Michael R. Bardsley

Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract

Populations of interstitial cells of Cajal (ICC) are altered in several gastrointestinal neuromuscular disorders. ICC are identified typically by ultrastructure and expression of Kit (CD117), a protein that is also expressed on mast cells. No other molecular marker currently exists to independently identify ICC. The expression of ANO1 (DOG1, TMEM16A), a Ca(2+)-activated Cl(-) channel, in gastrointestinal stromal tumors suggests it may be useful as an ICC marker. The aims of this study were therefore to determine the distribution of Ano1 immunoreactivity compared with Kit and to establish whether Ano1 is a reliable marker for human and mouse ICC. Expression of Ano1 in human and mouse stomach, small intestine, and colon was investigated by immunofluorescence labeling using antibodies to Ano1 alone and in combination with antibodies to Kit. Colocalization of immunoreactivity was demonstrated by epifluorescence and confocal microscopy. In the muscularis propria, Ano1 immunoreactivity was restricted to cells with the morphology and distribution of ICC. All Ano1-positive cells in the muscularis propria were also Kit positive. Kit-expressing mast cells were not Ano1 positive. Some non-ICC in the mucosa and submucosa of human tissues were Ano1 positive but Kit negative. A few (3.2%) Ano1-positive cells in the human gastric muscularis propria were labeled weakly for Kit. Ano1 labels all classes of ICC and represents a highly specific marker for studying the distribution of ICC in mouse and human tissues with an advantage over Kit since it does not label mast cells.

A Functional Family-Wide Screening of SP/KLF Proteins Identifies a Subset of Suppressors of KRAS-Mediated Cell Growth

SP/KLF (Specificity protein/Krüppel-like factor) transcription factors comprise an emerging group of proteins that may behave as tumour suppressors. Incidentally, many cancers that display alterations in certain KLF proteins are also associated with a high incidence of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) mutations. Therefore in the present paper we investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive family-wide screening of the 24 SP/KLF members, we discovered that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU (5-bromo-2-deoxyuridine) incorporation in KRAS oncogenic-mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and KLF16 also increase apoptosis in these cells. Using KLF11 as a representative model for mechanistic studies, we demonstrate that this protein inhibits the ability of cancer cells to form both colonies in soft agar and tumour growth in vivo. Molecular studies demonstrate that these effects of KLF11 are mediated, at least in part, through silencing cyclin A via binding to its promoter and leading to cell-cycle arrest in S-phase. Interestingly, similar to KLF11, KLF14 and KLF16 mechanistically share the ability to modulate the expression of cyclin A. Collectively, the present study stringently defines a distinct subset of SP/KLF proteins that impairs KRAS-mediated cell growth, and that mechanistically some members of this subset accomplish this, at least in part, through regulation of the cyclin A promoter.

Loss of Kitlow progenitors, reduced stem cell factor and high oxidative stress underlie gastric dysfunction in progeric mice

Gastrointestinal functions decline with ageing leading to impaired quality of life, and increased morbidity and mortality. Neurodegeneration is believed to underlie ageing‐associated dysmotilities but the mechanisms have not been fully elucidated. We used progeric mice deficient in the anti‐ageing peptide Klotho to investigate the contribution of key cell types of the gastric musculature to ageing‐associated changes in stomach function and the underlying mechanisms. Klotho expression, enteric neurons, interstitial cells of Cajal (ICC), smooth muscle cells and electrical activity were assessed by immunofluorescence, confocal microscopy, 3‐dimensional reconstruction, flow cytometry, quantitative RT‐PCR, Western immunoblotting and intracellular recordings. Gastric emptying of solids was analysed by the [13C]octanoic acid breath test. Circulating and tissue trophic factors were measured by enzyme immunoassays and quantitative RT‐PCR. The role of oxidative stress was investigated in organotypic cultures. Klotho expression was detected in gastric glands, myenteric neurons and smooth muscle cells. Progeric Klotho‐deficient mice had profound loss of ICC and ICC stem cells without a significant decrease in neuron counts, expression of neuronal nitric oxide synthase or smooth muscle myosin. Slow wave amplitude and nitrergic inhibitory junction potentials were reduced while solid emptying was unchanged. Klotho‐deficient mice were marantic and had low insulin, insulin‐like growth factor‐I and membrane‐bound stem cell factor. Klotho deficiency accentuated oxidative stress and ICC loss. We conclude that Klotho‐deficient, progeric mice display a gastric phenotype resembling human ageing and involving profound ICC loss. Klotho protects ICC by preserving their precursors, limiting oxidative stress, and maintaining nutritional status and normal levels of trophic factors important for ICC differentiation.

Protein Kinase Cγ Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Activation of the 5-hydroxytryptamine receptor 2B (5-HT2B), a Gq/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by LY294002 decreased base-line proliferation but had no effect on 5-HT2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT2B receptor was inhibited by the phospholipase C inhibitor U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT2B receptor activation in primary cell cultures obtained from PKCγ−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT2B receptor activation. The data demonstrate that the 5-HT2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca2+]i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.

Generalized neuromuscular hypoplasia, reduced smooth muscle myosin and altered gut motility in the klotho model of premature aging.

Background  Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging‐associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make‐up of the small intestinal and colonic neuromuscular apparatus.

FAM96A is a novel pro-apoptotic tumor suppressor in gastrointestinal stromal tumors.

The ability to escape apoptosis is a hallmark of cancer‐initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome‐activating protein and investigate its potential pro‐apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two‐hybrid screen and further studied by deletion mutants, glutathione‐S‐transferase pull‐down, co‐immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock‐down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), “fibroblast‐like cells” (FLCs) and ICC stem cells (ICC‐SCs) was investigated by Northern blotting, reverse transcription—polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC‐SCs stably transduced to re‐express FAM96A was studied by xeno‐ and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC‐SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC‐SCs. Re‐expression of FAM96A in GIST cells and transformed ICC‐SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro‐apoptotic tumor suppressor that is lost during GIST tumorigenesis.

Hedgehog pathway dysregulation contributes to the pathogenesis of human gastrointestinal stromal tumors via GLI-mediated activation of KIT expression

Gastrointestinal stromal tumors (GIST) arise within the interstitial cell of Cajal (ICC) lineage due to activating KIT/PDGFRA mutations. Both ICC and GIST possess primary cilia (PC), which coordinate PDGFRA and Hedgehog signaling, regulators of gastrointestinal mesenchymal development. Therefore, we hypothesized that Hedgehog signaling may be altered in human GIST and controls KIT expression. Quantitative RT-PCR, microarrays, and next generation sequencing were used to describe Hedgehog/PC-related genes in purified human ICC and GIST. Genetic and pharmacologic approaches were employed to investigate the effects of GLI manipulation on KIT expression and GIST cell viability. We report that Hedgehog pathway and PC components are expressed in ICC and GIST and subject to dysregulation during GIST oncogenesis, irrespective of KIT/PDGFRA mutation status. Using genomic profiling, 10.2% of 186 GIST studied had potentially deleterious genomic alterations in 5 Hedgehog-related genes analyzed, including in the PTCH1 tumor suppressor (1.6%). Expression of the predominantly repressive GLI isoform, GLI3, was inversely correlated with KIT mRNA levels in GIST cells and non-KIT/non-PDGFRA mutant GIST. Overexpression of the 83-kDa repressive form of GLI3 or small interfering RNA-mediated knockdown of the activating isoforms GLI1/2 reduced KIT mRNA. Treatment with GLI1/2 inhibitors, including arsenic trioxide, significantly increased GLI3 binding to the KIT promoter, decreased KIT expression, and reduced viability in imatinib-sensitive and imatinib-resistant GIST cells. These data offer new evidence that genes necessary for Hedgehog signaling and PC function in ICC are dysregulated in GIST. Hedgehog signaling activates KIT expression irrespective of mutation status, offering a novel approach to treat imatinib-resistant GIST.

Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes.

163 Hyperglycemia-Induced Hyperplasia of Interstitial Cells of Cajal (ICC) and ICC Stem Cells (ICC-SC) Is Associated With Accelerated Gastric Emptying in Obese Diabetic LeprDb/Db Mice

Background & Aims: ICC depletion is the most common cellular change in diabetic gastroparesis. However, in up to 22% of diabetic patients with gastric symptoms, gastric emptying (GE) is accelerated, rather than delayed. The fate of ICC in these patients is unclear. Previously we reported ICC and ICC-SC hyperplasia in hyperinsulinemic, diabetic Lepr mice. Here, we studied the relationship between GE of solids and ICC/ICC-SC numbers, and investigated the contribution of insulin/IGF1-dependent Kit ligand (Kitl) expression and hyperglycemia-induced ERK MAPK activation to ICC/ICC-SC hyperplasia. Methods: Lepr (n=32) and agesexand strain-matched controls (n=30) were studied 13-65 weeks after the onset of diabetes. ICC and ICC-SC were quantified by flow cytometry. Serum insulin was measured by enzyme immunoassay. Oxidative stress was determined by measuring serum malondialdehyde (MDA) and gastric 8OHdG. GE of solids was analyzed by 13C breath test. Gene expression was studied by qRT-PCR and WB. Effects of high glucose were investigated in murine gastric ICC primary cultures, a conditionally immortalized cell line derived from murine gastric ICC (ICL2A), and in an ICC-SC line isolated from the mouse stomach (2XSCS2F10). ERKMAPK signaling was evaluated byWB and pharmacological inhibition of MAPKK with PD98059. Cell proliferation and apoptosis were determined byMTS assay andCaspase Glo-3/7 assay, respectively. Results: Leprmice had significantly higher blood glucose (median[IQR]: 515[462;577] vs. 124[115;134] mg/dL; P,0.001) and serum insulin levels. Serum MDA and gastric 8OHdG were moderately increased (2.4and 2.2-fold, respectively, P,0.001). In Lepr mice, ICC and ICC-SC increased 2.0±0.1-fold (P,0.001) and 1.5±0.3-fold (P,0.05), respectively, and GE was accelerated (T1/2: 67±10 vs. 100±11 min; P,0.05). GE was similarly accelerated in Kit mice with generalized ICC hyperplasia due to an activating Kit mutation. In Lepr mice, total/soluble Kitl mRNA and Kitl protein were reduced. High glucose (500 mg/dL) significantly increased the numbers of primary ICC and stimulated proliferation and ERK MAPK phosphorylation in ICL2A and 2XSCS2F10 cells. PD98059 (20 μM) inhibited cell proliferation induced by high glucose without affecting basal proliferation. In contrast, osmotic stress induced by mannitol had no effect on ICC numbers, cell proliferation and ERK MAPK phosphorylation. ICC and ICC-SC were resistant to apoptosis induced by hyperglycemia at levels seen in Lepr mice. Conclusions: In the absence of major oxidative stress, hyperglycemia induces ICCSC and ICC hyperplasia via the ERK MAPK pathway even in the presence of reduced Kitl signaling. ICC hyperplasia, in turn, leads to accelerated GE and may contribute to gastric symptoms in a subset of diabetic patients. Grant support: NIH DK58185, DK68055.

T2049 Hydrogen Sulfide Ameliorates Murine Experimental Colitis Through Specific Effects on Diverse Classes of Infiltrating Immune Cells

Background: Hydrogen sulfide (H2S) has both proand anti-inflammatory effects. Aim: To quantify the effect of H2S on the immune cells in normal mouse colonic tissue and during acute and chronic colitis. Methods: In a 3x2 designed experiment mice either remained naive or received an enema of TNBS 24h or 6d prior to tissue collection and either received NaHS injections (100 mmol/kg, daily, i.p.) or served as control for NaHS injection. Colons were dissected into mucosal/submucosal and external muscle layers, separately digested to single cells, and stained with one of 5 different combinations of a total of 21 antibodies (7 per experiment) targeting standard cell surface lineage marker proteins. Non-immune antibodies of matching isotypes coupled to the same fluorochromes were used as controls. 50,000 cells in each sample were analyzed by multiparameter flow cytometry with gating strategies to identify 20 cell types. Data are presented as the mean±SEM proportion of total cells and analyzed by two-way ANOVA with TNBS treatment and NaHS as independent variables. Results: In the mucosa of mice naive to TNBS, five days of NaHS treatment increased the proportion of myeloid dendritic cells and eosinophils from 0.08±0.03% and 0.79±0.06% to 0.40±0.06% and 1.3±0.1%, respectively (N=3, P<0.05). In the external muscle layers of mice naive to TNBS, NaHS treatment increased the proportion of CD19+ B cells from 0.21±0.07% to 1.4±0.8% (N=4-6; P<0.05). NaHS treatment reduced the weight loss of TNBS-treated mice (85±2% vs 93±3% of basal weight, N=10-12; P<0.05) and also the proportion of CD45+ cells in the mucosa at the 6 d time point (17±2% vs. 12±2%, N= 10-12; P<0.05) but not at 24 h. NaHS treatment reduced the proportion of mast cells (at 24 h: 0.29±0.07% vs 0.08±0.04%, N=9; P<0.05; at 6 d: 0.23±0.07% vs 0.02±0.01%, N= 6-10; P<0.05). The proportion of natural killer cells was reduced at the 6d time point (1.62±0.04% vs 0.5±0.2%, N=3; P<0.05). In the external muscle layers NaHS treatment increased the proportion of B cells and T cells and decreased the proportion of natural killer cells and eosinophils at the 6d timepoint. Conclusion: H2S released from NaHS upregulates some immune cell types in normal animals but is strongly anti-inflammatory in the TNBS model of murine colitis. The anti-inflammatory action of H2S is complex and involves multiple cell types in both the mucosa and the muscle layers. Supported by a grant from the Minnesota Partnership for Biotechnology and Medical Genomics, and NIH grants DK76665, DK58185.