A new twist on trefoils. Focus on "TFF3 modulates NF-{kappa}B and a novel regulatory molecule of NF-{kappa}B in intestinal epithelial cells via a mechanism distinct from TNF-{alpha}"

Kim E. Barrett

Division of Gastroenterology, Department of Medicine and Biomedical Sciences Ph.D. Program, University of California San Diego School of Medicine, San Diego, California

THE INTESTINAL EPITHELIUM, particularly in the colon, faces unique challenges in maintaining cell and tissue homeostasis. Unlike most other epithelia in the body, it is constantly barraged with a soup of microbes and their products. These have the potential to activate inflammation and immune responses. Indeed, the normal physiological state of the intestine appears to be one of controlled inflammation, with a variety of immune and inflammatory effector cells located in the lamina propria immediately adjacent to the epithelium, even in health. This may reflect a state of readiness, where sufficient numbers of phagocytes and other antimicrobial effectors are present to deal quickly with any possible microbial invasion before it becomes systemic. However, in susceptible individuals, the regulatory controls on this "physiological" inflammation break down, likely due to either immune defects and/or defects in the barrier properties of the epithelium itself, and chronic, pathological inflammation ensues (15). This chronic inflammation, which is driven by immune responses to the enteric flora, establishes a vicious cycle because tissue injury, particularly to the epithelium, further compromises the ability of the gut to exclude immunostimulatory molecules (3, 11, 12). There is now substantial evidence that this type of cycle is centrally involved in the pathogenesis of important digestive diseases such as ulcerative colitis and Crohn’s disease, which may also predispose sufferers to subsequent development of colon cancer (7). However, the mechanisms that normally allow individuals to restrain immune responses to the enteric flora, and thus limit inflammation, are still poorly understood.

Inflammatory responses in the gut (as in other tissue sites) as well as progression to malignancy, are pivotally controlled by the transcription factor NF-{kappa}B (8). This factor is normally held in an inactive state in the cell cytosol as a complex with its inhibitor, I{kappa}B{alpha}. In response to a variety of extracellular signals, including those delivered by microbial products or inflammatory cytokines, I{kappa}B{alpha} is phosphorylated, and thereby targeted for ubiquitination and subsequent degradation by the proteosome. This frees NF-{kappa}B to translocate to the nucleus, where it binds to the promoter regions and evokes expression of a series of target genes that perpetuate the inflammatory response, including chemokines such as IL-8 that stimulate the influx of inflammatory cell types. In addition, NF-{kappa}B can trigger the production of signals that limit epithelial apoptosis, and may be involved in the predisposition to malignancy that accompanies chronic intestinal inflammation (6). Thus NF-{kappa}B can be considered a master regulator that can both initiate and perpetuate intestinal inflammation. On the other hand, the intestine also synthesizes products that tend to limit inflammation and improve the integrity of the epithelial barrier. One group of such products is the trefoil factor family (TFF), members of which are synthesized by goblet cells throughout the small and large intestine, both at baseline and more actively when the gut is injured, such as at the site of an ulceration (1, 16). TFFs are compact extracellular peptides with distinctive domains that utilize disulfide bonds to fold into a trefoil shape. As such, they are highly resistant to proteolysis, a characteristic that is likely critical to allow for their survival in the protease-rich environment of the intestinal lumen. TFFs had previously been considered to act predominantly to stabilize the mucous layer that overlies the intestinal epithelium, and which is also secreted by goblet cells (5). However, more recently, additional roles of TFFs have emerged that suggest direct effects, perhaps mediated by membrane receptors, on both epithelial and other cell types. For example, mice engineered to lack specific TFFs have increased susceptibility to intestinal or gastric injury, and exogenous administration of purified TFFs to animals can accelerate healing in various models where colitis is induced artificially (4, 9, 10, 1618). Furthermore, TFFs were shown to reduce the recruitment of inflammatory cells into the intestine as well as the expression of adhesion molecules that is needed to drive such recruitment (13). There is reason to conclude, therefore, that TFFs are one group of intestinal factors that allow the healthy gut to restrain inflammation and live harmoniously with its abundant flora. At odds with this scenario, however, was the report from an author of the article in focus that one TFF, TFF3, could transiently activate NF-{kappa}B in intestinal epithelial cells and thereby protect them from apoptosis (2). The current article reports studies that appear to resolve this paradox (Ref. 20; see p. C1085 of this issue).

Zhu and Tan studied the possibility that TFF3 may not only activate NF-{kappa}B in intestinal epithelial cells, but also stimulate the activity of signals that would limit the activity of this transcription factor and its downstream, proinflammatory targets. They focused on a possible role for a basic helix-loop-helix transcription factor known as Twist, which is the mammalian homologue of a Drosophila protein that binds to and alters the activity of dorsal, a Drosophila NF-{kappa}B homologue (19). Mice lacking twist or a related protein, dermo-1, displayed elevated levels of proinflammatory cytokines and died perinatally from cachexia (14). Indeed, in their elegant studies, Zhu and Tan now show that TFF3 leads to the persistent expression of Twist in intestinal epithelial cell lines (20). Moreover, they compared the mechanisms and outcomes ofNF-{kappa}B signaling induced by TFF3 with that induced by a classic stimulus of NF-{kappa}B, the proinflammatory cytokine, TNF-{alpha}. The authors show that divergent signaling and consequences of these two stimuli reflect reciprocal effects on Twist-TFF3 increases its availability, whereas TNF-{alpha} reduces it.

The studies described were undertaken in both a human colonic epithelial cell line as well as a rat small intestinal line. The authors first confirmed their prior observations that TFF3 activated NF-{kappa}B, although with more transient kinetics that seen with TNF-{alpha}. NF-{kappa}B actually encompasses a series of dimeric transcription factors made up of various subunits. Interestingly, NF-{kappa}B activated by TFF3 was composed of homodimers of p65 subunits, whereas that activated by TNF-{alpha} consisted of homodimers of p65 complexed with p50. These differences were accompanied by varying effects of TFF3 and TNF-{alpha} on I{kappa}B{alpha} degradation. TFF3 caused only partial degradation of this inhibitor, which was rapidly resynthesized. TNF-{alpha}, on the other hand, caused a more profound degradation of the inhibitor. The signaling differences between the two agonists also had functional significance: only TNF-{alpha}, and not TFF3, induced the synthesis of the chemokine, IL-8, by intestinal epithelial cells. The data imply that if the kinetics of NF-{kappa}B DNA binding in response to a given agonist are sufficiently transient, then the transcription factor will no longer be capable of triggering an inflammatory response. The authors therefore sought to test whether Twist might account for these differences because Twist can bind to NF-{kappa}B, thereby preventing its ability to activate the transcription of proinflammatory genes. In fact, TNF-{alpha} was shown to evoke a rapid and long-lasting reduction in Twist levels, mediated by proteosomal degradation. On the other hand, as previously mentioned, TFF3 markedly and persistently increased Twist expression, apparently via the ability of this factor to stimulate the activation of the ERK isoforms of mitogen-activated protein kinases. Finally, the functional significance of this response was demonstrated in studies where both basal and stimulated expression of Twist was inhibited by a specific siRNA for this molecule. In the cells in which Twist had been downregulated, TFF3 gained the ability to stimulate IL-8 secretion, presumably downstream of its now unopposed activation of NF-{kappa}B.

The studies of Zhu and Tan are significant because of the insights they provide into the close regulation of both pro- and anti-inflammatory signaling in intestinal epithelial cells (Fig. 1). It is clear that there is an exquisite balancing act that allows the healthy intestine to produce "just enough" inflammation to stand ready to counter microbial threats, but not so much that overwhelming inflammation and damage to the intestinal barrier ensues. Thus the reciprocal regulation of NF-{kappa}B by inflammatory cytokines vs. protective molecules such as TFF3 may be vital to intestinal homeostasis and the appropriate recovery from occasional intestinal injury. It would be very interesting to assess whether TFF3 can overcome Twist degradation induced by TNF-{alpha}, and doubtless such studies are in hand. Likewise, it will be fascinating to uncover the extent to which the pathways delineated here may be dysregulated in patients suffering from chronic inflammatory bowel diseases. At a minimum, the studies reported shed new light on the regulation of intestinal inflammation, and are important in that they may ultimately suggest novel therapeutic targets for conditions in which currently available therapies are suboptimal (12).



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Fig. 1. Pathways for reciprocal regulation of inflammatory responses in intestinal epithelial cells, as delineated by the studies of Zhu and Tan (20) reported in this issue. Both TNF-{alpha} and trefoil factor family 3 (TFF3) can activate the transcription factor, NF-{kappa}B, which hypothetically can translocate to the nucleus to activate transcription of proinflammatory gene products. However, these events are also regulated by the differential ability of TNF-{alpha} and TFF3 to modulate Twist, a transcription factor that is capable of binding to NF-{kappa}B and antagonizing its effects. TNF-{alpha} causes proteosomal degradation of Twist, whereas TFF3 induces its expression via activation of the ERK isoforms of mitogen-activated protein kinases. Of interest, the expression of TFF3 itself is induced in response to intestinal injury, suggesting that this molecule may play a critical adaptive role in the response to injury by shutting off the inflammatory cascade and restoring tissue homeostasis.

 

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The author’s work is supported by National Institutes of Health Grants DK-33491 and AT-01180.


    ACKNOWLEDGMENTS
 
The author thanks Glenda Wheeler for assistance with manuscript submission.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. E. Barrett, UCSD Medical Center 8414, 200 W. Arbor Dr. (for courier, use CTF-A108, 210 Dickinson St.), San Diego, CA 92103-8414 (e-mail: kbarrett{at}ucsd.edu)


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