Rat Pepsinogen Research Articles

Human gastric cancer risk screening: From rat pepsinogen studies to the ABC method.

We examined the development of gastric cancer risk screening, from rat pepsinogen studies in an experimental rat gastric carcinogenesis model induced with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and human pepsinogen studies in the 1970s and 1980s to the recent “ABC method” for human gastric cancer risk screening. First, decreased expression or absence of a major pepsinogen isozyme, PG1, was observed in the rat gastric mucosa from the early stages of gastric carcinogenesis to adenocarcinomas following treatment with MNNG. In the 1980s, decreases in PGI in the human gastric mucosa and serum were identified as markers of atrophic gastritis. In the 1990s, other researchers revealed that chronic infection with Helicobacter pylori (Hp) causes atrophic gastritis and later gastric cancer. In the 2000s, a gastric cancer risk screening method combining assays to detect serum anti-Hp IgG antibody and serum PGI and PGII levels, the “ABC method”, was established. Eradication of Hp and endoscopic follow-up examination after the ABC method are recommended to prevent gastric cancer.

Immunocytochemistry and in situ hybridization studies of pepsinogen C-producing cells in developing rat fundic glands.

The ontogeny of pepsinogen C-producing cells in rat fundic glands was studied by means of light and electron microscopy using an antiserum raised against a synthetic peptide based on rat pepsinogen C. To confirm the immunocytochemistry results, the expression of rat pepsinogen C messenger RNA (mRNA) in the fundic gland was also examined by in situ hybridization using a digoxigenin-labeled RNA probe. In adult rats, pepsinogen C was produced by chief cells, mucous neck cells, and intermediate mucopeptic cells. Pepsinogen C-producing cells appeared in embryos as early as 18.5 days' gestation. The development of these cells could be classified into four stages: (1) 18.5 days' gestation to 0.5 days after birth; (2) 0.5 days to 2 weeks after birth; (3) 3-4 weeks after birth; (4) 4-8 weeks after birth. In embryos and young animals, pepsinogen C-producing cells were mucopeptic cells. By 4 weeks after birth, mucous neck cells could be distinguished morphologically. The maturation stages of the chief cells could be traced by electron microscopy along the longitudinal axis of the rat fundic gland by double-staining with anti-pepsinogen C antibody and periodic acid-thiocarbohydrazide-silver proteinate. Positive reactions for pepsinogen C and pepsinogen C mRNA expression were detected in mucous neck cells. Therefore, we conclude that mucous neck cells are precursor cells of chief cells. Mucous neck cells, intermediate cells, and chief cells are in the same differentiating cell lineage.

Glucocorticoids Inhibit the Proliferation of Mucosal Cells and Enhance the Expression of a Gene for Pepsinogen and Other Markers of Differentiation in the Stomach Mucosa of the Adult Rat

Adrenalectomy caused a significant decrease in the mucosal level of pepsinogen in the adult rat stomach. This decrease was mainly due to a reduction in the level of the main component of rat pepsinogen isozymogens, namely, Pgl and in the level of expression of Pgl mRNA, with a maximal effect being evident 48 hours after operation. Immunohistochemical analysis revealed that the level of expression of Pgl in the pepsinogen-producing cells was decreased throughout the stomach mucosa and this decrease was especially marked in the immature chief cells located in the glandular neck of the fundic mucosa. Adrenalectomy also increased the proliferation of mucosal cells and suppressed the expression of cathepsin E and class III mucin, markers of differentiated stomach mucosa. All these changes were reversed by hydrocortisone replacement and the extent of the recovery of the level of Pgl mRNA was dependent on the dose of hydrocortisone, indicating the transcriptional control of the Pgl gene by hydrocortisone. The observed results suggest that the continuous presence of glucocorticoids is necessary for active transcription of Pgl gene in fully differentiated stomach mucosa and that glucocorticoids are important regulators of both the function and the morphology of stomach mucosal cells.

DNA methylation of the pepsinogen 1 gene during rat glandular stomach carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine or catechol.

The methylation patterns of the rat pepsinogen 1 (Pg1) gene in preneoplastic and neoplastic stomach lesions induced by genotoxic N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the non-genotoxic carcinogen catechol were investigated. Male WKY/Ncrj rats were given MNNG in their drinking water (50 mg/l) for 30 weeks or 0.8% catechol throughout the experiment (60 weeks). MNNG induced Pg1 altered pyloric glands (PAPG), adenomatous hyperplasias and well-differentiated adenocarcinomas. Catechol also induced PAPG and adenomatous hyperplasias although cancers did not develop. Adenomatous hyperplasias and adenocarcinomas all consisted of gastric type cells resembling surface mucous cells or pyloric gland cells with little or no Pg1 expression. In MNNG-induced stomach cancers generally lacking Pg1, altered Pg1 gene methylation was observed with both CCGG and GCGC sites being methylated more than normal pyloric mucosa. MNNG or catechol-induced adenomatous hyperplasias also demonstrated essentially the same methylation changes in the CCGG, but not in the GCGC sites. In the mucosa containing PAPG in groups treated with MNNG or catechol the methylation patterns of the Pg1 gene were quite similar to those of normal pyloric mucosa, although the CCGG sites tended to demonstrate slightly increased methylation. The results suggest that the altered methylation of the Pg1 gene observed in stomach cancers is acquired early in the carcinogenic process and progressive methylation changes occur with tumor development.

Immunohistochemical and Biochemical Identification of Pepsinogen Isozymes in the Hamster Lungs: Induction by Polychlorinated Biphenyls

Pepsinogens are acid protease enzymes of pepsin usually found in gastric mucosa. In the present study, we demonstrated the presence of pepsinogen isozymes in male Syrian golden hamster lung tissues by a combined immunohistochemical and biochemical approach. Immunohistochemically, using rat pepsinogen 1 antibody, pepsinogen positive cells were observed mainly in the epithelia of the terminal bronchioles. They demonstrated morphological features of Clara cells. The pepsinogen isozyme pattern of lung tissue determined by polyacrylamide gel electrophoresis was similar to that of stomach mucosa. Treatment of hamsters with polychlorinated biphenyls at a dose of 500 mg/kg body weight ip caused a 2.8-fold increase in pepsinogen content (p less than 0.01) as well as increase in numbers of pepsinogen positive cells in the lung.

Heterotypic induction of stomach-like glands in the urogenital sinus endoderm under the influence of stomach mesenchyme of foetal rats.

Urogenital sinus endoderm of 16.5-day rat foetuses was combined with stomach mesenchyme and the recombinants were either treated with testosterone and grown in vitro or cultured beneath the kidney capsule of adult male rats of the same strain. It was found that testosterone stimulated mitosis in the urogenital endoderm. In recombinants grown under the kidney capsule a stratified squamous epithelium and stomach-like glands were induced under the influence of the forestomach and glandular stomach mesenchymes. However, the induced glands expressed neither rat pepsinogen nor rat ventral prostatic antigen. They did not produce mRNA for the prostatic steroid-binding protein C1. Thus, stomach mesenchyme of rat foetuses induces organ-specific morphogenesis but not functional differentiation in the heterologous endoderm, indicating that cytodifferentiation does not always accompany morphogenesis.

Primary structure and transcriptional regulation of rat pepsinogen C gene

The entire rat pepsinogen C gene has been isolated from a rat genomic library, using the rat pepsinogen C cDNA as a probe. Southern blot analysis showed that there exists at least two rat pepsinogen C genes. The nucleotide sequences of the coding regions and the 5'- and 3'-flanking regions of one of the rat pepsinogen C genes have been determined. This gene is split into 9 exons interrupted by eight intervening sequences. The 5'-flanking region is similar to that of the human pepsinogen C gene, but only the former has the core sequence of the Sp1 binding site. The amount of transcripts of the rat pepsinogen C genes was found to increase during development, and a similar increase was shown to be induced by injection of hydrocortisone. As a candidate of a factor which regulates the transcription, we found a 25-kDa protein by Southwestern blotting. It binds to a specific site in the 5'-flanking region of the gene only in the presence of Mg2+ ion, and it is present in the nuclear fraction of the gastric mucosa but not of the liver.

Assignment of human pepsinogen C (PGC) gene to chromosome 6.

cDNA of rat pepsinogen C (PGC) hybridizes to, among others, a 3.2-kb band in Southern blot analysis of BamHI-cleaved human genomic DNA. This property was employed to localize the human PGC gene. Use of flow-sorted human chromosomes and 12 human x mouse somatic cell hybrid lines demonstrated that the gene is located on chromosome 6.

Changes in rat and human pepsinogen phenotypes induced by N'-methyl-N'nitro-N-nitrosoguanidine.

Changes have been studied in human and rat pepsinogen phenotypes induced by N'-methyl-N'nitro-N-nitrosoguanidine (MMNG) in in vitro rat experiments and in vivo cultures of human and rat isolated gastric chief cells. In vivo the fastest migrating electrophoretic band decreased or disappeared as early as 3 weeks after the start of MNNG treatment. The changes, observed in 17 of 32 rats receiving MNNG, were permanent and consistently associated with pronounced histopathologic changes seen 10 months later (17 of 17). Comparable phenotypic changes were observed after 7 days only in MNNG-treated rat chief cell cultures. In human chief cell cultures a decrease of the Pg3 band, which is consistent with the "carcinogenic" phenotype, was observed in two of six preparations treated with MNNG. This early preceding change in phenotype preceding tumor formation may be useful as a diagnostic tool for the onset of gastric cancer.

Gastric antisecretory activity of cycloheximide due to inhibition of protein synthesis

Treatment of rats with cycloheximide 1 h before carbachol dose-dependently reduced the secretagogue-stimulated gastric acid secretion in pylorus ligated rats, and partially blocked carbachol- or histamine-induced activation of rat gastric (H + + K +)-ATPase which includes translocation of reserve intracellular (H + + K +)-ATPase into the apical membrane of the parietal cells and induction of a KCl pathway. Time-course studies showed that the drug was effective only when administered at least 30 min before the secretagogues. Puromycin showed the same effect as cycloheximide. Pulse labelling studies with [ 35S]methionine led to identification of two most actively synthesized polypeptides in rat gastric mucosa; the proteins of 38 000 and 14 000 molecular weight. The larger polypeptide was identified as rat pepsinogen. The identity of the smaller protein is not known yet. We suggest that synthesis of nascent polypeptide(s) is required for certain steps of the acid secretory process leading to the activation of the acid pump.

Enzyme-linked immunosorbent assay of serum pepsinogen I

A hybridoma monoclonal antibody against human pepsinogen I was used to develop an enzyme-linked immunosorbent assay for pepsinogen I in serum. In the two-step competitive procedure using antimouse immunoglobulin F(ab') 2 fragment coupled to alkaline phosphatase, the measurable assay range was 8–256 μg/l. No cross-reactivity with rat pepsinogen I, human pepsinogen II, gastrin I, bombesin, somatostatin and peptide YY was shown. However, there was slight cross-reactivity (0.09%) with porcine pepsinogen. The coefficients of variation within and between series were 7.6% and 13.0%. This enzyme-linked immunosorbent assay for serum pepsinogen I correlated positively with radioimmunoassay ( r = 0.87, n = 92). The concentration range of serum pepsinogen I in 354 healthy controls was 15–100 μg/l with a lognormal distribution. Serum pepsinogen I levels were significantly higher in the subjects who developed active duodenal ulcer or active gastric ulcer, but significantly lower in those who had gastric cancer, than in control subjects.

大腸菌におけるペプシノゲン蛋白産生

Several plasmids have been constructed which direct the synthesis of rat pepsinogen or prepepsinogen in Escherichia coli. When plasmids with the E. coli lac promoter was introduced into E. coli, a protein identified as pepsinogen or prepepsinogen by immunoreactivity and SDS-PAGE was synthesized in the form of fused protein with β-galactosidase. They constituted up to 2.5% of the total cellular protein and formed insoluble aggregate in E. coli. They were solublized by urea extraction, but no pepsin activity was detected in these proteins. In these proteins, both intra and intermolecular activation from pepsinogen to pepsin was impaired. This impairment of the activation step was probably due to the structural change of the N-terminal regions of these proteins rather than the degeneration by urea extraction.

Serum pepsinogen levels in normal and experimental peptic ulcer rats measured by radioimmunoassay.

Antiserum to the rat pepsinogen (PG) purified by us was raised in the rabbit. We established a radioimmunoassay system for PG and measured the content of it in the digestive and urinary organs. Immunoreactive PG in serum was confirmed to be PG by high-performance liquid chromatography using an ion exchange column. Serum PG levels in pregnant or lactating rats were higher than normal. The maximum level during lactation was 6 or 7 times the normal value, and began to decrease from about the 16th day after delivery. Serum PG in infant rats was elevated along with the content of mucosal PG. In the measurement of serum PG levels in experimental peptic ulcer rats, no significant change was observed in Shay and cysteamine ulcers produced in the forestomach or/and the duodenum. However, serum PG levels rose significantly in rats with indomethacin, stress and ethanol ulcers, which represent injuries on the glandular portion.

Nucleotide sequence of a nearly full-length cDNA coding for pepsinogen of rat gastric mucosa.

A nearly full-length rat pepsinogen cDNA was isolated from a rat gastric mucosa cDNA library and its nucleotide sequence was determined. The cDNA comprises 1370 base pairs (bp), including the 5'-non-coding region (60 bp), the coding nucleotide sequence (1176 bp) and the 3'-non-coding region (131 bp). The predicted amino acid sequence of rat prepepsinogen (392 residues) contains a 16-residue signal sequence followed by the pepsionogen moiety of 376 residues. Rat pepsinogen has an amino acid composition characteristic of C-type pepsinogens and is much more homologous in amino acid sequence with C-type pepsinogens than A-type pepsinogens. These results indicate that the major form of rat pepsinogen can be classified as a C type pepsinogen.

Quantitative aspects of mucus glycoprotein biosynthesis in rat gastric mucosa

The synthesis of the polypeptide backbone of mucus glycoproteins in rat stomach was studied. CsCl centrifugation of the homogenate of [3H]serine pulse-chase labelled stomach or mucosal scrapings showed that [3H]serine was mainly incorporated into molecules having a density identical to that of proteins and that only 8-12% was incorporated into macromolecules with the density of mucus glycoproteins. [3H]-Galactose, however, was almost exclusively incorporated into macromolecules with a density identical to that of mucus glycoproteins. Electrophoretic analysis of the CsCl fraction containing the mucus glycoprotein revealed that 78% of the [3H]serine-labelled macromolecules had an electrophoretic behaviour identical to that of mucus glycoproteins. Thus, only a small portion (about 6-10%) of incorporated [3H]serine was present in the backbone of the mucus glycoprotein. Translation in a wheat germ cell-free system of total RNA derived from both whole stomach and superficial mucosal scrapings, using either [35S]methionine or [35S]cysteine as radioactive amino acid, yielded a wide range of proteins. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, one major translation product of whole stomach RNA had an apparent Mr (43000) identical to that of rat pepsinogen. As this polypeptide could not be found amongst the translation products of RNA from scrapings it probably was pepsinogen. The present data provide strong evidence that the backbone polypeptide of mucus glycoproteins only accounts for a small part of the proteins synthesized by mucus-producing cells.

The N-terminal sequence of rat pepsinogen

The amino-acid sequence of 96 residues in the N-terminal region of rat pepsinogen I was determined and the first 46 residues were found to constitute the activation peptide segment. There was high degree of homology between the activation segments of rat pepsinogen and some pepsinogens A (pig, cow, Japanese monkey and human). However, the number of residues substituted between rat and the other pepsinogens were considerably larger than those among pepsinogens A. In the N-terminal 24 residues of active pepsin, homology (88%) between rat pepsin and human gastricsin was higher than that (50%) between rat pepsin and pepsin A from human or pig. This strongly suggests that rat pepsin should be classified as pepsin C.