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In pyloric metaplasia, mature gastric chief cells reprogram via an evolutionarily conserved process termed paligenosis to re-enter the cell cycle and become spasmolytic polypeptide-expressing metaplasia (SPEM) cells. Here, we use single-cell RNA sequencing (scRNA-seq) following injury to the murine stomach to analyze mechanisms governing paligenosis at high resolution. Injury causes induced reactive oxygen species (ROS) with coordinated changes in mitochondrial activity and cellular metabolism, requiring the transcriptional mitochondrial regulator Ppargc1a (Pgc1α) and ROS regulator Nf2el2 (Nrf2). Loss of the ROS and mitochondrial control in Ppargc1a -/- mice causes the death of paligenotic cells through ferroptosis. Blocking the cystine transporter SLC7A11(xCT), which is critical in lipid radical detoxification through glutathione peroxidase 4 (GPX4), also increases ferroptosis. Finally, we show that PGC1α-mediated ROS and mitochondrial changes also underlie the paligenosis of pancreatic acinar cells. Altogether, the results detail how metabolic and mitochondrial changes are necessary for injury response, regeneration, and metaplasia in the stomach., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gastrointestinal tract organs harbor reserve cells, which are endowed with cellular plasticity and regenerate functional units in response to tissue damage. However, whether the reserve cells in gastrointestinal tract exist as long-term quiescent cells remain incompletely understood. In the present study, we systematically examine H2b-GFP label-retaining cells and identify a long-term slow-cycling population in the gastric corpus but not in other gastrointestinal organs. The label-retaining cells, which reside near the basal layers of the corpus, comprise a subpopulation of chief cells. The identified quiescent cells exhibit induction of Atf4 and its target genes including Atf3, a marker of paligenosis, and activation of the unfolded protein response, but do not show elevated expression of Troy, Lgr5, or Mist. External damage to the gastric mucosa induced by indomethacin treatment triggers proliferation of the quiescent Atf4 + population, indicating that the gastric corpus harbors a specific cell population that is primed to facilitate stomach regeneration., (© 2023. The Author(s).)

Background & Aims: Acute and chronic gastric injury induces alterations in differentiation within the corpus of the stomach called pyloric metaplasia. Pyloric metaplasia is characterized by the death of parietal cells and reprogramming of mitotically quiescent zymogenic chief cells into proliferative, mucin-rich spasmolytic polypeptide-expressing metaplasia (SPEM) cells. Overall, pyloric metaplastic units show increased proliferation and specific expansion of mucous lineages, both by proliferation of normal mucous neck cells and recruitment of SPEM cells. Here, we identify Sox9 as a potential gene of interest in the regulation of mucous neck and SPEM cell identity in the stomach., Methods: We used immunostaining and electron microscopy to characterize the expression pattern of SRY-box transcription factor 9 (SOX9) during murine gastric development, homeostasis, and injury in homeostasis, after genetic deletion of Sox9 and after targeted genetic misexpression of Sox9 in the gastric epithelium and chief cells., Results: SOX9 is expressed in all early gastric progenitors and strongly expressed in mature mucous neck cells with minor expression in the other principal gastric lineages during adult homeostasis. After injury, strong SOX9 expression was induced in the neck and base of corpus units in SPEM cells. Adult corpus units derived from Sox9-deficient gastric progenitors lacked normal mucous neck cells. Misexpression of Sox9 during postnatal development and adult homeostasis expanded mucous gene expression throughout corpus units including within the chief cell zone in the base. Sox9 deletion specifically in chief cells blunts their reprogramming into SPEM., Conclusions: Sox9 is a master regulator of mucous neck cell differentiation during gastric development. Sox9 also is required for chief cells to fully reprogram into SPEM after injury., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Adult stem cells constantly react to local changes to ensure tissue homeostasis. In the main body of the stomach, chief cells produce digestive enzymes; however, upon injury, they undergo rapid proliferation for prompt tissue regeneration. Here, we identified p57 Kip2 (p57) as a molecular switch for the reserve stem cell state of chief cells in mice. During homeostasis, p57 is constantly expressed in chief cells but rapidly diminishes after injury, followed by robust proliferation. Both single-cell RNA sequencing and dox-induced lineage tracing confirmed the sequential loss of p57 and activation of proliferation within the chief cell lineage. In corpus organoids, p57 overexpression induced a long-term reserve stem cell state, accompanied by altered niche requirements and a mature chief cell/secretory phenotype. Following the constitutive expression of p57 in vivo, chief cells showed an impaired injury response. Thus, p57 is a gatekeeper that imposes the reserve stem cell state of chief cells in homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In vivo administration of dopamine (DA) receptor (DR)-related drugs modulate gastric pepsinogen secretion. However, DRs on gastric pepsinogen-secreting chief cells and DA D 2 receptor (D 2 R) on somatostatin-secreting D cells were subsequently acquired. In this study, we aimed to further investigate the local effect of DA on gastric pepsinogen secretion through DRs expressed on chief cells or potential D 2 Rs expressed on D cells. To elucidate the modulation of DRs in gastric pepsinogen secretion, immunofluorescence staining, ex vivo incubation of gastric mucosa isolated from normal and D 2 R -/- mice were conducted, accompanied by measurements of pepsinogen or somatostatin levels using biochemical assays or enzyme-linked immunosorbent assays. D 1 R, D 2 R, and D 5 R-immunoreactivity (IR) were observed on chief cells in mouse gastric mucosa. D 2 R-IR was widely distributed on D cells from the corpus to the antrum. Ex vivo incubation results showed that DA and the D1-like receptor agonist SKF38393 increased pepsinogen secretion, which was blocked by the D1-like receptor antagonist SCH23390. However, D2-like receptor agonist quinpirole also significantly increased pepsinogen secretion, and D2-like receptor antagonist sulpiride blocked the promotion of DA. Besides, D2-like receptors exerted an inhibitory effect on somatostatin secretion, in contrast to their effect on pepsinogen secretion. Furthermore, D 2 R - / - mice showed much lower basal pepsinogen secretion but significantly increased somatostatin release and an increased number of D cells in gastric mucosa. Only SKF38393, not quinpirole, increased pepsinogen secretion in D 2 R - / - mice. DA promotes gastric pepsinogen secretion directly through D1-like receptors on chief cells and indirectly through D 2 R-mediated suppression of somatostatin release.

A single transcription factor, MIST1 (BHLHA15), maximizes secretory function in diverse secretory cells (like pancreatic acinar cells) by transcriptionally upregulating genes that elaborate secretory architecture. Here, we show that the scantly studied MIST1 target, ELAPOR1 (endosome/lysosome-associated apoptosis and autophagy regulator 1), is an evolutionarily conserved, novel mannose-6-phosphate receptor (M6PR) domain-containing protein. ELAPOR1 expression was specific to zymogenic cells (ZCs, the MIST1-expressing population in the stomach). ELAPOR1 expression was lost as tissue injury caused ZCs to undergo paligenosis (i.e., to become metaplastic and reenter the cell cycle). In cultured cells, ELAPOR1 trafficked with cis -Golgi resident proteins and with the trans -Golgi and late endosome protein: cation-independent M6PR. Secretory vesicle trafficking was disrupted by expression of ELAPOR1 truncation mutants. Mass spectrometric analysis of co-immunoprecipitated proteins showed ELAPOR1 and CI-M6PR shared many binding partners. However, CI-M6PR and ELAPOR1 must function differently, as CI-M6PR co-immunoprecipitated more lysosomal proteins and was not decreased during paligenosis in vivo. We generated Elapor1 -/- mice to determine ELAPOR1 function in vivo. Consistent with in vitro findings, secretory granule maturation was defective in Elapor1 -/- ZCs. Our results identify a role for ELAPOR1 in secretory granule maturation and help clarify how a single transcription factor maintains mature exocrine cell architecture in homeostasis and helps dismantle it during paligenosis. NEW & NOTEWORTHY Here, we find the MIST1 (BHLHA15) transcriptional target ELAPOR1 is an evolutionarily conserved, trans -Golgi/late endosome M6PR domain-containing protein that is specific to gastric zymogenic cells and required for normal secretory granule maturation in human cell lines and in mouse stomach.

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 β-cells. In summary, GPR56 shows a broad, not cell-type restricted expression in humans.

Background & Aims: The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organized inside glands and crypts. Regeneration depends on Wnt/β-catenin pathway activation, but to understand homeostasis and its dysregulation in disease, we need to identify the signaling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen-, and gastric acid-producing cells., Methods: We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief, and parietal cells. We localized the source of the growth factors in the tissue of origin., Results: We show that epidermal growth factor is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with bone morphogenetic factor/Noggin signals, epidermal growth factor controls the differentiation of foveolar cells vs parietal or chief cells. We also show that epidermal growth factor is likely to underlie alteration of the gastric mucosa in the precancerous condition atrophic gastritis., Conclusions: Use of our recently established mucosoid cultures in combination with analysis of the tissue of origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signaling microenvironment in the human gastric glands., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gastric chief cells differentiate from mucous neck cells and develop their mature state at the base of oxyntic glands with expression of secretory zymogen granules. After parietal cell loss, chief cells transdifferentiate into mucous cell metaplasia, designated spasmolytic polypeptide-expressing metaplasia (SPEM), which is considered a candidate precursor of gastric cancer. We examined the range of microRNA (miRNA) expression in chief cells and identified miRNAs involved in chief cell transdifferentiation into SPEM. Among them, miR-148a was strongly and specifically expressed in chief cells and significantly decreased during the process of chief cell transdifferentiation. Interestingly, suppression of miR-148a in a conditionally immortalized chief cell line induced up-regulation of CD44 variant 9 (CD44v9), one of the transcripts expressed at an early stage of SPEM development, and DNA methyltransferase 1 (Dnmt1), an established target of miR-148a. Immunostaining analyses showed that Dnmt1 was up-regulated in SPEM cells as well as in chief cells before the emergence of SPEM in mouse models of acute oxyntic atrophy using either DMP-777 or L635. In the cascade of events that leads to transdifferentiation, miR-148a was down-regulated after acute oxyntic atrophy either in xCT knockout mice or after sulfasalazine inhibition of xCT. These findings suggest that the alteration of miR-148a expression is an early event in the process of chief cell transdifferentiation into SPEM., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Many differentiated epithelial cell types are able to reprogram in response to tissue damage. Although reprogramming represents an important physiological response to injury, the regulation of cellular plasticity is not well understood. Damage to the gastric epithelium initiates reprogramming of zymogenic chief cells into a metaplastic cell lineage known as spasmolytic polypeptide-expressing metaplasia (SPEM). The present study seeks to identify the role of xCT, a cystine/glutamate antiporter, in chief cell reprogramming after gastric injury. We hypothesize that xCT-dependent reactive oxygen species (ROS) detoxification is required for the reprogramming of chief cells into SPEM., Methods: Sulfasalazine (an xCT inhibitor) and small interfering RNA knockdown were used to target xCT on metaplastic cells in vitro. Sulfasalazine-treated wild-type mice and xCT knockout mice were analyzed. L635 or DMP-777 treatment was used to chemically induce acute gastric damage. The anti-inflammatory metabolites of sulfasalazine (sulfapyridine and mesalazine) were used as controls. Normal gastric lineages, metaplastic markers, autophagy, proliferation, xCT activity, ROS, and apoptosis were assessed., Results: xCT was up-regulated early as chief cells transitioned into SPEM. Inhibition of xCT or small interfering RNA knockdown blocked cystine uptake and decreased glutathione production by metaplastic cells and prevented ROS detoxification and proliferation. Moreover, xCT activity was required for chief cell reprogramming into SPEM after gastric injury in vivo. Chief cells from xCT-deficient mice showed decreased autophagy, mucus granule formation and proliferation, as well as increased levels of ROS and apoptosis compared with wild-type mice. On the other hand, the anti-inflammatory metabolites of sulfasalazine did not affect SPEM development., Conclusions: The results presented here suggest that maintaining redox balance is crucial for progression through the reprogramming process and that xCT-mediated cystine uptake is required for chief cell plasticity and ROS detoxification., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Neonatal- Maternal Separation (NMS) deprives mammals from breastfeeding and maternal care, influencing growth during suckling- weaning transition. In the gastric mucosa, Mist1 (encoded by Bhlha15 gene) and moesin organize the secretory apparatus for pepsinogen C in zymogenic cells. Our current hypothesis was that NMS would change corticosterone activity through receptors (GR), which would modify molecules involved in zymogenic cell differentiation in rats. We found that NMS increased corticosterone levels from 18 days onwards, as GR decreased in the gastric mucosa. However, as nuclear GR was detected, we investigated receptor binding to responsive elements (GRE) and observed an augment in NMS groups. Next, we demonstrated that NMS increased zymogenic population (18 and and 30 days), and targeted Mist1 and moesin. Finally, we searched for evolutionarily conserved sequences that contained GRE in genes involved in pepsinogen C secretion, and found that the genomic regions of Bhlha15 and PgC contained sites highly likely to be responsive to glucocorticoids. We suggest that NMS triggers GR- GRE to enhance the expression and to prime genes that organize cellular architecture in zymogenic population for PgC function. As pepsinogen C- pepsin is essential for digestion, disturbance of parenting through NMS might alter functions of gastric mucosa in a permanent manner.

During human gastric carcinogenesis, intestinal metaplasia is frequently seen in the atrophic stomach. In mice, a distinct type of metaplasia known as spasmolytic polypeptide-expressing metaplasia (SPEM) is found in several inflammatory and genetically engineered models. Given the diversity of long- and short-term models of mouse SPEM, it remains unclear whether all models have a shared or distinct molecular mechanism. The origin of SPEM in mice is presently under debate. It is postulated that stem or progenitor cells acquire genetic alterations that then supply metaplastic cell clones, whereas the possibility of transdifferentiation or dedifferentiation from mature gastric chief cells has also been suggested. In this study, we report that loss of chief cells was sufficient to induce short-term regenerative SPEM-like lesions that originated from chief cell precursors in the gastric neck region. Furthermore, Lgr5 + mature chief cells failed to contribute to both short- and long-term metaplasia, whereas isthmus stem and progenitor cells efficiently contributed to long-term metaplasia. Interestingly, multiple administrations of high-dose pulsed tamoxifen induced expansion of Lgr5 expression and Lgr5-CreERT recombination within the isthmus progenitors apart from basal chief cells. Thus we conclude that short-term SPEM represents a regenerative process arising from neck progenitors following chief cell loss, whereas true long-term SPEM originates from isthmus progenitors. Mature gastric chief cells may be dispensable for SPEM development. NEW & NOTEWORTHY Recently, dedifferentiation ability in gastric chief cells during metaplasia development has been proposed. Our findings reveal that lesions that were thought to be acute metaplasia in fact represent normal regeneration supplied from neck lineage and that isthmus stem/progenitors are more responsible for sustained metaplastic changes. Cellular plasticity in gastric chief cells may be more limited than recently highlighted.

Spasmolytic polypeptide-expressing metaplasia (SPEM) develops in patients with chronic atrophic gastritis due to infection with Helicobacter pylori; it might be a precursor to intestinal metaplasia and gastric adenocarcinoma. Lineage tracing experiments of the gastric corpus in mice have not established whether SPEM derives from proliferating stem cells or differentiated, post-mitotic zymogenic chief cells in the gland base. We investigated whether differentiated cells can give rise to SPEM using a nongenetic approach in mice. Mice were given intraperitoneal injections of 5-fluorouracil, which blocked gastric cell proliferation, plus tamoxifen to induce SPEM. Based on analyses of molecular and histologic markers, we found SPEM developed even in the absence of cell proliferation. SPEM therefore did not arise from stem cells. In histologic analyses of gastric resection specimens from 10 patients with adenocarcinoma, we found normal zymogenic chief cells that were transitioning into SPEM cells only in gland bases, rather than the proliferative stem cell zone. Our findings indicate that SPEM can arise by direct reprogramming of existing cells-mainly of chief cells., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Glucagon-like peptide-2 (GLP-2) affects multiple facets of gastrointestinal physiology and have been used to treat patients with short bowel syndrome, but the distribution of its receptor (GLP2R) in human remains poorly understood. Gastric tissue samples of non-obese patients (NOB, n=10) and obese patients without diabetes (OB, n=31) and with diabetes (OWD, n=12) were used to evaluate GLP2R expression and distribution. Immunostaining with a validated antibody, as well as fluorescence in situ hybridization, showed that GLP2R expression was significantly increased in gastric chief cells in OB and OWD patients. PKCζ expression was also significantly increased. This is the first evidence of increased GLP2R expression in chief cells of patients with severe obesity regardless of diabetes status.

Iron pill gastritis has been shown to be associated with superficial gastric erosion and deposition of iron in lamina propria and gastric antral glands. However, iron absorption in gastric parietal and chief cells is rare. We present a case of a 62-year-old man with iron deficiency anemia. His past medical history is significant for Billroth II surgery. His medications include ferrous sulphate 325mg. Esophagogastroduodenoscopy showed diffuse circumferential abnormal mucosa at the gastro-jejunal anastomosis. The mucosa was erythematous and violaceous. Biopsy showed reactive gastropathy with iron deposits predominantly in macrophages, parietal cells, and chief cells. These findings were confirmed by iron stain and later by electron micrography of the gastric mucosa that showed iron deposits in mitochondria and cytoplasm of the parietal and chief cells., (© 2017 by the Association of Clinical Scientists, Inc.)

Parietal cell atrophy is considered to cause metaplasia in the stomach. We developed mice that express the diphtheria toxin receptor specifically in parietal cells to induce their death, and found this to increase proliferation in the normal stem cell zone and neck but not to cause metaplastic reprogramming of chief cells. Furthermore, the metaplasia-inducing agents tamoxifen or DMP-777 still induced metaplasia even after previous destruction of parietal cells by diphtheria toxin. Atrophy of parietal cells alone therefore is not sufficient to induce metaplasia: completion of metaplastic reprogramming of chief cells requires mechanisms beyond parietal cell injury or death., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The plasticity of gastric chief cells is exemplified by their ability to transdifferentiate into spasmolytic polypeptide-expressing metaplasia (SPEM) after parietal cell loss. We sought to determine if chief cell maturity is a limiting factor in the capacity to transdifferentiate. Mist1 - /- mice, previously shown to form only immature chief cells, were treated with DMP-777 or L635 to study the capability of these immature chief cells to transdifferentiate into a proliferative metaplastic lineage after acute parietal cell loss. Mist1 -/- mice treated with DMP-777 showed fewer chief cell to SPEM transitions. Mist1 -/- mice treated with L635 demonstrated significantly fewer proliferative SPEM cells compared with control mice. Thus immature chief cells were unable to transdifferentiate efficiently into SPEM after acute parietal cell loss. To determine whether chief cell age affects transdifferentiation into SPEM, we used tamoxifen to induce YFP expression in chief cells of Mist1 CreER/+ ;Rosa YFP mice and subsequently treated the cells with L635 to induce SPEM at 1 to 3.5 mo after tamoxifen treatment. After L635 treatment to induce acute parietal cell loss, 43% of all YFP-positive cells at 1 mo posttamoxifen were SPEM cells, of which 44% of these YFP-positive SPEM cells were proliferative. By 2 mo after tamoxifen induction, only 24% of marked SPEM cells were proliferating. However, by 3.5 mo after tamoxifen induction, only 12% of marked chief cells transdifferentiated into SPEM and none were proliferative. Thus, as chief cells age, they lose their ability to transdifferentiate into SPEM and proliferate. Therefore, both functional maturation and age limit chief cell plasticity., New & Noteworthy: Previous investigations have indicated that spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach arises from transdifferentiation of chief cells. Nevertheless, the intrinsic properties of chief cells that influence transdifferentiation have been largely unknown. We now report that the ability to transdifferentiate into SPEM is impaired in chief cells that lack full functional maturation, and as chief cells age, they lose their ability to transdifferentiate. Thus chief cell plasticity is dependent on both cell age and maturation.

Background & Aims: Gastric cancer develops in the context of parietal cell loss, spasmolytic polypeptide-expressing metaplasia (SPEM), and intestinal metaplasia (IM). We investigated whether expression of the activated form of Ras in gastric chief cells of mice leads to the development of SPEM, as well as progression of metaplasia., Methods: We studied Mist1-CreERT2Tg/+;LSL-K-Ras(G12D)Tg/+ (Mist1-Kras) mice, which express the active form of Kras in chief cells on tamoxifen exposure. We studied Mist1-CreERT2Tg/+;LSL-KRas (G12D)Tg/+;R26RmTmG/+ (Mist1-Kras-mTmG) mice to examine whether chief cells that express active Kras give rise to SPEM and IM. Some mice received intraperitoneal injections of the Mitogen-activated protein kinase kinase (MEK) inhibitor, selumetinib, for 14 consecutive days. Gastric tissues were collected and analyzed by immunohistochemistry, immunofluorescence, and quantitative polymerase chain reaction., Results: Mist1-Kras mice developed metaplastic glands, which completely replaced normal fundic lineages and progressed to IM within 3-4 months after tamoxifen injection. The metaplastic glands expressed markers of SPEM and IM, and were infiltrated by macrophages. Lineage tracing studies confirmed that the metaplasia developed directly from Kras (G12D)-induced chief cells. Selumetinib induced persistent regression of SPEM and IM, and re-established normal mucosal cells, which were derived from normal gastric progenitor cells., Conclusions: Expression of activated Ras in chief cells of Mist1-Kras mice led to the full range of metaplastic lineage transitions, including SPEM and IM. Inhibition of Ras signaling by inhibition of MEK might reverse preneoplastic metaplasia in the stomach., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Oxyntic atrophy in the stomach leads to chief cell transdifferentiation into spasmolytic polypeptide expressing metaplasia (SPEM). Investigations of preneoplastic metaplasias in the stomach are limited by the sole reliance on in vivo mouse models, owing to the lack of in vitro models for distinct normal mucosal lineages and metaplasias. Utilizing the Immortomouse, in vitro cell models of chief cells and SPEM were developed to study the characteristics of normal chief cells and metaplasia. Chief cells and SPEM cells isolated from Immortomice were cultured and characterized at both the permissive (33°C) and the nonpermissive temperature (39°C). Clones were selected on the basis of their transcriptional expression of specific stomach lineage markers (named ImChief and ImSPEM) and protein expression and growth were analyzed. The transcriptional expression profiles of ImChief and ImSPEM cells were compared further by using gene microarrays. ImChief cells transcriptionally express most chief cell markers and contain pepsinogen C and RAB3D-immunostaining vesicles. ImSPEM cells express the SPEM markers TFF2 and HE4 and constitutively secrete HE4. Whereas ImChief cells cease proliferation at the nonpermissive temperature, ImSPEM cells continue to proliferate at 39°C. Gene expression profiling of ImChief and ImSPEM revealed myelin and lymphocyte protein 2 (MAL2) as a novel marker of SPEM lineages. Our results indicate that the expression and proliferation profiles of the novel ImChief and ImSPEM cell lines resemble in vivo chief and SPEM cell lineages. These cell culture lines provide the first in vitro systems for studying the molecular mechanisms of the metaplastic transition in the stomach.

RNA-binding proteins and corresponding post-transcriptional controls play critical roles in gene expression. The poly-(C) binding proteins, PCBPs (αCPs, hnRNPEs), comprise a well-characterized family of abundant RNA-binding proteins that impact on RNA processing in the nucleus as well as mRNA stability and translation in the cytoplasm. Here we demonstrate that PCBP1 and PCBP2 are abundantly expressed in the gastric epithelium with prominent enrichment in specific cell types within the gastric glandular mucosa. The spatial and intracellular patterns of PCBP1 and PCBP2 expression in these regions are highly correlated. Remarkably, we observe that these proteins are present in the nuclear and cytoplasmic compartments of zymogenic chief cells while they are restricted to the nuclear compartment in acid-secreting parietal cells and poorly expressed in pit cells that line the gland exit. This specificity of expression patterns and subcellular localization of PCBP1 and PCBP2, along with their appearance in the precursor tissues of the gastric epithelium during early postnatal development, suggests these RNA-binding proteins play specific roles in cell differentiation and organismal development within the gastric glandular epithelium., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Mucous metaplasia (MM) is an aberrant secretory phenotype that arises during Helicobacter-induced gastric carcinogenesis. HSPA5, a key modulator of the unfolded protein response (UPR) activated by endoplasmic reticulum (ER) stress is overexpressed in gastric cancer (GC). We studied activation of the UPR in MM and GC in humans and mice. We assessed RNA and protein levels of ER stress markers (HSPA5, XBP1, and CHOP) in human GC, and correlated with Helicobacter pylori (H. pylori) status, then surveyed HSPA5 in normal gastric mucosa and gastric pre-neoplasia including gastritis and intestinal metaplasia (IM). The role of H. pylori infection in the UPR was assessed by co-culture with AGS GC cells. ER stress markers in metaplasia and dysplasia from transgenic K19-Wnt1/C2mE mice and C57Bl/6 mice with chronic Helicobacter felis (H. felis) infection were compared. HSPA5 was overexpressed in 24/73 (33%) of human GC. Induction of HSPA5 and XBP1 splicing was associated with H. pylori-associated GC (P=0.007 for XBP1 splicing). HSPA5 was overexpressed in MM but not gastritis in patients with H. pylori infection. Stimulation of AGS cells with CagA-positive H. pylori suppressed HSPA5 expression and XBP1 splicing. In the normal gastric mucosa of human and mouse, HSPA5 was constitutively expressed in MIST1-positive chief cells. Increased Hspa5 and Chop expression were found in dysplasia of C57Bl/6 mice with chronic H. felis infection but was absent in spontaneous gastric dysplasia in K19-Wnt1/C2mE mice with concomitant loss of Mist1 expression, similar to that observed in H. pylori-associated human GC. Induction of the UPR in the milieu of Helicobacter-induced chronic inflammation and MM may promote neoplastic transformation of Helicobacter-infected gastric mucosa.

KRAS-induced actin-interacting protein (KRAP), originally identified as one of the deregulated genes expressed in colorectal cancer, participates under physiological conditions in the regulation of systemic energy homeostasis and of the exocrine system. We have recently found that KRAP is a molecule associated with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization of IP(3)R in the liver and the pancreas. However, the expression of KRAP and its precise function in other tissues remain elusive. In this study, we aimed to identify the KRAP-expressing cells in mouse stomach and kidneys and to examine the relevance of KRAP expression in the regulation of IP(3)R localization in these tissues. In the stomach, double immunohistochemical staining for KRAP and IP(3)R demonstrated that KRAP was expressed along with the apical regions in the mucous cells and the chief cells, and IP(3)R3 was dominantly co-localized with KRAP in these cells. Furthermore, IP(3)R2 was also co-localized with IP(3)R3 in the chief cells. It is of note that the proper localization of IP(3)R3 and IP(3)R2 in the chief cells and of IP(3)R3 in the mucous cells were significantly abrogated in KRAP-deficient mice. In the kidneys, KRAP was expressed in both the apical and the basal regions of the proximal tubular cells. Intriguingly, KRAP deficiency abrogated the localization of IP(3)R1 in the proximal tubular cells. Finally, co-immunoprecipitation study in the stomachs and the kidneys validated the physical association of KRAP with IP(3)Rs. These findings demonstrate that KRAP physically associates with IP(3)Rs and regulates the proper localization of IP(3)Rs in the mucous cells and the chief cells of the stomach and in the proximal tubular cells of the kidneys.

Despite their divergent developmental ancestry, plasma cells and gastric zymogenic (chief) cells share a common function: high-capacity secretion of protein. Here we show that both cell lineages share increased expression of a cassette of 269 genes, most of which regulate endoplasmic reticulum (ER) and Golgi function, and they both induce expression of the transcription factors X-box binding protein 1 (Xbp1) and Mist1 during terminal differentiation. XBP1 is known to augment plasma cell function by establishing rough ER, and MIST1 regulates secretory vesicle trafficking in zymogenic cells. We examined morphology and function of plasma cells in wild-type and Mist1(-/-) mice and found subtle differences in ER structure but no overall defect in plasma cell function, suggesting that Mist1 may function redundantly in plasma cells. We next reasoned that MIST1 might be useful as a novel and reliable marker of plasma cells. We found that MIST1 specifically labeled normal plasma cells in mouse and human tissues, and, moreover, its expression was also characteristic of plasma cell differentiation in a cohort of 12 human plasma cell neoplasms. Overall, our results show that MIST1 is enriched upon plasma cell differentiation as a part of a genetic program facilitating secretory cell function and also that MIST1 is a novel marker of normal and neoplastic plasma cells in mouse and human tissues.

Background & Aims: We investigated the role of bone morphogenetic protein (BMP) signaling in the regulation of gastric epithelial cell growth and differentiation by generating transgenic mice that express the BMP inhibitor noggin in the stomach., Methods: The promoter of the mouse H+/K+-ATPase β-subunit gene, which is specifically expressed in parietal cells, was used to regulate expression of noggin in the gastric epithelium of mice. The transgenic mice were analyzed for noggin expression, tissue morphology, cellular composition of the gastric mucosa, gastric acid content, and plasma levels of gastrin. Tissues were analyzed by immunohistochemical, quantitative real-time polymerase chain reaction, immunoblot, microtitration, and radioimmunoassay analyses., Results: In the stomachs of the transgenic mice, phosphorylation of Smad 1, 5, and 8 decreased, indicating inhibition of BMP signaling. Mucosa were of increased height, with dilated glands, cystic structures, reduced numbers of parietal cells, and increased numbers of cells that coexpressed intrinsic factor, trefoil factor 2, and Griffonia (Bandeiraea) simplicifolia lectin II, compared with wild-type mice. In the transgenic mice, levels of the H+/K+-ATPase α-subunit protein and messenger RNA were reduced, whereas those of intrinsic factor increased. The transgenic mice were hypochloridric and had an increased number of Ki67- and proliferating cell nuclear antigen-positive cells; increased levels of plasma gastrin; increased expression of transforming growth factor-α, amphiregulin, and gastrin; and activation of extracellular signal-regulated kinase 2., Conclusions: Inhibiting BMP signaling in the stomachs of mice by expression of noggin causes loss of parietal cells, development of transitional cells that express markers of mucus neck and zymogenic lineages, and activation of proliferation. BMPs are therefore important regulators of gastric epithelial cell homeostasis., (Copyright © 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The lack of reliable molecular markers for normal differentiated epithelial cells limits understanding of human gastric carcinogenesis. Recognized precursor lesions for gastric adenocarcinoma are intestinal metaplasia and spasmolytic polypeptide expressing metaplasia (SPEM), defined here by ectopic CDX2 and TFF2 expression, respectively. In mice, expression of the bHLH transcription factor MIST1, normally restricted to mature chief cells, is down-regulated as chief cells undergo experimentally induced metaplasia. Here, we show MIST1 expression is also a specific marker of human chief cells. SPEM, with and without MIST1, is present in human lesions and, akin to murine data, likely represents transitional (TFF2(+)/MIST1(+) = "hybrid"-SPEM) and established (TFF2(+)/MIST1(-) = SPEM) stages. Co-visualization of MIST1 and CDX2 shows similar progressive loss of MIST1 with a transitional, CDX2(+)/MIST1(-) hybrid-intestinal metaplasia stage. Interinstitutional analysis and comparison of findings in tissue microarrays, resection specimens, and biopsies (n > 400 samples), comprising the entire spectrum of recognized stages of gastric carcinogenesis, confirm MIST1 expression is restricted to the chief cell compartment in normal oxyntic mucosa, rare in established metaplastic lesions, and lost in intraepithelial neoplasia/dysplasia and carcinoma of various types with the exception of rare chief cell carcinoma ( approximately 1%). Our findings implicate MIST1 as a reliable marker of mature, healthy chief cells, and we provide the first evidence that metaplasia in humans arises at least in part from the chief cell lineage.

Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. MIST1, an evolutionarily conserved transcription factor, is required for the formation of large, specialized secretory vesicles in gastric zymogenic (chief) cells (ZCs) as they differentiate from their mucous neck cell progenitors. Here, we show that MIST1 binds to highly conserved CATATG E-boxes to directly activate transcription of 6 genes, including those encoding the small GTPases RAB26 and RAB3D. We next show that RAB26 and RAB3D expression is significantly downregulated in Mist1(-)(/)(-) ZCs, suggesting that MIST1 establishes large secretory granules by inducing RAB transcription. To test this hypothesis, we transfected human gastric cancer cell lines stably expressing MIST1 with red fluorescent protein (RFP)-tagged pepsinogen C, a key secretory product of ZCs. Those cells upregulate expression of RAB26 and RAB3D to form large secretory granules, whereas control, non-MIST1-expressing cells do not. Moreover, granule formation in MIST1-expressing cells requires RAB activity because treatment with a RAB prenylation inhibitor and transfection of dominant negative RAB26 abrogate granule formation. Together, our data establish the molecular process by which a transcription factor can directly induce fundamental cellular architecture changes by increasing transcription of specific cellular effectors that act to organize a unique subcellular compartment.

The orderly differentiation of cell lineages within gastric glands is regulated by a complicated interplay of local mucosal growth factors and hormones. Histamine secreted from enterochromaffin-like cells plays an important role in not only stimulated gastric acid secretion but also coordination of intramucosal growth and lineage differentiation. We have examined histidine-decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis, to evaluate the influence of histamine on differentiation of fundic mucosal lineages and the development of metaplasia following induction of acute oxyntic atrophy. Stomachs from HDC-deficient mice and wild-type mice were evaluated at 8 wk and 12 mo of age. DMP-777 was administrated orally to 6-wk-old mice for 1 to 14 days. Sections of gastric mucosa were stained with antibodies against Mist1, intrinsic factor, H/K-ATPase, trefoil factor 2 (TFF2), chromogranin A, and Ext1 and for the cell cycle marker phospho-histone H3. HDC-deficient mice at 8 wk of age demonstrated a prominent increase in chief cells expressing Mist1 and intrinsic factor. Importantly Mist1-positive mature chief cells were present in the midgland region as well as at the bases of fundic glands, indicating a premature differentiation of chief cells. Mice dually deficient for both HDC and gastrin showed a normal distribution of chief cells in fundic glands. Treatment of HDC-deficient mice with DMP-777 led to loss of parietal cells and an accelerated and exaggerated emergence of mucous cell metaplasia with the presence of dual intrinsic factor and TFF2-expressing cells throughout the gland length, indicative of the emergence of spasmolytic polypeptide-expressing metaplasia (SPEM) from chief cells. These findings indicate that histamine, in concert with gastrin, regulates the appropriate differentiation of chief cells from mucous neck cells as they migrate toward the bases of fundic glands. Nevertheless, histamine is not required for emergence of SPEM following acute oxyntic atrophy.

The physiology of gastric epithelial cells is often studied by using cancer cell lines, which may or may not provide information relevant to normal cells. Because few models exist to study chief cell physiology in vitro, our purpose was to develop primary cultured chief cells from rodent species that are structurally and functionally similar to native chief cells. For this, isolated chief cells from the rat stomach, purified by counterflow elutriation and density gradient centrifugation, were grown in media with growth factors. Purity and the continuity of tight junctions were determined, and permeability, viability, transepithelial resistance (TER), cell number and proliferation, and pepsinogen secretion in response to carbachol were measured. When plated in media alone or with basic fibroblast growth factor, the isolated chief cells attached by 2 days and were confluent by 4 days after seeding. However, tight junctions were discontinuous, TER was less than 300 Omega cm(2), and permeability was high. In contrast, chief cells incubated with hepatocyte growth factor (HGF) were confluent in 3 days and had a TER greater than 2,000 Omega cm(2), continuous tight junctions, and low permeability. EGF was intermediate. HGF facilitated monolayer development by increasing cell number, which occurred by the proliferation of chief cells. Chief cell cultures, grown with HGF, consisted of more than 99% gastric intrinsic factor-expressing cells and showed robust pepsinogen secretion. Coexpression studies for neck and chief cell markers suggest that the cultures are a mixture of mature, immature, and transitional zone cells. This model will be useful for investigating mechanisms that regulate chief cell physiology in health and disease.

In the mammalian gastrointestinal tract, the cell fate decisions that specify the development of multiple, diverse lineages are governed in large part by interactions of stem and early lineage progenitor cells with their microenvironment, or niche. Here, we show that the gastric parietal cell (PC) is a key cellular component of the previously undescribed niche for the gastric epithelial neck cell, the progenitor of the digestive enzyme secreting zymogenic (chief) cell (ZC). Genetic ablation of PCs led to failed patterning of the entire zymogenic lineage: progenitors showed premature expression of differentiated cell markers, and fully differentiated ZCs failed to develop. We developed a separate mouse model in which PCs localized not only to the progenitor niche, but also ectopically to the gastric unit base, which is normally occupied by terminally differentiated ZCs. Surprisingly, these mislocalized PCs did not maintain adjacent zymogenic lineage cells in the progenitor state, demonstrating that PCs, though necessary, are not sufficient to define the progenitor niche. We induced this PC mislocalization by knocking out the cytoskeleton-regulating gene Cd2ap in Mist1(-/-) mice, which led to aberrant E-cadherin localization in ZCs, irregular ZC-ZC junctions, and disruption of the ZC monolayer by PCs. Thus, the characteristic histology of the gastric unit, with PCs in the middle and ZCs in the base, may depend on establishment of an ordered adherens junction network in ZCs as they migrate into the base.

Corpus-predominant infection with Helicobacter pylori (HP) results in the activation of programmed cell death pathways in surface, parietal, and chief cells. At present, mechanisms that regulate these pathways to result in HP-associated pathology are not fully understood. Because it is not known which survival and death pathways are present in gastric epithelial cells, we used an antibody panel to evaluate the expression of BCL-2 family prosurvival proteins or multi-Bcl-2 homology (BH)-domains (group 1) or BH3-only (group-2) proapoptotic proteins in the stomachs of uninfected or HP-infected C57BL/6 mice. This strategy identified BCL-2, BAK, and BAD as the major prosurvival and proapoptotic proteins, in surface cells and BAD as the only BCL-2 family protein expressed in parietal cells. Chief cells express altogether different effectors, including BCL-X(L)/BCL-2, for survival but have no constitutively expressed proapoptotic proteins. In model chief cells, however, the group 1 proapoptotic protein BCL-X(S) was expressed after exposure to proinflammatory cytokines concomitant with reduced viability, demonstrating that chief cells can transcriptionally regulate the induction of proapoptotic proteins to execute apoptosis. During HP infection, no additional BCL-2 family proteins were expressed in epithelial cells, whereas those present either remained unchanged or were reduced as cell deletion occurred over time. Additional studies demonstrated that the posttranslational regulation of BAD in surface and parietal cells was negatively affected by HP infection, a result that may be directly related to an increase in apoptosis during infection. Thus, gastric epithelial cells express cell-specific prosurvival and proapoptotic pathways. From the results presented here, mechanisms that regulate HP-related changes in the survival and death profile of gastric epithelial cells can be predicted and then tested, with the ultimate goal of elucidating important therapeutic targets to inhibit the progression of HP-related pathology in the stomach.

Huntingtin interacting protein 1 related (Hip1r) is an F-actin- and clathrin-binding protein involved in vesicular trafficking. In this study, we demonstrate that Hip1r is abundantly expressed in the gastric parietal cell, predominantly localizing with F-actin to canalicular membranes. Hip1r may provide a critical function in vivo, as demonstrated by extensive changes to parietal cells and the gastric epithelium in Hip1r-deficient mice. Electron microscopy revealed abnormal apical canalicular membranes and loss of tubulovesicles in mutant parietal cells, suggesting that Hip1r is necessary for the normal trafficking of these secretory membranes. Accordingly, acid secretory dynamics were altered in mutant parietal cells, with enhanced activation and acid trapping, as measured in isolated gastric glands. At the whole-organ level, gastric acidity was reduced in Hip1r-deficient mice, and the gastric mucosa was grossly transformed, with fewer parietal cells due to enhanced apoptotic cell death and glandular hypertrophy associated with cellular transformation. Hip1r-deficient mice had increased expression of the gastric growth factor gastrin, and mice mutant for both gastrin and Hip1r exhibited normalization of both proliferation and gland height. Taken together, these studies demonstrate that Hip1r plays a significant role in gastric physiology, mucosal architecture, and secretory membrane dynamics in parietal cells.

Background & Aims: Loss of gastric parietal cells is a critical precursor to gastric metaplasia and neoplasia. However, the origin of metaplasia remains obscure. Acute parietal cell loss in gastrin-deficient mice treated with DMP-777 leads to the rapid emergence of spasmolytic polypeptide/trefoil factor family 2 (TFF2)-expressing metaplasia (SPEM) from the bases of fundic glands. We now sought to characterize more definitively the pathway for emergence of SPEM., Methods: Emerging SPEM lineages in gastrin-deficient mice treated with DMP-777 were examined for immunolocalization of TFF2, intrinsic factor, and Mist1, and morphologically with electron microscopy. Emerging SPEM was isolated with laser-capture microdissection and RNA was analyzed using gene microarrays. Immunohistochemistry in mouse and human samples was used to confirm up-regulated transcripts., Results: DMP-777-induced SPEM was immunoreactive for TFF2 and the differentiated chief cell markers, Mist1 and intrinsic factor, suggesting that SPEM derived from transdifferentiation of chief cells. Microarray analysis of microdissected SPEM lineages induced by DMP-777 showed up-regulation of transcripts associated with G1/S cell-cycle transition including minichromosome maintenance deficient proteins, as well as a number of secreted factors, including human epididymis 4 (HE4). HE4, which was absent in the normal stomach, was expressed in SPEM of human and mouse and in intestinal metaplasia and gastric cancer in human beings., Conclusions: Although traditionally metaplasia was thought to originate from normal mucosal progenitor cells, these studies indicate that SPEM evolves through either transdifferentiation of chief cells or activation of a basal cryptic progenitor. In addition, induction of metaplasia elicits the expression of secreted factors, such as HE4, relevant to gastric preneoplasia.

Leptin is a hormone that plays important roles in nutritional status and in obesity. By means of immunocytochemistry, two populations of leptin-secreting cells were found in the lower half of the gastric mucosa. One consists of numerous large cells located around the gastric pits, the Chief epithelial cells, whereas the second refers to much smaller cells, strongly stained, few in number, and scattered between the gastric pits, the endocrine cells. By double immunostaining, leptin and pepsinogen were colocalized in the Chief cells, whereas the endocrine cells were positive only for leptin. Immunoelectron microscopy showed that leptin is present along the rough endoplasmic reticulum-Golgi-granules secretory pathways of the Chief and endocrine cells. On the other hand, leptin-receptor (long and short forms) immunolabelings, although absent in the gastric epithelial cell plasma membranes, were present in enterocytes at the level of their apical and basolateral membranes. Duodenal, jejunal, and ileal enterocytes displayed similar labelings for the leptin receptor. Thus, exocrine and endocrine secretions of leptin together with the presence of leptin receptors on enterocyte plasma membranes constitute a gastroenteric axis that coordinates the role played by leptin in the digestive tract.

In the present study, we investigated whether activation of protease-activated receptor type 2 (PAR-2) with SLIGRL (SL)NH2, a short mimetic agonistic peptide, directly stimulates pepsinogen secretion from gastric-isolated, pepsinogen-secreting (chief) cells. Immunostaining of gastric-dispersed chief cells with a specific anti-PAR-2 antibody demonstrated expression of PAR-2 receptors on membrane and cytoplasm. SL-NH2 and trypsin potently stimulated pepsinogen secretion (EC50 = 0.3 nM) and caused Ca2+ mobilization (EC50 = 0.6 nM). In contrast to SL-NH2, the scramble peptide LSIGRL-NH2 failed to stimulate pepsinogen release. Exposure to SL-NH2 also resulted in ERK1/2 phosphorylation and activation. Exposure of chief cells to phosphotyrosine kinase inhibitors and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one, a selective MEK inhibitor, significantly reduced secretion induced by SL-NH2. Pepsinogen secretion induced by SL-NH2 was desensitized by pretreating the cells with the mimetic peptide and trypsin, and exposure to SL-NH2 abrogates pepsinogen secretion induced by carbachol and CCK-8, but not secretion induced by secretin and vasointestinal peptide. Exposure to Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substance P) but not to calcitonin gene-related peptide increased pepsinogen release. The neurokinin-1 receptor antagonist, N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzyl ester, inhibited substance P-stimulated pepsinogen secretion, whereas it did not affect secretion induced by SL-NH2. Collectively, these data indicate that PAR-2 is expressed on gastric chief cells and that its activation causes a Ca2+-ERK-dependent stimulation of pepsinogen secretion.

Cross talk between signal transduction pathways augments pepsinogen secretion from gastric chief cells. A-kinase anchoring proteins (AKAPs) associate with regulatory subunits of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (PP2B) and localize this protein complex to specific cell compartments. We determined whether an AKAP-signaling protein complex exists in chief cells and whether this modulates secretion. In Western blots, we identified AKAP150, a rodent homologue of human AKAP79 that coimmunoprecipitates with PKA, PKC, and actin. The association of PKA and PP2B was demonstrated by affinity chromatography. Confocal microscopy revealed colocalized staining at the cell periphery for AKAP150 and PKC. Ht31, a peptide that competitively displaces PKA from the AKAP complex, but not Ht31P, a control peptide, inhibited 8-Br-cAMP-induced pepsinogen secretion. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and/or calcium. However, Ht31, but not Ht31P, inhibited carbachol- and A23187-stimulated augmentation of secretion from cells preincubated with cholera toxin. These data suggest the existence in chief cells of a protein complex that includes AKAP150, PKA, PKC, and PP2B. Disruption of the AKAP-PKA linkage impairs cAMP-mediated pepsinogen secretion and cross talk between signaling pathways.

Background and Aim: The circulating peptide leptin produced by fat cells acts on central receptors to control food intake and body weight homeostasis. Contrary to initial reports, leptin expression has also been detected in the human placenta, muscles, and recently, in rat gastric chief cells. Here we investigate the possible presence of leptin and leptin receptor in the human stomach., Methods: Leptin and leptin receptor expression were assessed by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot analysis on biopsy samples from 24 normal individuals. Fourteen (10 healthy volunteers and four patients with non-ulcer dyspepsia and normal gastric mucosa histology) were analysed for gastric secretions. Plasma and fundic mucosa leptin content was determined by radioimmunoassay., Results: In fundic biopsies from normal individuals, immunoreactive leptin cells were found in the lower half of the fundic glands. mRNA encoding ob protein was detected in the corpus of the human stomach. The amount of fundic leptin was 10.4 (3.7) ng leptin/g mucosa, as determined by radioimmunoassay. Intravenous infusions of pentagastrin or secretin caused an increase in circulating leptin levels and leptin release into the gastric juice. The leptin receptor was present in the basolateral membranes of fundic and antral gastric cells. mRNA encoding Ob-RL was detected in both the corpus and antrum, consistent with a protein of approximately 120 kDa detected by immunoblotting., Conclusion: These data provide the first evidence of the presence of leptin and leptin receptor proteins in the human stomach and suggest that gastric epithelial cells may be direct targets for leptin. Therefore, we conclude that leptin may have a physiological role in the human stomach, although much work is required to establish this.

Lasp-1 is a unique LIM and src homology 3 (SH3) domain-containing protein that was initially identified as a 40 kDa cAMP-dependent phosphoprotein in the HCl-secreting gastric parietal cell. Because cAMP is a potent stimulator of parietal cell acid secretion, we have hypothesized that changes in lasp-1 phosphorylation might be involved in the regulation of ion transport-related activities, perhaps by modulating interactions among cytoskeletal and/or vesicle-associated proteins. In this study, we demonstrate that the cAMP-dependent acid secretory agonist, histamine, induces a rapid, sustained rise in parietal cell lasp-1 phosphorylation and this increase in phosphorylation is closely correlated with the acid secretory response. In addition, elevation of intracellular cAMP concentrations appear to induce a partial redistribution of lasp-1 from the cell cortex, where it predominates along with the gamma-isoform of actin in unstimulated cells, to the beta-actin enriched, apically-directed intracellular canalicular region, which is the site of active proton transport in the parietal cell. Additional studies demonstrate that although lasp-1 mRNA and protein are expressed in a wide range of tissues, the expression is specific for certain actin-rich cell types present within these tissues. For example, gastric chief cells, which contain relatively little F-actin and secrete the enzyme, pepsinogen, by regulated exocytosis, do not appear to express lasp-1. Similarly, lasp-1 was not detected in pancreatic acinar cells, which secrete enzymes by similar mechanisms and also contain relatively low levels of F-actin. Lasp-1 also was not detectable in proximal tubules in the kidney, in gastrointestinal smooth muscle, heart or skeletal muscle. In contrast, expression was prominent in the cortical regions of ion-transporting duct cells in the pancreas and in the salivary parotid gland as well as in certain F-actin-rich cells in the distal tubule/collecting duct. Interestingly, moderate levels of expression were also detected in podocytes present in renal glomeruli and in vascular endothelium. In primary cultures of gastric fibroblasts, lasp-1 was present mainly within the tips of lamellipodia and at the leading edges of membrane ruffles. Taken together these results support the hypothesis that the lasp-1 plays an important role in the regulation of dynamic actin-based, cytoskeletal activities. Agonist-dependent changes in lasp-1 phosphorylation may also serve to regulate actin-associated ion transport activities, not only in the parietal cell but also in certain other F-actin-rich secretory epithelial cell types.