Differential expression of KCNQ1 and ATP4A genes according to the sex and age in the stomach.

We compared the expression of six genes in stomach tissue samples between healthy men and women in different age groups to study sexually dimorphic gene expression. Real-Time RT-PCR was used to compare gene expression between men and women. Our results showed that the expression of KCNQ1 (p = 0.01) was significantly higher in non-menopausal women compared to post-menopausal women. In addition, the expression level of the ATP4A gene in men under 35 years was significantly higher than in men above 50 (p = 0.026). Sexually and age dimorphic gene expression in some genes throughout life may affect gastric function.

The gastric proton pump in gobiid and mudskipper fishes. Evidence of stomach loss?

Stomach loss has occurred independently multiple times during gnathostome evolution with notable frequency within the Teleostei. Significantly, this loss of acid-peptic digestion has been found to correlate with the secondary genomic loss of the gastric proton pump subunits (atp4a, atp4b) and pepsinogens/pepsins (pga, pgc). Gastric glands produce gastric juice containing the acid and pepsin and thus their presence is a hallmark feature of a digestive system capable of acid-peptic digestion. However, in gobiid fishes although oesogaster and gastric glands have been identified histologically, their functional significance has been questioned. In the present study we address whether the gastric proton pump is present and expressed in gastric glands of the goby Neogobius species (Gobiidae) and in members of the family Oxudercidae, a group of amphibious gobiid fishes commonly known as mudskippers (genera: Periophthalmus, Boleophthalmus, Periophthalmodon and Scartelaos). We confirmed the presence of gastric glands and have immunohistochemically localized gastric proton pump expression to these glands in Neogobius fluviatilis and Periophthalmus novemradiatus, Periophthalmus barbarus and Boleophthalmus boddarti. Genome analysis in Neogobius melanostomus, Periophthalmus magnuspinnatus, Scartelaos histophorus, Boleophthalmus pectinirostris, and Periophthalmodon schlosseri revealed the presence of both atp4a and atp4b subunit orthologues in all species in a conserved genomic loci organization. Moreover, it was possible to deduce that the complete open reading frame and the key functional amino acid residues are present. The conserved expression of the gastric proton pump provides clear evidence of the potential for gastric acid secretion indicating that acid digestion is retained in these gobiid fishes and not lost.

Panax notoginseng attenuates hypoxia-induced glycolysis in colonic mucosal epithelial cells in DSS-induced colitis.

Background: Colonic mucosal injuries are an important manifestation of ulcerative colitis (UC), which is related to hypoxia-induced glycolysis in colonic mucosal epithelial cells (cmECs). Panax notoginseng (PN) promotes the repair of colonic mucosal injuries by inhibiting hypoxia-induced glycolysis in cmECs; However, the mechanism by which this occurs is not completely clear. Here, we are to investigate the effects of PN on glucose metabolism in cmECs in colitis and the underlying mechanism. Methods: A model of dextran sulfate sodium-induced colitis rats was used in this research, and the severity of colitis was assessed by pathology, disease activity index (DAI), and weight changes. The content of intracellular pyruvate, intracellular lactate, adenosine triphosphate (ATP), reactive oxygen species (ROS), mitochondrial ROS (mtROS), myeloperoxidase (MPO) activity, superoxide dismutase (SOD) activity, and inflammatory cytokines was detected by assay kits. The expression levels of proteins were detected by western blotting. The expression levels of the ATP4a gene were detected by quantitative polymerase chain reaction (QT-PCR). Results: The colonic mucosal injuries of the colitis rats were significantly worse than those of the control group. Specifically, the hypoxia-induced glycolysis and potential of hydrogen (pH) in the colonic lumen were increased, and the expression of ATP4a was downregulated in the colitis rats. PN (1.0 g/kg) promoted the repair of colonic mucosal injuries, and reversed the pH in the colonic lumen. Further, PN increased the expression of ATP4a proteins, the content of ATP, and the SOD activity, and decreased the expression of pyruvate dehydrogenase lipoamide kinase isozyme and hypoxia-inducible factor 1-alpha proteins, the content of ROS, and MPO activity in cmECs in colitis. PN also increased the expression of ATP4a, cytochrome P450 family 21 subfamily a member 2, and hydroxy-delta-5-steroid dehydrogenase, 3 beta and steroid delta-isomerase 2 proteins in the mitochondria, and decreased the content of mtROS in cmECs. Conclusions: PN alleviated the pH in the colonic lumen and hypoxia-induced glycolysis in cmECs by reducing the hypoxia-induced glycolysis caused by the downregulation of ATP4a protein, thereby promoting the repair of colonic mucosal injuries in colitis.

Case Report: CMV Infection and Same Mechanism-Originated Intestinal Inflammation Compatible With Bowel/Crohn's Disease Is Suggested in ATP4A Mutated-Driven Gastric Neuroendocrine Tumors.

Mutations in the ATP4A proton pump prevent gastric acidification and explain the chronic autoimmune gastritis scenario that conducts the gastric neuroendocrine tumor (gNET) formation. Here, we wanted to investigate the co-occurrence cytomegalovirus (CMV) infection and intestinal inflammation that presented all members of a family affected with gNET and carrying an ATP4A mutation. Intestinal inflammation persisted after CMV eradication and anemia treatment. The inflammation was compatible with a ileitis/Crohn's disease and was originated by the same autoimmune mechanism described in the tumorigenesis of gNETS. The same secondary disease but no the CMV infection was observed in all members affected with gNET and carrying the ATP4A mutation. Our results suggest that the ATP4A malfunction not only explained gNETs but also the co-occurring disease and opportunistic infections, which allowed to link autoimmune pathologies and gNETs in a unique mechanism. Our results open a new window to better understand not only gastric neoplasms formation but the co-occurring autoimmune disorders and the inflammatory mechanism that compose a premalignant scenario for other tumor formation. Our findings are important since contribute to describe the genetic landscape of the Inflammatory Bowel/Crohn's disease and alert clinicians to monitor patients with gastric neoplasms mediated by achlorhydria mechanisms for concomitant secondary pathologies.

Identification and Analysis of Key Genes Driving Gastric Cancer Through Bioinformatics.

Objective: The aim of this study was to use bioinformatic analyses to identify key genes and pathways driving gastric cancer (GC). Materials and Methods: The gene expression profiles, from human gastric tissue samples were downloaded from the Gene Expression Omnibus (GSE)29272 dataset. These data revealed 284 differentially expressed genes (DEGs) that included a group upregulated in cancer tissues (n = 142) and another group that were downregulated in cancer tissues. (n = 142). These DEGs were identified using the GEO2R. We used multiple online analysis tools, including, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction networks, gene expression profiling interactive analysis (GEPIA), and the cBio Cancer Genomics Portal (cBioportal) database. Next, we identified the most significant DEGs using the Kaplan-Meier plotter (KM-plotter) database. Multiple bioinformatic platforms were used to identify candidate prognostic marker genes. We then analyzed freshly frozen GC tissues for the expression of these marker genes to validate the informatic findings. Results: We identified three DEGs related to overall survival from our analyses of the GEO data. Next, we analyzed these three DEGs in GEPIA and the cBioportal database and found that the biglycan (BGN) gene was related to invasion and metastases of GCs. This finding of differential gene expression was confirmed in a separate laboratory analysis of normal and GC tissues. In this analysis we found that high levels of BGN expression were correlated with GC clinicopathological characteristics, including microvascular tumor thrombus (p = 0.018), lymph node metastases (p = 0.013), and vessel invasion (p = 0.004). Conclusions: BGN expression levels appear to be an independent prognostic factor for predicting the survival times of GC patients.

Proton Pump Inhibitor Use, Hypergastrinemia, and Gastric Carcinoids-What Is the Relationship?

Neuroendocrine tumors (NETs) throughout the body are the focus of much current interest. Most occur in the gastrointestinal tract and have shown a major increase in incidence over the past 30 years, roughly paralleling the world-wide increase in the use of proton pump inhibitor (PPI) drugs. The greatest rise has occurred in gastric carcinoids (g-NETs) arising from enterochromaffin-like (ECL) cells. These tumors are long known to occur in auto-immune chronic atrophic gastritis (CAG) and Zollinger-Ellison syndrome (ZES), with or without multiple endocrine neoplasia type-1 (MEN-1), but the incidences of these conditions do not appear to have increased over the same time period. Common to these disease states is persistent hypergastrinemia, generally accepted as causing g-NETs in CAG and ZES, and postulated as having similar tumorigenic effects in PPI users. In efforts to study the increase in their occurrence, g-NETs have been classified in a number of discussed ways into different grades that differ in their incidence and apparent pathogenesis. Based on a large amount of experimental data, tumorigenesis is mediated by gastrin's effects on the CCK2R-receptor on ECL-cells that in turn leads to hyperplasia, dysplasia, and finally neoplasia. However, in all three conditions, the extent of response of ECL-cells to gastrin is modified by a number of genetic influences and other underlying risk factors, and by the duration of exposure to the hormonal influence. Data relating to trophic effects of hypergastrinemia due to PPI use in humans are reviewed and, in an attached Appendix A, all 11 reports of g-NETs that occurred in long-term PPI users in the absence of CAG or ZES are summarized. Mention of additional suspected cases reported elsewhere are also listed. Furthermore, the risk in humans may be affected by the presence of underlying conditions or genetic factors, including their PPI-metabolizer phenotype, with slow metabolizers likely at increased risk. Other problems in estimating the true incidence of g-NETs are discussed, relating to non-reporting of small tumors and failure of the Surveillance, Epidemiology, and End Results Program (SEER) and other databases, to capture small tumors or those not accorded a T1 rating. Overall, it appears likely that the true incidence of g-NETs may be seriously underestimated: the possibility that hypergastrinemia also affects tumorigenesis in additional gastrointestinal sites or in tumors in other organ systems is briefly examined. Overall, the risk of developing a g-NET appears greatest in patients who are more than 10 years on drug and on higher doses: those affected by chronic H. pylori gastritis and/or consequent gastric atrophy may also be at increased risk. While the overall risk of g-NETs induced by PPI therapy is undoubtedly low, it is real: this necessitates caution in using PPI therapy for long periods of time, particularly when initiated in young subjects.

Reactivation of Atp4a concomitant with intragenic DNA demethylation for cancer inhibition in a gastric cancer model.

Accumulating evidence suggests that aberrant DNA methylation and gene silencing of tumor suppressors are pervasive in gastric malignancies, supporting reactivation of tumor suppressors through DNA demethylation as a potential therapeutic opportunity. Atp4a is an important tumor suppressor gene, encoding H+, K+-ATPase, and mediating gastric acid secretion in the stomach. Using transgenic gastric cancer model K19-Wnt1/C2mE (Gan) mice, by combining the transcriptome and MeDIP (methylated DNA immunoprecipitation) sequencing, together with qRT-PCR, we showed that Atp4a was expressed at low levels in tumor tissues and multiple GC cells, while both 5-aza-CdR and 18ß-glycyrrhetinic acid (GRA) pharmacological treatment triggered Atp4a activation with downregulation of DNMT1. In addition, CpG island (CGI) search showed that the CpG rich region is absent in the promoter region but present in exons 9-14 of Atp4a. Methylation specific PCR (MSP) indicated that Atp4a was fully or partly methylated in multiple GC cells. Further MassArray suggested that the demethylation in the CpG site 75, 183, 196, 262-268 might be responsible for the reactivation of Atp4a. Our research identified that GRA, a bioactive component found in abundance in Radix Glycyrrhiza, reactivated Atp4a expression and inhibited gastric tumorigenesis as a potential demethylation agent.

Weipiling ameliorates gastric precancerous lesions in Atp4a-/- mice.

BACKGROUND: Altered cellular metabolism is considered to be one of the hallmarks of cancer (Coller, Am J Pathol 184:4-17, 2014; Kim and Bae, Curr Opin Hematol 25:52-59, 2018). However, few studies have investigated the role of metabolism in the development of gastric precancerous lesions (GPLs). Weipiling (WPL), a traditional Chinese medicine formula for treatment of GPLs. In this study, we evaluated the amelioration of GPLs by WPL and investigated the possible role of WPL in regulating glucose metabolism. METHODS: Firstly, the major components of WPL are chemically characterized by HPLC analytical method. In this study, we chose the Atp4a-/- mouse model (Spicer etal., J Biol Chem 275:21555-21565, 2000) for GPL analysis. Different doses of WPL were administered orally to mice for 10 weeks. Next, the pathological changes of gastric mucosa were assessed by the H&E staining and AB-PAS staining. In addition, TUNEL staining was used to evaluate apoptosis, and we further used immunohistochemically labelled CDX2, MUC2, ki-67, PTEN, and p53 proteins to assess the characteristic changes of gastric mucosa in precancerous lesions. The levels of such transporters as HK-II, PKM2, ENO1, MPC1, and LDHA were determined by Western blot analysis. Finally, we assessed the expression of mTOR, HIF-1α, AMPK, Rheb, TSC1 and TSC2 protein in the gastric mucosa of Atp4a-/-mice. RESULTS: In this work, we evaluated the protective effect of WPL on gastric mucosa in mice with precancerous lesions. The aberrant apoptosis in gastric mucosa of gastric pre-cancerous lesions was controlled by WPL (P<0.05). Furthermore, WPL suppressed the expression of CDX2, MUC2, ki-67, PTEN and p53, as the levels of these proteins decreased significantly compared with the model group (P<0.05). In parallel, WPL significantly suppressed the expression of transporters, such as HK-II, PKM2, ENO1, MPC1 and LDHA (P<0.05). In addition, mTOR, HIF-1a, AMPK, Rheb, TSC1 and TSC2 protein levels in gastric mucosa of Atp4a-/- mice in the high- and low-dose WPL groups were significantly lower than those in the model group (P<0.05), while the expression of TSC1 and TSC2 protein was significantly higher (P<0.05). CONCLUSIONS: Conclusively, WPL could ameliorate GPLs in Atp4a-/- mice by inhibiting the expression of transporters and suppressing the aberrant activation of mTOR/HIF-1α.

Measurement of Autoantibodies to Gastric H+,K+-ATPase (ATP4A/B) Using a Luciferase Immunoprecipitation System (LIPS).

The Luciferase Immuno Precipitation System (LIPS) enables the detection of specific serum antibodies by immunoprecipitation of recombinant antigens tagged with a luciferase reporter. Here we describe LIPS assays for the quantification of autoantibodies to the H+, K+-ATPase A (ATP4A) and B (ATP4B) subunits, two serological markers of autoimmune atrophic gastritis and pernicious anemia. In particular, we will describe the expression of luciferase-tagged recombinant ATP4A and ATP4B, their immunoprecipitation with test sera, the recovery and washing of immune-complexes with a protein-A coated resin, and the quantification of autoantibodies by addition of a luciferase substrate and the measurement of the light output from captured luciferase-tagged antigens.

ATP4A autoimmunity in pediatric patients with type 1 diabetes and its relationship to blood count, iron metabolism, and vitamin B12.

OBJECTIVE: We aimed to assess the prevalence of autoantibodies against the 4A subunit of the gastric proton pump (ATP4A) in pediatric type 1 diabetes (T1D) patients and explore the relationship between ATP4A positivity and blood cell count, iron turnover, and vitamin B12 concentration. SUBJECTS: The study included 94 (59% female) T1D children (aged 12.5 ± 4.1 years, T1D duration 4.2 ± 3.6 years, HbA1c 7.3 ± 1.5% (57 ± 12.6 mmol/mol) with no other autoimmune diseases. METHODS: ATP4A antibodies were measured in T1D patients using a radioimmunoprecipitation assay. Blood cell count, iron concentration, total iron binding capacity, ferritin, transferrin, hepcidin, and vitamin B12 concentration were measured in all the study participants. RESULTS: A total of 16 (17%) children were ATP4A positive. Serum concentrations of ferritin were significantly lower in ATP4A positive than in antibody negative subjects (P = .034). Overall the levels of ATP4A antibodies (ATP4A Index) correlated positively with the age at T1D diagnosis (r = 0.228, P = .026) and negatively with ferritin levels (r = -0.215, P = .037). In ATP4A positive patients, the ATP4A Index correlated positively with age at diagnosis (r = 0.544, P = .032) and negatively with vitamin B12 levels (r = -0.685, P = .004). CONCLUSIONS: ATP4A antibodies were present in a significant proportion of children with T1D. Higher ATP4A levels in T1D children are associated with lower, yet still fitting within the normal range, levels of vitamin B12, and ferritin. Routine screening of T1D children for gastric autoimmunity (ATP4A) should be considered with follow-up of those positive for vitamin B12 and iron deficiency.

A cumulative effect involving malfunction of the PTH1R and ATP4A genes explains a familial gastric neuroendocrine tumor with hypothyroidism and arthritis.

BACKGROUND: Type I gastric neuroendocrine tumors (gNETs) classically arise because of hypergastrinemia and involve destruction of parietal cells, which are responsible for gastric acid secretion through the ATP4A proton pump and for intrinsic factor production. METHODS: By whole exome sequencing, we studied a family with three members with gNETs plus hypothyroidism and rheumatoid arthritis to uncover their genetic origin. RESULTS: A heterozygous missense mutation in the ATP4A gene was identified. Carriers of this variant had low ferritin and vitamin B12 levels but did not develop gNETs. A second heterozygous mutation was also uncovered (PTH1R p.E546K). Carriers exhibited hypothyroidism and one of them had rheumatoid arthritis. Gastrin activates parathyroid hormone like hormone/parathyroid hormone 1 receptor (PTH1R) signaling, which is involved in gastric cell homeostasis. Activation of parathyroid hormone/PTH1R, which is upregulated by thyrotropin in the thyroid, is also involved in RANKL expression, which regulates bone homeostasis. Thyrotropin and RANKL expression were deregulated in PTH1R mutation carriers, suggesting a link between the PTH1R gene, hypothyroidism, rheumatoid arthritis, and gastric disease. Only patients with both mutations developed gNETs plus hypothyroidism and rheumatoid arthritis. CONCLUSION: Both mutations suggest that a collaborative mechanism is operative in this family, in which mutations in these genes affect the function and viability of parietal cells and lead to the achlorhydria that drives hypergastrinemia and the formation of gNETs.

A knockin mouse model for human ATP4aR703C mutation identified in familial gastric neuroendocrine tumors recapitulates the premalignant condition of the human disease and suggests new therapeutic strategies.

By whole exome sequencing, we recently identified a missense mutation (p.R703C) in the human ATP4a gene, which encodes the proton pump responsible for gastric acidification. This mutation causes an aggressive familial type I gastric neuroendocrine tumor in homozygous individuals. Affected individuals show an early onset of the disease, characterized by gastric hypoacidity, hypergastrinemia, iron-deficiency anemia, gastric intestinal metaplasia and, in one case, an associated gastric adenocarcinoma. Total gastrectomy was performed as the definitive treatment in all affected individuals. We now describe the generation and characterization of a knockin mouse model for the ATP4a(R703C) mutation to better understand the tumorigenesis process. Homozygous mice recapitulated most of the phenotypical alterations that were observed in human individuals, strongly suggesting that this mutation is the primary alteration responsible for disease development. Homozygous mice developed premalignant condition with severe hyperplasia, dysplasia and glandular metaplasia in the stomach. Interestingly, gastric acidification in homozygous mice, induced by treatment with 3% HCl acid in the drinking water, prevented (if treated from birth) or partially reverted (if treated during adulthood) the development of glandular metaplasia and dysplasia in the stomach and partially rescued the abnormal biochemical parameters. We therefore suggest that, in this model, achlorhydria contributes to tumorigenesis to a greater extent than hypergastrinemia. Furthermore, our mouse model represents a unique and novel tool for studying the pathologies associated with disturbances in gastric acid secretion.

ATP4 and ciliation in the neuroectoderm and endoderm of Xenopus embryos and tadpoles.

During gastrulation and neurulation, foxj1 expression requires ATP4a-dependent Wnt/ß-catenin signaling for ciliation of the gastrocoel roof plate (Walentek et al. Cell Rep. 1 (2012) 516-527.) and the mucociliary epidermis (Walentek et al. Dev. Biol. (2015)) of Xenopus laevis embryos. These data suggested that ATP4a and Wnt/ß-catenin signaling regulate foxj1 throughout Xenopus development. Here we analyzed whether foxj1 expression was also ATP4a-dependent in other ciliated tissues of the developing Xenopus embryo and tadpole. We found that in the floor plate of the neural tube ATP4a-dependent canonical Wnt signaling was required for foxj1 expression, downstream of or in parallel to Hedgehog signaling. In the developing tadpole brain, ATP4-function was a prerequisite for the establishment of cerebrospinal fluid flow. Furthermore, we describe foxj1 expression and the presence of multiciliated cells in the developing tadpole gastrointestinal tract. Our work argues for a general requirement of ATP4-dependent Wnt/ß-catenin signaling for foxj1 expression and motile ciliogenesis throughout Xenopus development.

ATP4A gene regulatory network for fine-tuning of proton pump and ion channels.

The ATP4A encodes α subunit of H(+), K(+)-ATPase that contains catalytic sites of the enzyme forming pores through cell membrane which allows the ion transport. H(+), K(+)-ATPase is a membrane bound P-type ATPase enzyme which is found on the surface of parietal cells and uses the energy derived from each cycle of ATP hydrolysis that can help in exchanging ions (H(+), K(+) and Cl(-)) across the cell membrane secreting acid into the gastric lumen. The 3-D model of α-subunit of H(+), K(+)-ATPase was generated by homology modeling. It was evaluated and validated on the basis of free energies and amino acid residues. The inhibitor binding amino acid active pockets were identified in the 3-D model by molecular docking. The two drugs Omeprazole and Rabeprazole were found more potent interactions with generated model of α-subunit of H(+), K(+)-ATPase on the basis of their affinity between drug-protein interactions. We have generated ATP4A gene regulatory networks for interactions with other proteins which involved in regulation that can help in fine-tuning of proton pump and ion channels. These findings provide a new dimension for discovery and development of proton pump inhibitors and gene regulation of the ATPase. It can be helpful in better understanding of human physiology and also using synthetic biology strategy for reprogramming of parietal cells for control of gastric ulcers.