Jennifer A. Timmons

Polyamines regulate E-cadherin transcription through c-Myc modulating intestinal epithelial barrier function

The integrity of the intestinal epithelial barrier depends on intercellular junctions that are highly regulated by numerous extracellular and intracellular factors. E-cadherin is found primarily at the adherens junctions in the intestinal mucosa and mediates strong cell-cell contacts that have a functional role in forming and regulating the epithelial barrier. Polyamines are necessary for E-cadherin expression, but the exact mechanism underlying polyamines remains elusive. The current study was performed to determine whether polyamines induce E-cadherin expression through the transcription factor c-Myc and whether polyamine-regulated E-cadherin plays a role in maintenance of the epithelial barrier integrity. Decreasing cellular polyamines reduced c-Myc and repressed E-cadherin transcription as indicated by a decrease in levels of E-cadherin promoter activity and its mRNA. Forced expression of the c-myc gene by infection with adenoviral vector containing c-Myc cDNA stimulated E-cadherin promoter activity and increased E-cadherin mRNA and protein levels in polyamine-deficient cells. Experiments using different E-cadherin promoter mutants revealed that induction of E-cadherin transcription by c-Myc was mediated through the E-Pal box located at the proximal region of the E-cadherin promoter. Decreased levels of E-cadherin in polyamine-deficient cells marginally increased basal levels of paracellular permeability but, remarkably, potentiated H(2)O(2)-induced epithelial barrier dysfunction. E-cadherin silencing by transfection with its specific small interfering RNA also increased vulnerability of the epithelial barrier to H(2)O(2). These results indicate that polyamines enhance E-cadherin transcription by activating c-Myc, thus promoting function of the epithelial barrier.

Polyamines and Gut Mucosal Homeostasis

The epithelium of gastrointestinal (GI) mucosa has the most rapid turnover rate of any tissue in the body and its integrity is preserved through the dynamic balance between cell migration, proliferation, growth arrest and apoptosis. To maintain tissue homeostasis of the GI mucosa, the rates of epithelial cell division and apoptosis must be highly regulated by various extracellular and intracellular factors including cellular polyamines. Natural polyamines spermidine, spermine and their precursor putrescine, are organic cations in eukaryotic cells and are implicated in the control of multiple signaling pathways and distinct cellular functions. Normal intestinal epithelial growth depends on the available supply of polyamines to the dividing cells in the crypts, and polyamines also regulate intestinal epithelial cell (IEC) apoptosis. Although the specific molecular processes controlled by polyamines remains to be fully defined, increasing evidence indicates that polyamines regulate intestinal epithelial integrity by modulating the expression of various growth-related genes. In this review, we will extrapolate the current state of scientific knowledge regarding the roles of polyamines in gut mucosal homeostasis and highlight progress in cellular and molecular mechanisms of polyamines and their potential clinical applications.

Gut microbiota, tight junction protein expression, intestinal resistance, bacterial translocation and mortality following cholestasis depend on the genetic background of the host

Failure of the intestinal barrier is a characteristic feature of cholestasis. We have previously observed higher mortality in C57BL/6J compared with A/J mice following common bile duct ligation (CBDL). We hypothesized the alteration in gut barrier function following cholestasis would vary by genetic background. Following one week of CBDL, jejunal TEER was significantly reduced in each ligated mouse compared with their sham counterparts; moreover, jejunal TEER was significantly lower in both sham and ligated C57BL/6J compared with sham and ligated A/J mice, respectively. Bacterial translocation to mesenteric lymph nodes was significantly increased in C57BL/6J mice vs. A/J mice. Four of 15 C57BL/6J mice were bacteremic; whereas, none of the 17 A/J mice were. Jejunal IFN-γ mRNA expression was significantly elevated in C57BL/6J compared with A/J mice. Western blot analysis demonstrated a significant decrease in occludin protein expression in C57BL/6J compared with A/J mice following both sham operation and CBDL. Only C57BL/6J mice demonstrated a marked decrease in ZO-1 protein expression following CBDL compared with shams. Pyrosequencing of the 16S rRNA gene in fecal samples showed a dysbiosis only in C57BL/6J mice following CBDL when compared with shams. This study provides evidence of strain differences in gut microbiota, tight junction protein expression, intestinal resistance and bacterial translocation which supports the notion of a genetic predisposition to exaggerated injury following cholestasis.

Candidate genes for limiting cholestatic intestinal injury identified by gene expression profiling

The lack of bile flow from the liver into the intestine can have devastating complications including hepatic failure, sepsis, and even death. This pathologic condition known as cholestasis can result from etiologies as diverse as total parenteral nutrition (TPN), hepatitis, and pancreatic cancer. The intestinal injury associated with cholestasis has been shown to result in decreased intestinal resistance, increased bacterial translocation, and increased endotoxemia. Anecdotal clinical evidence suggests a genetic predisposition to exaggerated injury. Recent animal research on two different strains of inbred mice demonstrating different rates of bacterial translocation with different mortality rates supports this premise. In this study, a microarray analysis of intestinal tissue following common bile duct ligation (CBDL) performed under general anesthesia on these same two strains of inbred mice was done with the goal of identifying the potential molecular mechanistic pathways responsible. Over 500 genes were increased more than 2.0‐fold following CBDL. The most promising candidate genes included major urinary proteins (MUPs), serine protease‐1‐inhibitor (Serpina1a), and lipocalin‐2 (LCN‐2). Quantitative polymerase chain reaction (qPCR) validated the microarray results for these candidate genes. In an in vitro experiment using differentiated intestinal epithelial cells, inhibition of MUP‐1 by siRNA resulted in increased intestinal epithelial cell permeability. Diverse novel mechanisms involving the growth hormone pathway, the acute phase response, and the innate immune response are thus potential avenues for limiting cholestatic intestinal injury. Changes in gene expression were at times found to be not only due to the CBDL but also due to the murine strain. Should further studies in cholestatic patients demonstrate interindividual variability similar to what we have shown in mice, then a “personalized medicine” approach to cholestatic patients may become possible.

W1704 Polyamines Enhance Intestinal Epithelial Restitution by Increasing Stim1-Mediated CA2+ Influx Through Store-Operated CA2+ Channel After Wounding

gastric epithelial cells as susceptible to both alcohol-induced cell damage (P=0.07) and apoptosis (P=0.06) as the gastric endothelial cells. Conversely, forced overexpression of survivin by transient transfection rendered gastric endothelial cells as resistant to both alcohol-induced cell damage (P=0.08) and apoptosis (P=0.06) as mock-transfected gastric epithelial cells. Moreover, overexpression of a threonine-34 to glutamate phosphorylation mimic mutant survivin construct rendered gastric endothelial cells significantly more resistant to alcohol-induced damage (P<0.02) and apoptosis (P<0.02) vs. mock-transfected gastric epithelial cells. CONCLUSIONS: 1) Disparate survivin expression levels can explain, in part, the discrepancy between gastric epithelial and endothelial cell susceptibility to alcoholinduced injury. 2) A negatively charged amino acid substitution at position 34, or the phosphorylation modification that naturally occurs on threonine-34 producing a negative charge at this position, increases the potency of survivin in mediating protection against alcohol-induced gastric mucosal cellular injury. (Supported by NIH R01AA14946)

577 Reduced HuR Association With Occludin mRNA Plays an Important Role in Pathogenesis of Gut Barrier Dysfunction During Septic Stress

Gut barrier dysfunction occurs in various critical illnesses, leading to the translocation of luminal toxic substances and bacteria to the blood stream. Occludin in the tight junctions (TJs) is an integral membrane protein that forms the sealing element of TJs and its normal expression is critical for maintaining epithelial barrier function during stress and is tightly regulated at multiple levels. The RNA-binding protein HuR binds to many labile mRNAs bearing Uor AU-rich elements and modulates their stability and translation. Our previous studies show that HuR directly interacts with occludin mRNA and regulates occludin expression in cultured intestinal epithelial cells (IECs) and that HuR silencing represses occludin translation In Vitro. This study further determines whether HuR-mediated occludin expression plays a role in the pathogenesis of gut barrier dysfunction during septic stress. Methods: Studies were conducted in A/J mice, and septic stress was induced by the method of cecal ligation and puncture (CLP). Gut permeability was detected by using the fluorescent tracer FITC-dextran; and levels of occludin mRNA and protein were examined by quantitative real-time PCR and Western blotting analyses. HuR binding to occludin mRNA was examined by ribonucleoprotein immunoprecipiatation assays. Results: Exposure to CLP for 8 h decreased occludin protein levels, and maximal reduction in occludin (by ~85%) occurred 24 and 48 h, then it began to recover gradually thereafter. Consistently, gut barrier dysfunction as indicated by an increase (by >2-fold) in the mucosal permeability to FITC-dextran also occurred 8 h after CLP and maintained for additional 40 h. CLP failed to reduce total occludin mRNA levels, but it repressed occludin mRNA association with HuR (by ~80%). At 72 h after CLP, the levels of [HuR/occludin mRNA] complex increased partially (by ~33%) as compared with those observed in 24 and 48 h, which was associated with partial recovery of gut barrier function. In addition, depletion of cellular polyamines by treatment with DFMO (a specific inhibitor of polyamine biosynthesis) not only repressed HuR binding to occludin mRNA but also delayed the recovery of gut barrier function in mice exposed to CLP. In cultured IECs, polyamine depletion inhibited HuR phosphorylation, reduced HuR association with occludin mRNA, and repressed occludin translation, thus disrupting the barrier function. Conclusions: CLP-induced repression of occludin expression occurs at the posttranscriptional level and that reduced HuR association with occludin mRNA plays an important role in gut barrier dysfunction during critical stress.

674 Concomitant Knockout of M1 Muscarinic Receptors (CHRM1) in Azoxymethane-Treated Chrm3−/− Mice Nullifies Reductions in Colon Tumor Number and Size

G A A b st ra ct s (HP). Animals were analyzed 3 and 12 months after inoculation. Morphology of the fundus was examined in sections stained with H&E. Expression of MIP-2, TNF-alfa, INF-gamma, DCAMKL1, villin, activation-induced cytidine deaminase (AID), TFF2 and COX-2 genes were measured by QRT-PCR. Distribution of parietaland DCAMKL1-positive cells was analyzed immunohistochemistry. Western blots were used to assess the phosphorylation of Smad1-5-8 and STAT3. RESULTS: The mucosa of the TG-mice exhibited decreased phosphorylation of Smad1-5-8, confirming inhibition of BMP signaling. Histological analysis showed increased height, dilated glands, decreased parietal cell number and amodest increase in inflammatory cells. The TG-mice also exhibited a 2to 3-fold increase in the expression of the inflammatory genes MIP-2, INF-gamma TNF-alfa, and COX-2. Inhibition of BMP signaling led to increased expression of the gastric progenitor cell (GPC) markers DCAMKL1 and villin, and of AID and TFF2, molecules associated with gastric carcinogenesis. Infection of the TG-mice with HP, led to a dramatic increase in inflammation, characterized by enhanced cytokine gene expression and by a robust influx of inflammatory cells. HP-infected non-TG mice exhibited inflammatory changes that were less severe then those seen in the infected TG-animals. Intraepithelial neoplasia was seen in TG-mice infected for 12 months but not in age matched, non-infected TG-mice, or HP-infected non-TG-mice. HP infection also led to enhanced expression of AID, TFF2, villin and DCAMKL1 and to the phosphorylation and activation of the oncogenic molecule STAT3. CONCLUSIONS: Loss of gastric BMP signaling leads to a pro-inflammatory state, resulting in extreme responses and accelerated development of intraepithelial neoplasia with HP infection, suggesting that BMPs normally temper gastric inflammatory responses. Low BMP signaling and heightened inflammation stimulate GPCs, including increased marker expression and cell number. Thus BMPs play an important role in modulating inflammatory responses in the development of gastric cancer.