Regulation of mammalian gastrin/CCK receptor (CCK2R) expression in vitro and in vivo

The gastrin/CCK receptor (CCK2R) mediates the physiological functions of gastrin in the stomach, including stimulation of acid secretion and cellular proliferation and migration, but little is known about the factors that regulate its expression. We identified endogenous CCK2R expression in several cell lines and used luciferase promoter–reporter constructs to define the minimal promoter required for transcription in human gastric adenocarcinoma, AGS, and rat gastric mucosa, RGM1, cells. Consensus binding sites for SP1, C/EBP and GATA were essential for activity. Following serum withdrawal from RGM1 and AR42J cells, endogenous CCK2R mRNA abundance and the activity of a CCK2R promoter–reporter construct were significantly elevated. Transcription of CCK2R was also increased in AGS-GR and RGM1 cells by gastrin through mechanisms partly dependent upon protein kinase C (PKC) and mitogen/extracellular signal-regulated kinase (MEK). Gastrin significantly increased endogenous CCK2R expression in RGM1 cells, and CCK2R protein expression was elevated in the stomach of hypergastrinaemic animals. In mice with cryoulcers in the acid-secreting mucosa, CCK2R expression increased progressively in the regenerating mucosa adjacent to the ulcer repair margin, evident at 6 days postinjury and maximal at 13 days. De novo expression of CCK2R was observed in the submucosa beneath the repairing ulcer crater 6–9 days postinjury. Many of the cells in mucosa and submucosa that expressed CCK2R in response to cryoinjury were identified as myofibroblasts, since they coexpressed vimentin and smooth muscle α-actin but not desmin. The data suggest that increased CCK2R expression might influence the outcome of epithelial inflammation or injury and that the response may be mediated in part by myofibroblasts

The Role of Proteasome Beta Subunits in Gastrin-Mediated Transcription of Plasminogen Activator Inhibitor-2 and Regenerating Protein1

The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and regenerating protein 1(Reg1). Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR) to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP) assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role

Distinct miRNA profiles in normal and gastric cancer myofibroblasts and significance in Wnt signaling

Stromal cells influence epithelial function in both health and disease. Myofibroblasts are abundant stromal cells that influence the cellular microenvironment by release of extracellular matrix (ECM) proteins, growth factors, proteases, cytokines, and chemokines. Cancer-associated myofibroblasts (CAMs) differ from adjacent tissue (ATMs) and normal tissue myofibroblasts (NTMs), but the basis of this is incompletely understood. We report now the differential expression of miRNAs in gastric cancer CAMs. MicroRNA arrays identified differences in the miRNA profile in gastric and esophageal NTMs and in CAMs from stomach compared with NTMs. miR-181d was upregulated in gastric CAMs. Analysis of differentially regulated miRNAs indicated an involvement in Wnt signaling. Examination of a microarray data set then identified Wnt5a as the only consistently upregulated Wnt ligand in gastric CAMs. Wnt5a stimulated miR-181d expression, and knockdown of miR-181d inhibited Wnt5a stimulation of CAM proliferation and migration. Analysis of miR-181d targets suggested a role in chemotaxis. Conditioned medium from CAMs stimulated gastric cancer cell (AGS) migration more than that from ATMs, and miR-181d knockdown reduced the effect of CAM-CM on AGS cell migration but had no effect on AGS cell responses to ATM conditioned media. The data suggest that dysregulation of miRNA expression in gastric CAMs, secondary to Wnt5a signaling, accounts at least in part for the effect of CAMs in promoting cancer cell migration. stromal cells have emerged in recent years as important determinants of epithelial cell function in the gastrointestinal mucosa in health and disease (7, 23, 25). There are multiple stromal cell types, including inflammatory and immune cells, fibroblasts, pericytes, and myofibroblasts. The latter are sparse in many tissues, but in the gut there is normally a sheath of myofibroblasts that surrounds intestinal crypts and gastric glands. They may arise from activation of fibroblasts, for example, by TGFβ, by transdifferentiation of mesenchymal stem cells (26), or by epithelial-mesenchymal transition (20). Physiologically, they play a role in wound healing. They may also influence tumor progression (26). Myofibroblasts are often operationally defined as expressing α-smooth muscle actin (SMA), vimentin, and fibroblast activation protein and are negative for cytokeratin and usually desmin (7). An emerging body of evidence from multiple experimental platforms supports the idea that there are marked differences between different myofibroblast populations in both health and disease. For example, microarray studies reveal differences between myofibroblasts from different regions of the normal gastrointestinal tract (12). Moreover, there are marked differences in cancer at the levels of transcripts, proteins, and functions. Previously, we showed that myofibroblasts from gastric or esophageal cancer differ from their counterparts in adjacent tissue with evidence that myofibroblasts from advanced gastric tumors promote more aggressive phenotypes in cancer cells (3, 13, 14, 17). We also showed that esophageal cancer-associated myofibroblasts (CAMs) exhibit increased secretion of the chemokine-like peptide chemerin, which plays a role in mesenchymal stem cell recruitment (17). MicroRNAs (miRNAs) are short RNAs of ∼22 nucleotides that act posttranscriptionally to determine mRNA stability and translation (1). They regulate an impressive diversity of biological processes and importantly may contribute to cancer initiation and progression. In stomach and esophagus, previous studies have identified differentially expressed miRNAs (8, 11, 19). However, it is not known whether miRNAs contribute to the differences in function of different myofibroblast populations. In view of differences in the secretomes and proteomes of gastric or esophageal cancer-derived myofibroblasts compared with their respective adjacent tissue myofibroblasts (ATMs), in the present study we sought to determine whether there might also be differences in their miRNA expression profiles compared both with each other and with normal tissue myofibroblasts (NTMs). We now report that gastric and esophageal NTM miRNA profiles are readily distinguishable, that gastric CAMs differ from their respective NTMs in their miRNA profiles, and that Wnt5a (which is upregulated in gastric CAMs) may act in part via miR-181d to influence mesenchymal-epithelial signaling

Non-Invasive Exploration of Neonatal Gastric Epithelium by Using Exfoliated Epithelial Cells

Background & Aims: In preterm infants, exfoliated gastric epithelial cells can be retrieved from aspirates sampled through the naso-gastric feeding tube. Our aims were to determine (1) whether the recovery of exfoliated cells is feasible at any time from birth through the removal of the nasogastric tube, (2) whether they can be grown in culture in vitro, and (3) whether the physiological state of exfoliated cells expressing H+/K+-ATPases reflects that of their counterparts remaining in situ at the surface of the gastric epithelium in neonatal rat pups. Methods: In infants, gastric fluid aspirates were collected weekly after birth or every 3 hours over 24-h periods, and related to clinical parameters (Biocollection PROG/09/18). In rat pups submitted to a single fasting/refeeding cycle, we explored circadian exfoliation with the cellular counter-parts in the gland. All samples were analyzed by confocal imaging and Enzyme-Linked Immunosorbent Assay. Results: Epithelial cells were identified by microscopy using membrane-bound anti-H+/K+ ATPases antibody, assessed for nucleus integrity, and the expression of selected proteins (autophagy, circadian clock). On 34 infants, the H+/K+-ATPasepositive cells were consistently found quiescent, regardless of gestational age and feeding schedule from day-5 of life to the day of removal of the naso-gastric tube. By logistic regression analysis, we did find a positive correlation between the intensity of exfoliation (cellular loss per sample) and the postnatal age (p,0.001). The H+/K+ ATPase-positive cell

The distribution of cholecystokinin-8 in the central nervous system of turtles: An immunohistochemical and biochemical study

Immunohistochemical techniques, radioimmunoassay (RIA) and high performance liquid chromatography (HPLC) were used to: (1) determine the regional distribution and amounts of cholecystokinin-8 (CCKS)-like immunoreactivity in the turtle central nervous system, and (2) chemically characterize the CCK8-like material present in the turtle central nervous system. High levels of CCK8-like immunoreactivity were found in the turtle central nervous system, with the highest levels being present in the hypothalamus and neurohypophysis. Moderate levels of the CCK8-like material were found in all other regions of the turtle nervous system except the cerebellum, the olfactory bulbs and the dorsal ventricular ridge of the telencephalon, which contained low levels. The bulk (87%) of the CCK8-like material in turtle central nervous system co-eluted with CCK8-sulfate in gradient elution HPLC. The distribution of CCK8-like immunoreactivity (CCK8LI) observed using immunohistochemistry was consistent with the results of the RIA studies. Numerous CCK8LI-containing neurons and fibers were observed in the hypothalamus and neurohypophysis. Neurons and fibers containing CCK8 were, however, more sparsely distributed outside the hypothalamus. The immunohistochemical data provided evidence for the existence of two major CCK8-containing pathways in turtles that have been previously described in mammals: a pathway from the supraoptic and paraventricular magnocellular nuclei to the external zone of the median eminence and neurohypophysis and a pathway from dorsal root ganglia to the dorsal horn of the spinal cord. Overall, the present results, in conjunction with several previous studies, indicate that CCK8 has had a relatively stable evolutionary history as a CNS neuropeptide among land vertebrates. The molecular structure of CCK8 appears to have been largely (if not entirely) conserved, as has its concentration in many brain regions. A noteworthy exception to such conservatism in the localization of CCK8 is that the concentration of CCK8 in the telencephalon, particularly in the telencephalic cortex, is much lower in turtles than in mammals. The present results therefore suggest that CCK8 may not have become a prominent peptide in the telencephalic cortex (or its anatomical equivalents) until the evolution of neocortex in the mammalian lineage.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25605/1/0000152.pd