Abstract
Gastric units in the glandular epithelium of the mouse stomach contain several types of continuously renewing epithelial cells. Acid-producing parietal cells are derived from a multipotent stem cell that also gives rise to mucus-producing pit cells and pepsinogen- and intrinsic factor-producing zymogenic cells. We used nucleotides -1035 to +24 of the mouse H+/K(+)-ATPase beta subunit gene (H+/K(+)-ATPase beta subunit-1035 to +24) to examine the consequences of expressing simian virus 40 T antigen (SV 40 TAg) in the normally rare, nonproliferating, short-lived pre-parietal cell progenitor. Light and electron microscopic morphologic studies plus multilabel immunohistochemical analyses of postnatal day (P) 14-80-day transgenic mice revealed that SV40 TAg produces a 50-70-fold amplification of pre-parietal cells which become the predominant cell type in gastric units. Differentiation to mature parietal cells is blocked, resulting in hypochlorhydria and an associated systemic iron deficiency. SV40 TAg-induced pre-parietal proliferation is accompanied by apoptosis. Examination of adult transgenic mice homozygous for p53 wild type or p53 null alleles established that the apoptosis occurs through a p53-independent pathway. H+/K(+)-ATPase beta subunit -1035 to +24/SV40 Tag is not expressed during differentiation of the zymogenic lineage. Nonetheless, P28-P80 transgenic mice exhibit an apparent block in the conversion of pre-zymogenic to zymogenic cells. This block appears to be quite specific: conversion of preneck to neck cells and neck to pre-zymogenic cells is not affected. Comparison of normal and transgenic mice that are p53+/+ or p53-/- confirmed that the loss of mature zymogenic cells is not dependent upon p53. Although H+/K(+)-ATPase beta subunit -1035 to +24 is not active in pit cell progenitors or their differentiated descendants, there is a 2-3-fold increase in mature pit cells in transgenic animals. Our findings (i) demonstrate an approach for amplifying and characterizing pre-parietal or other progenitor cell populations in gastric units, (ii) reveal an SV40 TAg-inducible, p53-independent apoptotic mechanism that operates in a committed epithelial progenitor cell, and (iii) provide a transgenic mouse model for defining factors that may mediate progression through specific points in the differentiation programs of the parietal and zymogenic cell lineages or that may influence decisions about allocation to the pit cell lineage.
Highlights
Summary ofHistopathologic Changes in Gastric Units-Fig. 3 shows toluidine blue-stained, semithin sections of gastric units located in the zymogenic zone of a P42 transgenic mouse and its normal FVB/N littermate
A comparison ofpanels A and B reveals the major histopathologic changes encountered in intrinsic factor-positive zymogenic cells [7, 23]
K, normal mouse stomach stained with rabbit anti-intrinsic factor sera
Summary
AnimalsFVB/N mice were purchased from Taconic Farms. C57BIJ6 p53_1_ mice and p53+/+ controls were obtained from GenPharm. p53 genotyping was performed using PCR, tail DNA, plus the following primer pairs: 5'-GTGTTTCATTAGTTCCCCACCCTTGAC-3' and 5'-ATGGGAGGCTGCCAGTCCTAACCC-3' (p53 wild type allele) or 5'-GGGAATTCTGGGACAGCCAAGTCTGT-3' and 5'-TTTACGGAGCCCTGGCGCTCGATGT-3' (p53 null allele).Generation of Transgenic MiceA 2.7-kilobase BamHl fragment, spanning nucleotides 2533-5235 of the SV40 genome and encoding the large T antigen, was isolated from pLITAg [15] and inserted into the BamHl site ofpBS/HKATP, yielding pBS/HKATP!rAg. (pBS/HKATP contains nucleotides -1035 to +24 of the mouse H+/K+ -ATPase {3 subunit gene in pBluescript II SK+ [7].) The 3785-base pair H+/K+ -ATPase- 1035 to +24/SV40 TAg insert in pBS/ HKATP!rAg was released with Eagl and EcoRI, purified by agarose gel electrophoresis followed by glass bead extraction (Geneclean II, Bio 101), and used for pronuclear injection of FVB/N oocytes [16].2 S. FVB/N mice were purchased from Taconic Farms. P53 genotyping was performed using PCR, tail DNA, plus the following primer pairs: 5'-GTGTTTCATTAGTTCCCCACCCTTGAC-3' and 5'-ATGGGAGGCTGCCAGTCCTAACCC-3' (p53 wild type allele) or 5'-GGGAATTCTGGGACAGCCAAGTCTGT-3' and 5'-TTTACGGAGCCCTGGCGCTCGATGT-3' (p53 null allele). A 2.7-kilobase BamHl fragment, spanning nucleotides 2533-5235 of the SV40 genome and encoding the large T antigen, was isolated from pLITAg [15] and inserted into the BamHl site ofpBS/HKATP, yielding pBS/HKATP!rAg. (pBS/HKATP contains nucleotides -1035 to +24 of the mouse H+/K+ -ATPase {3 subunit gene in pBluescript II SK+ [7].) The 3785-base pair H+/K+ -ATPase- 1035 to +24/SV40 TAg insert in pBS/ HKATP!rAg was released with Eagl and EcoRI, purified by agarose gel electrophoresis followed by glass bead extraction (Geneclean II, Bio 101), and used for pronuclear injection of FVB/N oocytes [16].
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