Tingxi Yu

Polyamines Regulate the Stability of JunD mRNA by Modulating the Competitive Binding of Its 3′ Untranslated Region to HuR and AUF1

ABSTRACT 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.

Competitive Binding of CUGBP1 and HuR to Occludin mRNA Controls Its Translation and Modulates Epithelial Barrier Function

The present study shows that RNA-binding proteins CUGBP1 and HuR jointly regulate the translation of occludin and play a crucial role in the maintenance of tight junction integrity.

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.

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.

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.

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.

986 Mir-29b and Mir-503 Differentially Modulate Intestinal Epithelial Barrier Function by Altering Claudin-1 Expression

G A A b st ra ct s uncertain (e.g. ulcer disease, esophagitis). Analyzing data through 2009, we then identified individuals within the cohort who had a repeat EGD within three years of their index EGD. Results: Approximately 11.6% of Medicare beneficiaries in the sample underwent an index EGD between 2004 and 2006 (n = 108,785). Of these, 33.4% (n = 36,331) had at least one repeat EGD within three years of their index EGD. Most likely to undergo a repeat EGD were patients with an index diagnosis of varices (61.0%) and Barretts esophagus (58.5%). However, the absolute contribution of these two diagnoses to all repeat exams was very small (0.5% and 6.5%). Index diagnoses suggesting that a repeat EGD was routinely indicated comprised only 22.6% of repeat exams. In contrast, index diagnoses that did not suggest the need for a repeat EGD comprised 59.9% of repeat exams. Common diagnostic categories in this latter group included gastritis/duodenitis (21.0% of repeat exams), abdominal pain/dyspepsia (9.6%), or nonspecific EGD findings (9.2%). Conclusion: One in three Medicare beneficiaries undergoing an EGD had a repeat EGD within three years. Most of these repeated exams were performed for patients with a diagnosis at index endoscopy that does not imply that a repeat exam would be necessary. These findings raise questions about whether EGD is overutilized, particularly in terms of repetitive exams.

Mo1959 Induced JunD Represses Intestinal Epithelial Cell Proliferation by Increasing MicroRNA-29b After Polyamine Depletion

G A A b st ra ct s (p<0.05). Conclusions: Pediatric AP patients are kept fasting for extended periods of time and the initial diet is typically clear liquids despite the lack of evidence to support this practice. Tolerance of nutrition continues to be monitored by serum lipase levels even though it is known to correlate poorly with disease severity. Pediatric AP patients with more severe disease are more likely to receive PN and spend longer periods fasting. This occurs despite evidence in adults suggesting that early enteral nutrition is associatedwith improved outcomes in severe AP. Because of this discrepancy, there is a need for further research in pediatric AP regarding the potential benefit of EN and its early institution.

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.