| INTRODUCTIONNF - κB is a transcription factor involved in important biological processes in vivo . The physiological roles of NF - κB are both cell type - and stimulus -dependent. Previous investigations have shown that activation of NF - κB by extracellular stimuli is predominantly mediated by a distinctive signal pathway linked to degradation of IκBα. Activated NK - κB complex is translocated into the nucleus and binds to the promoter regions of its targeted genes.In recent times, a growing body of evidence suggests the existence of auto-regulatory loops for a net negative regulation of NK - κB functions in mammalian cells. For example, activation of NK - κB results not only in up - regulation of genes involving inflammation and cell survival but also in synthesizing/re - synthesizing NK - κB - dependent, negative regulators of NK - κB signaling. The negative regulatory loop of NK - κB ensures transient generation of intracellular signaling to prevent uncontrolled - NK - κB activation. It is critical to limit inflammatory injury by terminating and blocking proinflammatory cytokine — induced NK - κB activity in vivo.Twist protein is a basic helix - loop - helix transcription factor. Previously, Sosic et al. have demonstrated that (1) Twist protein interacts with transcription factor NK - κB;(2) Twist is essential for the down - regulation of NK - κB activity in vivo;and (3) NK - κB regulates Twist gene expression. Thus, Twistprotein is a NK - kB associated protein. It plays an important role in the regulation of the negative regulatory loop of NK - kB pathway and the attenuation of inflammatory responses. Recently, Twist has been shown to be present in mouse mammary epithelial cell lines, 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. These peptides contain distinctive cystine - rich " trefoil" domains and are resistant to proteolytic degradation. Three mammalian TFFs have been identified, namely, pS2 ( TFF1) , SP (TFF2) , and intestinal trefoil factor (ITF or TFF3 ). In contrast to TGF - alpha and EGF, TFFs are expressed rapidly in response to injury. It has been shown that intestinal epithelium is the targeted tissue of TFFs.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. Secretion of TFF3 is evoked by certain neurotransmitters and inflammatory mediators. Previous studies 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. However, it is still unknown whether TFF3 modulates inflammatory responses in intestinal epithelial cells. In the present study, we examined (1) how TFF3 activates NK -kB 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 NK - kB - targeted proinflam-matory cytokines such as IL - 8 in intestinal epithelial cells.MATERIALS AND METHODSReagentsAll cell culture media were obtained from GIBCO Invitrogen. Polyclonalantibodies against Twist , IkBo: , NF - kB p50, and p65 were purchased from Santa Cruz Biotechnology . Anti -phosphotyrosine mAb (clone 4G10) was purchased from Upstate Biotechnology . HRP - conjugated goat anti — rabbit IgG and anti - mouse IgG were obtained from Jackson Immuno Research Laboratories, Inc. mAb against GAPDH was purchased from Abeam, Inc . Chemicals and molecular biology reagents were purchased from Sigma Chemical Company. Reagents for electrophoresis and Western blotting were supplied by Bio - Rad Laboratories . Precasted 4 - 20% iGels were purchased from Gradipore Ltd . Quanti Kine R Human IL - 8 Immunoassay Kit was purchased from R and D Systems . Protease inhibitor cocktail tablets were supplied by Roche Molecular Bio-chemicals . All tissue culture plasticwares were supplied by Costar . U126 and PD98059 (selective inhibitors for ERK kinase) and MG132 ( a potent protea-some inhibitor) were obtained from Biomol . ECL System and [*y - 2P] -ATP were supplied by Amersham Pharmacia Biotech .Preparation of Rat Recombinant TFF3 in YeastRat TFF3 was expressed from Pichia pastoris yeast using a method modified from our previous protocol. The recombinant protein was purified with a FPLC (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 (Zhu et al. , unpublished data).Cell CultureHT - 29 cells ( human intestinal epithelial cell line derived from colonic ad-enocarcinoma) and IEC - 18 cells ( a nontransformed rat intestinal epithelial cell line) were purchased from American Type Culture Collection (Rockville, MD, USA) and cultured under condition required by ATCC.Western blottingExtracted protein were resolved on SDS - PAGE gel along with molecularweight standards, then transferred onto a nitrocellulose membrane. The membranes containing sample proteins were used for immunodetection of Twist protein. Briefly, blots preincubated with primary antibody against Twist protein (1: 500) , the blot was washed with PBS -T, and then incubated with PBS -T containing 1:10000 diluted HRP - conjugated goat anti - rabbit IgG . Immune complexes on the blot were visualized using the ECL system. Blots were stripped and reprobed with mAb against GAPDH (1:10000) following a standard procedure.ImmunoprecipitationLysate proteins was treated with rabbit anti - IxBa antibodies at 4X. overnight , and the immune complexes were precipitated with protein A/G - Sepha-rose beads. The beads were thoroughly washed, resuspended in SDS sample buffer, and boiled. Then, the proteins were resolved on 4 -20% SDS r PAGE gel, electrotransferred to a nitrocellulose membrane, and probed with mouse anti - phosphotyrosine monoclonal antibody ( 1:1000 ). The blot was then treated with HRP - conjugated goat anti - mouse IgG and finally detected by ECL reagent.Preparation of Nuclear Extracts from CellsCells were lysized with 500 jxl of buffer A, scraped cell off Petri dishes, and incubated at 4^. The nuclei were collected by a brief centrifugation. The supernatant was removed. The nuclear pellet was washed with buffer A, resuspended in 150 |xl of buffer B and incubated for 15 minutes on ice. Following centrifugation at 14,000 x g for 10 minutes at 4X., the supernatant ( nuclear protein fraction) was diluted in 300 ul buffer C and stored at -80t. Protein concentration was measured by the Bradford's method.Electrophoretic Mobility Shift Assay (EMSA) and Supershift AssayThe NF - kB consensus oligonucleotide was labeled by [ -y - 32P] ATP with T4 polynucleotide kinase. Nuclear extracts, 32P - labeled oligonucleotide probe were added to gel shift binding buffer, and the mixture was incubated at RT for20 min. Electrophoresis was done, dried, and analyzed using a phosphorimaging system. Supershift experiments wereN performed as follows. Nuclear extracts were incubated with 32P - labeled oligonucleotide . 1 /xg of antibody against p50, p65, p52, c - Rel, or RelB subunit of NF - kB was subsequently added, samples were incubated and electrophoresis was performed. To confirm the specificity of NF - kB DNA binding activity, a competitive experiment was done by adding unlabelled oligonucleotide to the reaction mixture.ELISA assay for quantization IL -8 in conditioned mediumThe cells were seeded, and were cultured to reach confluent monolayer . After every groups cells containing different stimulators were incubated in the serum deprived medium for indicated times. Medium was collected and IL - 8 was detected using Quantikine human IL - 8 immunoassay kit (R&D). 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.Immiinohistochemical stainingRat intestinal tissues were sectioned with a cryostat system, fixed, and air -dried. Slides were hydrated and incubated with 1% BSA. Next, slides were incubated with rabbit pAb against Twist, washed, and incubated with biotinylat-ed goat anti -rabbit IgG. After washing, slides were incubated with FITC -labeled streptavidin, washed, and covered with FTuorSave reagent. Finally, slides were visualized under a fluorescence microscope.siRNA - mediated Twist gene silencingThe siRNA sequences of double stranded siRNA targeting the human Twist mRNA (Gene Bank Accession No: NM000474) are: TGG GAT CAA ACT GGC CTG CAA and TAA GAA CAC CTT TAG AAA TAA. These sequences were submitted to BLAST search to ensure that only the Twist gene was targeted by the Twist siRNA, and control sequences ( Non - silencing labeled Control siRNA) were not homologous to any known genes. HT29 cells were transfectedusing RNAi Human/Mouse Control Kit (Qiagen, Valencia, CA). For transfec-tion, siRNA (1. 9mg/ ml) was transfected with the RNAiFect reagent ( Qiagen, Valencia, CA). Cells were used for designed experiments on the fourth day after transfection.StatisticsData were expressed as means + s. e. m. Analysis of variance and one way analysis of variance (ANOVA) followed by Fisher's protected least significant difference post hoc test were performaced to assess the significant of differences;P < 0.05 was considered significant.RESULTS1. TFF3 activates NK - kB in intestinal epithelial cells in a manner different from TNFHere, we examined the kinetics of TFF3 - induced NK - kB activation, after HT -29 cells were treated with TFF3. EMSA assay showed a low basal level of NK - kB activity in resting intestine cells.To compare the effect of TFF3 and TNF on the degradation of kBa, HT -29 cells were also treated cells with TNF or TFF3 followed by detection of IicBa protein with Western blot. The data suggested that TFF3 induces partial degradation of Ser - 32 and Ser - 36 of IicBa but without induction of its tyrosine phosphorylation in intestinal epithelial cells. The effect is associated with transient activation of p65/p65 homodimer of NK - kB. In contrast to TFF3, TNF induced more degradation of IkBo: and prolonged activation of p50/p65 hert-erodimer of NK - kB in intestinal epithelial cells.2. TFF3 does not induce EL -8 production in intestinal epithelial cellsPersistent activation of NK - kB in intestinal epithelial cells results in up -regulation of several NK - kB targeted proinflammatory cytokines such as IL - 8. As we have shown that TFF3 induces transient activation of NK - kB in intesti-nal epithelial cells, however, whether transient activation of NK - kB induces inflammatory mediators is not clear. Thereafter, we measured IL - 8 in culture cell medium treated by TFF3 or TNF with ELISA assay. As shown, TNF strongly induced IL - 8 release in HT - 29 cells, whereas TFF - 3 showed no effect. The data indicated that transient activation of NK - kB by TFF3 did not result in IL — 8 production in intestinal epithelial cells.3. Twist is constitutively expressed in intestinal epithelial cells.To determine whether intestinal epithelium expresses Twist, Western blot a-nalysis was performed. Data indicated that Twist protein was present in both HT -29 and IEC -18 cells with skeletal muscle tissue as the positive control. Furthermore , the localization of Twist protein in rat small intestinal tissue was visualized by immunofluorescence microscopy using anti - Twist polyclonal antibody, data showed Twist exists in cytosol of intestinal epithelial cells and some cells in the lamina propria stained with anti - Twist pAb. The data suggests that Twist protein is present in intestinal epithelial cells in vivo.4. Effect of TNF on the expression of Twist protein in intestinal epithelial cells.Recently, Sosic et al demonstrated that Twist plays an important role in the attenuation of inflammation in multiple tissues such as muscles and skin. Our Western blot analysis revealed that in IEC -18 cells and in HT —29 cells suggests that TNF induces the degradation of Twist in intestinal epithelial cells.We found pretreatment of cells with MG132, a potent inhibitor of protesome resulted in blocking the effect of TNF on the expression of Twist protein. This result suggests that TNF - triggered degradation of Twist in intestinal epithelial cells is mediated by proteasome.5. Effect of TFF3 on the expression of Twist protein in intestinal epithelial cells.Previous investigations have demonstrated that TFF3 is up - regulated in intestinal inflammatory conditions such as inflammatory bowel disease. However,the role of TFF3 in inflammation remains unclear. We first investigated whether TFF3 regulates Twist expression. Our Western blot analysis revealed that TFF3 increased the expression of Twist protein in intestinal epithelial cells. Pretreat-ment of cells with U126 resulted in blocking the effect of TFF3 on expression of Twist protein. In addition, we obtained similar results by using PD98059, another potent ERK inhibitor. Together, these data suggest that ERK kinase mediates this TFF3 action.6. Twist plays a role in blocking EL -8 production in TFF3 -stimulated intestinal epithelial cells.We demonstrated treatment with siRNA for twist resulted in attenuation of Twist protein expression in intestinal epithelial cells. To examine whether Twist protein modulates IL - 8 expression in TFF3 - stimulated cells, HT - 29 cells were transfected with Twist siRNA. Conditioned - medium was then collected for IL - 8 assay. As shown, HT - 29 cells constitutively secrete IL - 8 protein. The basal level of IL - 8 in cells with silenced Twist was not changed compared to the control group, suggesting that constitutive IL - 8 expression in HT - 29 cells is not modulated by Twist protein. In contrast, TFF3 markedly induced IL - 8 secretion in HT - 29 cells with silenced Twist, indicating endogenous Twist protein plays a role in balancing IL - 8 expression during transient NK - kB activation in intestinal epithelial cells by TFF3.DISCUSSIONSA growing body of evidence suggests that transiently activated NK - kB in non - inflammatory states and persistently activated NK - kB during inflammation play different pathophysiological roles in vivo. During the initial phase of inflammation , proinflammatory cytokines and mediators induce prolonged NK - kB activation in various inflammatory cells and endothelium. The activated NK - kB further up - regulates the expression of a number of proinflammatory molecules. Thus, persistent NK - kB activation during the early phase of inflammation amplifies inflammatory response in vivo.In contrast, transient activation of NK - kB prior to inflammatory stimulation results in the anti -inflammatory response. For example, several investigators have found that pretreatment of rats with LMW - HA induces hepatoprotec-tion against inflammatory insults via transient activation of NK - kB. In the intestine , preconditioning with lipopolysaccharide results in transient activation of NK - kB and induces protective mechanisms against intestinal dysfunction.In the present study, we demonstrated that TFF3 - induced NK - kB activation is a transient event. The transient activation of NK - kB by TFF3 is followed by induction of Twist protein, a new NK - kB associated negative regulatory molecule for NK - kB pathway. Recent studies have shown that Twist interacts with NK - kB. It represses NK - kB activity and attenuates the inflammatory response via suppression of NK — kB activity. We showed that silencing expression of Twist in TFF3 - treated cell results in the induction of IL - 8, a NK - kB regulated proinflammatory cytokine. Thus, TFF3 - induced transient activating NK - kB is associated with strengthening the negative regulatory loop of NK - kB , which inhibits pro - inflammatory 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 its ability to release proinflammatory cytokines such as IL - 1 and IL - 8. Previously, we and others have shown that inflammatory mediators induce NK - kB activation in the intestine. The activated NK - kB further up - regulates the expression of a number of proinflammatory molecules. Meanwhile, NK - kB is controlled by a negative control loop. However, it is not clear whether the loop is regulated during the inflammation. Here, we reported for the first time that Twist protein, a novel molecule in the loop, is present in intestinal epithelial cells in vivo. TNF induces markedly degradation of Twist protein in intestinal epithelial cells by a proteasome - dependent mechanism. The effect is associated with TNF — induced NK - kB 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 maintaining NK - kB activity. Persistent ac-tivation of NK - kB causes prolonged expression of genes including both induction of and activation by NK - kB.Previous studies have shown that TFF3 is expressed in goblet cells and secreted onto intestinal lumen in normal circumstance. It targets intestinal epithelial cells. In contrast to TNF, TFF3 induces up - regulation 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 down - regulation 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 NK — kB in vivo. 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, since TNF induces degradation of Twist in intestinal epithelial cells, which is associated with prolonged NK - kB activation, to prevent cytokine - induced down - regulation of Twist protein may be a novel strategy for blocking inflammation in the GI tract.Previously, we and others have demonstrated that in response to TFF3 stimulation, intestinal epithelial cells release nitric oxide and prostaglandins. TFF3 activates several intracellular molecules such as NK - kB and ERK in intestinal epithelial cells. In the present study, we found that TFF3 enhances Twist protein level in intestinal epithelial cells. We further showed that the selective inhibitor of ERK kinase attenuates the effect of TFF3 on up - regulation 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, IkBo: , Twist, and NK - kB 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.ConclusionWe found that TFF3 activates intestinal epithelial NK - kB in a mechanism distinctive from TNF. We demonstrated for the first time that intestinal epithelial cells constitutively express Twist protein, a novel negative regulator for NK - kB pathway. We showed that TNF, which induces prolonged NK - kB activation, induces degradation of Twist protein in intestinal epithelial cells. The TNF effect is mediated by the proteasome activity. In contrast, TFF3, which activates NK - kB in a transient event, up — regulates 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 NK - kB activated intestinal epithelial cells.Further understanding of these mechanisms will provide new insights into the controlling process involved in activation of NK - kB in inflammation, and may lead to develop a new pharmaceutical strategy to block GI inflammation.
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