The initial finding of this study was reduced SP and pS2 expression in ITF null mice. This suggested the possibility of a regulatory effect on SP and pS2 transcription by ITF, which was confirmed by an increase in steady-state trefoil mRNA in gastrointestinal cell lines treated with hSP or hITF and increased SP and pS2 promoter activity in transfected gastric cell lines after stimulation. Signaling studies indicated that transcriptional activation occurs through a Ras/MEK/MAPK pathway, which also involves EGF-R phosphorylation and kinase activity.
While we have shown involvement of classic Ras/MAPK signaling pathways in gastric cell lines, whether this effect, or any function of trefoil peptides, occurs through binding to a specific receptor is still unresolved. Saturable porcine SP binding activity within isolated rat intestinal membranes, with 2 affinity states, has been described (46
). Subsequently, an ITF-derived fusion protein and 125
I-labeled rat ITF was shown to bind gut tissue sections in vitro, with strong signals in the gastric neck region and Paneth’s cells (48
). A trefoil peptide–binding protein of approximately 28 kDa has been detected after cross-linking studies (50
). At this time, however, these entities are incompletely characterized, and molecular cloning of these proteins has not been reported. We have found in this study that ITF does not appear to directly bind the EGF-R or colocalize with the EGF-R under nonpermissive conditions but does colocalize with the transferrin receptor to clathrin-coated pits. Trefoil-mediated activation of the EGF-R, therefore, may occur indirectly, possibly by recruitment of adaptor molecules or Ras. The trefoil peptides may also activate after endocytosis. These findings do not necessarily invoke a specific receptor, but suggest an alternative hypothesis where trefoil-mediated events occur following internalization of the peptides.
We have shown in this study that the increase in SP and pS2 mRNA following trefoil peptide stimulation is most likely a transcriptional response. SP
gene promoters were activated 5- to 10-fold in response to ITF. The cis
-acting elements mediating this transcriptional enhancement were not identified in this study but were present within the 820-bp and 1,100-bp SP
promoters, respectively. Previously, an enhancer element responsive to signals initiated by Ras, EGF, and phorbol ester was identified in the human pS2
promoter, and this enhancer activity was allocated to the imperfect palindrome GGTCACGGTGGCC, present 450 bp upstream of the initiator ATG (26
). This sequence is not found in the human SP
promoter described here or in the ITF promoter sequences reported to date; nor do the SP/pS2
promoters contain canonical MAPK response elements such as AP-1, SRE, or CRE motifs. Therefore, identification of the enhancer elements necessary for trefoil interregulation will be a focus of further studies.
The rise in steady-state trefoil mRNA after stimulation was augmented by cycloheximide; this feature, together with the rapidity of stimulation, suggests that the trefoils may act as immediate-early genes. Regulation of gastric trefoil expression by EGF-R ligands, some of which are also immediate-early genes capable of cross-induction (35
), has been proposed (23
); indeed, pS2 transcription is markedly induced by EGF in the breast cancer cell line MCF-7 (26
). In the present study, using cells of gastrointestinal tract origin, EGF was a modest stimulant of trefoil expression (Figure ) at the concentration used. Instead, trefoil peptides themselves appear to act via EGF-R to initiate a signal transduction cascade terminating in trefoil gene induction. The finding that the EGFR-trefoil relationship mediates transcriptional responses to ITF provides insight into otherwise paradoxical observations. In healing gut mucosa, induction of EGF-R has been described (41
), but no single EGF-R ligand has been demonstrated to be critical for gastrointestinal healing. Thus, TGF-α null mice appear to have normal healing after induction of gastric ulcers (36
). In contrast, the colonic erosions induced by oral dextran sodium sulfate, while promptly repaired in wild-type mice, are fulminant in ITF null mice, leading to death of the animal. This defect can be reversed by rescue with topical ITF (11
How does the gastric mucous neck cell, the site of SP
gene expression, “see” surface-expressed pS2, or more problematic, ITF, a product of the intestinal goblet cell? Though the dynamics of gastric mucus flow are essentially unknown, it is possible that pS2 generated and secreted by the surface mucous cells may be swept proximally to the gland neck. Although ITF is expressed and secreted in the base of gastric glands, peptide levels are only approximately 1% those found in the intestine (16
). However, it is possible that this level may be sufficient to sustain SP
induction. Alternatively, substantial ITF is expressed in the duodenum and may bathe the gastric antrum as a component of duodenogastric refluxate. Circulating trefoil peptides may also be responsible for this cross-regulation. SP and pS2 expression by endocrine cells of the gut has been reported (23
), and systemic administration of SP was able to protect rats from gastric damage caused by indomethacin (13
). Consistent with this possibility is the observation of increased ITF expression in uninjured gastric mucosa lying opposite injured and regenerating gastric mucosa (Taupin, D.R., et al., unpublished observations).
A further possibility is that the degree of trefoil expression is programmed in pluripotent cells in the proliferative zone of the gastric gland. In this context, autocrine stimulation of SP expression might be partly dependent on the expression of ITF (or pS2) by that cell, dictating subsequent expression by differentiated progeny. Thus, cells of relatively undifferentiated morphology in regenerating gastric glands are capable of expressing the full trefoil repertoire (21
). Detailed characterization of epithelial stem cells present in different regions of the gastrointestinal tract may provide further insight.
In aggregate, these data provide a paradigm for the rapid self-sustaining induction of trefoil transcription after mucosal injury via EGF-R activation and through the Ras/MEK/MAP kinase signaling pathway, leading to activation of trefoil genes through cis
-acting regulatory regions. A role for trefoil-induced signals in the maintenance of metaplastic gut lineages also appears likely. As MAP kinase activation appears to be necessary for cell migration (37
), effects of trefoil peptides on cell restitution may also occur through this pathway. Nevertheless, the possibility of trefoil peptide internalization suggests that coupling to alternate signaling pathways is also possible.