Overexpression of glycine-extended gastrin in transgenic mice results in increased colonic proliferation

Abstract

Gastrin is a peptide hormone involved in the growth of both normal and malignant gastrointestinal tissue. Recent studies suggest that the glycine-extended biosynthetic intermediates mediate many of these trophic effects, but the in vivo relevance of glycine-extended gastrin (G-Gly) has not been tested. We have generated mice (MTI/G-GLY) that overexpress progastrin truncated at glycine-72 to evaluate the trophic effects of G-Gly in an in vivo model. MTI/G-GLY mice have elevated serum and colonic mucosal levels of G-Gly compared with wild-type mice. MTI/G-GLY mice had a 43% increase in colonic mucosal thickness and a 41% increase in the percentage of goblet cells per crypt. MTI/G-GLY mice exhibited increased colonic proliferation compared with wild-type controls, with an expansion of the proliferative zone into the upper third of the colonic crypts. Continuous infusion of G-Gly into gastrin-deficient mice for two weeks also resulted in elevated G-Gly levels, a 10% increase in colonic mucosal thickness, and an 81% increase in colonic proliferation when compared with gastrin-deficient mice that received saline alone. To our knowledge, these studies demonstrate for the first time that G-Gly's contribute to colonic mucosal proliferation in vivo.

Figure 1

Map of the MTI/G-GLY transgene. The MTI/G-GLY transgene was generated by PCR mutagenesis of the human gastrin cDNA by placing a stop codon after Gly-72. It was then inserted into the BglII site of EV142, a plasmid containing the mouse metallothionein promoter and the human growth hormone poly(A) tail (31). Mice expressing the MTI/G-GLY transgene have elevated levels of G-Gly in the serum and in many organs, including the colon and the lung.

Figure 2

MTI/G-GLY transgenic mice express human G-Gly. (a) Northern blot analysis using a human gastrin cDNA probe reveals that human gastrin is being transcribed in the MTI/G-GLY mouse colon (C), kidney (K), liver (Li), stomach (S), lung (Lu), and pancreas (P). MTI/G-GLY is also transcribed in a transfected cell line (T1). RNA from hGAS kidney (–) is used as a negative control, and liver RNA from hGAS transgenic mice (+) is used as a positive control. (b) GAPDH controls from same blot. (c) RT-PCR reaction was performed using antisense primers (GLY) that are proximal to Gly-72, resulting in a 173-bp PCR product (open arrow), or primers (GAS) that are distal to Gly-72, resulting in a 322-bp PCR product (filled arrow). The colons of MTI/G-GLY mice produce the MTI/G-GLY transgene transcript but not the full-length gastrin transcript. The stomachs of MTI/G-GLY mice produce both transcripts. Liver RNA from hGAS mice (which overexpress human progastrin in the liver) was used as a control.

Figure 3

MTI/G-GLY mice have increased numbers of goblet cells in the colon. Three-month-old mice were sacrificed. On routine histology, the MTI/G-GLY mice (a) appear to have an increased number of goblet cells compared with wild-type mice (b). This result was confirmed by Alcian blue staining, which revealed an increased number of goblet cells in the MTI/G-GLY mice (c) compared with wild-type control (d).

Figure 4

MTI/G-GLY mice have increased colonic proliferation with no change in gastric proliferation. Three-month-old mice were injected with BrdU (50 mg/kg intraperitoneally) one hour before sacrifice. There is increased BrdU labeling in the MTI/G-GLY mice (a), with a migration of the proliferative zone from the base of the crypts, when compared with age-controlled wild-type mice (b). There was no difference in the BrdU labeling index in the stomachs of MTI/G-GLY mice (c) compared with wild-type mice (d).

Figure 5

Infusion of G-Gly into gastrin-deficient mice results in increased colonic proliferation. (a) Infusion of G-Gly results in a significant increase in the BrdU labeling index in gastrin-deficient mice when compared with infusion of saline or amidated G-17. (b) Mice receiving G-Gly have hypertrophied colonic crypts when compared with those receiving saline (c).