A paradoxical reduction in susceptibility to colonic injury upon targeted transgenic ablation of goblet cells

Abstract

MethodsPreparation of mITF promoter. To obtain a sufficiently large segment of 5′ flanking promoter region to confer goblet cell-specific expression of the transgene, a Hiroshi Itoh and Paul L. Beck contributed equally to this work.Received for publication January 5, 1999, and accepted in revised form October 21, 1999.Goblet cells are the major mucus-producing cells of the intestine and are presumed to play an important role in mucosal protection. However, their functional role has not been directly assessed in vivo.In initial studies, a 5′ flanking sequence of the murine intestinal trefoil factor (ITF) gene was found to confer goblet cell-specific expression of a transgene. To assess the role of goblet cells in the intestine, we generated transgenic mice in which ∼60% of goblet cells were ablated by the expression of an attenuated diphtheria toxin (DT) gene driven by the ITF promoter; other cell lineages were unaffected. We administered 2 exogenous agents, dextran sodium sulfate (DSS) and acetic acid, to assess the susceptibility of mITF/DT-A transgenic mice to colonic injury. After oral administration of DSS, 55% of control mice died, whereas DT transgenic mice retained their body weight and less than 5% died. Similarly, 30% of the wild-type mice died after mucosal administration of acetic acid, compared with 3.2% of the transgenic mice. Despite the reduction in goblet-cell number, the total amount of ITF was increased in the mITF/DT-A transgenic mice, indicating inducible compensatory mechanisms. These results suggest that goblet cells contribute to mucosal protection and repair predominantly through production of trefoil peptides. Construction of transgene and generation of β-galactosidase ITF transgenic mice. The β-galactosidase gene was obtained from pSV-β-galactosidase vector (Promega Corp., Madison, Wisconsin, USA) by digestion with BamHI and HindIII. The resulting 3.7 kb of β-galactosidase gene was inserted into the BamHI and HindIII sites of pBluescript II KS(+). The 6.3 kb of XhoI-digested 5′ end of the mITF promoter was then inserted into XhoI sites of pBluescript II KS(+) containing the β-galactosidase gene. The resulting plasmid was digested with BamHI, yielding a 10-kb DNA fragment consisting of the 6.35-kb mITF promoter followed by the β-galactosidase gene (3.7 kb). This fragment was gel purified, dialyzed, and injected into the mouse oocytes. The resulting offspring were screened both by Southern blotting (using the above β-galactosidase fragment as a probe) and by PCR using the following primers directed at the inserted β-galactosidase gene: sense: 5′-CCTGAGGCCGATACTGTCGTC-3′; antisense: 5′-CCAGATAACTGCCGTCACTCC-3′. PCR was performed according to the manufacturer's guidelines (Promega Corp.) using 0.5 µL of Taq DNA polymerase (Promega Corp.) in a 50-µL reaction containing 1.5 mM MgCl 2 , 0.2 mM dNTP, 0.8 µM of each primer, and approximately 1 µg of extracted tail DNA. PCR conditions included an initial 10 minutes at 95°C followed by 36 cycles each of 95°C for 1 minute, 58°C for 1 minute, 72°C f...

Absence of bacterially induced RELMβ reduces injury in the dextran sodium sulfate model of colitis

“…However, recent studies have questioned this because disruption of goblet cells reduces susceptibility of mice to colonic injury (16). Our results reconcile these seemingly disparate observations by showing that the absence of goblet cell RELMβ dramatically reduces susceptibility to DSS-induced colitis.…”

“…Herein, we show that RELMβ is an activator of the innate immune response and that disruption of this gene protects against dextran sodium sulfate-induced (DSS-induced) colitis in mice. These results provide a potential explanation for the results described by Itoh et al (16) showing that mice with a reduction in colonic goblet cells are protected from DSS-induced colitis.…”

“…Second, as with the induction of RELMβ by bacterial colonization, DSS-induced colitis also develops in SCID mice, eliminating the role of adaptive immunity as a factor in this model (28). Finally, the ablation of goblet cells in the colon reduces mucosal damage in DSS-induced colitis (16).…”

“…As the interface between the bacteria-laden intestinal lumen and the mucosal immune system, the intestinal epithelium has been hypothesized to play a critical role in the development of IBD. Indeed, the ablation of goblet cells in the colon reduces the development of colitis in mice (16), suggesting that this cell lineage may be of particular relevance to the pathogenesis of IBD.…”

Actin reorganization as the molecular basis for the regulation of apoptosis in gastrointestinal epithelial cells

“…This is consistent with the current thinking that goblet cells have a part in the protection rather than the pathogenesis of intestinal disorders. 24 It may be noted that a strong association between abnormal apoptosis, increased goblet cell numbers and GI disorders has been described previously. 25 Similar biochemical, morphological or histological changes were not noted in wild-type, villin or gelsolin single-knockout mice (Supplementary Figure 7A).…”