Kinase suppressor of ras regulates the fate of intestinal epithelial cells exposed to TNFA

GASTROENTEROLOGY Vol. 118, No.4

A556 AGA ABSTRACTS

2918

2920

EXPRESSION OF EGF AND ERBB RECEPTOR PROTEINS IN JEJUNAL EPITHELIUM.

EFFECTS OF THE POLYPHENOL RESVERATROL ON THE CO· LON CARCINOMA CELL LINE CACO-2.

Laurie E. Wallace, Brian M. Chung, James A. Hardin, Donald G. Gall, Univ of Calgary, Calgary, AB, Canada. Controversy surrounds the cellular localization of the epidermal growth factor (EGF) receptor in jejunal intestinal epithelial cells. A number of studies suggest the EGF receptor is located on the basolateral membrane of small intestinal enterocytes. In contrast, we have previously demonstrated that luminal EGF induces an acute increase in jejunal nutrient transport function. The EGF receptor (EGF-R) belongs to a family of receptors including: EGF-R or erbBI, erbB2, erbB3 and erbB4. Aim: To examine EGF and erbB receptor expression in jejunal epithelium. Methods: NZW rabbits (I Kg) were fasted and jejunal segments were fixed in 10% formalin, cryosectioned and then probed with anti-EGF-R, anti-erbB2, anti-erbB3 and anti-erbB4 antibodies using immunocytochemistry and fluorescent microscopy. In separate experiments, brush border membrane (BBM) and basolateral membrane (BLM) vesicles were prepared from jejunal tissue and EGF-R protein expression was measured by western immunoblot. Results: EGF-R immunofluorescent staining was observed on the apical brush border along the entire length of the villus. EGF-R immunoreactivity was also localized to enterocyte basolateral membranes though staining was most prominent in the upper villus. No EGF-R labeling was visible in the crypts. ErbB4 staining was present on the brush border but only extended 2/3 of the way down the villus. ErbB4 was also localized to the basal aspect of jejunal enterocytes with the strongest staining seen on the upper villus. ErbB4 immunoreactivity was not present in the crypts. ErbB2 and erbB3 immunoreactivity was not observed in jejunal enterocytes. The presence of EGF-R in BBM and BLM was confirmed by western immunoblot. Conclusions: EGF-R and erbB4 are present on both the brush border and basolateral membranes of jejunal enterocytes. These findings suggest that jejunal enterocytes can respond to both luminal and circulating EGF.

Freya Wolter, Bora Akoglu, Wolfgang F. Caspary, Juergen Stein, 2nd Dept of Medicine, J W Goethe Univ, Frankfurt, Germany; J W Goethe Univ, Frankfurt, Germany. Background and Aims: Resveratrol (3,5,4' -trihydroxystilbene), a naturally occurring molecule which is present at high levels in grapes and red wine, is classified as an anti-fungicide phytoalexin. The compound has been shown to inhibit cyclooxygenase-l (COX-I) and the endoperoxidase activity of COX-2 in promyelocitic HL-60 cells, where it also arrests the cell cycle at the S/G2_phase transition. At present little is known about similar effects on growth and apoptosis of colon cancer cells. The purpose of the present study was (l) to investigate the effect of resveratrol on cell growth and differentiation of the colon adenocarcinoma cell line Caco- 2 and (2) to characterize the precise mechanism of action of the compound on cell cycle and apoptosis. Methods: Caco-2 cells grown in standard culture media were incubated with different concentrations of resveratrol (1-40 pM) or vehicle (DMSO, <0.1 %) for 24 hours. Cytotoxicity was excluded by lactate dehydrogenase release. Cell proliferation was measured by thymidine incorporation. Apoptosis was characterized by DNA-fragmentation, cell cycle kinetics by flow cytometry, and protein levels of Cyclin D I, CDK4, pRB and BcI-xL were determined by Western blotting. Results: Resveratrol increased apoptosis at a concentration of 25 pM and 50 p.M 1.7:t0.26 and 2.9:t0.95 fold, respectively, (p
2919 ENHANCED SURVIVAL AND MUCOSAL REPAIR FOLLOWING DSS COLITIS IN TRANSGENIC MICE THAT OVEREXPRESS BOVINE GROWTH HORMONE. Kristen L. Williams, C. Randall Fuller, Levinus A. Dieleman, Christopher M. DaCosta, Kimberly S. Clark, R. Balfour Sartor, Pauline K. Lund, Univ of North Carolina, Chapel Hill, NC. Introduction and Aims: The growth-promoting effects of growth hormone (GH) on the intestine are well documented. GH has been used in recent clinical trials to treat patients with short bowel syndrome or Crohn' s disease (CD). However, the specific effects of GH on inflamed intestine are not defined. MTl-bGH-TG mice on C57BI6/SJL background overexpress GH and insulin-like growth factor-I (IGF-I) in plasma and provide a model of chronic GH and IGF-I excess. The present study tested the hypothesis that chronic GH and IGF-I excess in MTl-bGH-TG will alter susceptibility to injury, severity and duration of inflammation, degree of mucosal damage andlor mucosal repair during colitis induced by dextran sulfate sodium (DSS). Methods: Six MTl-bGH-TG and WT litterrnate pairs were treated with 3% and 4% DSS for 5 days followed by 0, 3, 7, or 10 days ofrecovery. Blinded histological colitis scores were assessed on H&E stained sections of distal colon based on three parameters: severity of inflammation, extent of inflammation, and crypt damage. These three scores are added together to yield a total colitis score. Colon was also scored for regeneration in the recovery phase. Results: All of the animals survived five days of DSS treatment and 100% of MTl-bGH-TG survived the entire recovery period. WT showed 50% mortality during recovery from 4% DSS and 25% mortality during recovery from 3% DSS. After 5 days of 3% DSS treatment, MTl-bGH-TG and WT did not differ significantly in total colitis score (TG= I 1.7:t 1.3, WT=12.1 :t1.9) or in any of the subscores. However, at 7 days of recovery from DSS treatment, MTl-bGH-TG had improved total colitis scores (TG=9.5:t1.6, WT=I9.4:t4.1, p<0.05), reflecting reduced inflammation (TG=3.8:t0.58, WT=7.4:t2.0, p<0.05) and reduced mucosal damage (TG=2.4:t0.47, WT=6.1:t 1.8, p=0.050). Enhanced recovery of TG was associated with increased plasma IGF-I (TG= 1113.0:t54.5 nglml vs WT=5I 1.1:t61.8 nglml, p<0.05). Colonic IGF-I expression was increased in DSS treated TG and WT relative to water controls but did not differ between TG and WT. Conclusions: GH does not alter susceptibility and severity of acute DSS induced colitis but improves survival and mucosal repair during recovery phase. These effects are associated with increased plasma levels of IGF-I. These findings support the concept that improving colonic repair by GH can decrease chronic intestinal inflammation and has potential therapeutic merit for mD patients.

2921 EVIDENCE FOR CO-ORDINATED IN VIVO EXPRESSION OF TREFOIL PEPTIDES IN GASTROINTESTINAL EPITHELIA REGENERATING AFTER DAMAGE. Richard Poulsom, Stefan V. Dubois, Andrew A. Fagbemi, Nicholas Borley, Bryan Warren, Nicholas A. Wright, Imperial Cancer Research Fund, London, United Kingdom; Imperial Coli Sch of Medicine, London, United Kingdom; Univ of Oxford, Oxford, United Kingdom. Introduction- Recent observations (Taupin, J Clin Invest 1999; 103; R3I) have shown that TFF2lhSP and TFF3/1TF show cross-induction in gastric epithelial cell lines in vitro, and that both TFF2lhSP and TFFlIpS2 expression are reduced in TFF3/ITF null mice, indicating co-ordinated regulation of these genes, whose products are motogens for gastrointestinal epithelial cells. In normal human tissues, trefoil peptides show a fairly constant tissue distribution: TFF2 is localised to deep glands in the gastric antrum, mucous neck cells in the gastric body, and Brunner's gland acini, while TFFI is seen in foveolar pit cells in both antrum and body, and a small proportion of mucous neck cells. TFF3 is localised to small and large intestinal goblet cells. In these tissues, TFFI seems to co-localise with MUC 5AC, TFF2 with MUC6 and TFF3 with MUC2 respectively. AimTo assess whether cross-induction of TFF genes occurs in damaged human gastrointestinal tissues. Methods- We examined a series of tissues in which the common theme was regeneration after damage, using immunohistochemistry and in situ hybridisation with 35S riboprobes. Results- In normal tissues trefoil peptide genes appear selectively in most mucous cell lineages, without evidence of co-ordinated expression. In surgical excision specimens from infants with necrotizing enterocolitis there was constitutive expression of TFF3 in goblet cells; TFFI was observed in the restituting tongues of epithelium repairing areas of ulceration. In ulcers in adults the migrating epithelium was capable of expressing all three trefoil peptide genes. Conclusion- Our observations in damaged tissues suggest that there is co-ordinate regulation of trefoil peptide genes; supported by our previous observations of apparently ectopic TFFI expression in goblet cells in Crohn's disease tissues, and induction of TFF3 in the later stages of healing of experimental gastric ulcers. EGF is known to increase TFFI gene expression, and it is possible that may be the first step in the proposed stimulation of cis-acting regulatory regions by trefoil peptides themselves.

2922 KINASE SUPPRESSOR OF RAS REGULATES THE FATE OF INTESTINAL EPITHELIAL CELLS EXPOSED TO TNFA. Fang Yan, Sutha K. John, D. Brent Polk, Vanderbilt Univ, Nashville, TN. Cellular survival is determined by the balance between antiapoptotic and proapoptotic signals. Tumor necrosis factor (TNF)a regulates cell survival through activation of extracellular signal regulated kinase (ERKIIERK2)1 MAP kinase and nuclear factor (NF) KB, as antiapoptotic signals. The TNFa-induced proapoptotic signals include activation of stress-activatedl Jun protein kinase (SAPKlJNK). We have reported that kinase suppressor of Ras (KSR) is an essential upstream regulator of TNFa-stimulated

AGAA557

April 2000

ERKIMAP kinase, and plays a central role in TNFa signal transduction. In the present study, we evaluated the role of KSR in the proliferative and apoptotic response of intestinal cells to TNFa. We studied young adult mouse colon (YAMC) cells expressing a dominant negative, kinase inactive (ki) KSR. Proliferation assays were performed on cells treated with TNFa (IOOng/ml, 24 h) by counting crystal-violet stained cells. Apoptosis was detected by (I) DNA staining of cells treated with TNFa for 6 hours by 4,6-diamidino-2-phenylindole (DAPI), and (2) Western blot analysis with anti-poly ADP-ribose polymerase (PARP) p85 fragment, a cleavage product of caspase-3. As expected TNFa induced antiproliferative effects in YAMC cells without inducing apoptosis. However, kiKSR expression altered the cellular response to TNFa causing a 97% cell loss. DAPI staining showed apoptotic nuclei within 6 hours of TNFa treatment in kiKSR expressing cells, but not in untreated cells, or TNFa-treated cells expressing endogenous KSR. Treatment of kiKSR expressing cells, but not wild type KSR cells with TNFa also induced PARP cleavage. We studied TNFa antiapoptotic signals, activation of ERKIMAP kinase, inhibitor of NFKB (IKBa) degradation, and NFKB-activated apoptosis inhibitor gene expression, c-inhibitor of apoptosis-2 (c-IAP2), by Western blot analysis. TNFa stimulated ERKllERK2 activation, IKBa degradation, and c-IAP2 expression in YAMC cells expressing endogenous KSR. However, these antiapoptotic activities were all inhibited in the YAMC cells expressing the dominant negative kiKSR. Interestingly, TNFa activation of SAPKlJNK, a proapoptotic signal, was not inhibited by kiKSR expression. In summary, expression of a dominant negative KSR in intestinal epithelial cells shifts the balance from antiapoptotic to proapoptotic TNFa signal transduction pathways. These findings have significant implications for understanding intestinal cell fate as well as the pathogenesis of inflammatory bowel disease.

2923 A NOVEL EPIDERMAL GROWTH FACTOR-LIKE MOLECULE CONTAINING TWO FOLLISTATIN MODULES STIMULATES TYROSINE PHOSPHORYLATION OF ERBB-4 IN MKN28 GASTRIC CANCER CELLS. Masaoki Yonezawa, Tohru Uchida, Ken Wada, Tomonori Akamatsu, Akira Mizoguchi, Choitsu Sakamoto, Taku Tsukui, Kei Sinoki, Jun Satou, 3rd Dept of internal medicine, Nippon Med Sch, Tokyo, Japan; 2nd Dept of Internal Medicine, Kobe Univ Sch, Kobe, Japan; Nippon Med Sch, Tokyo, Japan; Dept of Anatomy, Kyoto Univ Sch of Medicine, Kyoto, Japan. Epidermal growth factor (EGF) and EGF-related peptides, including transforming growth factor-a(TGF-a), amphiregulin, heparin binding-EGF-like growth factor and betacellulin, are expressed in the gastrointestinal tract. EGFlNeuregulin (NRG) family are known to exert various effects on gastrointestinal function. Due to the biological importance of this extended family of ligands, we have searched for additional homologues using RT-PCR with degenerate primers designed from consensus sequence of the EGF-like domains of EGFINRG family growth factors. In this sturdy, we have isolated a gene from stomach fibroblasts encoding a novel protein, termed tomoregulin (TR), containing two follistatin modules which might bind TGF{3-related growth factors and a single EGF-like domain which is closely related to EGFINRG family growth factors. Sequence analysis revealed cytoplasmic domains of TR contained potential G-protein activating motifs. Hydropathy analysis of the TR precursor sequence revealed that pro-TR is a transmembrane protein. Analysis of conditioned media from CHO-KI cells overexpressing TR eDNA by western blotting showed that TR is cleaved and released into media as two soluble proteins distinguished by the presence or absence of the EGF-like domain. Northern blot analysis using TR cDNA as a hybridization probe showed that expression of TR mRNAs was also observed in adult central nervous system tissues and middle to late stages of embryos as well as in gastric fibroblasts. These results indicate TR may have important roles in normal development of embryos and maintenance of adult central nervous system. In MKN 28 gastric cancer cells, TR failed to stimulate tyrosine phosphorylation of EGF receptor, erbB-2 and erbB-3. However, soluble TR stimulated erbB-4 tyrosine phosphorylation, although it was weak compared to NRG-induced erbB-4 tyrosine phosphorylation. These results suggest that TR released from gastric fibroblasts as a soluble protein might stimulate erbB-4 tyrosine phosphorylation and exert multiple effects in the gastric mucosa.

2924 EXTRACELLULAR TIDOLIDISULFIDE REDOX STATE REGU· LATES GLUTAMINE AND GROWTH FACTOR-INDUCED CACO-2 CELL PROLIFERATION. Thomas r. Ziegler, Carolyn r. Jonas, Li h. Gu, Dean p. Jones, emory Univ, Atlanta, GA. Background: Glutamine, keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I) have gut-trophic effects in animal models. In vivo, both glutamine and KGF increase gut mucosal levels of glutathione (GSH), the central regulator of thiol redox state. In vitro, increased cellular GSH and a more reduced thiol redox status are associated with increased epithelial cell proliferation. Our aim was to determine whether extracellular thiol redox state controls human intestinal cell proliferation in response to glutamine and growth factors in vitro. Methods: CaCo-2 cells were treated for 24 hr with L-glutamine (10 mM), KGF (10 ng/ml) or IGF-I (100 ng/ml) under a range of extracellular thiol redox conditions (Eh) obtained by varying the cysteine and cystine concentrations in DMEM. Glutamine effects were compared to responses to L-alanine (10 mM) and the nonmetabolizable glutamine analog, 6-diazo-5-oxo-L-norIeucine (DON, 10 mM). All treatments were compared to control cells cultured in DMEM with 4 mM glutamine. Proliferation was measured by DNA synthesis using BrdU incorporation and intracellular GSH concentrations by HPLC. Results: A more reducing thioI/disulfide redox environment increased CaCo-2 BrdU incorporation in control medium. Glutamine (10 mM) increased DNA synthesis by 200 to 250% at more oxidizing redox states (Eh 0 to -109 mV), but had no effect at more reducing Eh conditions (-131 and -150 mY). L-alanine and DON did not change CaCo-2 DNA synthesis at any redox condition. KGF and IGF-I increased DNA synthesis by 50 to 100% during more oxidizing redox states (Eh -46 to -80 mV and 0 to -80 mY, for KGF and IGF-I, respectively). Extracellular thiol redox state and glutamine/growth factor treatment did not alter intracellular GSH levels during increased cell proliferation. Conclusions: CaCo-2 cell proliferation in DMEM is stimulated by a more reduced extracellular thioI/disulfide redox state. Treatment with the amino acid glutamine and the peptides KGF and IGF-I each induce CaCo-2 cell proliferation. The trophic response to glutamine is nutrient-specific and requires glutamine metabolism. Extracellular thiol redox status regulates cell proliferation in response to glutamine and these peptide growth factors, an effect not mediated by increased intracellular GSH. The extracellular reducing environment is regulates cell proliferation induced by gut-trophic nutrients and growth factors in CaCo-2 cells. Supported by NIH RROO039, DK54823 and ES09047.

2925 EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) BLOCKADE IMPROVES CLINICAL AND BIOCHEMICAL FEATURES OF MENETRIER'S DISEASE. 1. Steven Burdick, Robert V. Coffey, E. Chun, G. Lindberg, G. Tanner, K. Washington, 1. R. Goldenring, The Univ of Texas Southwestern Med Ctr, Dallas, TX; Vanderbilt Univ, Nashville, TN; Vanderbilt, Nashville, TN; Univ of Texas Southwestern, Dallas, TX; Med ColI of Georgia, Augusta,

GA. Aim: To determine the clinical and biochemical response to epidermal growth factor receptor (EGFR) blockade in protein losing gastropathy (Menetrier's) disease. Background: Enhanced EGFR signaling has been implicated in the pathogenesis of Menetrier's Disease (Gastro 103:19501963,1992) Hypothesis:We hypothezied that delivery of a neutralizing monoclonal antibody to ectodomain of human EGFR(C225; Imclone)would improve signs and symptoms of Menetrier's disease. Methods: A 50 yo man with Menetrier's disease presented with refractory vomiting, hypoalbuminemia, and absence of parietal cells on multiple snare biopsies of the gastric fundus. He had failed conventional therapy, and was at excessive surgical risk due to primary pulmonary hypertension. Compassionate use for C225 was obtained. After one month of weekly intravenous infusion of C225 (250mg/m 2)he was re-evaluated. Results: See table below for graphic data. One day after start of treatment, TGF a levels in serum and gastric juice increased 3-fold (serum 15.6 vs 43.7 pg/cc; gastric juice 63.8 vs 200.8 pg/cc); active, phosphorylated MAP kinase in gastric biopsies decreased completely while MAP kinase immunoreactivity was unchanged; finally total AKT protein and active phosphorylated AKT increased 2.5 fold- these latter parameters may reflect, respectively, decreased proliferation of mucus cells and reemergence of parietal cells. Conclusion: EGFR blockade provided objective evidence for successful treatment of Menetrier's disease. PreRx Episodes of Vomiting/Month Quality of LifeHealth Function Domain Psycho/Social Domain Serum A1bumnin (mg/dl) a 1Anlt-trypsln Stool (mg/dl) Parietal Cells Gastric

Polt-Rx

280

5

11% 7.9%

39% 50% 3.1

2 296 negative

108 positive