Adhesion GPCR GPR56 Expression Profiling in Human Tissues.

Despite the immense functional relevance of GPR56 (gene ADGRG1) in highly diverse (patho)physiological processes such as tumorigenesis, immune regulation, and brain development, little is known about its exact tissue localization. Here, we validated antibodies for GPR56-specific binding using cells with tagged GPR56 or eliminated ADGRG1 in immunotechniques. Using the most suitable antibody, we then established the human GPR56 tissue expression profile. Overall, ADGRG1 RNA-sequencing data of human tissues and GPR56 protein expression correlate very well. In the adult brain especially, microglia are GPR56-positive. Outside the central nervous system, GPR56 is frequently expressed in cuboidal or highly prismatic secreting epithelia. High ADGRG1 mRNA, present in the thyroid, kidney, and placenta is related to elevated GPR56 in thyrocytes, kidney tubules, and the syncytiotrophoblast, respectively. GPR56 often appears in association with secreted proteins such as pepsinogen A in gastric chief cells and insulin in islet ß-cells. In summary, GPR56 shows a broad, not cell-type restricted expression in humans.

Role of pepsin and pepsinogen: linking laryngopharyngeal reflux with otitis media with effusion in children.

OBJECTIVES/HYPOTHESIS: To analyze the relationship between laryngopharyngeal reflux (LPR) represented by pepsin and pepsinogen, and pathogenesis of otitis media with effusion (OME). STUDY DESIGN: Prospective case-control study. METHODS: Children with OME who required adenoidectomy and tympanostomy/tympanostomy tubes placement were enrolled in OME group, whereas children with adenoid hypertrophy (AH) who required adenoidectomy and individuals who required cochlear implantation (CI) were enrolled in AH and CI groups, respectively. Pepsinogen mRNA and protein levels were assessed by real-time fluorescence-based quantitative polymerase chain reaction and immunohistochemistry in adenoid specimens from the OME and AH groups. Pepsin and pepsinogen concentrations were evaluated by enzyme-linked immunosorbent assay in middle ear fluid and plasma from the OME and CI groups. RESULTS: The levels of pepsinogen protein expressed in cytoplasm of epithelial cells and clearance under epithelial cells in adenoid specimens from the OME group were significantly higher than those in the AH group. Furthermore, the concentrations of pepsin and pepsinogen in the OME group were 51.93±11.58 ng/mL and 728±342.6 ng/mL, respectively, which were significantly higher than those in the CI group (P<.001). In addition, the concentrations of pepsin in dry ears were significantly lower than those in serous and mucus ears in the OME group (F=22.77, P<.001).Finally, the concentration of pepsinogen in middle ear effusion was positively correlated with the expression intensity of pepsinogen protein in cytoplasm of epithelial cells (r=0.73, P<.05) in the OME group. CONCLUSIONS: Pepsin and pepsinogen in middle ear effusion are probably caused by LPR and may be involved in the pathogenesis of OME. LEVEL OF EVIDENCE: 3b.

RUNX3 expression correlates with chief cell differentiation in human gastric cancers.

RUNX3 is a novel tumor suppressor in gastric carcinogenesis and an important factor for differentiation of chief cells in the normal gastric fundic mucosa. In this study, we confirmed RUNX3 immunolocalization in the fundic gland (bottom part) but minimum in surface mucous cell epithelium (top part) in the isolated gland from fundic mucosa. We also analyzed RUNX3 expression by immunohistochemistry in 102 gastric cancers and made a histological assessment of the expression of differentiation markers to evaluate interrelations. Among them, 45 and 57 cases were judged to be RUNX3 positive and negative, respectively, and 33 and 69 cases were pepsinogen I positive and negative, with no link to histological types. RUNX3 expression was significantly associated with that of pepsinogen I (P<0.001), but not mucins, including MUC5AC and MUC6, or the parietal or intestinal phenotypes. In conclusion, the present study showed, for the first time to our knowledge, a relation between RUNX3 and pepsinogen I expression in human gastric cancers. RUNX3 is strongly associated with chief cell phenotypic expression in human gastric cancers, as well as in normal gastric mucosa, and could be considered to play an important role in maintaining the chief cell phenotype.

A novel fusion protein system for the production of native human pepsinogen in the bacterial periplasm.

Human pepsinogen is the secreted inactive precursor of pepsin. Under the acidic conditions present in the stomach it is autocatalytically cleaved into the active protease. Pepsinogen contains three consecutive disulfides, and was used here as a model protein to investigate the production of aspartic proteases in the Escherichia coli periplasm. Various N-terminal translocation signals were applied and several different expression vectors were tested. After fusion to pelB, dsbA or ompT signal peptides no recombinant product could be obtained in the periplasm using the T7 promoter. As a new approach, human pepsinogen was fused to E. coli ecotin (E. coli trypsin inhibitor), which is a periplasmic homodimeric protein of 142 amino acids per monomer containing one disulfide bridge. The fusion protein was expressed in pTrc99a. After induction, the ecotin-pepsinogen fusion protein was translocated into the periplasm and the ecotin signal peptide was cleaved. Upon acid treatment, the fusion protein was converted into pepsin, indicating that pepsinogen was produced in its native form. In shake flasks experiments, the amount of active fusion protein present in the periplasm was 100 microg per litre OD 1, corresponding to 70 microg pepsinogen. After large scale cultivation, the fusion protein was isolated from the periplasmic extract. It was purified to homogeneity with a yield of 20%. The purified protein was native. Acid-induced activation of the fusion protein proceeded very fast. As soon as pepsin was present, the ecotin part of the fusion protein was rapidly digested, followed by a further activation of pepsinogen.

BMP-2 modulates the proliferation and differentiation of normal and cancerous gastric cells.

Bone morphogenetic protein 2 (BMP-2), a member of the transforming growth factor beta super-family, has been shown to act as an antiproliferative agent for a variety of cell lines by activating signaling cascades that cause cell cycle arrest. However, the biological effect and mechanism of action of BMP-2 on gastric cells remain unknown. In the present study, we showed that recombinant human BMP-2 dose-dependently inhibited the growth of OUMS37 rat gastric cells and MKN74 human gastric cancer cells. The antiproliferation seems to be due to cell cycle arrest in the G1-phase, which was revealed by flow cytometric assays. BMP-2 increased the level of p21/WAF1/CIP1, suggesting that BMP-2-mediated inhibition of cell proliferation may be induced through p21/WAF1/CIP1. In addition, BMP-2 increased the expression of pepsinogen II, a differentiation marker of the stomach, in MKN74 cells. These results indicate that BMP-2 plays important roles in modulating the proliferation and differentiation of gastric epithelial cells.

Gastric cancer cell lines as models to study human digestive functions.

The present investigation aims at defining the functional status of several gastric cancer cell lines in order to assess their usefulness as adequate cellular models to study the regulation of gastric digestive functions. Compared to AGS, Hs746t and KATO-III cells, NCI-N87 exhibited an unique differentiation status. They formed coherent monolayers expressing E-cadherin and ZO-1 junctional proteins; their integrity and epithelial morphology were maintained at post-confluency for up to 10 days. All cell lines synthesized PAS-reactive (mucous-type) glycoconjugates. However, only NCI-N87 cells expressed MUC6 glycoprotein suggesting a mucopeptic phenotype. Immunostaining, enzymatic assays, Western blotting and Reverse Transcriptase polymerase chain reaction (RT-PCR) revealed that all cell lines contained varying levels of pepsinogen (Pg5) and human gastric lipase (HGL). Only NCI-N87 cells were able to express zymogens at higher levels, in granule-like structures, and to efficiently secrete both HGL and Pg5. The addition of epidermal growth factor (EGF) to post-confluent NCI-N87 cells, which exhibit an abundant membrane staining for EGF-receptors, modulated HGL activity without affecting Pg5. In conclusion, this investigation enlightens the potential usefulness of the gastric cell line NCI-N87 as a model for elucidating the cellular and molecular mechanisms involved in the regulation of human gastric epithelial functions.

Significación biológica y clínica de las enzimas proteolíticas y sus inhibidores en los carcinomas humanos / Biological and clinical significance of proteolytic enzymes and their inhibitors in human carcinomas

Las enzimas proteolíticas han adquirido recientemente un gran interés en fisiopatología tumoral debido a su papel potencial en la degradación de los componentes principales de la matriz extracelular y membrana basal facilitando, de esa forma, la invasión tumoral y las metástasis. Las enzimas proteolíticas que son expresadas por los carcinomas humanos se agrupan en cuatro grandes familias: metaloproteinasas, aspartil-proteinasas, cisteín-proteinasas y serín-proteinasas. La expresión tumoral de la mayoría de estas enzimas ha sido asociada con un comportamiento más agresivo de los carcinomas humanos y un pronóstico desfavorable de los pacientes. Sin embargo, su expresión tumoral no siempre implica un pronóstico adverso, ya que enzimas como el activador tisular del plasminógeno, el antígeno prostático específico y el pepsinógeno C han sido asociadas con un pronóstico favorable en pacientes con cáncer de mama. Además, existen datos que sugieren que estas expresiones tumorales pueden estar rela cionadas con una vía específica de respuesta hormonal, lo que resulta demostrativo del complejo papel que pueden desempeñar las enzimas proteolíticas en la fisiopatología tumoral. Por otra parte, el resultado final de la acción de las enzimas pro teolíticas dependerá en gran medida del balance con sus inhibidores naturales, producidos por las propias células tumorales para coordinar su acción invasiva, o bien por las células estromales como mecanismo de defensa ante la progresión tumoral. Finalmente, existen datos recientes que indican que la utilización de inhibidores sintéticos de las enzimas proteolíticas puede representar una nueva y prometedora alternativa terapéutica para los carcinomas humanos (AU)

Establishment of culture systems of human gastric epithelium for the study of pepsinogen and gastric lipase synthesis and secretion.

A main purpose of gastric secretion pertains to the digestion of dietary proteins and involves the release of pepsinogens by the fundic and antral mucosa. Over the last decade, data on human gastric physiology has expanded to equally include a significant role in fat digestion. Characteristics of human gastric lipase (HGL) such as optimum acid pH, resistance to proteolysis and non requirement of bile salts or cofactors, are advantageous in gastric lipolysis. Furthermore, the importance of HGL increases in the context of perinatal physiology and pathological situations where secretion of HGL could compensate, to some extent the depressed pancreatic activities. It is therefore important to understand the regulatory mechanisms involved in the synthesis and secretion of human gastric digestive enzymes. The establishment of an organ culture technique as well as a novel primary culture system of human gastric epithelium permitted us to demonstrate that Pg5 and HGL are colocalized in human chief cells and both digestive enzymes are efficiently synthesized and secreted in explants and primary cultures. Pepsin activity rises at the cellular level while its secretion remains constant. In contrast, cellular lipase activity drastically diminishes while being preferentially secreted. This nonparallelism supports the concept that Pg5 and HGL are differently regulated in culture. Furthermore, EGF downregulates HGL expression at the mRNA level via the p42/44(MAPK) pathway without affecting Pg5. Future studies should be designed to fully understand the cellular and molecular mechanisms involved in regulating HGL activity in normal and pathological conditions.

Molecular cloning of neonate/infant-specific pepsinogens from rat stomach mucosa and their expressional change during development.

To clarify the nature of rat neonate/infant-specific pepsinogens, we carried out their purification and molecular cloning. Prochymosin was found to be the major neonatal pepsinogen. The general proteolytic activity of its active form, chymosin, was, however, lower than those of pepsins A and C which are predominant in adult animals. Molecular cloning of rat prochymosin cDNA was achieved along with cDNA for another neonate-specific pepsinogen, pepsinogen F, although determination of pepsinogen F in neonatal gastric mucosa was unsuccessful, presumably due to its lack of proteolytic activity or different proteolytic specificity. Northern blot analysis confirmed that genes for prochymosin and pepsinogen F are expressed only at neonatal/infant stages and the switching of gene expression to that of pepsinogen C occurred at late infant stages. A phylogenetic tree based on nucleotide sequences showed clearly that pepsinogens fall into four major groups, namely prochymosin and pepsinogen F of the neonate/infant and pepsinogens A and C of adult animals. Although, to date, prochymosin and pepsinogen F were believed to be expressed in only a limited number of mammals, the present results suggest that they might be expressed at the neonatal/infant stage in a variety of mammals.

Serum pepsinogen in gastric and duodenal ulcer patients before and after proximal gastric vagotomy

O pepsinogenio serico em situacao basal e sob o estimulo betazolico foi avaliado em 69 pacientes: 14 normais, nove ulcerosos duodenais nao operados, 36 apos vagotomia gastrica proximal devido a ulcera duodenal e dez ulcerosos gastricos, antes e apos serem submetidos a vagotomia gastrica proximal. O seguimento medico foi de 38.7 meses para os pacientes operados por ulcera duodenal e de um ano para os dez ulcerosos gastricos. Dos 36 operados por ulcera duodenal, 22 nao apresentavam recidiva ulcerosa, enquanto 14 a apresentava. Tanto os niveis de pepsinogenio serico basal como estimulado foram estatisticamente elevados nos ulcerosos duodenais nao operados, quando comparados aos controles e aos pacientes submetidos a vagotomia gastrica proximal. O pepsinogenio serico nao mostrou sensibilidade para diferenciar os ulcerosos gastricos do grupo controle. O pepsinogenio serico diferenciou pacientes com e sem recidiva ulcerosa apos vagotomia gastrica proximal devido a ulcera duodenal, tanto em situacao basal, como apos estimulo betazolico. O pepsinogenio serico discrimina pacientes ulcerosos duodenais dos individuos normais e reduz-se significantemente apos vagotomia gastrica proximal, apesar de nao ser indicador sensivel de ulcera gastrica ou recidiva ulcerosa apos vagotomia gastrica proximal.