1Molecular and Cellular Pathobiology Program, Childrens Memorial Research Center, Childrens Memorial Hospital, Chicago; and 2Departments of Pathology and Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
Submitted 19 April 2005 ; accepted in final form 6 July 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
trefoil factor 3; signal transduction
Recent studies have suggested the existence of autoregulatory loops for a net negative regulation of NF-B functions in mammalian cells (3, 4, 6, 10, 38, 43, 44). For example, activation of NF-
B results not only in upregulation of genes involved in inflammation and cell survival but also in synthesizing/resynthesizing NF-
B-dependent negative regulators of NF-
B signaling, such as I
B
, Bcl-3, A20, nitric oxide, and prostaglandin E2 (6, 10, 25, 26, 29, 39, 43, 44). Newly synthesized I
B
and Bcl-3 are translocated into the nucleus and serve to terminate NF-
B action (3, 6), whereas A20, nitric oxide, and prostaglandin E2 are responsible for blocking the NF-
B pathway (12, 13, 28). Overexpression of I
B
results in cardioprotection in trauma (7). Mice lacking A20 suffer from severe systemic inflammation (28, 43). Taken together, the negative regulatory loop of NF-
B ensures transient generation of intracellular signaling to prevent uncontrolled NF-
B activation. It is critical to limit inflammatory injury by terminating and blocking proinflammatory cytokine-induced NF-
B activity in vivo.
Twist protein is a basic helix-loop-helix transcription factor. In mammals, Twist contributes to the morphogenesis of cranial neural tube and participates in the regulation of muscular differentiation (9). Previously, Sosic et al. (43) have demonstrated that 1) Twist protein interacts with transcription factor NF-B; 2) Twist is essential for the downregulation of NF-
B activity in vivo; and 3) NF-
B regulates Twist gene expression. Thus Twist protein is a NF-
B-associated protein. It plays an important role in the regulation of the negative regulatory loop of NF-
B pathway and the attenuation of inflammatory responses (42). Recently, Twist has been shown to be present in mouse mammary epithelial cell lines (18), suggesting that it may be involved in the regulation of physiological or pathophysiological processes in epithelial tissues.
The trefoil factor (TFF) family is a group of extracellular peptides that were originally found in the gastrointestinal (GI) tract (37, 51, 53). These peptides contain distinctive cystine-rich "trefoil" domains and are resistant to proteolytic degradation (16, 37, 51). Three mammalian TFFs have been identified, namely, pS2 (TFF1), SP (TFF2), and intestinal trefoil factor (ITF or TFF3). TFFs are expressed in several tissues of the body but are most pronounced in the GI tract. They are usually associated with the mucous layer in the GI tract. Under normal circumstances, TFF1 and TFF2 are expressed in the human stomach, whereas TFF3 is expressed in the small and large intestines. The expression of TFF has been demonstrated to be upregulated in GI tract ulcers (2). In contrast to TGF- and EGF, TFFs are expressed rapidly in response to injury. It has been shown that intestinal epithelium is the targeted tissue of TFFs (17).
TFF3 is predominantly expressed in mucous epithelia. A major source for TFF3 is goblet cells of the small and large intestine. Maximal TFF3 expression in the GI tract was observed in the distal portions of the ileum and the colon (31). Secretion of TFF3 is evoked by certain neurotransmitters and inflammatory mediators (33). Among TFFs, the physiological functions of TFF3 have been well characterized. Previous studies (35) demonstrated that TFF3 enhances restitution in intestinal epithelial cells and sustains mucosa integrity. We showed that TFF3 protects epithelial cells against reactive oxygen species-induced injury (45). In addition, we found that TFF3 also induces activation of NF-B in intestinal epithelial cells and demonstrated that TFF3 prevents apoptosis of intestinal epithelial cells via NF-
B pathway (8). Pretreatment of the GI mucosa with TFF3 protects epithelium against various injuries (5, 24, 45). However, it is still unknown whether TFF3 modulates inflammatory responses in intestinal epithelial cells. In the present study, we examined 1) how TFF3 activates NF-
B in intestinal epithelial cells, 2) whether Twist protein is expressed in intestinal epithelial cells and regulated by TFF3 and TNF, and 3) whether Twist protein plays a role in modulation of expression of NF-
B-targeted proinflammatory cytokines, such as IL-8 in intestinal epithelial cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of rat recombinant TFF3 in yeast. Rat TFF3 was expressed from Pichia pastoris using a method modified from our previous protocol (8). The recombinant protein was purified with a fast-performance liquid chromatography (AKTA FPLC, Amersham Pharmacia Biotech). The purified TFF3 peptide was visualized as a single band with the silver staining technique. Its biological activity was assessed by the cell migration assay using IEC-18 cells. The peptide induced an approximately threefold increase in cell migration.
Cell culture. HT-29 and IEC-18 cells were purchased from American Type Culture Collection (Rockville, MD) and cultured in a water-saturated atmosphere with 5% CO2 at 37°C. HT-29 cells (passages 2035 after receipt from American Type Culture Collection) were maintained in Dulbeccos modified Eagles minimum essential medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FBS. IEC-18 cells were maintained in Dulbeccos modified Eagles minimum essential medium supplemented with 10% heat-inactivated FBS, insulin (0.1 U/ml), 1% nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Western blot analysis. Cells were lysed in a buffer containing 2 mM Tris-Cl (pH 7.6), 30 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, complete protease inhibitor cocktail (1 tablet/10 ml), and 1% Nonidet P-40. Total cell lysate was processed through sonication at 4°C and followed by centrifugation at 10,000 g for 10 min at 4°C. Supernatants were mixed with an equal volume of 2x Laemmli buffer and boiled for 5 min. Thirty micrograms of protein were resolved on 420% SDS-PAGE gel along with molecular weight standards. The proteins were then transferred onto a nitrocellulose membrane as described before (57). The membranes containing sample proteins were used for immunodetection of Twist protein. Briefly, blots preincubated with PBS containing 5% skim milk were reacted with primary antibody against Twist protein (1:500) for 1 h at room temperature. After incubation, the blot was washed four times with PBS containing 0.05% Tween 20 (PBST), and then incubated with PBST containing 1:10,000 diluted HRP-conjugated goat anti-rabbit IgG for 1 h at room temperature. After additional washing with PBST, immune complexes on the blot were visualized using the ECL system. Blots were stripped and reprobed with MAb against GAPDH (1:10,000) following a standard procedure (45).
Immunoprecipitation.
Lysate containing 0.4 mg of proteins was treated with 1 µg of rabbit anti-IB
antibodies in 800 µl of lysate buffer at 4°C overnight, and the immune complexes were precipitated with protein A/G-Sepharose beads. The beads were thoroughly washed, resuspended in SDS sample buffer, and boiled for 5 min. After being boiled, the proteins were resolved on 420% SDS-PAGE gel, electrotransferred to a nitrocellulose membrane, and probed with mouse anti-phosphotyrosine monoclonal antibody (1:1,000). The blot was then treated with HRP-conjugated goat anti-mouse IgG and finally detected using ECL reagent.
Preparation of nuclear extracts from cells. After the medium was removed, cells (5 x 106) were washed with cold PBS containing 0.5 mM DTT, directly treated with 500 µl of buffer A containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.1% Nonidet P-40, scraped off petri dishes, and incubated for 15 min at 4°C. The nuclei were collected by a brief centrifugation using a microcentrifuge (8). The supernatant was removed. The nuclear pellet was washed with buffer A and resuspended in 150 µl of buffer B containing 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.5 mM DTT, 0.42 M NaCl, 0.2 mM EDTA, 25% glycerol, and 0.5 mM PMSF, and then incubated for 15 min on ice. Following a 10-min centrifugation at 14,000 g at 4°C, the supernatant (nuclear protein fraction) was diluted in buffer C containing (in mM) 20 HEPES, pH 7.9, 50 KCl, 0.5 DTT, 0.2 EDTA, 0.5 PMSF to 300 µl (5 x 106 cells), and stored at 80°C. Protein concentration was measured using Bradford method.
EMSA and supershift assay.
NF-B DNA-binding activity was determined by EMSA as described before (8). Briefly, the NF-
B consensus oligonucleotide was labeled by [
-32P]ATP (3,000 Ci/mmmol, 10 mCi/ml) with T4 polynucleotide kinase. Nuclear extracts (5 µg) were added to 10 µl of gel shift binding buffer [40% glycerol, 10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 0.5 mM DTT, 0.01 mg/ml poly(dI-dC)]. 32P-Labeled oligonucleotide probe (1 µl) was added and the mixture was incubated at room temperature for 20 min. Electrophoresis was done on a 6% polyacrylamide gel with 0.5x Tris-borate-EDTA buffer. The gel was dried and analyzed using a phosphorimaging system. Supershift experiments were performed as follows. Nuclear extracts were incubated with 32P-labeled oligonucleotide (1 µl) at room temperature for 20 min. Antibody (1 µg) against p50, p65, p52, c-Rel, or RelB subunit of NF-
B was subsequently added, samples were incubated at room temperature for 15 min, and electrophoresis was performed. To confirm the specificity of NF-
B DNA binding activity, a competitive experiment was done by adding 1 µl (1,750 pmol/ml) of unlabeled oligonucleotide to the reaction mixture.
ELISA assay for quantitation IL-8 in conditioned medium. Cells were harvested by trypsinization and seeded at 5 x 104 cells/well in 96-well tissue culture plates. Cells were further incubated for 2 days to reach confluence. The medium of confluent monolayers was changed to serum-deprived medium, and cells were cultured overnight. The medium was then changed to fresh serum-deprived medium containing different stimulators and incubated for the indicated times. Medium was collected and IL-8 was detected using Quantikine human IL-8 immunoassay kit (R & D Systems). The protocol provided by the manufacturer was followed. Standard curves were generated for IL-8 using the standard provided in the kit, and the concentration of IL-8 in the cell supernatant was determined by interpreting from the appropriate standard curve.
Immunohistochemical staining. Rat intestinal tissues were sectioned with a cryostat system, fixed with acetone for 10 min at 20°C, and air dried. Slides were rinsed with PBS and incubated with PBS containing 1% BSA for 10 min. Next, slides were incubated with rabbit polyclonal antibody (PAb) against Twist (1:500) for 30 min, washed, and incubated with biotinylated goat anti-rabbit IgG for 30 min. After being washed, slides were incubated with FITC-labeled streptavidin for 30 min, washed, and covered with FluorSave reagent (Calbiochem, San Diego, CA). The staining was performed at room temperature. Finally, slides were visualized under a fluorescence microscope.
siRNA-mediated Twist gene silencing. The siRNA duplexes targeting the human Twist mRNA (GenBank accession no. NM_000474) were designed with the most efficient siRNA-2 duplex design protocol and synthesized by Qiagen (Valencia, CA). The targeting sequences of double-stranded siRNA are TGG GAT CAA ACT GGC CTG CAA and TAA GAA CAC CTT TAG AAA TAA. These sequences were submitted to a Basic Local Alignment Search Tool to ensure that only the TWIST gene was targeted by the TWIST siRNA, and control sequences (nonsilencing labeled control siRNA) were not homologous to any known genes. HT-29 cells were transfected using RNAi Human/Mouse Control kit (Qiagen). In brief, HT-29 cells were seeded onto six-well plates (4 x 104 cells/well) 24 h before siRNA treatment. For transfection, siRNA (1.9 µg/ml) was transfected with the RNAiFect reagent (Qiagen), according to the manufacturers instructions. Twenty-four hours after transfection, culture medium was changed. Cells were used for designed experiments on the fourth day after transfection.
Statistics. Data were expressed as means ± SE, ANOVA and one-way ANOVA, followed by Fishers protected least-significant differences post hoc test to assess the significance of differences. P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TFF3 activates NF-B in intestinal epithelial cells in a manner different from TNF.
Our recent investigation suggests that TFF3 activates NF-
B in intestinal epithelial cells (8). Here, we further examined the kinetics of TFF3-induced NF-
B activation. HT-29 cells were treated with TFF3 (2.5 µM) for 0.5, 1, 2, 4, 8, and 24 h respectively. At the end of the treatment, nuclear proteins were extracted and an EMSA was performed with 32P-labeled NF-
B consensus probe. As shown in Fig. 1A, a low basal level of NF-
B activity was detected in resting HT-29 cells. NF-
B binding activity was induced within 30 min after treatment and rapidly fell thereafter, reaching control levels by 60 min. The specificity of DNA/NF-
B binding was confirmed with a 100-fold excess of unlabeled NF-
B consensus oligonucleotide (data not shown). Incubation of nuclear extracts with anti-p65 antibody resulted in the abrogation in NF-
B/DNA complex, whereas anti-p50 antibody had no effect (Fig. 1A). In addition, the NF-
B/DNA complex was not reduced by antibodies against p52, c-Rel, or RelB (data not shown). Together, the data suggests that NF-
B activated by TFF3 in human intestinal epithelial cells was composed of p65 homodimer. In contrast, TNF induced strong NF-
B activation in HT-29 cells, and the effect persisted for 24 h (Fig. 1B). Supershift assay revealed that TNF-activated NF-
B in intestinal epithelial cells contained p50/p65 heterodimers (Fig. 1B).
|
|
TFF3 does not induce IL-8 production in intestinal epithelial cells.
Persistent activation of NF-B in intestinal epithelial cells results in upregulation of several NF-
B-targeted proinflammatory cytokines such as IL-8. However, whether transient activation of NF-
B induces inflammatory mediators is not clear. Because we have shown that TFF3 induces transient activation of NF-
B in intestinal epithelial cells, we further examined the ability of TFF3 to induce the release of IL-8 by intestinal epithelial cells. For this experiment, we subjected HT-29 cells to stimulation with TFF3 (2.5 µM) or TNF (10 ng/ml) for 1 h. We then removed the medium, washed the cells with PBS, added fresh standard medium, and chased for 23 h. Thereafter, we measured IL-8 in culture medium using ELISA. As shown in Fig. 3, TNF strongly induced IL-8 release in HT-29 cells, whereas TFF-3 showed no effect. The data indicated that transient activation of NF-
B by TFF3 did not result in IL-8 production in intestinal epithelial cells.
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast, transient activation of NF-B before inflammatory stimulation results in the anti-inflammatory response. For example, several investigators (41, 54, 56) have found that transient activation of NF-
B is required for the heart to tolerate ischemia-reperfusion-induced myocardial stunning and myocardial infarction. Pretreatment of rats with low-molecular-weight hyaluronic acid induces hepatoprotection against inflammatory insults via transient activation of NF-
B (52). In the intestine, preconditioning with lipopolysaccharide results in transient activation of NF-
B and induces protective mechanisms against intestinal dysfunction (40).
In the present study, we have demonstrated that TFF3-induced NF-B activation is a transient event. The transient activation of NF-
B by TFF3 is followed by induction of Twist protein, a NF-
B-associated negative regulatory molecule for the NF-
B pathway. Previous studies have shown that Twist interacts with NF-
B (43). It represses NF-
B activity and attenuates the inflammatory response via suppression of NF-
B activity (43). We showed that silencing expression of Twist in TFF3-treated cells results in the induction of IL-8, a NF-
B-regulated proinflammatory cytokine. Thus TFF3-induced transient activating NF-
B is associated with strengthening of the negative regulatory loop of NF-
B, which inhibits proinflammatory cytokine expression in intestinal epithelial cells and protects against inflammation of the GI mucosa.
Intestinal epithelial cells play a unique role in the GI inflammation through their ability to release proinflammatory cytokines such as IL-1 and IL-8. Previously, we and others have shown that inflammatory mediators induce NF-
B activation in the intestine (14, 15, 22, 48). The activated NF-
B further upregulates the expression of several proinflammatory molecules, such as endothelial cell adhesion molecules and macrophage inflammatory protein-2, which result in neutrophilic infiltration and mucosal injury (1, 27, 34). NF-
B is controlled by a negative control loop. However, it is not clear whether the loop is regulated during the inflammation. Herein we report for the first time that Twist protein, a novel molecule in the loop, is present in intestinal epithelial cells in vivo. TNF induces marked degradation of Twist protein in intestinal epithelial cells by a proteasome-dependent mechanism. The effect is associated with TNF-induced NF-
B activity in the cells, which suggests that 1) cytokines regulate the expression of molecules in the loop and 2) reduction of Twist protein by TNF may result in maintenance of NF-
B activity. Persistent activation of NF-
B causes prolonged expression of genes, including both induction of and activation by NF-
B.
Previous studies (37, 51, 53) have shown that TFF3 is expressed in goblet cells and secreted onto intestinal lumen in normal circumstances. It targets intestinal epithelial cells. In contrast to TNF, TFF3 induces upregulation of Twist in intestinal epithelial cells. Thus TFF3 probably plays an important role in maintaining Twist protein in intestinal epithelial cells, which may contribute to downregulation of inflammation in vivo.
The physiological function of Twist in intestinal epithelial cells is not clear. Previously, Twist has been found to play an important role in negative regulation of NF-B in vivo (43). Thus we hypothesize that endogenous Twist in intestinal epithelial cells is involved in controlling proinflammatory cytokine expression during inflammation. This hypothesis is further supported by our observation that TFF3 induces IL-8 production after elimination of Twist protein from intestinal epithelial cells. Furthermore, because TNF induces degradation of Twist in intestinal epithelial cells, which is associated with prolonged NF-
B activation, prevention of cytokine-induced downregulation of Twist protein may be a novel strategy for blocking inflammation in the GI tract.
In addition to participation in negative regulation of the NF-B signaling pathway, induction of Twist has been shown to play an essential role in the prevention of cells from undergoing apoptosis (30). Interestingly, we (8) and others (50) showed that TFF3 protects intestinal epithelial cells against apoptosis. Taken together, TFF3-induced Twist protein may mediate the antiapoptotic effect of TFF3 in intestinal epithelial cells.
Previously, we and others have demonstrated that intestinal epithelial cells are the target of TFF3 (51). TFF3 binds to intestinal epithelial cells (11, 46, 47). In response to TFF3 stimulation, intestinal epithelial cells release nitric oxide and prostaglandins (45, 47). TFF3 activates several intracellular molecules such as NF-B and ERK in intestinal epithelial cells (8, 49). In the present study, we found that TFF3 enhances Twist protein levels in intestinal epithelial cells. We further showed that the selective inhibitor of ERK kinase attenuates the effect of TFF3 on the upregulation of Twist protein, suggesting that TFF3 activates a distinctive signal pathway involved in the modulation of Twist protein. These observations indicate that multiple intracellular regulatory molecules, including ERK, I
B
, Twist, and NF-
B complex may participate to mediate the effect of TFF3. Dissection of the distinctive signal pathway linking these molecules is the subject of our ongoing research.
In summary, we found that TFF3 activates intestinal epithelial NF-B by a mechanism distinct from TNF. We demonstrated for the first time that intestinal epithelial cells constitutively express Twist protein, a novel negative regulator of the NF-
B pathway. We showed that TNF, which induces prolonged NF-
B activation, induces degradation of Twist protein in intestinal epithelial cells. The TNF effect is mediated by the proteasome activity. In contrast, TFF3, which activates NF-
B in a transient event, upregulates Twist protein in intestinal epithelial cells. The effect of TFF3 is mediated by endogenous ERK activity. In addition, we showed that Twist protein plays an important role in silencing IL-8 production in NF-
B- activated intestinal epithelial cells. Further understanding of these mechanisms will provide new insights into the controlling process involved in the activation of NF-
B in inflammation and may lead to the development of a new pharmaceutical to block GI inflammation.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Alison MR, Chinery R, Poulsom R, Ashwood P, Longcroft JM, and Wright NA. Experimental ulceration leads to sequential expression of spasmolytic polypeptide, intestinal trefoil factor, epidermal growth factor and transforming growth factor alpha mRNAs in rat stomach. J Pathol 175: 405414, 1995.[CrossRef][ISI][Medline]
3. Arenzana-Seisdedos F, Thompson J, Rodriguez MS, Bachelerie F, Thomas D, and Hay RT. Inducible nuclear expression of newly synthesized IB
negatively regulates DNA-binding and transcriptional activities of NF-
B. Mol Cell Biol 15: 26892696, 1995.[Abstract]
4. Auphan N, DiDonato JA, Rosette C, Helmberg A, and Karin M. Immunosuppression by glucocorticoids: inhibition of NF-B activity through induction of I kappa B synthesis. Science 270: 286290, 1995.[Abstract]
5. Babyatsky MW, deBeaumont M, Thim L, and Podolsky DK. Oral trefoil peptides protect against ethanol- and indomethacin-induced gastric injury in rats. Gastroenterology 110: 489497, 1996.[CrossRef][ISI][Medline]
6. Brasier AR, Lu M, Hai T, Lu Y, and Boldogh I. NF-B-inducible BCL-3 expression is an autoregulatory loop controlling nuclear p50/NF-
B1 residence. J Biol Chem 276: 3208032093, 2001.
7. Carlson DL, White DJ, Maass DL, Nguyen RC, Giroir B, and Horton JW. IB overexpression in cardiomyocytes prevents NF-
B translocation and provides cardioprotection in trauma. Am J Physiol Heart Circ Physiol 284: H804H814, 2003.
8. Chen YH, Lu Y, De Plaen IG, Wang LY, and Tan XD. Transcription factor NF-B signals antianoikic function of trefoil factor 3 on intestinal epithelial cells. Biochem Biophys Res Commun 274: 576582, 2000.[CrossRef][ISI][Medline]
9. Chen ZF and Behringer RR. Twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev 9: 686699, 1995.[Abstract]
10. Chiao PJ, Miyamoto S, and Verma IM. Autoregulation of IB
activity. Proc Natl Acad Sci USA 91: 2832, 1994.
11. Chinery R and Cox HM. Immunoprecipitation and characterization of a binding protein specific for the peptide, intestinal trefoil factor. Peptides 16: 749755, 1995.[CrossRef][ISI][Medline]
12. Conte E, Bonaiuto C, Nesci C, Crimi N, Vancheri C, and Messina A. Nuclear factor-B activation in human monocytes stimulated with lipopolysaccharide is inhibited by fibroblast conditioned medium and exogenous PGE2. FEBS Lett 400: 315318, 1997.[CrossRef][ISI][Medline]
13. DAcquisto F, Sautebin L, Iuvone T, Di Rosa M, and Carnuccio R. Prostaglandins prevent inducible nitric oxide synthase protein expression by inhibiting nuclear factor-B activation in J774 macrophages. FEBS Lett 440: 7680, 1998.[CrossRef][ISI][Medline]
14. De Plaen IG, Tan XD, Chang H, Qu XW, Liu QP, and Hsueh W. Intestinal NF-B is activated, mainly as p50 homodimers, by platelet-activating factor. Biochim Biophys Acta 1392: 185192, 1998.[ISI][Medline]
15. De Plaen IG, Tan XD, Chang H, Wang L, Remick DG, and Hsueh W. Lipopolysaccharide activates nuclear factor B in rat intestine: role of endogenous platelet-activating factor and tumour necrosis factor. Br J Pharmacol 129: 307314, 2000.[CrossRef][ISI][Medline]
16. Dignass A, Lynch-Devaney K, Kindon H, Thim L, and Podolsky DK. Trefoil peptides promote epithelial migration through a transforming growth factor -independent pathway. J Clin Invest 94: 376383, 1994.[ISI][Medline]
17. Hoffmann W, Jagla W, and Wiede A. Molecular medicine of TFF-peptides: from gut to brain. Histol Histopathol 16: 319334, 2001.[ISI][Medline]
18. Howe LR, Watanabe O, Leonard J, and Brown AM. Twist is up-regulated in response to Wnt1 and inhibits mouse mammary cell differentiation. Cancer Res 63: 19061913, 2003.
19. Hu X. Proteolytic signaling by TNF: caspase activation and IkappaB degradation. Cytokine 21: 286294, 2003.[CrossRef][ISI][Medline]
20. Hu X, Bryington M, Fisher AB, Liang X, Zhang X, Cui D, Datta I, and Zuckerman KS. Ubiquitin/proteasome-dependent degradation of D-type cyclins is linked to tumor necrosis factor-induced cell cycle arrest. J Biol Chem 277: 1652816537, 2002.
21. Imbert V, Rupec RA, Livolsi A, Pahl HL, Traenckner EB, Mueller-Dieckmann C, Farahifar D, Rossi B, Auberger P, Baeuerle PA, and Peyron JF. Tyrosine phosphorylation of IB-
activates NF-
B without proteolytic degradation of I
B-
. Cell 86: 787798, 1996.[CrossRef][ISI][Medline]
22. Jobin C, Panja A, Hellerbrand C, Iimuro Y, Didonato J, Brenner DA, and Sartor RB. Inhibition of proinflammatory molecule production by adenovirus-mediated expression of a nuclear factor kappaB super-repressor in human intestinal epithelial cells. J Immunol 160: 410418, 1998.
23. Karin M and Lin A. NF-B at the crossroads of life and death. Nat Immun 3: 221227, 2002.[CrossRef][ISI]
24. Kindon H, Pothoulakis C, Thim L, Lynch-Devaney K, and Podolsky DK. Trefoil peptide protection of intestinal epithelial barrier function: cooperative interaction with mucin glycoprotein. Gastroenterology 109: 516523, 1995.[CrossRef][ISI][Medline]
25. Laherty CD, Hu HM, Opipari AW, Wang F, and Dixit VM. The Epstein-Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B. J Biol Chem 267: 2415724160, 1992.
26. Laherty CD, Perkins ND, and Dixit VM. Human T cell leukemia virus type I Tax and phorbol 12-myristate 13- acetate induce expression of the A20 zinc finger protein by distinct mechanisms involving nuclear factor kappa B. J Biol Chem 268: 50325039, 1993.
27. Laskowski I, Pratschke J, Wilhelm MJ, Gasser M, and Tilney NL. Molecular and cellular events associated with ischemia/reperfusion injury. Ann Transplant 5: 2935, 2000.[Medline]
28. Lee EG, Boone DL, Chai S, Libby SL, Chien M, Lodolce JP, and Ma A. Failure to regulate TNF-induced NF-B and cell death responses in A20-deficient mice. Science 289: 23502354, 2000.
29. Lowenstein CJ, Alley EW, Raval P, Snowman AM, Snyder SH, Russell SW, and Murphy WJ. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc Natl Acad Sci USA 90: 97309734, 1993.
30. Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, Sartorelli V, Kedes L, Doglioni C, Beach DH, and Hannon GJ. Twist is a potential oncogene that inhibits apoptosis. Genes Dev 13: 22072217, 1999.
31. Matsuoka Y, Pascall JC, and Brown KD. Quantitative analysis reveals differential expression of mucin (MUC2) and intestinal trefoil factor mRNAs along the longitudinal axis of rat intestine. Biochim Biophys Acta 1489: 336344, 1999.[ISI][Medline]
32. Meng L, Mohan R, Kwok BH, Elofsson M, Sin N, and Crews CM. Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo anti-inflammatory activity. Proc Natl Acad Sci USA 96: 1040310408, 1999.
33. Moro F, Levenez F, Durual S, Plaisancie P, Thim L, Giraud AS, and Cuber J. Secretion of the trefoil factor TFF3 from the isolated vascularly perfused rat colon. Regul Pept 101: 3541, 2001.[CrossRef][ISI][Medline]
34. Olson TS and Ley K. Chemokines and chemokine receptors in leukocyte trafficking. Am J Physiol Regul Integr Comp Physiol 283: R7R28, 2002.
35. Podolsky DK. Mechanisms of regulatory peptide action in the gastrointestinal tract: trefoil peptides. J Gastroenterol 35, Suppl 12: 6974, 2000.[ISI][Medline]
36. Sands BE, Ogata H, Lynch-Devaney K, deBeaumont M, Ezzell RM, and Podolsky DK. Molecular cloning of the rat intestinal trefoil factor gene. Characterization of an intestinal goblet cell-associated promoter. J Biol Chem 270: 93539361, 1995.
37. Sands BE and Podolsky DK. The trefoil peptide family. Annu Rev Physiol 58: 253273, 1996.[CrossRef][ISI][Medline]
38. Scheinman RI, Cogswell PC, Lofquist AK, and Baldwin AS Jr. Role of transcriptional activation of IB
in mediation of immunosuppression by glucocorticoids. Science 283286, 1995.
39. Schmedtje JF Jr, Ji YS, Liu WL, DuBois RN, and Runge MS. Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. J Biol Chem 272: 601608, 1997.
40. Schwarz NT, Engel B, Eskandari MK, Kalff JC, Grandis JR, and Bauer AJ. Lipopolysaccharide preconditioning and cross-tolerance: the induction of protective mechanisms for rat intestinal ileus. Gastroenterology 123: 586598, 2002.[CrossRef][ISI][Medline]
41. Shinmura K, Xuan YT, Tang XL, Kodani E, Han H, Zhu Y, and Bolli R. Inducible nitric oxide synthase modulates cyclooxygenase-2 activity in the heart of conscious rabbits during the late phase of ischemic preconditioning. Circ Res 90: 602608, 2002.[Medline]
42. Sosic D and Olson EN. A new twist on Twistmodulation of the NF-B pathway. Cell Cycle 2: 7678, 2003.[Medline]
43. Sosic D, Richardson JA, Yu K, Ornitz DM, and Olson EN. Twist regulates cytokine gene expression through a negative feedback loop that represses NF-B activity. Cell 112: 169180, 2003.[CrossRef][ISI][Medline]
44. Sun SC, Ganchi PA, Ballard DW, and Greene WC. NF-kappa B controls expression of inhibitor IB
: evidence for an inducible autoregulatory pathway. Science 259: 19121915, 1993.[ISI][Medline]
45. Tan XD, Chen Y, Liu Q, Gonzalez-Crussi F, and Liu X. Prostanoids mediate the protective effect of trefoil factor 3 in oxidant-induced intestinal epithelial cell injury: role of cyclooxygenase-2. J Cell Sci 113: 21492155, 2000.
46. Tan XD, Hsueh W, Chang H, Wei KR, and Gonzalez-Crussi F. Characterization of a putative receptor for intestinal trefoil factor in rat small intestine: identification by in situ binding and ligand blotting. Biochem Biophys Res Commun 237: 673677, 1997.[CrossRef][ISI][Medline]
47. Tan XD, Liu QP, Hsueh W, Chen YH, Chang H, and Gonzalez-Crussi F. Intestinal trefoil factor binds to intestinal epithelial cells and induces nitric oxide production: priming and enhancing effects of mucin. Biochem J 338: 745751, 1999.[CrossRef][ISI][Medline]
48. Tan XD, Sun X, Gonzalez-Crussi FX, Gonzalez-Crussi F, and Hsueh W. PAF and TNF increase the precursor of NF-B p50 mRNA in mouse intestine: quantitative analysis by competitive PCR. Biochim Biophys Acta 1215: 157162, 1994.[ISI][Medline]
49. Taupin D, Wu DC, Jeon WK, Devaney K, Wang TC, and Podolsky DK. The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor- and MAP kinase-dependent interregulation. J Clin Invest 103: R31R38, 1999.[ISI][Medline]
50. Taupin DR, Kinoshita K, and Podolsky DK. Intestinal trefoil factor confers colonic epithelial resistance to apoptosis. Proc Natl Acad Sci USA 97: 799804, 2000.
51. Thim L. Trefoil peptides: from structure to function. Cell Mol Life Sci 53: 888903, 1997.[CrossRef][ISI][Medline]
52. Wolf D, Schumann J, Koerber K, Kiemer AK, Vollmar AM, Sass G, Papadopoulos T, Bang R, Klein SD, Brune B, and Tiegs G. Low-molecular-weight hyaluronic acid induces nuclear factor-B-dependent resistance against tumor necrosis factor-
-mediated liver injury in mice. Hepatology 34: 535547, 2001.[CrossRef][ISI][Medline]
53. Wright NA, Hoffmann W, Otto WR, Rio MC, and Thim L. Rolling in the clover: trefoil factor family (TFF)-domain peptides, cell migration and cancer. FEBS Lett 408: 121123, 1997.[CrossRef][ISI][Medline]
54. Xuan YT, Tang XL, Banerjee S, Takano H, Li RC, Han H, Qiu Y, Li JJ, and Bolli R. Nuclear factor-B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res 84: 10951109, 1999.
55. Yamamoto Y and Gaynor RB. Role of the NF-B pathway in the pathogenesis of human disease states. Curr Mol Med 1: 287296, 2001.[CrossRef][Medline]
56. Zhao TC, Taher MM, Valerie KC, and Kukreja RC. p38 Triggers late preconditioning elicited by anisomycin in heart: involvement of NF-B and iNOS. Circ Res 89: 915922, 2001.[Medline]
57. Zhu YQ, Lu Y, and Tan XD. Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C. Am J Physiol Cell Physiol 285: C1294C1303, 2003.