Cathepsin L expression is modulated by GATA-4 and Cdx2 during intestinal epithelial cell differentiation

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Fibroblast Growth Factors Promote Hepatocytc Stem Cell Renewal in Embryonic Liver Cultures Sandeep Sekhon, Xinping Tan, William Bowen, George Michalopoulos, Satdarshan Pal S. Monga

Paneth Ceils Secrete A Soluble Isoform Of Maltase-Glucoamylase (MGAs) With Full Starch Digesting Capacity In Membrane MGA (MGAm) Null Mice Buford L. Nichols Jr., Stephen E. Avery, Nancy F. Butte, Ursula Luginbuehl, Erwin E. Sterchi, Milton Finegold

Fibroblast growth factors (FGFs) have been shown to play a definite role in hepatic induction. Stage and tissue specific expression of FGF 1,4 and 8 are necessary for initiation of hepatic bud from the foregut endoderm FGF is also routinely used to maintain and propagate embryonic stem ceils in culture. The aim of this study was to investigate the effect of FGF 1, 4 and 8 on liver development utilizing an in vitro embryonic liver culture system and recotnbinant FGF proteins. Livers were harvested from day 10 embryos (El0) and cultured as an organ in DMEM supplemented with 10% FCS (control) with or without additional FGF 1 or FGF 4 or FGF 8. After 72 hours, the cultured livers were fixed and processed and 4/zm sections were utilized for histologic and immunohistochemical evaluation. Proliferation (PCNA and Ki-67) and apoptosis (TUNEL) assay was also performed. The major difference in the histology of the FGF 1/4/8 treated cultures was in the arrangement of cells in sheet like architecture only as compared to the controls that displayed cells in both ductular and sheet like arrangements. There was no significant change in number of apoptotic cells in presence of FGF 1/4 but there was a decrease in number of apoptotic nuclei in FGF8 treated cultures. Almost all cells in all FGF cultures exhibited PCNA positivity and a sigmficant number of cells were Ki-67 (S-phase) positive. There was a striking difference in the number of c-kn positive stem cells in the presence of any of the three FGFs used as compared to the controls. These stem cells were also positive for a-fetoprotein and albumin and very few were positive for CK-19. There was an overall decrease in number of CK-19 positive cells in presence of the FGFs. To conclude, FGF 1, 4 and 8 are excellent growth factors to maintain and propagate liver stem cells in development. These factors support prohferation and affect apoptosis and thus promote liver stem cell renewal. Also, lineage analysis shows a preponderance of unipotential stem cells (more hepatocytic than biliary) in the FGF treated cultures that suggest existence of selective hepatocyte stem ceils, h appears that FGFs promotes self-renewal of such a unipotential stem cell population in the liver.

Background: Although starch is the major energy substrate in the diet, nothing is known about the phenotype of adult MGA deficiency. MGA is the enzyme that converts the dominant 1-4 linkages of solubilized starch to glucose. We generated a knockout of MGAm to study MGA deficiency phenotype. Methods: A selection cassette containing Lac-Z was inserted in place of exon 2 of MGAm KO was confirmed by Q-PCR. X-gal was used to stain tissue Lac-Z expression. Glucoamylase activity (GA) was measured by Dahlqvist assays with Polycose substrate. Soluble forms were isolated by 100,000 G centrifugation. In vivo starch utilization was measured as prandial kCal/g/hr in calorimeters on amylopectin and amylose diets. lmmunohistology (IH) used affinity purified polyclonal antibodies against C-terminal. Maize fragments were identified by polarized light microscopy. KO null and Wildtype (WT)5' cDNA were sequenced. Results: Q-PCR and X-gal staining confirmed the MGAm KO. Jejunal GA was decreased by 40% but ileal GA was intact in KO nulls. 28% of ileal GA was in KO and WT homogenate supematants. Amylopectin and amylose utilizations were identical in KO null and WT by calorimetry. IH showed absence of enterocyte staining in KO nulls but intense staining of Paneth cell granules in KO and WT. Staining was faintly present in some goblets. No staining was detected in colonic mucosa. IH revealed abundant MGAs staining in ileal and colonic lumens. Washings of small intestine revealed GA in nulls and WT. Colonic null and WT homogenates had GA. 66% of small intestine luminal and colonic homogenate GA was in supernatants. By IH and polarized light, infranatant ileal lumen MGAs was bound to starch granules and maize cell fragments. The KO null cDNA sequence revealed excisional splicing of MGAm exon 2, which codes the binding domain. Conclusion: KO of MGAm revealed the presence of MGKs, a secreted isoform, produced by Paneth cells. The MGAs activity was sufficient to maintain normal starch utilization in KO nulls. MGAs was present by IH in ileal and colonic lumens and GA was present in supernatants of KO null ileal washings and colonic homogenates. These results suggests that Paneth cells play a novel but major role in mouse starch digestion by secreting GA in a spliced isoform, MGAs, into the ileal lumen that is preserved in the lumen of the colon.

$952 Potential of Bone Marrow Cells for Epithelial Replacement Therapy in the Murine Gut Satish K. Singh, Selvi Kr/shnau, Daisuke Kohayashi, Hiroshi Mashimo

$955 Gastric Acid Neutralization and Protease Inhibition Improves Peroral Recombinant Aden~Associated Virus-Mediated Gene Delivery to the Intestines Guohong Shao, Kristin C. Baekstrom, Qin Huang, Thomas J. Sferra

BACKGROUND/AIMS: There is evidence that hematogenously injected bone marrow stem cells may sporadically differentiate into gut epithelial cells. We hypothesized that a large number of bone marrow cells could differentiate into epithelial cells when injected locally in the adult murine gut. METHODS: Bone marrow ceils were harvested from femurs and tibias of transgenic ROSA26 mice (Jackson Labs). These ceils constitutively express betagaIactosidase which serves as a discernible marker after injection into host (C57BL/6Jx129SV/ J)F1 mice (Jackson Labs). The derived ROSA26 bone marrow cells were sypgeneic with our host mice, negating the need for immunosuppression. After panning on plastic petridish, non-adherent bone marrow cells (5 x 104 ceils) were microinjected into the gastric and proximal duodenal wall of 6-8 week old adult mice. These tissues were harvested at 1 and 4 weeks post-injection for immunohistochemical staining. RESULTS: Derived ROSA26 bone marrow cells stained strongly blue for beta-galactnsidase. These cells persisted in vivo at the injection site when assessed at 1 and 4 weeks. There was no associated inflammation or tumors. Moreover, injected ceils preferentially engrafted into the deeper regions of the gastric and duodenal mucosa. These cells formed some gland-like structures, and respected local tissue architecture. Beta-galactosidase positive cells within the mucosa expressed markers of epithelial differentiation including cytokeratin and e-cadherin. In addition, certain betagalactosidase positive gastric epithelial cells stained positive for H +-K §-ATPase and the gastrin receptor. CONCLUSIONS: Bone marrow cells implanted into the stomach and duodenal wall engraft, and are capable of differeutiating into epithelial cells. This approach holds promise for long-term implantation and epithelial replacement, and may lead to treatments for a wide range of ulcerative, degenerative, and genetic diseases of the gut.

Gene transfer to the gastrointestinal tract mediated by recombinant adeno-associated virus (rAAV) vectors has many potential applications including complementation of single gene disorders, genetic immunization, and production of therapeutic molecules to act at local or distant sites. In vitro studies indicate that rAAV serotype 2 vectors are adversely affected by conditions within the GI tract (e.g. low intragastric pH, secreted proteases). The objective of this study was to evaluate whether acid neutralization and protease inhibition can improve rAAV gene delivery to the intestines. Methods: An rAAV type 2 vector (10 ~~infectious units) carrying a [3-galactosidase expression cassette was administered with or without sodium bicarbonate (NaHCO3; 1% solution) and aprotinin (protease inhibitor; 3 trypsin inhibitory units) to fasted, adult, FVB/N mice via a feeding tube. Groups of mice were sacrificed at 1.5 hrs and 2 wks post-vector administration. The small intestines were harvested and divided into thirds. Lumenal contents of each segment were evaluated for the presence of functional rAAV by measuring the ability to transduce HeLa cells (e.g. mediate [3-galactosidase expression). Intestinal tissues were evaluated for gene transfer by: (i) a PCR assay for vector genomes and (ii) histochemical staining for [3-galactosidase activity. Results: At 1.5 hrs postrAAV administration, mice (n = 2) receiving rAAV plus NaHCO3/aprotinin had greater amounts of functional rAAV within the lumen of the small intestine than mice receiving rAAV alone (n = 2). The [3-galactosidase level following transduction of HeLa cells for all segments in the NaHCO3/aprotinin group was 277 ( +/-216 S.D.) times (X) the [3-galactosidase level in control, nontransduced HeLa cells and the rAAV-alone group was 9X (+A16 S.D.) controls. The distal intestinal segment of each mouse contained 58 and 176X control levels in the NaHCO3/aprotinin group and 1.0 and 1.2X controls in the rAAV-alone group. At 2 wks post-vector administration, vector genomes were present only within the intestinal tissue of mice receiving the NaHCO3/aprutinin. [3-galactosidase positive cells were not present in either group of mice. Conclusions: Peroral delivery of rAAV type 2 vectors to the intestines can be enhanced by neutralization of gastric acid and inhibition of gastrointestinal proteases. Further studies are necessary to determine whether increases in transgene expression can be achieved.

$953 Primary Cultures of Fully Differentiated Mouse Intestinal Mucosa Akifumi Ootani, Kazuma Fujimoto, Shuji Toda, Hajime Sugihara The factors controlling the proliferation and differentiation of the intestinal mucosa are unknown and have proved difficult to identify because of a lack of in vitro models for studying the proliferating cells of the mucosa. The aim of this study was to develop a novel method for preparing a viable fully differentiated normal enterocytes. METHODS: Minced intestines of newborn mouse were cultured in three-dimensional collagen gel system at an air-liquid interface environment for 7 days. RESULTS: The polarized epithelial cells organized the tubule structure with outer n'tyofibroblast lining. The epithelial ceils consisted of absorptive enterocytes and goblet cells. The former showed a tall columnar cell shape with blush border and immunoreactivity for absorptive cell marker, CD10. The latter showed mucous containing cytoplasm with basal nuclei and positive staining for goblet cell marker, MUC2. 13romodeoxyuridine uptake study showed that active growth of these cells. Interestingly, the viable epithelial cells were observed invariably with underlying myofibroblasts. TenascinC, fibronectin-derived anti-adhesive peptide, was expressed in apical and basolateral side of the fully differentiated epithelial cells. When the tissue fragments were cultured under immersed conditions, none of the viable ceils were observed. CONCLUSIONS: This investigation establishes for the first time that mouse intestinal mucosa can be maintained with fully differentiation in vitro. Our results suggest that intestinal myofibroblasts play a crucial role in the survival, proliferation, and differentiation of intestinal epithelial cells, and that the expression of tenascin-C may be involved in the regeneration and maintenance of the intestinal tissue architecture under epithehal-mesenchymal interaction.

$956 Cathepsin L Expression Is Modulated by GATA-4 and Cdx2 During Intestinal Epithelial Cell Differentiation Richard K. Kim, Joao P. Teixeira, Eun Ran Suh, Peter G. Traber, Francois Boudreau INTRODUCTION: Intestinal epithelial cell differentiation is thought to be orchestrated by the combinatory action of several transcription factors. We have preciously shown that conditional expression of Cdx2 in undifferentiated intestinal epithelial cells regulates proliferation and differentiation. Moreover, we have recently reported that transcriptional activation of Sucrase-lsomaltase, an enterocyte gene specifically induced during differentiation, is modulated by the synergistic interaction of HNF- la, Cdx2 and GATA-4 transcription factors. AIM: To investigate the combined role of Cdx2 and GATA-4 on gene expression during intestinal epithelial cell differentiation. METHODS AND RESULTS: The profile of GATA-4 expression was first evaluated by Western during the course of IEC-6/Cdx2 differentiation. GATA-4 protein expression was strongly induced following the activation of Cdx2 expression. A microarray analysis of genes differentially expressed during IEC-6/Cdx2 differentiation identified Cathepsin L as a putative induced target of GATA-4 and Cdx2. The expression of catalytic Cathepsin L was induced during IEC-6/Cdx2 differentiation as confirmed by

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Western. A portion of the rat Cathepsin L promoter was amplified by PCR and subdoned into a luciferase-reporter vector. Co-transfecttons of NIH 3T3, Caco2 and IEC-6 ceils with either GATA-4 or Cdx2 expression vectors in the presence of Cathepsin Uluciferase construct specifically activated the promoter. Two putative GATA DNA regulatory elements were identified within the promoter. EMSA analysis confirmed that both sites were able to interact specifically with the GATA-4 protein. Finally, we stably transfected undifferentiated IEC-6/ Cdx2 with an inducible vector containing the first 530 nt of GATA-4 cDNA cloned in an ant[sense orientation. Induction of the ant[sense GATA-4 resulted in a reduction of GATA4 protein level as well as catalytic Cathepsin L in 3 independent established clones. CONCLUSION: Our results suggest that GATA-4 and Cdx2 are important factors for the modulation of Cathepsin L expression during intestinal cell differentiation. Given the role of Cathepsin L in extracefiuLar matrix remodeling and its well-established increase in expression in different cancer types including colon, we hypothesize that Cathepsin L plays an important role in intestinal epithelial cell maintenance. *The first two authors have equally contributed to this work.

$959 The CDX2-Site Polymorphism in the Vitamin D Receptor Gene and Expression of Genes for Intestinal Calcium Absorption Juharl R. Wahers, Mohammed Khanji, Natalie Barley, Orli Rhodes-Kendler, Umma R'hair Background: Intestinal absorption of dietary calcium is necessary for the development and maimenance of skeletal mineralization. There is considerable variation in calcium absorption and there is a greater risk of osteoporotic fractures in individuals taking low calcium diets who have low fractional absorption. Blood levels of the active vitamin D metabolite, 1,25(OH)2Dj, account for only part of the variation, suggesting that other [actors contribute to differences in the intestinal expression of genes involved in calcium transport. The vitamin D receptor (VDR) mediates 1,25(OH)2D3 transcriptional effects on expression of calcium transport proteins. Recently a single nucleotide poiymorphism (SNP) has been identified in the promoter of the VDR gene in a consensus response element for the caudal-related homeobox transcription factor CDX2, highly expressed in intestine. This SNP has been shown to affect transcription of the VDR gene and to be associated with differences in bone mineral density in a Japanese population. We hypothesized that the SNP would affect intestinal expression of VDR and consequently that of vitamin D-dependent genes involved in calcium absorption. Methods: The VDR genotype (G or A) was determined by PCR and sequencing m 82 British patients with known bone mineral density, and in 32 subjects with normal endoscopies where the expression of RNA for calcium transport proteins had been determined in duodenal mucosal biopsies Results: The aflelic frequencies of this SNP were 0.76 G : 0.24 A, a lower proportion of A than found in the Japanese study. Consequently in our population, there were relatively few AA homozygotes, the group shown previously to have better bone mineralization, and no significant differences in measurements of lumbar spine or femoral neck bone mineral density could be demonstrated. In the patients who had duodenal biopsies, the GG and AG genotypes were not associated with significant differences in mean levels of expression of the three calcium transport genes, ECAC2/CAT1, calbindin-D9k or PMCA1. However greater sensitivity to 1,25(OH)2D3 levels was found in the GG group, where the relationship with ECAC2/CAT1 and calbindin-D9k was significantly stronger. Conclusion: The CDX-2 site polymorphism in the VDR gene produces differencies in sensitivity of VDR-responaive genes to 1,25(OH):D3 which can affect calcium transport in the intestine.

$957 PDX-1 Interacts with the Lactase Promoter and Functions as a Repressor Zhi Wang, Eric Sibley Background & Aim: Lactase-phlorizin hydrolase gene expression is spatially restricted along the anterior-posterior gut axis Lactase transcription is maximal in the distal duodenum and jejunum in adult mammals. In the small intestine, PDX-1, pancreas duodenum homeobox 1 protein, is expressed maximally in the proximal duodenum. We hypothesize that PDX-1 functions to repress lactase expression in the proximal duodenum The study aimed to determine the role of PDX-1 in regulating lactase gene promoter activity. Methods: 3.0 kb and 100 bp 5' flanking fragments of the factase gene promoter were cloned upstream of a firefly luciferase reporter cDNA. Caco-2 cells were co-transfected with the lactase promoterreporter constructs in the presence of a PDX-1 expression vector (pCMV-PDX1) or the empty vector control and assayed for luciferase activity Nuclear protein bound to a candidate lactase promoter PDX-1 binding site sequence (-71 to -53) was analyzed by electrophoretic mobility shift assays (EMSA) and by gel supershifts with PDX-1 antibody. EMSAs were performed with a radiolabeled oligouucleotide probe corresponding to the putative PDX-1 binding site incubated in the presence of rat jejunal nuclear extract or in vitro translated PDX-I protein. The specificity of DNA-protein interactions was determined by incubation in the presence of unlabeled wild-type or mutant binding site competitor oligonucleotides. Results: PDX-1 represses lactase promoter activity driven by the 3.0 kb and 100 bp promoterreporter constructs by 40% and 70% respectively relative to the empty vector control. A candidate PDX-1 DNA binding site was identified in the 100 bp lactase promoter sequence. EMSAs performed with the binding site probe and in vitro translated PDX-1 or jejunal nuclear extract results in a specific DNA/Protein complex that is competed away by excess unlabeled wild-type oligonucleotide but not by an oligonucleotide in which the core PDX1 binding sequence, TAAT, is mutated. The DNA/Protein complex is supershified by PDXi antibody. Site-directed mutagenesis of the PDX-I core binding site in the lactase promoterreporter constructs disrupts PDX-1 mediated repression of lactase promoter activity. Conclusion: PDX-1 can bind to a specific sequence in the rat lactase gene promoter and functions to repress Lactase promoter activity. PDX-1 repression may function to specify the anterior boundary of lactase gene expression in the small intestine. PDX- 1 is thus a candidate regulator o[ anterior spatial restriction in the gut,

$960 Mutations in HNF-lot That Result in Maturity Onset Diabetes of The Young Retain Wild-Type Activation of Target Genes But Exhibit a Selective Defect in Cooperative Interactions with Endodermal Transcription Factors Joyce K. Divine, Theodore C. Simon Genes expressed in the intestinal epithelium and hepatocyte are activated by a common set of endodermal transcnption factors of the Cdx, C/EBP, FOXA, GATA, HNF-1, HNF-4, and HNF-6 famdies. The rat liver fatty acid binding protein geue (Fabpl) contains functional binding sites for all these transcription [actors in a compact sequence in the proximal promoter. Factors from all these families activate an Fabpl transgene in cultured cells, and multiple factors exhibit striking cooperative transgene activation. HNF-la activates the Fabpl transgene seventeen fold greater in the presence of endodermal factors from these families that are present in hepatocytes than in their absence. Numerous studies conducted by different investigators have confirmed that cooperative act[rational synergy occurs between many of these endodermal factors in cultured cell assays with several target genes. However, the in vivo relevance of this synergy has not yet been established. Maturity onset diabetes of the young (MODY) results from mutations in HNF-la, HNF-I[3, HNF-4a, and other transcription factors. We have found that HNF-la MODY mutants activate the Fabpl transgene as well as wild-type, but are completely defective in cooperative activation with the group of hepatic endodermal factors. Four different mutations in HNF-la have been identified with specific defects in cooperative interactions resulting from a single amino acid substitution, and these mutations are in different domains of the protein. HNF-la exhibits individual cooperative activation of the Fapbl transgene with other factors in the hepatic endodermal factor mixture, but the MODY mutants are defective only in specific interactions with certain factors. Thus, a complete defect in cooperativity is achieved by loss of interaction with a subset of factors. These results indicate that a defect in cooperativity between specific endodermal transcription factors gives rise to an in vivo phenotype.

$958 Differential HNF-loL-lndependent Activation of The Human Lactase-Phlorizin Hydrolase Promoter Between GATA-4 and GATA-5 Is Due to The Zinc Finger and Basic Regions Tjalling Bosse, Herbert M. Van Wering, Lauren N Dowling, Robert K. Montgomery, Pechard J. Grand, Stephen D Krasinski The GATA-4, -5, -6 subfamily of GATA factors are all expressed in the intestinal epithelium and are known to activate the promoters of target intestinal genes, including lactase-phlorizin hydrolase (LPH). However, little is known about the independent and overlapping functions of individual GATA family members. We previously demonstrated that both GATA-4 and GATA-5 can synergistically activate the LPH promoter by physical association with HNFlc~, but that only GATA-4 is capable of activating the LPH promoter independently of HNFlo~ (Gastroenterology 2002;122:A90). The goal of these studies was to define the structures within GATA-4 and delineate the mechanism by which GATA-4 activates the LPH promoter independently of HNF-le~ GATA-4 and GATA-5 each contain a pair of activation domains at the N-terminus, a pair of zinc fingers and an adjacent basic region in the middle of the proteins, and a C-terminal domain Since these structures are conserved, a domain swapping approach was undertaken in which the zinc finger/basic regions of GATA-4 and GATA-5 were interchanged resulting in a GATA-5 protein containing the zinc finger and basic region of GATA-4 (G5ZnG4), and a GATA-4 protein containing the zinc finger and basic region of GATA-5 (G4ZnGS) The chimeric proteins were structurally intact, as demonstrated by their ability to bind GATA sites on DNA (EMSAs) and interact appropriately with epitopespecific antibodies (supershifis). Transient co-transfection assays in HeLa cells, which do not synthesize HNF-ls and are therefore a model for HNF-la-independent GATA activation, revealed that the human LPH promoter was activated by wild type GATA-4 and G5ZnG4, but not by wild type GATA-5 or G4ZnG5 (P < 0.01). This finding demonstrates that HNFle~-independent activation by GATA-4 is due to its zinc finger and basic region. Competition affinity EMSAs using a mixture of GATA-4 and GATA-5, which are distinguishable by different mobility due to different sizes, and a probe that binds both GATAs, revealed that the two GATA sites in the human LPH promoter preferentially bind GATA-4 over GATA5. Conclusion: HNF-la-independent activation of the human LPH promoter by GATA-4 is due to its zinc finger and basic region, which may impart preferential binding to specific DNA targets such as those in the LPH promoter.

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$961 Bile acids regulate MUC4 mucin gene expression at the transcriptional level in esophagus cancer cells. PI3K appears as the major signaling pathway Christophe Mariette, Michael Perrats, Pascal Figny, Nicolas ]onckheere, Emmanuelle Leteurtre, Jean-Pierre Aubert, Jean-Pierre Triboulet, Isabelle Van Seuinngen Purpose: Abnormal gastroesophageal reflux and bile acids have been linked to the presence of Barrett's esophagus premalignant lesion that is accompanied by a metaplastic process vath an increase of mucin-producing goblet cells, that eventually leads to esophagus adenocarcinoma. Methods: Having previously shown that bile acids and their conjugates regulate MUC4 mucin gene expression and transcription in esophagus adenocarcinoma cancer cells, we undertook to identify the intracefiular signaling pathways that are mggered by bile acids to regulate MUC4. To this end, we used specific pharmacological inhibitors of MAPK (PD98059), PKC (GF109203X), PKA (ICf5720) and PI3K (wortmannin) kinases. Effects on MUC4 expression were measured by R'r -PCR, transient transfection assays and immunohistochemistry. Results: OE33 cells express MUC4 apomucin and transcripts of MUC4 were also detected as one of the major mucin gene expressed in these cells along with MUC1. The promoter of MUC4 is very active and contains positive regulatory elements for most of the bile acids. Taurodeoxycholic acid was shown to strongly activate MUC4 expression both at the transcriptional and translational levels, inhibition of PI3K by wortmannin led to at least 50% inhibition of the bile acid-mediated transact[rating effect previously seen on both proximal and distal promoters of MUC4 inhibition of MAPK by PD98059 on the other hand, led to a substantial potentiation of the effects of glycochofic, taurochofic and taurodeoxycholic

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