Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract

BACKGROUND: Sonic hedgehog (Shh) is an important endodermal morphogenetic signal during the development of the vertebrate gut. It controls gastrointestinal patterning in general, and gastric gland formation in particular. We have previously shown that Shh regulates gastric gland proliferation in the adult but detailed analysis of its expression along the adult gastrointestinal tract has never been undertaken. We therefore studied Shh expression along the normal human and rodent adult gastrointestinal tract as well as in intestinal metaplasia of the stomach, gastric and intestinal metaplasia of the oesophagus, and gastric heterotopia in Meckel's diverticulum. METHODS: The studies were performed with in situ hybridisation and by immunohistochemistry using an antibody that recognises the Shh precursor form. RESULTS: We found that in the normal gastrointestinal tract, high levels of Shh were expressed in the fundic glands of the stomach. Shh expression was also found in fundic gland metaplasia and heterotopia. However, Shh expression was lost in intestinal metaplasia of the stomach. CONCLUSION: We found a strong correlation between Shh expression and fundic gland differentiation. Our current study therefore provides evidence that in addition to its role in gastric epithelial development, Shh plays a unique role in gastric epithelial differentiation in adults.     

Expression of retinoid receptors in multiple cell lineages in the gastric mucosae of mice and humans

BACKGROUND AND AIM: In mice and humans, the gastric epithelial progenitors undergo proliferation and bipolar migration from the isthmus associated with their differentiation into mucus-, acid- and pepsinogen-secreting cell lineages. Little is known about factors that control the dynamics of these isthmal progenitor cells. Retinoids have long been known as chemopreventive agents against gastric mucosal damage and carcinogenesis. The aim of the present study was to examine the cellular localization of the various retinoid receptors proteins (RAR and RXR) in the gastric epithelium of mice and humans. METHODS: Gastric antral biopsies of normal individuals and the oxyntic and antral regions of the mouse stomach were processed for immunohistochemistry using anti-RAR and anti-RXR antibodies. To label the progenitor cell zone, some sections were also probed with antibodies specific for proliferating cell nuclear antigen. RESULTS: The immunoprobed oxyntic mucosal sections of the mice showed that RXRbeta protein was present in the epithelial isthmal cells, neck cells, zymogenic cells and some pit and parietal cells. In addition, RARbeta was found in isthmal and neck cells, and RARgamma was mainly found in neck cells. In the mouse antrum, only RXRbeta was detected in the isthmal cells and their pit and gland cell descendents. In humans, immunoprobed antral sections showed that RARbeta, RARgamma, RXRalpha and RXRgamma proteins are expressed in the isthmal, pit and gland cells. CONCLUSIONS: Retinoid receptors are expressed in multiple cell lineages of the mouse and human gastric epithelium and may, therefore, account for the possible effects of retinoids on gastric epithelial cell proliferation and differentiation.     

Helicobacter pylori eradication restored sonic hedgehog expression in the stomach

BACKGROUND/AIMS: Sonic hedgehog (Shh) is a morphogen involved in many aspects of patterning of the gut during embryogenesis and in gastric fundic gland homeostasis in the adult. Shh expression is reportedly to be reduced in Helicobacterpylori-associated gastritis. The aim of this study was to assess the restoration of Shh expression after H. pylori eradication. METHODOLOGY: Twenty H. pylori-positive patients who underwent upper gastrointestinal endoscopy before and after the eradication were studied. Biopsy specimens were taken from the greater curvature of the middle third of the stomach body. The specimens were evaluated for the severity of acute and chronic inflammation and for that of mucosal atrophy, based on the updated Sydney system. Immunohistochemistry for Shh and H(+)-, K(+)-ATPase was also performed; the percentages of positively stained epithelial cells for the two molecules were expressed as the Shh index and H(+)-, K(+)-ATPase index, respectively. RESULTS: There was a significant decrease in the acute and chronic inflammation scores and also in the mucosal atrophy score following the eradication. The Shh and H(+)-, K(+)-ATPase index were significantly increased following the eradication. CONCLUSIONS: Suppressed Shh expression in the gastric mucosa by H. pylori infection was significantly restored following eradication of the infection.     

Role of Sonic Hedgehog signaling during progression from inflammation to cancer in the stomach

Despite advances in treatment and the declining incidence, gastric cancer remains the second leading cause of cancer-related deaths in the world. Understanding the progression from inflammation to cancer in the stomach is crucial in the development of novel therapies and strategies for treating this disease. Chronic inflammation of the stomach is typically caused by Helicobacter pylori (H. pylori) and resulting lesions may lead to gastric cancer. During the progression from inflammation to cancer, the stomach epithelium changes with evidence of the disruption of normal epithelial cell differentiation and infiltrating inflammatory cells. Coincident with the development of atrophic gastritis and metaplasia, is the loss of the gastric morphogen Sonic Hedgehog (Shh). Given its critical role as a regulator of gastric tissue homeostasis, the disruption of Shh expression during inflammation correlates with the loss of normal epithelial cell differentiation, but this has only recently been rigorously tested in vivo using a unique mouse model of targeted gastric Shh deletion. While pre-neoplastic lesions such as atrophic gastritis and intestinal metaplasia are associated with the loss of Shh within the acid-secreting glands of the stomach, there is a clear link between elevated Shh and signaling to gastric cancers. The current review focuses on the effects of aberrant Shh expression and its role in the development of gastric cancer, specifically in response to H. pylori infection.     

Trefoil factor 1 is required for the commitment programme of mouse oxyntic epithelial progenitors

BACKGROUND: Trefoil factor 1 (TFF1/pS2) is a major secretory product of the stomach and TFF1 knockout mice constantly develop adenomas and occasional carcinomas in the pyloric antrum. AIM: To analyse the role of TFF1 in the differentiation of gastric epithelial cell lineages using oxyntic mucosae from normal and TFF1 knockout mice. METHODS: The various cell lineages were labelled using specific markers of pit, neck, parietal, and enteroendocrine cells. Patterns of TFF1, TFF2, and TFF3 expressions were defined using western blotting, immunohistochemistry, and/or immunogold electron microscopy. RESULTS: In normal mice, starting from postnatal day 1 (P1), TFF1 and TFF2 were produced by mucus secreting cells of the developing epithelium. At P7, TFF3 expression occurred in pit and parietal cells. When oxyntic glands were compartmentalised, at P21 and in older mice, TFF1 and TFF2 were expressed in pit and neck cells, respectively, and TFF3 was no longer in parietal cells but became a feature of zymogenic cells. In TFF1 deficient mice, alteration of oxyntic epithelial differentiation became obvious at P21, showing significant amplification of pit cells at the expense of parietal cells. At the molecular level, lack of TFF1 induced dramatic inhibition of TFF2 expression and more precocious TFF3 expression. CONCLUSION: In the oxyntic mucosa, all three TFFs are produced in a lineage specific manner and TFF1 is essential in maintaining the normal commitment programme of epithelial progenitors.     

Expression and function of sonic hedgehog pathway components in pituitary adenomas: evidence for a direct role in hormone secretion and cell proliferation

CONTEXT: Sonic hedgehog (Shh) belongs to a family of signaling proteins involved in development and has been recently implicated in cancer. Shh signaling is active in the corticotrophs of the adult pituitary gland, where it cross-talks with the CRH pathway and regulates ACTH secretion. Because developmental pathways are involved in pituitary tumorigenesis, we hypothesized that Shh may be important in pituitary tumors. OBJECTIVE: The objective of this study was to examine the expression and function of Shh-pathway components in pituitary adenomas. METHODS: Using immunohistochemistry, we determined the expression of Shh and its receptors Patched 1 (Ptc1) and Patched 2 (Ptc2) in 55 human pituitary adenomas compared with the normal pituitary gland. The AtT-20 and GH3 pituitary tumor cell lines were used as models for studying the role of Shh on cell proliferation and hormone secretion. The effect of Shh on hormone secretion was confirmed in primary cultures of normal rat pituitaries and human pituitary tumors. RESULTS: Ptc1 and Ptc2 were present, whereas Shh was down-regulated in pituitary adenomas and completely absent in Cushing tumors. Shh inhibited cell proliferation in AtT-20 corticotrophinoma cells and the Shh-specific inhibitor cyclopamine increased proliferation in GH3 mammosomatotrophinoma cells. On the other hand, exogenous administration of Shh increased hormone secretion from normal rat pituitaries, pituitary cell lines, and 10 different pituitary tumors. CONCLUSIONS: Our results suggest that Shh might maintain pituitary cells in a nonproliferative state. We conclude that Shh is a newly described hypophysiotropic cytokine and its down-regulation may be involved in the pathogenesis of pituitary adenomas.     

H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

INTRODUCTION H pylori is an important pathogen associated with gastritis and peptic ulcers[1]. It has also been defined as a carcinogen[2]. The mechanisms of pathogenic and carcinogenic effects of H pylori infection are under intensive investigation. Rese…     

Study of Sonic hedgehog signaling pathway related molecules in gastric carcinoma

AIM: To study the expression of Sonic hedgehog pathway-related molecules, Sonic hedgehog (Shh) and Glil in gastric carcinoma. METHODS: Expression of Shh in 56 gastric specimens including non-cancerous gastric tissues, gastric adenocarcinoma, gastric squamous cell carcinoma was detected by RT-PCR, in situ hybridization and immunohistochemistry. Expression of Glil was observed by in situ hybridization. RESULTS: The positive rate of Shh and Glil expression was 0.0%, 0.0% in non-cancerous gastric tissues while it was 66.7%, 57.8% respectively in gastric adenocarcinoma, and 100%, 100% respectively in gastric squamous cell carcinoma. There was a significant difference between the non-cancerous gastric tissues and gastric carcinoma (P<0.05). Elevated expression of Shh and Glil in gastric tubular adenocarcinoma was associated with poorly differentiated tumors while the expression was absent in gastric mucinous adenocarcinoma. CONCLUSION: The elevated expression of Shh and Glil in gastric adenocarcinoma and gastric squamous cell carcinoma shows the involvement of activated Shh signaling in the cellular proliferation of gastric carcinogenesis. It suggests Shh signaling gene may be a new and good target gene for gastric tumor diagnosis and therapy.     

Estrogen receptor α and hedgehog signal pathway developmental biology of gastric adenocarcinoma

Recent analyses revealed that estrogen receptor? a and hedgehog signal pathway both play important roles in gastric adenocarcinoma. Estrogen receptor? a has been shown to be involved in the regulation of proliferation, differentiation and tumorigenesis of gastrointestinal tract epithelium. Hedgehog family which includes Sonic (Shh), Indian (Ihh) and Desert (Dhh) hedgehogs, is involved in the vast majority of cellular processes and is fundamentally important in regulating cell proliferation, tissue generation and cell differentiation in adenocarcinoma. Experimental and mechanistic findings indicate that down-regulation of expression ER can induce expression of Shh in ER positive cancer cell lines. In this review, we summarize the implications of cellular and molecular mechanisms of estrogen receptor? a and hedgehog signal pathway related to the biochemistry and cell biology in gastric adenocarcinoma.     

The role of matrix metalloproteinase-7 in redefining the gastric microenvironment in response to Helicobacter pylori

BACKGROUND & AIMS: Interactions between epithelial and stromal cells are important determinants of mucosal organization, but the signaling mechanisms are understood incompletely. Matrix metalloproteinase (MMP)-7 is produced uniquely in epithelia, may act on growth factors and matrix proteins, and in the stomach is increased with Helicobacter pylori infection. We have studied the role of MMP-7 in signaling between epithelial cells and a key stromal cell type, the myofibroblast. METHODS: Immunohistochemistry and Western blotting were applied to gastric corpus biopsy specimens; primary cultures of human gastric glands and myofibroblasts were used to study the role of MMP-7 in regulating proliferation and migration of the latter, and MMP-7 substrates were identified by proteomic methods. RESULTS: Increased abundance of the myofibroblast marker alpha-smooth muscle actin was identified in H. pylori-positive biopsy specimens. Media from H pylori-infected gastric epithelial cultures stimulated proliferation and migration of primary human gastric myofibroblasts and antisense oligonucleotide treatment indicated a role for MMP-7. Proteomic methods identified insulin-like growth factor binding protein (IGFBP)-5 as a substrate for MMP-7 in medium from gastric myofibroblasts. Knockdown of IGFBP-5 by small interfering RNA or immunoneutralization of IGF-II, abolished myofibroblast responses to MMP-7. Proliferation of gastric epithelial cells also was stimulated by MMP-7-treated myofibroblasts via IGF-II. CONCLUSIONS: MMP-7 acts as an epithelial-derived signal increasing the bioavailability of IGF-II released from myofibroblasts. Because IGF-II acts on both stromal and epithelial cells, the findings suggest that increased MMP-7 expression contributes to redefining the niche occupied by dividing cells and leading to hyperproliferation in H pylori infection.     

Immunolocalization of cholecystokinin-2 receptors in rat gastric mucosa

BACKGROUND: Gastrin exerts trophic effects on the gastric mucosa by mechanisms not yet completely elucidated. Our aim was to localize the cholecystokinin-2 (CCK2) receptor in epithelial cells of foetal and adult rat stomachs in order to determine the cell types that are directly affected by gastrin. METHODS: Gastric tissue was subjected to indirect double immunofluorescence staining with antiserum against the C-terminal decapeptide of the CCK2 receptor and antibodies against 5' bromo-2-deoxyuridine, which had been injected into the rats I h before they were killed, the acid pump H,K-ATPase, the membrane-cytoskeletal linker ezrin, pepsin/pepsinogen or histidine decarboxylase. RESULTS: Undifferentiated foetal gastric epithelial cells expressed CCK2 receptors, whereas stem cells of adult gastric glands did not exhibit immunoreactivity. However, other epithelial cells in the progenitor zone of adult gastric glands did express CCK2 receptors. Some of these cells were faintly stained for H,K-ATPase; pepsin/pepsinogen was also detected in this region. Parietal cells in the isthmus/pit region of the glands contained ezrin, and some showed weak immunoreactivity for the CCK2 receptor. As expected, enterochromaffin-like cells also expressed CCK2 receptors. CONCLUSION: Our findings are consistent with the hypothesis that a CCK2 receptor mediates direct effects of gastrin on gastric epithelial cells during both stomach organogenesis and adult life.     

Immunolocalization of Cholecystokinin-2 Receptors in Rat Gastric Mucosa

Background: Gastrin exerts trophic effects on the gastric mucosa by mechanisms not yet completely elucidated. Our aim was to localize the cholecystokinin-2 (CCK 2 ) receptor in epithelial cells of foetal and adult rat stomachs in order to determine the cell types that are directly affected by gastrin. Methods: Gastric tissue was subjected to indirect double immunofluorescence staining with antiserum against the C-terminal decapeptide of the CCK 2 receptor and antibodies against 5' bromo-2-deoxyuridine, which had been injected into the rats 1 h before they were killed, the acid pump H,K-ATPase, the membranecytoskeletal linker ezrin, pepsin/pepsinogen or histidine decarboxylase. Results: Undifferentiated foetal gastric epithelial cells expressed CCK 2 receptors, whereas stem cells of adult gastric glands did not exhibit immunoreactivity. However, other epithelial cells in the progenitor zone of adult gastric glands did express CCK 2 receptors. Some of these cells were faintly stained for H,K-ATPase; pepsin/pepsinogen was also detected in this region. Parietal cells in the isthmus/pit region of the glands contained ezrin, and some showed weak immunoreactivity for the CCK 2 receptor. As expected, enterochromaffin-like cells also expressed CCK 2 receptors. Conclusion: Our findings are consistent with the hypothesis that a CCK 2 receptor mediates direct effects of gastrin on gastric epithelial cells during both stomach organogenesis and adult life.     

Wnt-reporter expression pattern in the mouse intestine during homeostasis

Background

The canonical Wnt signaling pathway is a known regulator of cell proliferation during development and maintenance of the intestinal epithelium. Perturbations in this pathway lead to aberrant epithelial proliferation and intestinal cancer. In the mature intestine, proliferation is confined to the relatively quiescent stem cells and the rapidly cycling transient-amplifying cells in the intestinal crypts. Although the Wnt signal is believed to regulate all proliferating intestinal cells, surprisingly, this has not been thoroughly demonstrated. This important determination has implications on intestinal function, especially during epithelial expansion and regeneration, and warrants an extensive characterization of Wnt-activated cells.

Methods

To identify intestinal epithelial cells that actively receive a Wnt signal, we analyzed intestinal Wnt-reporter expression patterns in two different mouse lines using immunohistochemistry, enzymatic activity, in situ hybridization and qRT-PCR, then corroborated results with reporter-independent analyses. Wnt-receiving cells were further characterized for co-expression of proliferation markers, putative stem cell markers and cellular differentiation markers using an immunohistochemical approach. Finally, to demonstrate that Wnt-reporter mice have utility in detecting perturbations in intestinal Wnt signaling, the reporter response to gamma-irradiation was examined.

Results

Wnt-activated cells were primarily restricted to the base of the small intestinal and colonic crypts, and were highest in numbers in the proximal small intestine, decreasing in frequency in a gradient toward the large intestine. Interestingly, the majority of the Wnt-reporter-expressing cells did not overlap with the transient-amplifying cell population. Further, while Wnt-activated cells expressed the putative stem cell marker Musashi-1, they did not co-express DCAMKL-1 or cell differentiation markers. Finally, gamma-irradiation stimulated an increase in Wnt-activated intestinal crypt cells.

Conclusion

We show, for the first time, detailed characterization of the intestine from Wnt-reporter mice. Further, our data show that the majority of Wnt-receiving cells reside in the stem cell niche of the crypt base and do not extend into the proliferative transient-amplifying cell population. We also show that the Wnt-reporter mice can be used to detect changes in intestinal epithelial Wnt signaling upon physiologic injury. Our findings have an important impact on understanding the regulation of the intestinal stem cell hierarchy during homeostasis and in disease states.     

Cdx1 induced intestinal metaplasia in the transgenic mouse stomach: comparative study with Cdx2 transgenic mice

BACKGROUND AND AIMS: Gastric intestinal metaplasia, which is mainly induced by Helicobacter pylori infection, is thought to be a precancerous lesion of gastric adenocarcinoma. Intestinal metaplastic mucosa expresses intestine specific homeobox genes, Cdx1 and Cdx2, in the human gastric mucosa. We and others have reported that ectopic expression of Cdx2 in the gastric epithelium generates intestinal metaplasia in the transgenic mouse model. METHODS: To clarify the differences in the roles of Cdx1 and Cdx2 in intestinal metaplasia, we generated transgenic mice expressing Cdx1 in the gastric mucosa and compared Cdx1 induced gastric mucosal morphological changes with Cdx2 induced intestinal metaplasia. RESULTS: The gastric mucosa in Cdx1 transgenic mice was completely replaced by intestinal metaplastic mucosa, consisting of all four intestinal epithelial cell types: absorptive enterocytes, goblet, enteroendocrine, and Paneth cells. Paneth cells, which were not recognised in Cdx2 transgenic mice, were in the upper portion of the intestinal metaplastic mucosa. Pseudopyloric gland metaplasia, which was induced in Cdx2 transgenic mice, was not recognised in Cdx1 transgenic mice. Proliferating cell nuclear antigen (PCNA) positive cells were diffusely scattered in Cdx1 induced intestinal metaplastic mucosa while PCNA positive cells in Cdx2 induced intestinal metaplastic mucosa were in the base of the metaplastic mucosa. Intestinal metaplastic mucosa of Cdx1 transgenic mouse stomach was significantly thicker than that of wild-type or Cdx2 transgenic mouse stomach. CONCLUSIONS: We have confirmed that Cdx1 induced gastric intestinal metaplasia but that it differed from Cdx2 induced intestinal metaplasia in differentiation, structure, and proliferation.     

Progesterone induction of calcitonin expression in the murine mammary gland

Progesterone, via its nuclear receptor, is mandatory not only for the induction and specification of mammary gland ductal side-branching and lobuloalveologenesis but also for carcinogen-induced mammary tumorigenesis. Notwithstanding these recent advances, a more comprehensive molecular explanation of progesterone-induced mammary morphogenesis is contingent upon the identification and characterization of mammary molecular targets that are responsive to the progesterone signal. Toward this goal, we report that calcitonin, a 32 amino acid peptide hormone involved in calcium homeostasis, is exclusively expressed in, and secreted from, luminal epithelial cells within the mammary gland of the pregnant mouse, and, importantly, its expression is progesterone-dependent. Conversely, the calcitonin receptor is present during all stages of post-natal mammary development examined, is localized to the myoepithelial cell lineage, and is not regulated by progesterone. Because calcitonin induction spatiotemporally correlates with increases in progesterone-induced mammary gland proliferation and structural remodeling, we posit that calcitonin - through its receptor - may be involved in one or both of these progesterone-dependent processes.     

M3受体在人胃和食管组织中的表达

目的研究发现石胆酰牛磺酸及其他一些胆汁酸是M3受体的部分激动剂，而胆汁反流到胃和食管又是一个普遍现象。本研究旨在探索胃、食管黏膜的M3受体表达情况。方法分别使用羊和兔抗人M3受体的多克隆IgG抗体A和B，采用免疫组化，在11例人胃和食管的不同部位黏膜对M3受体进行定位。结果使用抗体A发现胃泌酸腺表面黏液细胞、小凹细胞、颈部细胞和壁细胞，及食管鳞状上皮的棘层细胞表现为细胞质染色，使用抗体B发现除上述细胞的胞质染色外，人胃壁肌层平滑肌细胞表现为胞膜和胞质共同染色。结论研究揭示M3受体表达于人胃泌酸腺表面黏液细胞、小凹细胞、腺颈部细胞、壁细胞和食管鳞状上皮的棘层细胞。