contributing 11.053 person-years of follow-up. During the study period, a total of 27 CRC cases occurred among FDRs at a median age of 62 years (interquartile range: 57-78) compared to 5 expected CRC cases (p<0.001). The relative risk of CRC in FDRs compared to the general population was 5.4 (95%-CI: 3.7-7.8). No significant difference was seen between the relative risk for male and female FDRs regarding the CRC incidence. In 4 FDRs from 4 different pedigrees multiple histologically confirmed hyperplastic polyps were reported, satisfying the criteria for HPS. Based on the estimated HPS prevalence of 1:3000 in the general population the projected relative risk of HPS in FDRs would be 39 (95%-CI: 13- 121). In most FDRs endoscopy was not performed (281/347: 81%) suggesting that the true prevalence of CRC and HPS in FDRs may be underestimated. Conclusions: Our results showed that FDRs of HPS patients have an increased relative risk for both CRC and HPS. Hence, as long as no genetic substrate has been identified, screening colonoscopies for FDRs seem justified but needs to be prospectively evaluated. 748 ErbB4 Transactivation by TNF Mediates Colon Epithelial Cell Survival by TNF Alpha Converting Enzyme-Dependent Ligand Release Valda C. Hilliard, Mark R. Frey, Peter J. Dempsey, D. Brent Polk Background: Inflammatory bowel diseases (IBD) are marked by increased levels of inflammat- ory cytokines, such as tumor necrosis factor (TNF), and enhanced epithelial cell apoptosis. We have recently shown that expression of ErbB4, a member of the epidermal growth factor receptor family of receptor tyrosine kinases, is induced and phosphorylated in response to TNF and is also elevated in IBD. In this study, we tested the hypothesis that TNF transactivates ErbB4 through TNF alpha converting enzyme (TACE)-mediated ligand release, and that this transactivation protects colonic epithelial cells from inflammation-induced apoptosis. Methods: Conditionally immortalized TACE -/- mouse colon epithelial (MCE) cells expressing either wild type TACE or vector were treated with 100 ng/mL TNF; subsequently, ErbB4 phosphorylation was assessed by Western blot analysis of whole cell lysates using an antibody directed towards phospho-ErbB4 (pY-1284). ErbB4-expressing young adult mouse colon epithelial (YAMC) cells were pretreated with an ErbB4 blocking antibody (10 μg/mL) for 1 hr before the addition of TNF (100 ng/mL, 30 min) to determine whether extracellular ligands were required for ErbB4 transactivation. For survival studies, YAMC cells were pretreated with either the ErbB4 blocking antibody or with 10 μM TAPI-1 (a TACE specific inhibitor) for 1 hr before the addition of TNF and cycloheximide (1 μg/mL) for 5 hours. Apoptosis was then measured by Western blot analysis of cell lysates using anti-active caspase 3 and cleaved PARP antibodies, or by counting TUNEL stained cells. Results: ErbB4 was not phosphorylated in response to TNF in TACE -/- MCE cells; re-expression of wild type TACE restored TNF transactivation of ErbB4. TNF-induced phosphorylation of ErbB4 was also completely inhibited by pretreatment of cells with an ErbB4 blocking antibody, sup- porting our hypothesis that ligand release mediates ErbB4 transactivation by TNF. Both TACE activity and ErbB4 ligand binding were required for ErbB4-mediated anti-apoptotic effects in the presence of inflammatory cytokines. During TNF exposure, YAMC cells overex- pressing ErbB4 exhibited decreased TUNEL staining compared to cells expressing vector only. However, pretreatment with an ErbB4 blocking antibody or TACE inhibitor reversed the ability of ErbB4 to protect from apoptosis. These results were corroborated by immunob- lotting with antibodies against the apoptotic markers cleaved PARP and active caspase 3. Conclusions: TNF transactivates ErbB4 through TACE-dependent ligand cleavage, which is required for ErbB4 protection against inflammatory cytokine-induced apoptosis in colon epithelial cells. 749 Induced MicroRNA-222 in Intestinal Epithelial Cells Represses CDK4 Translation Following Polyamine Depletion Lan Xiao, Rao N. Jaladanki, Tongtong Zou, Lan Liu, Tingxi Yu, Yu-Hong Cui, Myriam Gorospe, Jian-Ying Wang Maintenance of gut mucosal integrity is important to preservation of normal digestive and barrier functions and is tightly regulated by multiple factors including polyamines. Besides regulating transcription, polyamines also potently modulate gene expression posttranscrip- tionally, but their exact mechanism remains unknown. microRNAs (miRNAs) are potent posttranscriptional regulators that affect mRNA turnover and translation through imperfect base pairing. miRNA-222 (miR-222) is one of the best studied miRNAs and was shown to play a pivotal role in the posttranscriptional control of several target mRNAs. CDK4 (cyclin- dependent kinase-4) is essential for the G1-to-S phase transition during the cell cycle and its expression requires cellular polyamines. This study determines whether polyamines modulate CDK4 translation by altering miR-222 activity in normal intestinal epithelial cells (IEC-6 line). Methods: Cellular polyamines were depleted by inhibiting ODC (key enzyme for polyamine biosynthesis) with DFMO but increased by overexpressing ODC gene. Levels of miR-222 were measured by real-time PCR analysis; its interaction with CDK4 mRNA was examined by pmir-Glo reporter system and biotin labeled miR-222 pull-down assay. CDK4 translation was examined by using the chimeric luciferase (Luc)-CDK4 CR reporter gene assays and by examining the distribution of CDK4 mRNA in polyribosomes. Functions of miR-222 were investigated by its silencing and overexpression. Results: Depletion of cellular polyamines by treatment with DFMO increased miRNA-222 levels (by ~2-folds), although elevated levels of polyamines by ODC overexpression failed to reduce basal level of miR- 222. miR-222 directly bound to CDK4 mRNA via two seeding regions (spanning positions: 264-284, and 690 -710) in its open reading frame, and this binding affinity increased significantly (by ~3-folds) following polyamine depletion but it was prevented by exogenous polyamine putrescine given together with DFMO. Polyamine depletion also repressed CDK4 mRNA translation and decreased its protein levels. The miR-222 silencing by using specific siRNA decreased [miR-222/CDK4 mRNA] complex and enhanced CDK4 translation, thus promoting cell proliferation in polyamine-deficient cells. In contrast, miR-222 overexpression in normal IECs increased its binding to CDK4 mRNA and repressed CDK4 translation, thus inhibiting cell proliferation. Conclusions: These results indicate that 1) polyamines are necessary for repression of miR-222 expression and 2) polyamine depletion inhibits CDK4 translation by increasing miR-222 interaction with CDK4 mRNA. S-103 AGA Abstracts 750 Germ Free Mice Show Apoptosis but Dramatically Reduced Overt Mucosal Damage After Doxorubicin Rachael J. Rigby, Pauline K. Lund, Christopher M. Dekaney Introduction: A rate limiting factor in administration of chemotherapeutic agents is induction of widespread damage to the intestinal mucosa. We recently reported that a single injection of doxorubicin (Dox) given to mice induced significant, but transient increases in apoptosis in the stem cell zone of the jejunum, followed by mucosal damage involving decreases in crypt proliferation, crypt number, and villus height (Dekaney 2009 AJP G461-70). Damage was followed by repair associated with crypt hypertrophy, Paneth cell hyperplasia and, ultimately, return of the intestinal mucosa to normal morphology. Prior studies suggest that treatment with Dox causes increases in permeability of the intestinal epithelial barrier (Sun 1998 Scand J Gastroenterol 415-22). This study tested the hypothesis that bacteria in the intestinal lumen contribute to apoptosis and mucosal damage after Dox administration. Methods: Germ free (GF) or conventionally raised (CONV) but specific pathogen free mice were injected with a single dose (20 mg/kg) of Dox. Jejunum was harvested at 0 - 168h later. Apoptosis, crypt depth, villus height, and gross mucosal damage were evaluated. Results: Dox treatment rapidly induced similar levels of apoptosis per crypt in both GF (2.9±0.9) and CONV (2.9±0.5) animals. In contrast to CONV mice, GF mice showed no significant change in crypt depth or villus height throughout the entire time course after Dox, even though at baseline GF crypts are significantly smaller than crypts in CONV animals. Overall, despite Dox-induced apoptosis in GF animals, we found no indication of mucosal damage in GF mice following Dox treatment. Discussion: Intestinal bacteria are not required for induction of apoptosis by the chemotherapeutic agent Dox. However, bacteria do play a critical role in subsequent mucosal damage and crypt loss following Dox- induced apoptosis; presumably by penetrating a more permeable intestinal epithelial barrier and worsening and perpetuating mucosal damage and loss due to Dox induced apoptosis. These findings are significant because they suggest that controlling the intestinal microbiota during chemotherapy with Dox or other agents may reduce damage to the intestinal mucosa. This in turn might lead to more efficacious anticancer therapies with fewer adverse effects due to loss of intestinal function. 751 Transgenic Protein Kinase D1 Stimulates the Rate of Epithelial Cell Proliferation in Intestinal Crypts James Sinnett-Smith, Robert K. Kui, Steven H. Young, Carlos Huang, Nora Rozengurt, Enrique Rozengurt Background: The sequential proliferation, lineage-specific differentiation, crypt-villus migra- tion, and cell death of the epithelial cells of the intestinal mucosa is tightly regulated by regulatory peptides, differentiation signals, and luminal stimuli, including nutrients and pathogenic/commensal organisms. Despite its importance for understanding normal homeostasis and pathogenesis of disease states, the intracellular signal transduction mechan- isms involved remain incompletely understood. Here, we tested the hypothesis that PKD1 plays a key role in intestinal crypt cell proliferation In Vivo. Results: To clarify the role of PKD1 in intestinal epithelial cell proliferation In Vivo, we generated transgenic mice that express elevated PKD1 protein in the distal small intestinal and proximal colonic epithelium. We verified that the catalytic activity of PKD1 was strikingly higher in extracts from ileal mucosa of transgenic mice as compared with non-transgenic littermates. Furthermore, trans- genic PKD1 exhibited constitutive phosphorylation at the activation loop residues, Ser744 and Ser748 and on the autophosphorylation site, Ser916. These results indicate that trans- genic PKD1 is functional in the intestinal epithelium. In order to determine whether PKD1 signaling stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine (BrdU) incorporated into the cell nuclei of crypt cells of the ileum and proximal colon, where PKD1 protein is maximally expressed. Our results demonstrated a highly statistically significant increase (p<0.005) in DNA synthes- izing cells in the crypts of the PKD1 transgenic mice as compared with non-transgenic littermates. The proliferating cells in the crypt are localized to positions consistent with those corresponding to stem cells and transit amplifying cells. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the nontransgenic littermates (p< 0.006). We found a significant increase in the localization of β-catenin in the nucleus of crypt epithelial cells of PKD1 transgenic mice, as compared with non-transgenic littermates. As with BrdU labeling, we noted nuclear catenin in basal columnar cells of the crypt. Conclusion: Transgenic PKD1 expression increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. These results support the hypothesis that PKD1 signaling plays a role in a pathway leading to proliferation in intestinal epithelial cells. 752 Epidermal Growth Factor (EGF) Prevents Osmotic Stress and Dextran Sodium Sulfate (DSS)-Induced Disruption of Tight Junctions (TJ) in CACO-2 Cell Monolayers: Role of ERK-Mediated Prevention of JNK2 Activation Geetha Samak, Radhakrishna (RK) Rao EGF is known to protect the intestinal mucosal epithelium from various injurious factors. Our previous studies demonstrated that osmotic stress and DSS disrupt TJ and induce barrier dysfunction in Caco-2 cell monolayers by a c-Jun N-terminal kinase (JNK)-dependent mechanism. In the present study, we evaluated the effect of EGF on osmotic stress and DSS-induced disruption of TJs. Methods: Caco-2 cell monolayers were exposed to osmotic stress (0.35M mannitol) or DSS (2%, w/v) for 1-3 hrs in the presence or absence of 30 nM EGF. Barrier function was evaluated by measuring transepithelial electrical resistance (TER) and unidirectional flux of FITC-inulin. TJ integrity was assessed by confocal microscopy for occludin, ZO-1, claudin-4 and F-actin. Activation of JNK and ERK was assessed by immunob- lot and immunofluorescence analysis for p-JNK and p-ERK. Role of ERK in EGF-mediated TJ protection was determined by evaluating the effect of U0126 on EGF-mediated TJ AGA Abstracts