Noncoding Vault RNA1-1 Impairs Intestinal Epithelial Renewal and Barrier Function by Interacting With CUG-binding Protein 1.

BACKGROUND & AIMS: Small noncoding vault RNAs (vtRNAs) are involved in many cell processes important for health and disease, but their pathobiological functions in the intestinal epithelium are underexplored. Here, we investigated the role of human vtRNA1-1 in regulating intestinal epithelial renewal and barrier function. METHODS: Studies were conducted in vtRNA1-1 transgenic (vtRNA1-1Tg) mice, primary enterocytes, and Caco-2 cells. Extracellular vesicles (EVs) were isolated from the serum of shock patients and septic mice. Intestinal organoids (enteroids) were prepared from vtRNA1-1Tg and littermate mice. Mucosal growth was measured by Ki67 immunostaining or BrdU incorporation, and gut permeability was assessed using the FITC-dextran assay. RESULTS: Intestinal tissues recovered from shock patients and septic mice evidenced mucosal injury and gut barrier dysfunction; vtRNA levels were elevated in EVs isolated from their sera. In mice, intestinal epithelial-specific transgenic expression of vtRNA1-1 inhibited mucosal growth, reduced Paneth cell numbers and intercellular junction (IJ) protein expression, and increased gut barrier vulnerability to lipopolysaccharide exposure. Conversely, in vitro silencing of vtRNA1-1 increased IJ protein levels and enhanced epithelial barrier function. Exposing enteroids to vtRNA1-1-rich EVs augmented paracellular permeability. Mechanistically, vtRNA1-1 interacted with CUG-binding protein 1 (CUGBP1) and increased CUGBP1 association with claudin-1 and occludin mRNAs, thereby inhibiting their expression. CONCLUSIONS: These findings indicate that elevated levels of vtRNA1-1 in EVs and mucosal tissues repress intestinal epithelial renewal and barrier function. Notably, this work reveals a novel role for dysregulation of the vtRNA1-1/CUGBP1 axis in the pathogenesis of gut mucosal disruption in critical illness.

The M1 muscarinic acetylcholine receptor in the crypt stem cell compartment mediates intestinal mucosal growth.

IMPACT STATEMENT: Localization of a specific subtype of the muscarinic acetylcholine receptor in the crypt stem cell compartment suggests a critical role in intestinal mucosal homeostasis. Here we demonstrate the localization of the M1 muscarinic acetylcholine receptor to the stem cell compartment and demonstrate increase morphometric and proliferative parameters when this is stimulated in vivo. These data provide novel information about this complex signaling microenvironment and offer potential future therapeutic targets for future study.

Role of the PI3K/AKT pathway and PTEN in otitis media.

Mucosal hyperplasia is common sequela of otitis media (OM), leading to the secretion of mucus and the recruitment of leukocytes. However, the pathogenic mechanisms underlying hyperplasia are not well defined. Here, we investigated the role of the AKT pathway in the development of middle mucosal hyperplasia using in vitro mucosal explants cultures and an in vivo rat model. The Akt inhibitor MK2206 treatment inhibited the growth of middle ear mucosal explants in a dose-dependent manner. In vivo, MK2206 also reduced mucosal hyperplasia. Unexpectedly, while PTEN is generally thought to act in opposition to AKT, the PTEN inhibitor BPV reduced mucosal explant growth in vitro. The results indicate that both AKT and PTEN are mediators of mucosal growth during OM, and could be potential therapeutic targets.

Potentiated serotonin signaling in serotonin re-uptake transporter knockout mice increases enterocyte mass and small intestinal absorptive function.

Genetic knockout of the serotonin reuptake transporter (SERT) potentiates serotonin signaling and increases crypt-cell proliferation, neuroplasticity, and mucosal surface area. However, it remains unknown whether these changes occur throughout the small intestine and whether they increase nutrient absorption. We hypothesized that serotonin-mediated mucosal growth would occur throughout the intestine and would increase enterocyte mass and absorptive function. Following institutional approval, intestinal segments spanning the bowel were harvested from 10 to 12 week-old SERT knockout (SERTKO) and wild-type (WT) C57Bl/6 mice. Histologic sections were used to measure villus height (VH), crypt depth (CD), and crypt proliferation index (CPI). Plasma citrulline was measured colorimetrically. Glucose and peptide absorption in isolated segments of small bowel were calculated using a previously described method for quantification after luminal instillation of substrate. At baseline, morphometric (VH/CD) and proliferative (CPI) parameters varied from jejunum to ileum. Enhanced 5-HT signaling significantly increased plasma citrulline levels and morphometric/proliferative parameters in all regions analyzed. Glucose absorption in WT mice varied throughout the small intestine, and SERTKO mice demonstrated significant increases in the middle and distal bowel. WT peptide absorption was similar throughout the small bowel, and SERTKO mice had significant increases in the proximal and distal bowel. Enhanced serotonin signaling results in increased morphometric and proliferative parameters throughout the small intestine, and results in increased enterocyte mass and intestinal absorptive function. These data further advance the concept that the serotonin system is an attractive therapeutic target for increasing functional intestinal mucosa.

Improved capacity to evaluate changes in intestinal mucosal surface area using mathematical modeling.

Quantification of intestinal mucosal growth typically relies on morphometric parameters, commonly villus height, as a surrogate for presumed changes in mucosal surface area (MSA). We hypothesized that using mathematical modeling based on multiple unique measurements would improve discrimination of the effects of interventions on MSA compared to standard measures. To determine the ability of mathematical modeling to resolve differences in MSA, a mouse model with enhanced serotonin (5HT) signaling known to stimulate mucosal growth was used. 5-HT signaling is potentiated by targeting the serotonin reuptake transporter (SERT) molecule. Selective serotonin reuptake inhibitor-treated wild-type (WT-SSRI), SERT-knockout (SERTKO), and wild-type C57Bl/6 (WT) mice were used. Distal ileal sections were H&E-stained. Villus height (VH), width (VW), crypt width (CW), and bowel diameter were used to calculate surface area enlargement factor (SEF) and MSA. VH alone for SERTKO and SSRI was significantly increased compared to WT, without a difference between SERTKO and WT-SSRI. VW and CW were significantly decreased for both SERTKO and WT-SSRI compared to WT, and VW for WT-SSRI was also decreased compared to SERTKO. These changes increased SEF and MSA for SERTKO and WT-SSRI compared to WT. Additionally, SEF and MSA were significantly increased for WT-SSRI compared to SERTKO. Mathematical modeling provides a valuable tool for differentiating changes in intestinal MSA. This more comprehensive assessment of surface area does not appear to correlate linearly with standard morphometric measures and represents a more comprehensive method for discriminating between therapies aimed at increasing functional intestinal mucosa.

Jnk2 deletion disrupts intestinal mucosal homeostasis and maturation by differentially modulating RNA-binding proteins HuR and CUGBP1.

Homeostasis and maturation of the mammalian intestinal epithelium are preserved through strict regulation of cell proliferation, apoptosis, and differentiation, but the exact mechanism underlying this process remains largely unknown. c-Jun NH2-terminal kinase 2 (JNK2) is highly expressed in the intestinal mucosa, and its activation plays an important role in proliferation and also mediates apoptosis in cultured intestinal epithelial cells (IECs). Here, we investigated the in vivo function of JNK2 in the regulation of intestinal epithelial homeostasis and maturation by using a targeted gene deletion approach. Targeted deletion of the jnk2 gene increased cell proliferation within the crypts in the small intestine and disrupted mucosal maturation as indicated by decreases in the height of villi and the villus-to-crypt ratio. JNK2 deletion also decreased susceptibility of the intestinal epithelium to apoptosis. JNK2-deficient intestinal epithelium was associated with an increase in the level of the RNA-binding protein HuR and with a decrease in the abundance of CUG-binding protein 1 (CUGBP1). In studies in vitro, JNK2 silencing protected intestinal epithelial cell-6 (IEC-6) cells against apoptosis and this protection was prevented by inhibiting HuR. Ectopic overexpression of CUGBP1 repressed IEC-6 cell proliferation, whereas CUGBP1 silencing enhanced cell growth. These results indicate that JNK2 is essential for maintenance of normal intestinal epithelial homeostasis and maturation under biological conditions by differentially modulating HuR and CUGBP1.