Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway.

The trefoil peptides, a recently recognized family of protease-resistant peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as growth factors, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil factor (RITF and HITF) to subconfluent nontransformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or nontransformed fibroblasts (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6-fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a 15-fold enhancement in restitution. No synergistic effect was observed by the addition of nonmucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGF beta bioactivity, trefoil peptide, and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGF-beta and was not affected by the presence of immunoneutralizing anti-TGF beta antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.

Identification and characterization of rat intestinal trefoil factor: tissue- and cell-specific member of the trefoil protein family.

The trefoil peptide family encompasses a group of small proteins that appear to assume a distinctive secondary structure that leads to intrinsic resistance to protease digestion. Induction of these peptides has been associated with response to injury in the gastrointestinal tract and related organs. Using an oligonucleotide derived from N-terminal amino acid sequencing of a transformed growth-inhibiting protein, a cDNA was cloned from rat intestinal villus epithelial cells that encodes a protein 81 amino acids in length with the characteristic trefoil peptide cysteine residue motif. Northern blot analysis demonstrates specific expression of a single transcript of 0.43 kilobase in small and large intestinal epithelium in rat and man. Indirect immunofluorescent staining with antiserum raised using a synthetic peptide based on the predicted C-terminal sequence of this protein, designated intestinal trefoil factor, demonstrated that it is primarily expressed and secreted onto the intestinal surface by goblet cells, suggesting that it may be an important component of intrinsic mechanisms for defending mucosal integrity.

Hepatocyte growth factor/scatter factor modulates intestinal epithelial cell proliferation and migration.

Various peptide growth factors have been found to regulate epithelial cell function within the mucosal epithelium of the gastrointestinal tract. In this study hepatocyte growth factor/scatter factor (HGF/SF) was found to stimulate intestinal epithelial cell proliferation: 2.5-fold in the non-transformed rat small intestinal epithelial cell line IEC-6 and 1.9-fold in the human colon cancer-derived HT-29 cell line. In addition, HGF/SF enhanced epithelial cell restitution, the initial step involved in gastrointestinal wound healing, in an in vitro model. Migration of IEC-6 in wounded monolayers was enhanced up to 7-fold. Enhancement of restitution by HGF could be completely abrogated by addition of immunoneutralizing anti-TGF beta 1, indicating that this process is mediated through a TGF beta-dependent pathway. These findings suggest that HGF exerts functional effects on intestinal epithelial cell populations and may play a role in the morphogenesis of the gastrointestinal tract and its remodeling following injury.

Rapid mitogen-activated protein kinase activation by transforming growth factor alpha in wounded rat intestinal epithelial cells.

BACKGROUND & AIMS: To define signaling events initiating healing after intestinal epithelial injury, activation of mitogen-activated protein kinase (MAPK) pathways was assessed after wounding using an in vitro model. METHODS: Proteins isolated from wounded monolayers of nontransformed intestinal epithelial cells (IEC-6) were analyzed for tyrosine phosphorylation and MAPK expression by Western blot. Extracellular signal-regulated kinase (ERK) 1, ERK2, and Raf-1 activities were assessed by immune complex kinase assays. RESULTS: Tyrosine phosphorylation of several proteins including ERK1 was substantially increased 5 minutes after injury. Another MAPK, c-Jun-N-terminal protein kinase (JNK), was also activated after wounding. Conditioned medium from wounded but not intact IEC-6 monolayers resulted in increased activity of ERK1, ERK2, and Raf-1 kinase. Wound-conditioned medium stimulated proliferation of subconfluent IEC-6 cells compared with conditioned medium from intact IEC-6 cultures and contained higher amounts of transforming growth factor (TGF)-alpha than supernatants of confluent IEC-6 cultures. Activation of ERK1 and ERK2 was partially inhibited by neutralizing anti-TGF-alpha. CONCLUSIONS: Wounding of intestinal epithelial cells results in activation of Raf-1, ERK1, ERK2, and JNK1 MAPKs and subsequent cell proliferation in vitro. Activation of ERK1 and ERK2 is mediated in part by TGF-alpha.

Trefoil peptide protection of intestinal epithelial barrier function: cooperative interaction with mucin glycoprotein.

BACKGROUND & AIMS: Goblet cells secrete a combination of trefoil peptides and mucin glycoproteins to form a continuous gel on the mucosal surface. The functional effects of these products remain uncertain. METHODS: Trefoil peptides and/or mucin glycoproteins were added to Transwell monolayers of the human colonic cancer-derived T84 cell line. Intact monolayers permitted penetration of < 4% of the inert marker [3H]mannitol at 4 hours. Exposure to the toxic lectin phytohemagglutinin (1 mg/mL), oleic acid (8 mmol/L) and taurocholic acid (12 mmol/L), or Clostridium difficile toxin A (0.7 microgram/mL) resulted in loss of barrier function with 36%, 62%, and 45% of [3H]mannitol penetration, respectively. RESULTS: Addition of recombinant human intestinal trefoil factor in physiological concentrations (1-5 micrograms/microL) resulted in attenuation of the damage to monolayer integrity by up to 52%. Protection was enhanced (up to 95%) by the copresence of human colonic mucin glycoproteins. Similar effects were observed when rat intestinal trefoil factor or human spasmolysin, another human trefoil peptide, were added alone or in the presence of human mucin glycoproteins. Conversely, mucin glycoproteins isolated from the rat colon or stomach facilitated protection when added with human spasmolysin or human intestinal trefoil factor. CONCLUSIONS: Trefoil peptides and mucin glycoproteins protect gastrointestinal mucosa from a variety of insults.

Characterization of human and rat intestinal trefoil factor produced in yeast.

Intestinal trefoil factor (ITF) from human (hITF) and rat (rITF) have been produced in Saccharomyces cerevisiae. The DNA encoding the two peptides were cloned by polymerase chain reactions (PCR) from a human normal colon library and a rat small intestinal epithelial cell library. Recombinant plasmids were constructed to encode a fusion protein consisting of a hybrid leader sequence and the rat and human ITF sequences, respectively. The leader sequence used serves to direct the fusion protein into the secretory (and processing) pathway of the cell. The secreted recombinant hITF was found in a monomer and a dimer form, whereas the rITF was only secreted as a dimer. The secreted peptides were purified by a combination of ionic exchange chromatography and preparative HPLC. From 8 L of yeast fermentation broth, 256 mg of hITF (monomer) and 133 mg of hITF (dimer) were isolated, and from 8.7 L of fermentation broth, 236 mg of rITF (dimer) was isolated. The structure of hITF (monomer), hITF (dimer), and rITF (dimer) was determined by amino acid analyses, peptide mapping, sequence analyses, and electrospray mass spectrometry analyses. In hITF (monomer) six of the seven cysteines are disulfide-linked to form 3 disulfide bridges. Mass analysis indicated that the last cysteine residue (Cys-57) did not exist as free (-SH) cysteine, but have reacted with cysteine to form an S-S linked cystine. Sequence and mass spectrometry analyses as well as peptide mapping showed that the dimer form of both hITF and rITF is mediated by a disulfide bridge between Cys-57 residues of two monomers.

Molecular cloning of the rat intestinal trefoil factor gene. Characterization of an intestinal goblet cell-associated promoter.

Intestinal trefoil factor (ITF) is a small peptide bearing the unique motif of intrachain disulfide bonds characteristic of the trefoil family. Previous work had localized expression of ITF primarily within goblet cells in the small and large bowel, making it a candidate gene for the study of the molecular basis of intestinal and goblet cell-specific gene expression. In order to study the regulation of ITF expression, we have cloned the rat ITF gene and sequenced 1.7 kilobases of the 5'-flanking region. RNase protection analysis demonstrated a single transcriptional start site. Various lengths of the 5'-flanking region were linked to the reporter gene luciferase and transfected into the colon cancer cell lines LS174T and Caco-2, representing, respectively, cells with and without goblet cell-like phenotype. Expression in the goblet cell-like LS174T colon cancer cell line was nearly 10-fold greater than expression in Caco-2 cells which exhibit columnar enterocyte-like phenotype. The pattern of goblet cell-associated selective transcription required only 153 base pairs of the rat ITF 5'-flanking sequence. Transfection of a construct of human growth hormone under the control of the rat ITF promoter in the N2 subclone of HT-29 cells demonstrated expression of the reporter gene only in those cells exhibiting a goblet cell phenotype as assessed by expression of immunoreactive mucin. These initial studies of the 5'-flanking region of the ITF gene demonstrate the presence of cis-regulatory elements capable of directing goblet cell specific expression.

Identification of human intestinal trefoil factor. Goblet cell-specific expression of a peptide targeted for apical secretion.

Trefoil peptides are a recently recognized group of small peptides abundantly produced at mucosal surfaces that offer the opportunity to define mechanisms of mucosal cell-specific differentiation and to illuminate new mechanisms for the preservation of mucosal integrity. We report the cDNA cloning of a 75-amino acid human trefoil factor expressed in small and large intestinal mucosas that is highly homologous to the intestinal trefoil factor, with 70% identity at the amino acid level of the predicted mature protein. This human intestinal trefoil factor is also homologous, although to a lesser extent, to trefoil peptides expressed at other sites in the gastrointestinal tract in man, exhibiting absolute conservation of the P domain motif (CX9CX9CX4CCX9WCF) that defines this family of peptides. These findings indicate a high degree of evolutionary conservation of organ/region-specific members of this peptide family. In situ hybridization of intestinal trefoil factor demonstrates a high degree of expression in mature small intestine villus and colonic epithelial goblet cells. Immunogold staining demonstrates high concentrations of intestinal trefoil factor in the rough endoplasmic reticulum and theca of goblet cells as well as throughout the mucosal surface, consistent with vectorial secretion of this factor by goblet cells onto the intestinal luminal surface. In addition, intestinal trefoil factor was also localized within columnar epithelial cells by immunogold labeling despite the absence of mRNA. These observations suggest that peptide secreted by goblet cells might be taken up from the luminal surface and transcytosed by enterocytes. Human intestinal trefoil factor expression was also detected in the HT-29N2 and HT-29H2 subclones in conjunction with the emergence of the goblet cell phenotype, but not in the CaCO2 cell line that exhibits enterocytic phenotype. In summary, these findings confirm the existence of a highly conserved family of peptides that are abundantly expressed in distinctive regions throughout the gastrointestinal tract in a highly cell-specific pattern reflecting a goblet cell differentiation pathway. They form one of the more abundant constituents of the interface between the mucosa and "outside" environment and may provide a new paradigm of regulation of the integrity of epithelial surfaces as well as a previously unrecognized dimension of goblet cell function.