Regulated expression of the human gastrin gene in mice

Abstract

Gastrin is secreted from neuroendocrine cells residing in the adult antrum called G cells, but constitutively low levels are also expressed in the duodenum and fetal pancreas. Gastrin normally regulates gastric acid secretion by stimulating the proliferation of enterochromaffin-like cells and the release of histamine. Gastrin and progastrin forms are expressed in a number of pathological conditions and malignancies. However, the DNA regulatory elements in the human versus the mouse gastrin promoters differ suggesting differences in their transcriptional control. Thus, we describe here the expression of the human gastrin gene using a bacterial artificial chromosome (BAC) in the antral and duodenal cells of gastrin null mice. All 5 founder lines expressed the 253 kb human gastrin BAC. hGasBAC transgenic mice were bred onto a gastrin null background so that the levels of human gastrin peptide could be analyzed by immunohistochemistry and radioimmunoassay without detecting endogenous mouse gastrin. We have shown previously that chronically elevated gastrin levels suppress somatostatin. Indeed, infusion of amidated rat gastrin depressed somatostatin levels, stimulated gastric acid secretion and an increase in the numbers of G cells in the antrum and duodenum. In conclusion, human gastrin was expressed in mouse enteroendocrine cells and was regulated by somatostatin. This mouse model provides a unique opportunity to study regulation of the human gastrin promoter in vivo by somatostatin and possibly other extracellular regulators contributing to our understanding of the mechanisms involved in transcriptional control of the human gene.

Figure 1. Characterization of hGasBAC

Panel A. The genomic segment encompassing the human gastrin gene locus is represented as a horizontal line. Exons are represented as filled boxes and introns are unshaded. The various restriction sites spanning the 5′ and 3′ regions and the size of the genomic human gastrin gene hGas is indicated. Panel B. Long-range map showing the various sequence-tagged-site (STS) and restriction sites for clone characterization of the genomic segment.

Figure 1. Characterization of hGasBAC

Panel A. The genomic segment encompassing the human gastrin gene locus is represented as a horizontal line. Exons are represented as filled boxes and introns are unshaded. The various restriction sites spanning the 5′ and 3′ regions and the size of the genomic human gastrin gene hGas is indicated. Panel B. Long-range map showing the various sequence-tagged-site (STS) and restriction sites for clone characterization of the genomic segment.

Figure 2. Expression of human gastrin in the antrums of 5 founder lines

Immunohistochemical staining for gastrin in the antrum of A) WT (wildtype), B) GAS−/−, and BAC mice from founder lines C) 334 (BAC³³⁴), D) 425 (BAC⁴²⁵), E) 809 (BAC⁸⁰⁹), F) 341 (BAC³⁴¹) and G) 807 (BAC⁸⁰⁷).

Figure 3. Detection of the human gastrin in transgenic mice by immunoblot analysis

Antral tissue collected from 3 hGas Bac mice was analyzed by immunoblot analysis using an antibody that recognizes unprocessed and processed gastrin (Santa Cruz) at 1:1000 dilution. Shown is the precursor progastrin (ProGAS) form detected at 14 kDa.

Figure 4. Detection of human gastrin in antral tissue by RIA

A. Antral tissue levels of gastrin measured by RIA in hGasBAC founder lines 334 and 807 and compared to wild-type age-matched (controls) and gastrin knock-out mice (Gas^−/−) as positive and negative controls, respectively. B. Plasma gastrin concentrations in hGasBAC mouse lines 334 and 807 were compared to wild-type age-matched (controls) and gastrin knock-out mice (Gas^−/−).

Figure 5. Gastric acidity is increased in hGas Bac mice infused with gastrin

Gastric acidity in WT (wild type), gastrin infused WT (WT^GAS), hGasBAC (BAC), infused with rodent gastrin BAC (BAC^GAS) and gastrin-deficient (GAS−/−) mice. *P<0.05 compared to WT, #P<0.05 compared to BAC. The results are expressed as the mean ± SEM for 3 mice per group (unpaired t-test).

Figure 6. Hyperacidity decreases somatostatin levels in hGasBAC mice

A. qRT-PCR levels for somatostatin on WT and hGasBAC mice infused with rat gastrin. *P<0.05 compared to BAC; #P<0.05 compared to WT; n = 3 per group (unpaired t-test). B: Decrease in somatostatin modulates expression of human gastrin in hGasBAC mice. qRT-PCR levels for human gastrin on hGasBAC mice infused with rat gastrin. *P<0.05 compared to BAC; #P<0.05 compared to WT; n = 3 per group. ND: not detected. The PCR primers used in the analysis were specific for human and not mouse gastrin. Therefore no mouse gastrin mRNA was detected serving as a useful control analysis of expression of the human gastrin BAC mRNA.

Figure 6. Hyperacidity decreases somatostatin levels in hGasBAC mice

A. qRT-PCR levels for somatostatin on WT and hGasBAC mice infused with rat gastrin. *P<0.05 compared to BAC; #P<0.05 compared to WT; n = 3 per group (unpaired t-test). B: Decrease in somatostatin modulates expression of human gastrin in hGasBAC mice. qRT-PCR levels for human gastrin on hGasBAC mice infused with rat gastrin. *P<0.05 compared to BAC; #P<0.05 compared to WT; n = 3 per group. ND: not detected. The PCR primers used in the analysis were specific for human and not mouse gastrin. Therefore no mouse gastrin mRNA was detected serving as a useful control analysis of expression of the human gastrin BAC mRNA.

Figure 7. Up-regulation of gastrin cells in the antrum of gastrin infused WT and hGasBAC mice

A. Immunohistochemical staining for gastrin in antrums of A) WT (wild type), WT mice infused with rodent gastrin B) WT (WT^GAS), C) hGas BAC mice (BAC), D) gastrin infused BAC (BAC^GAS) and E) gastrin-deficient (Gas−/−) mice. B. Quantification of the number of gastrin positive cells per gland was determined by morphometry. *P<0.05 compared to WT. The results are expressed as the mean ± SEM for 3 mice per group (unpaired t-test).

Figure 8. Up-regulation of gastrin cells in the duodenum of gastrin infused hGasBAC mice

A. Immunohistochemical staining for gastrin in duodenums of A) WT (wild type), gastrin infused B) WT (WT^GAS), C) BAC, D) gastrin infused hGasBAC (BAC^GAS) and E) gastrin-deficient (Gas^−/−) mice. Duodenal G cells indicated (arrow). B. Quantification of the number of gastrin positive cells per villus was determined by morphometry. *P<0.05 compared to WT. The results are expressed as the mean ± SEM for 3 mice per group (unpaired t-test).