mutant STIM1 enhanced Ca2+ influx through SOCs and stimulated IEC migration after wounding. STIM1 interacted with TRPC1 and formed STIM1/TRPC1 complex, whereas inactivation of STIM1 by STIM1 silencing decreased SOC-mediated Ca2+ influx and inhibited epithelial restitution. In cells overexpressing EF-hand mutant STIM1, TRPC1 silencing also decreased STIM1/TRPC1 complex, reduced SOC-mediated Ca2+ influx, and repressed cell migration after wounding. Our findings demonstrate that induced STIM1 translocation to the plasma membrane promotes IEC migration after wounding by enhancing TRPC1-mediated Ca2+ signaling and provide new insight into the mechanism of intestinal epithelial restitution.

Induced ATF-2 represses CDK4 transcription through dimerization with JunD inhibiting intestinal epithelial cell growth after polyamine depletion.

Intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated by numerous factors including polyamines. Decreased levels of cellular polyamines increase activating transcription factor (ATF)-2, but the exact role and mechanism of induced ATF-2 in the regulation of intestinal epithelial cell (IEC) growth remain elusive. Cyclin-dependent kinase (CDK) 4 is necessary for the G1-to-S phase transition during the cell cycle, and its expression is predominantly controlled at the transcription level. Here, we reported that induced ATF-2 following polyamine depletion repressed CDK4 gene transcription in IECs by increasing formation of the ATF-2/JunD heterodimers. ATF-2 formed complexes with JunD as measured by immunoprecipitation using the ATF-2 and JunD antibodies and by glutathione S-transferase (GST) pull-down assays using GST-ATF-2 fusion proteins. Studies using various mutants of GST-ATF-2 revealed that formation of the ATF-2/JunD dimers depended on the COOH-terminal basic region-leucine zipper domain of ATF-2. Polyamine depletion increased ATF-2/JunD complex and inhibited CDK4 transcription as indicated by a decrease in the levels of CDK4-promoter activity and its mRNA. ATF-2 silencing not only prevented inhibition of CDK4 transcription in polyamine-deficient cells but also abolished repression of CDK4 expression induced by ectopic JunD overexpression. ATF-2 silencing also promoted IEC growth in polyamine-depleted cells. These results indicate that induced ATF-2/JunD association following polyamine depletion represses CDK4 transcription, thus contributing to the inhibition of IEC growth.

Post-transcriptional regulation of MEK-1 by polyamines through the RNA-binding protein HuR modulating intestinal epithelial apoptosis.

MEK-1 [MAPK (mitogen-activated protein kinase) kinase-1] is an important signal transducing enzyme that is implicated in many aspects of cellular functions. In the present paper, we report that cellular polyamines regulate MEK-1 expression at the post-transcriptional level through the RNA-binding protein HuR (Hu-antigen R) in IECs (intestinal epithelial cells). Decreasing the levels of cellular polyamines by inhibiting ODC (ornithine decarboxylase) stabilized MEK-1 mRNA and promoted its translation through enhancement of the interaction between HuR and the 3'-untranslated region of MEK-1 mRNA, whereas increasing polyamine levels by ectopic ODC overexpression destabilized the MEK-1 transcript and repressed its translation by reducing the abundance of HuR-MEK-1 mRNA complex; neither intervention changed MEK-1 gene transcription via its promoter. HuR silencing rendered the MEK-1 mRNA unstable and inhibited its translation, thus preventing increases in MEK-1 mRNA and protein in polyamine-deficient cells. Conversely, HuR overexpression increased MEK-1 mRNA stability and promoted its translation. Inhibition of MEK-1 expression by MEK-1 silencing or HuR silencing prevented the increased resistance of polyamine-deficient cells to apoptosis. Moreover, HuR overexpression did not protect against apoptosis if MEK-1 expression was silenced. These results indicate that polyamines destabilize the MEK-1 mRNA and repress its translation by inhibiting the association between HuR and the MEK-1 transcript. Our findings indicate that MEK-1 is a key effector of the HuR-elicited anti-apoptotic programme in IECs.

Repair of glutamate-induced excitotoxic neuronal damage mediated by intracerebroventricular transplantation of neural stem cells in adult mice.

OBJECTIVE: To investigate a possibility of repairing damaged brain by intracerebroventricular transplantation of neural stem cells (NSCs) in the adult mice subjected to glutamate-induced excitotoxic injury. METHODS: Mouse NSCs were isolated from the brains of embryos at 15-day postcoitum (dpc). The expression of nestin, a special antigen for NSC, was detected by immunocytochemistry. Immunofluorescence staining was carried out to observe the survival and location of transplanted NSCs. The animals in the MSG + NSCs group received intracerebroventricular transplantation of NSCs (approximately 1.0 x 10(5) cells) separately on day 1 and day 10 after 10-d MSG exposure (4.0 g/kg per day). The mice in control and MSG groups received intracerebroventricular injection of Dulbecco's minimum essential medium (DMEM) instead of NSCs. On day 11 after the last NSC transplantation, the test of Y-maze discrimination learning was performed, and then the histopathology of the animal brains was studied to analyze the MSG-induced functional and morphological changes of brain and the effects of intracerebroventricular transplantation of NSCs on the brain repair. RESULTS: The isolated cells were Nestin-positive. The grafted NSCs in the host brain were region-specifically survived at 10-d post-transplantation. Intracerebroventricular transplantation of NSCs obviously facilitated the brain recovery from glutamate-induced behavioral disturbances and histopathological impairs in adult mice. CONCLUSION: Intracerebroventricular transplantation of NSCs may be feasible in repairing diseased or damaged brain tissue.

[Cell cycle arrest and apoptosis induced by tubeimoside in HeLa cell].

BACKGROUND & OBJECTIVE: Tubeimoside, which is composed of tubeimoside I (79%) and II (21%), was isolated from the tubers of Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), a traditional Chinese medicine, "Tu-Bei-Mu". This study was designed to investigate the anti-tumor mechanism of tubeimoside. METHODS: Growth inhibition was measured by MTT assay. Induction of cell cycle arrest and apoptosis was determined by flow cytometry, fluorescence and electron microscopy, and gel electrophoresis of fragmented DNA. RESULTS: Tubeimoside display strong growth inhibitory effect in a dose- and time-dependant manner against HeLa cells with estimated IC50 values of 20.0, 18.8, and 8.8 mumol/L after 24, 48, and 72 h of treatment with tubeimoside. The flow cytometry profiles revealed that treatment with tubeimoside (5 h; 15, 30, 35 mumol/L) led to a dose-dependant shift from 9.80% up to 21.90%, and 27.00% in percentage of cells with a G2/M-like DNA content. On the other hand, treatment with tubeimoside (12 h, 15, 30, 35 mumol/L) led to a time-dependant shift from 8.20% up to 21.40%, 31.15%, and 34.55%, respectively. Exposure of HeLa cells to 40 mumol/L of tubeimoside induced nuclear shrinkage, chromation condensation and margination against nuclear envelope, subdiploid peak, and DNA fragmentation, characteristic as seen in apoptotic cells. CONCLUSION: Induction of cell cycle arrest and apoptosis may play an important role in the anti-tumor effect of tubeimoside.