Crypts Of Mice Research Articles

Abstract 1203: Coordinated regulation of intestinal radio-sensitivity by PUMA and p21

Abstract p53-upregulated modulator of apoptosis (PUMA) has been shown to be vital for radio-sensitivity in the small intestine. PUMA ablation results in drastic radio-resistance, lengthening the survival of mice exposed to 15 Gy whole body irradiation and blocking apoptosis in the crypts. In contrast, we and others have found that p21 ablation leads to slightly increased radio-sensitivity. To better elucidate the relative and perhaps linked roles of PUMA and p21 in the intestinal responses to irradiation, we produced PUMA/p21 double knockout (DKO) mice. Initial experiments revealed that DKO mice responded similarly to PUMA KO mice within the first 24 hours after 15 Gy ionizing radiation as measured by the DNA damage response and apoptosis in the crypts. Both groups showed more rapid, though less sustained, phosphorylation of H2AX than wild-type (WT) mice, while displaying significantly less apoptosis in the crypts. However, DKO mice were found to have similarly enhanced overall radio-sensitivity as p21 KO mice. Further studies revealed that DKO and PUMA KO mice may not be equally effective at repair despite their similar kinetics in the initial DNA damage response. At 48 and 72 hours post-irradiation, the crypts in DKO mice had a substantial number of TUNEL+ cells, while those in PUMA KO or WT mice had almost none. At 48 hours after irradiation, the crypts in p21 KO mice also had a large number of TUNEL+ cells, which declined by 72 hours. Additionally, the crypt cells in DKO and p21 KO mice, but not in WT or PUMA KO mice, were found to be positive for both γ-H2AX and the mitosis marker phospo-H3, indicating abrogated cell cycle control likely due to a futile attempt at proliferation. These data further suggest that the protection of intestinal epithelium against radiation injury through ablation of PUMA requires a functional p21 checkpoint in addition to inhibition of crypt apoptosis. Additional studies are underway in our laboratory to further elucidate how these two proteins might coordinately regulate intestinal injury and regeneration by modulating the survival and proliferation of intestinal stem cells and progenitors following irradiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1203.

Abstract 3446: Induction of colonic aberrant crypts in mice by an apparent N-nitroso compound fraction derived from hot dogs

Abstract Introduction: Nitrite-preserved meat products, e.g., hot dogs, may be a cause of colon cancer. This may occur because such products contain a possibly carcinogenic fraction called “total apparent N-nitroso compounds” (ANC). Feeding such products to humans and mice increased the fecal excretion of ANC. We tested here whether hot-dog-derived ANC could induce colonic aberrant crypts (ABC), a precursor lesion for colon cancer, in mice. Methods: We purified the ANC precursor (ANCP) fraction of commercial hot hogs by adsorption-desorption on silica gel and cation exchange resin, and treated the ANCP with nitrite to convert them to ANC. As a positive control group, adult female A/J mice were given 1 or 3 i.p. injections of 5 mg/kg of azoxymethane (AOM) [Experiment (Exp.) 1]. In Exps. 2 and 3, similar mice were continuously fed the purified hot dog ANC (initial dose, 100 nmol/g diet) in a high-fat or “high-fat stress” diet. In Exp. 2 the ANC dose initially dropped sharply, probably because the ANCP was unstable on thawing, but later in Exp 2 and throughout Exp 3 the ANCP level was stable and was maintained at 100 nmol/g diet. The mice were killed after 8 (for AOM tests) or 24-34 (for ANC tests) weeks of treatment. Feces of the ANC-treated mice contained up to 225 nmol ANC/g, indicating that the ANC reached the colon. The distal 2 cm of each colon was stained with methylne blue, the mucosal surfaces were scanned at 400 x magnification, and ABC were identified and counted. Results: Mean numbers of ANC/colon for 1 and 3 AOM injections, AOM controls, ANC (Exp 2), ANC (Exp. 3) and ANC controls were 28, 76, 14, 20, 14 and 5-8. Standard deviations were <50% of mean values. The effect of ANC was significant by the rank order test (P < 0.01) for both Exps. 2 and 3. Potency of ABC induction per mole ANC was 1.7% (Exp. 2) and 0.4% (Exp. 3) of that for AOM in Exp. 1. In Exp. 3 we noted that the ABC occurred as foci with 1-4 ABC/focus and a mean of 6.8 foci/colon. ANC treatment did not affect the number of foci. Conclusions: Hot-dog-derived ANC induced a significant number of ABC in the mouse colon, but the effect was relatively weak. These results provide a direct link between ANC in a nitrite-preserved meat product and effects in the colon. Partly supported by NIH grant RO1 CA-143460-01. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3446.

Abstract 4157: Genetic and dietary complementation in risk for mouse intestinal tumorigenesis

Abstract Dietary factors are significant in determining probability for intestinal tumor development even in the presence of strong genetic initiation. For example, a rodent Western-style diet (WD1), modeled to reproduce intake of nutrient risk factors in the human diet associated with sporadic colon cancer, potentiates the formation of intestinal tumors in Apc1638N/+ mice, even when the genetic initiation by Apc is augmented by targeted inactivation of the p21Waf1/cip1 locus. To investigate mechanisms of this synergistic effect, we profiled gene expression along the mouse crypt-villus axis (CVA) using cells isolated as a function of their position along this axis from WT, Apc1638N/+ and p21−/− mice fed control AIN76A diet, and WT mice fed the WD1. Modulation of sequence expression by the WD1 was enriched in the villus compartment, while changes induced by targeting either genetic locus (Apc1638N/+ or p21−/−) were largely in the crypt, where the overlap in altered gene sets was greatest for the genetic models. There was greater overlap among expression changes between Apc1638N/+ and p21−/− mice, than between these genetic mice models and mice fed the WD1, in both the villus and crypt. Paneth cell markers, whose deregulation has been reported in intestinal tumors, were up-regulated in the p21−/− mouse crypt and in WD1 mouse villus. Moreover, there was attenuated expression of secretory cell lineage markers (ie goblet and enteroendocrine cells), but enhanced expression of enterocyte markers, for both the genetic and dietary models. Genes that encode tricarboxylic acid (TCA) cycle enzymes were uniformly decreased in the crypt of both genetic models, with fewer changes in this pathway altered in the WD1 mouse. Gene ontology and biological pathway analysis also showed that Wnt pathway and c-myc target genes were overrepresented in the villus of the WD1 mouse. Supplementation of WD1 with calcium and vitamin D (WD2) reversed aberrantly up-regulated cyclin D1, cyclin D2, defensins and lysozymes in the WD1 villus back to control levels. In summary, complementary changes in cell reprogramming contributed by a western style diet and targeted inactivation of the p21 gene are important in the synergy of these factors in elevating probability of intestinal tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4157.

Abstract 4259: Over-expression of autocrine progastrin up-regulates DCAMKL+1/CD44 positive stem/progenitor cells, associated with surprising transformation of HEK-293 cells, in vitro and in vivo

Abstract Background and Aims. In recent years we have demonstrated that progastrin peptide (PG) exerts anti-apoptotic and proliferative effects on normal and cancerous epithelial cells, in vitro and in vivo, and increases risk of colon carcinogenesis in transgenic mice overexpressing PG (Cancer 2004; Oncogene 2007; CanRes 2007; Oncogene 2008; JBC 2009). In vivo studies with PG overexpressing hGAS mice suggest that there is an increase in the census of DCAMKL+1 and CD44 positive colonic crypt cells (Jin, et al, JCI 2009). It is, however, not known if PG can directly induce an increase in the number of stem/progenitor cells, in vitro, and if this increase translates into transformation of the cells. To address this novel possibility, we used PG responsive HEK-293 cells, which are incompletely transformed with negligible clonogenic/tumorigenic potential. Methods and Results. Mutant-hGAS-HEK293 clones (HEK-PG) were generated and confirmed to overexpress full-length (9KDa) PG. HEK293 clones transfected with empty vector (HEK-C) served as controls. A significant increase in proliferation of HEK-PG vs HEK-C cells was measured in vitro. Cells, stained for either the putative stem cell marker (DCAMKL+1) or progenitor cells marker (CD44), were examined by fluorescense confocal microscopy and quantified by Western Blot analysis. Expression of DCAMKL+1/CD44 increased 2-3 fold in HEK-PG clones vs HEK-C clones, suggesting for the first time, that PG directly enhances the number of stem/progenitor cells. Significant increase in DCAMKL+1/CD44 cells was confirmed in colonic crypts of Fabp-PG mice, overexpressing PG in colons. The most surprising finding was that overexpression of PG in HEK-PG clones imparted a clonogenic/tumorigenic/metastatic potential to the cells, in vitro and in vivo (using SCID mouse model). However, IEC-18-PG clones were not rendered clonogenic/tumorigenic, suggesting that overexpression of PG in the background of HEK-293 cells, represented the second hit required for transformation. IEC-18 cells are immortalized but apparently lack the first hit (initiating events) and thus remain non-transformed in the presence of PG over-expression. In associated studies we have measured activation of two powerful transcription factors, NFκB/β-catenin, in response to PG, which may represent the second hit for transformation of cells, in response to autocrine PG, in pre-initiated cells (such as HEK-293). Since a large % of GI cancers express autocrine PG, down-regulation of PG and/or its receptor (Annexin2), and/or its mediatory signaling pathways (NFκB/β-catenin) should prove to be a powerful method for reducing stem cell/progenitor cell populations and attenuating tumorigenic/metastatic potential of PG-expressing cancer cells. Supported by NIH grants R01CA114264 and R01CA097959 and a Sealy Center grant to P. Singh Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4259.

Comparison of Intestinal Warm Ischemic Injury in PACAP Knockout and Wild-Type Mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in the gastrointestinal tract and plays a central role in the intestinal physiology, mainly in the secretion and motility. The aim of our study was to compare the ischemic injury in wild-type and PACAP-38 knockout mice following warm mesenteric small bowel ischemia. Warm ischemia groups were designed with occlusion of superior mesenteric artery for 1, 3, and 6 h in wild-type (n = 10 in each group) and PACAP-38 knockout (n = 10 in each group) mice. Small bowel biopsies were collected after laparotomy (control) and at the end of the ischemia periods. To determine oxidative stress parameters, malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) were measured. Tissue damage was analyzed by qualitative and quantitative methods on hematoxylin/eosin-stained sections. In PACAP-38 knockout animals, tissue MDA increased significantly after 3 and 6 h ischemia (133.97 ± 6,2; 141.86 ± 5,8) compared to sham-operated (100.92 ± 3,6) and compared to wild-type results (112.8 ± 2,1; 118.4 ± 1.03 μmol/g, p < 0.05). Meanwhile, tissue concentration of GSH and activity of SOD decreased significantly in knockout mice compared to wild-type form (GSH, 795.97 ± 10.4; 665.1 ± 8,8 vs. 893.23 ± μmol/g; SOD, 94.4 ± 1.4; 81.2 ± 3.9 vs. 208.09 ± 3,7 IU/g). Qualitative and quantitative histological results showed destruction of the mucous, submucous layers, and crypts in knockout mice compared to wild-type tissues. These processes correlated with the warm ischemia periods. Our present results propose an important protective effect of endogenous PACAP-38 against intestinal warm ischemia, which provides basis for further investigation to elucidate the mechanism of this protective effect.

MiR‐103 modulates G1/S transition in IGF‐1 stimulated mouse intestinal crypt cell proliferation

microRNAs play extensive roles in cellular development. Analysis of the microRNA expression pattern during intestinal cell proliferation in early life is likely to unravel molecular mechanisms behind intestinal development and have implications for therapeutic intervention. In this study, we examined differences in microRNA expression and identified miR‐103 as a regulator in IGF‐1 stimulated proliferation of mouse intestinal crypt cells. Mouse intestinal crypt cells were cultured and treated with IGF‐1 for 24 h, microRNA microarrays showed that multiple microRNAs are regulated by IGF‐1, and miR‐103 was the most sharply down‐regulated among these microRNAs. Expression of miR‐103 in mouse intestinal crypt cells was confirmed by real‐time Q‐PCR. Sequence analyses showed that, among the 1040 predicted miR‐103 target genes, CCNE1, CREB1, IGFBP1, and CDKL2 contain complementary sequences to the miR‐103 seed region and are conserved between human and mouse. We further demonstrated that miR‐103 directly controls the expression level of the four genes by luciferase assay and immunoblotting assay, respectively. Taken together, our data suggest that in mouse crypt cells, miR‐103 is part of the G1/S transition regulation network, which targets CCNE1, CREB1, and CDKL2 during IGF‐1 stimulated proliferation. (Supported by Mead Johnson Nutrition)

The kinase TNIK is an essential activator of Wnt target genes

Wnt signalling maintains the undifferentiated state of intestinal crypt/progenitor cells through the TCF4/beta-catenin-activating transcriptional complex. In colorectal cancer, activating mutations in Wnt pathway components lead to inappropriate activation of the TCF4/beta-catenin transcriptional programme and tumourigenesis. The mechanisms by which TCF4/beta-catenin activate key target genes are not well understood. Using a proteomics approach, we identified Tnik, a member of the germinal centre kinase family as a Tcf4 interactor in the proliferative crypts of mouse small intestine. Tnik is recruited to promoters of Wnt target genes in mouse crypts and in Ls174T colorectal cancer cells in a beta-catenin-dependent manner. Depletion of TNIK and expression of TNIK kinase mutants abrogated TCF-LEF transcription, highlighting the essential function of the kinase activity in Wnt target gene activation. In vitro binding and kinase assays show that TNIK directly binds both TCF4 and beta-catenin and phosphorylates TCF4. siRNA depletion of TNIK followed by expression array analysis showed that TNIK is an essential, specific activator of Wnt transcriptional programme. This kinase may present an attractive candidate for drug targeting in colorectal cancer.

Functional Cross-talk between β-Catenin and NFκB Signaling Pathways in Colonic Crypts of Mice in Response to Progastrin

We recently reported a critical role of NFkappaB in mediating hyperproliferative and anti-apoptotic effects of progastrin on proximal colonic crypts of transgenic mice overexpressing progastrin (Fabp-PG mice). We now report activation of beta-catenin in colonic crypts of mice in response to chronic (Fabp-PG mice) and acute (wild type FVB/N mice) progastrin stimulation. Significant increases were measured in relative levels of cellular and nuclear beta-catenin and pbeta-cat45 in proximal colonic crypts of Fabp-PG mice compared with that in wild type littermates. Distal colonic crypts were less responsive. Interestingly, beta-catenin activation was downstream of IKKalpha,beta/NFkappaB, because treatment of Fabp-PG mice with the NFkappaB essential modulator (NEMO) peptide (inhibitor of IKKalpha,beta/NFkappaB activation) significantly blocked increases in cellular/nuclear levels of total beta-catenin/pbeta-cat45/and pbeta-cat552 in proximal colons. Cellular levels of pbeta-cat33,37,41, however, increased in proximal colons in response to NEMO, probably because of a significant increase in pGSK-3betaTyr216, facilitating degradation of beta-catenin. NEMO peptide significantly blocked increases in cyclin D1 expression, thereby, abrogating hyperplasia of proximal crypts. Goblet cell hyperplasia in colonic crypts of Fabp-PG mice was abrogated by NEMO treatment, suggesting a cross-talk between the NFkappaB/beta-catenin and Notch pathways. Cellular proliferation and crypt lengths increased significantly in proximal but not distal crypts of FVB/N mice injected with 1 nM progastrin associated with a significant increase in cellular/nuclear levels of total beta-catenin and cyclin D1. Thus, intracellular signals, activated in response to acute and chronic stimulation with progastrin, were similar and specific to proximal colons. Our studies suggest a novel possibility that activation of beta-catenin, downstream to the IKKalpha,beta/NFkappaB pathway, may be integral to the hyperproliferative effects of progastrin on proximal colonic crypts.

Identification of Early Intestinal Neoplasia Protein Biomarkers Using Laser Capture Microdissection and MALDI MS

Obtaining protein profiles from a homogeneous cell population in tissues can significantly improve our capability in protein biomarker discovery. In this study, unique protein profiles from the top and bottom sections of mouse crypts and Apc(Min+/-) adenomas were obtained using laser capture microdissection (LCM) combined with MALDI MS. Statistically significant protein peaks with differential expression were selected, and a set of novel protein biomarkers were identified. Immunohistochemistry was performed to confirm the differentially expressed protein biomarkers found by LCM combined with MALDI MS. To validate the relevance of the findings in human colorectal cancer (CRC), S100A8 was further confirmed in human CRC using immunohistochemistry. In addition, S100A8 was found to have an increased expression at different human CRC stages (Duke's A-D) compared with controls at both protein (n = 168 cases) and RNA (n = 215 cases) levels. Overall LCM combined with MALDI MS is a promising method to identify intestinal protein biomarkers from minute amounts of tissue. The novel protein biomarkers identified from the top and bottom crypts will increase our knowledge of the specific protein changes taking place during cell migration from the crypt bottom to top. In addition, the identified cancer protein biomarkers will aid in the exploration of colorectal tumorigenesis mechanisms as well as in the advancement of molecularly based diagnosis of colorectal cancer.

Methylamine dichloramine may play a role in the process of colorectal disease through architectural and oxidative changes in crypts in mice

Aims Methylamine dichloramine (CH 3NCl 2) produced by neutrophils may promote colon tumors and colitis via architectural and oxidative changes in crypts, which are secretory granulae composed of goblet cells located in the colorectal mucosal layer. We investigated whether CH 3NCl 2, in comparison with the other reactive oxygen species (ROS) such as H 2O 2 and HOCl, derived from primed neutrophils in inflammatory sites in the large intestine, is a biogenic factor for the induction of colorectal disease in mice. Main methods Male ICR-strain mice were administered each oxidant (0.5–0.7 μmol/mouse) by enema under anesthesia. The colorectal tissues were evaluated by histopathological and immunohistochemical analyses. Hemolysis and hemoglobin oxidation by the methylamine chloramines and HOCl were examined by adding them (50–400 μM) to a sheep erythrocyte suspension (1 × 10 8 cells/ml) and its lysate at pH 7 and 37 °C. Key findings CH 3NCl 2 oxidized erythrocyte hemoglobin more effectively than HOCl, indicating it has high cell permeability and selective oxidation ability. CH 3NCl 2 mainly induced atrophy of crypts at 6 h after administration, while the other ROS tested did not. Furthermore, 4-hydroxy-2-nonenal (4-HNE) showed positive immunostains throughout the mucosal layer, including around the basal regions of atrophied crypts, only with CH 3NCl 2, while positive immunostains were observed for 3-nitrotyrosine (3-NT) in the atrophied crypts and their surrounding lamina propria in the mucosal layer. Significance The results suggest that CH 3NCl 2derived from primed neutrophils may play the most important role in promoting the development of colon tumor formation and colitis by oxidative stress through its high degree of cell permeability.

Apo‐lactoferrin stimulates proliferation of mouse crypt cells by activating the MAPK signaling pathway

Lactoferrin (Lf), an iron‐binding protein in human milk, plays an important role in intestinal epithelial cell proliferation, but the underlying molecular mechanisms remain unknown. We have shown that the Lf receptor (LfR) mediates Lf endocytosis by Caco‐2 cells via a clathrin‐mediated pathway. The Ras‐dependent extracellular signal‐regulated mitogen‐activated protein kinase (MAPK) cascade is an important pathway transmitting extracellular signals that stimulate cell proliferation. We hypothesized that Lf stimulates proliferation of intestinal epithelial cells by activating Ras‐MAPK and that LfR is involved. Mouse crypt cells were isolated from d 10 pups. Confocal microscopy showed that LfR is expressed and localized at the plasma membrane of crypt cells, specifically binding iron‐free Lf (apo‐Lf) and iron‐saturated Lf (holo‐Lf). BrdU assay showed that Lf stimulates cell proliferation and that apo‐Lf has stronger effect than holo‐Lf. Both LfR antibody blocking and MEK inhibitor (U0126) treatment inhibited stimulation of crypt cell proliferation by apo‐Lf but not holo‐Lf. Western blot showed that apo‐Lf but not holo‐Lf initiates MAPK signaling by ERK‐phosphorylation. Further, both MEK inhibitor and LfR antibody blocking inhibited activation of MAPK signaling by apo‐Lf. Thus, apo‐Lf but not holo‐Lf stimulates intestinal proliferation via initiating MAPK signaling and LfR mediates this process.

Gp130-Mediated Stat3 Activation in Enterocytes Regulates Cell Survival and Cell-Cycle Progression during Colitis-Associated Tumorigenesis

Although gastrointestinal cancers are frequently associated with chronic inflammation, the underlying molecular links have not been comprehensively deciphered. Using loss- and gain-of-function mice in a colitis-associated cancer model, we establish here a link comprising the gp130/Stat3 transcription factor signaling axis. Mutagen-induced tumor growth and multiplicity are reduced following intestinal epithelial cell (IEC)-specific Stat3 ablation, while its hyperactivation promotes tumor incidence and growth. Conversely, IEC-specific Stat3 deficiency enhances susceptibility to chemically induced epithelial damage and subsequent mucosal inflammation, while excessive Stat3 activation confers resistance to colitis. Stat3 has the capacity to mediate IL-6- and IL-11-dependent IEC survival and to promote proliferation through G1 and G2/M cell-cycle progression as the common tumor cell-autonomous mechanism that bridges chronic inflammation to tumor promotion.

Role of PI3K/Akt signaling in TRAIL- and radiation-induced gastrointestinal apoptosis

Activation of the PI3K/Akt pathway is associated with tumorigenesis and resistance to apoptosis and ionizing radiation (IR). We sought to characterize the effects of physiologic and genetic manipulation of Akt signaling on IR-induced gastrointestinal (GI) apoptosis in mice. PI3K/Akt signaling is stimulated by insulin. We evaluated the time course of Akt stimulation by insulin and found it overlapped with protection from apoptosis induced by TRAIL (TNF-alpha Related Apoptosis Inducing Ligand) in cell lines. Mice were treated with insulin and glucose, and the kinetics of in vivo Akt stimulation were determined by phospho-Akt (S473) (P-Akt) immunofluorescence in the gut. Irradiation of mice by 5 Gy at 30 min after insulin/glucose administration induced apoptosis in the crypts of the ileum and colon after 6 hrs, but induced little apoptosis in the liver or esophagus. Pre-treatment with insulin and glucose did not significantly alter levels of IR-induced apoptosis in the gut. IR alone led to sustained increases in P-Akt in the gut at 6 hrs, a protective response that may have precluded additional protection from insulin/glucose. In Akt1 -/- mice, there was significantly more apoptosis in ileum crypts of irradiated mice compared to Akt1 +/+ mice, suggesting a role for the pathway in the GI tract in response to IR. Taken together, modulation of the PI3K/Akt pathway may sensitize or protect against cancer therapies in both tumor and normal tissues.

Activation of NF-κB is required for mediating proliferative and antiapoptotic effects of progastrin on proximal colonic crypts of mice, in vivo

Mice overexpressing progastrin (PG) in intestinal mucosa (fatty acid-binding protein (Fabp)-PG mice) are at an increased risk of proximal colon carcinogenesis in response to azoxymethane. Here, we report a significant increase in the length of proximal colonic crypts in Fabp-PG mice, associated with potent antiapoptotic effects of PG, which likely contributed to the previously reported increase in colon carcinogenesis in Fabp-PG mice. Phosphorylation of kinase of IkappaBalpha (IKKalpha/beta), inhibitor of kappaB (IkappaB)alpha and p65NF-kappaB was significantly elevated in proximal colonic crypts of Fabp-PG versus wild-type mice, which was associated with degradation of IkappaBalpha and nuclear translocation/activation of p65. Surprisingly, distal colonic crypt cells were not as responsive to elevated levels of PG in Fabp-PG mice. Annexin II, recently described as a high-affinity receptor for PG, strongly co-localized with PG intracellularly and on basolateral membranes of proximal crypt cells, providing evidence that annexin-II binds PG in situ in colonic crypt cells. Proliferative and antiapoptotic effects of PG on proximal crypts of Fabp-PG mice were attenuated to wild-type levels, on treatment with NEMO peptide (an inhibitor of nuclear factor-kappaB (NF-kappaB) activation), demonstrating for the first time a critical role of NF-kappaB in mediating hyperproliferative affects of PG on colonic crypts of Fabp-PG mice, in vivo. Thus, downregulation of NF-kappaB may significantly reduce the increased risk of colon carcinogenesis in response to PG.

S1647 NF-κB Mediates Anti-Apoptotic Effect of Progastrin (PG) On Proximal (P) Colonic Crypts of Mice: Intracellular Co-Localization of PG with PG/Gastrin Receptor, Annexin-II (Anx-II), in P Colonic Crypts

S1647 NF-κB Mediates Anti-Apoptotic Effect of Progastrin (PG) On Proximal (P) Colonic Crypts of Mice: Intracellular Co-Localization of PG with PG/Gastrin Receptor, Annexin-II (Anx-II), in P Colonic Crypts

T‐cell factor 4 (Tcf7l2) maintains proliferative compartments in zebrafish intestine

Previous studies have shown that Wnt signals, relayed through beta-catenin and T-cell factor 4 (Tcf4), are essential for the induction and maintenance of crypts in mice. We have now generated a tcf4 (tcf7l2) mutant zebrafish by reverse genetics. We first observe a phenotypic defect at 4 weeks post-fertilization (wpf), leading to death at about 6 wpf. The phenotype comprises a loss of proliferation at the base of the intestinal folds of the middle and distal parts of the intestine. The proximal intestine represents an independent compartment, as it expresses sox2 in the epithelium and barx1 in the surrounding mesenchyme, which are early stomach markers in higher vertebrates. Zebrafish are functionally stomach-less, but the proximal intestine might share its ontogeny with the mammalian stomach. Rare adult homozygous tcf4(-/-) 'escapers' show proliferation defects in the gut epithelium, but have no other obvious abnormalities. This study underscores the involvement of Tcf4 in maintaining proliferative self-renewal in the intestine throughout life.

PPARβ/δ Regulates Paneth Cell Differentiation Via Controlling the Hedgehog Signaling Pathway

All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

C-Myc Is Required for the Formation of Intestinal Crypts but Dispensable for Homeostasis of the Adult Intestinal Epithelium

In self-renewing tissues such as the skin epidermis and the bone marrow, Myc proteins control differentiation of stem cells and proliferation of progenitor cell types. In the epithelium of the small intestine, we show that c-Myc and N-Myc are expressed in a differential manner. Whereas c-Myc is expressed in the proliferating transient-amplifying compartment of the crypts, N-Myc is restricted to the differentiated villus epithelium and a single cell located near the crypt base. c-Myc has been implicated as a critical target of the canonical Wnt pathway, which is essential for formation and maintenance of the intestinal mucosa. To genetically assess the role of c-Myc during development and homeostasis of the mammalian intestine we induced deletion of the c-myc(flox) allele in the villi and intestinal stem cell-bearing crypts of juvenile and adult mice, via tamoxifen-induced activation of the CreER(T2) recombinase, driven by the villin promoter. Absence of c-Myc activity in the juvenile mucosa at the onset of crypt morphogenesis leads to a failure to form normal numbers of crypts in the small intestine. However, all mice recover from this insult to form and maintain a normal epithelium in the absence of c-Myc activity and without apparent compensation by N-Myc or L-Myc. This study provides genetic and molecular evidence that proliferation and expansion of progenitors necessary to maintain the adult intestinal epithelium can unexpectedly occur in a Myc-independent manner.

Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/β-Catenin Complex

T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by beta-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/beta-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/beta-catenin transcriptional complex by means of proteomics. A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the beta-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. In this study, we identified PARP-1 as a novel coactivator of the beta-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis.

Altered Epithelial Cell Lineage Allocation and Global Expansion of the Crypt Epithelial Stem Cell Population Are Associated with Ileitis in SAMP1/YitFc Mice

Crohn's disease is characterized by cycles of mucosal injury and ulceration followed by epithelial regeneration and restoration of normal epithelial function. In this study, we examined whether ileitis in SAMP1/YitFc mice, a recombinant-inbred line that spontaneously develops ileitis resembling human Crohn's disease, was associated with alterations in normal patterns of epithelial differentiation or changes in epithelial regeneration after experimental injury. Increased numbers of Paneth, goblet, and intermediate cells were present focally in the ileum of SAMP1/YitFc mice by 4 weeks of age, before any histological evidence of acute or chronic inflammation. This increase in secretory cells became more pronounced at sites of ileitis with increasing age and inflammation. Additionally, there was mispositioning of Paneth and intermediate cells along the crypt-to-villus unit. A concomitant reduction in the number of absorptive enterocytes was observed. In contrast to the ileal-specific changes in lineage allocation, crypt stem cell numbers began to increase in both the ileum and proximal jejunum at the onset of inflammation in SAMP1/YitFc mice. These data suggest that the alterations in epithelial cell differentiation and increases in the size of the crypt stem cell population observed in SAMP1/YitFc mice are regulated by distinct mechanisms. We speculate that these epithelial alterations may play a role in the pathogenesis of ileitis in this murine model of Crohn's disease.