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

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

, CCK2R immunoreactivity and DAPI staining in full-thickness corpus 2, 6, 9 and 13 days following cryoulcer generation

<p><b>Copyright information:</b></p><p>Taken from "Regulation of mammalian gastrin/CCK receptor (CCK2R) expression and "</p><p></p><p>Experimental Physiology 2007;93(2):223-236.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2253704.</p><p>© The Authors. Journal compilation © 2007 The Physiological Society</p> Note intense epithelial staining at ulcer repair margins at 13 days, and staining in the submucosa beneath the ulcer crater (indicated by white line) at 9 days. Scale bar represents 500 μ. Sections are representative from four individual animals at each time point. , CCK2R and smooth muscle α-actin immunoreactivity in gastric corpus epithelium at the repair margin of a 9 day ulcer. Upper panel, CCK2R (FITC); centre panel, smooth muscle α-actin (Texas Red); and lower panel, overlay. , CCK2R and vimentin immunoreactivity in gastric corpus submucosa beneath the repairing crater of a 9 day ulcer. Upper panel, CCK2R (FITC); centre panel, vimentin (Texas Red); and lower panel, overlay. , deconvolved images of gastric corpus epithelial cells at the repair margin of a 9 day ulcer. Upper left panel, CCK2R (FITC); upper right panel, smooth muscle α-actin (Texas Red); and lower panel, overlay. , deconvolved images of gastric corpus submucosal cells beneath the repairing crater of a 9 day ulcer. Upper panel, CCK2R (FITC); centre panel, vimentin (Texas Red); and lower panel, overlay. For and , scale bar represents 50 μm; for and , scale bar respresents 10 μm

, CCK2R expression in mucosa and submucosa 9 days following cryoulcer generation

<p><b>Copyright information:</b></p><p>Taken from "Regulation of mammalian gastrin/CCK receptor (CCK2R) expression and "</p><p></p><p>Experimental Physiology 2007;93(2):223-236.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2253704.</p><p>© The Authors. Journal compilation © 2007 The Physiological Society</p> Bracket indicates residual ulcer crater. , CCK2R expression in mucosa and submucosa 9 days following cryoulceration, 0.5 cm away from the ulcer site. C, regenerative mucosa, adjacent to cryoulcer. , submucosa below residual ulcer crater. , normal mucosa, 0.5 cm away from site of ulcer. , normal submucosa, 0.5 cm away from site of ulcer. For and , scale bar respresents 300 μm; for , scale bar represents 50 μm. Hybridizations were performed using a mixture (1:1:1) of the three oligonucleotides described in the Methods

, endogenous CCK2R mRNA abundance in AR42J cells in response to serum withdrawal for times indicated

<p><b>Copyright information:</b></p><p>Taken from "Regulation of mammalian gastrin/CCK receptor (CCK2R) expression and "</p><p></p><p>Experimental Physiology 2007;93(2):223-236.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2253704.</p><p>© The Authors. Journal compilation © 2007 The Physiological Society</p> Results are normalized to 18S rRNA and are expressed as percentage of full serum value (= 100%). = 4. * = 0.016, ** = 0.013, *** = 0.005 time 0, ANOVA. , CCK2R mRNA abundance in RGM1 cells in response to serum withdrawal for times indicated. Results are normalized to 18S rRNA and are expressed as percentage of full serum value (= 100%). = 3. * = 0.0016 time 0, ANOVA