265 Immunoreactivity for Ano1 Detects Depletion of Interstitial Cells of Cajal in Patients with Slow Transit Constipation

267 No-Induced Proteasomal Degradation of Mypt1 Enhances Giant Migrating Contractions and Tone in Colonic Inflammation Qingjie Li, Sushil K. Sarna Background and Aim: Inflammation induces diarrhea by enhancing colonic mass movements produced by giant migrating contractions (GMCs). The cellular mechanisms of stimulation of mass movements during inflammation are not understood. We tested the hypothesis that NO release during inflammation causes proteasomal degradation of Mypt1, which serves as the primary site for regulation of myosin light chain phosphatase activity, to enhance the generation of GMCs. Methods: We used rat colonic muscle strips. Results: Gradedinflammation by 17, 50, 65 and 130 mg/kg TNBS dose-dependently increased the expression of iNOS and decreased Mypt1 in colonic muscularis externa. The incubation of circular muscle strips with 1, 10, 25 and 100 μM GSNO, an NO donor, for 24 hours dose-dependently decreased the expression of Mypt1. qRT-PCR showed that GSNO did not affect Mypt1 mRNA expression. However, both cGMP antagonist Rp-8-pCPT-cGMP and PKG antagonist KT5823 blocked the depletion of Mypt1 by GSNO. Time-course study showed significant increase of pMypt1(S696) after 1-hour treatment with 25 μM GSNO. In co-immunoprecipitation (co-IP) assay, ubiquitin antibody pulled down large amount of pMypt1 at 1-hour, suggesting that Mypt1 phosphorylation induced by NO facilitated its ubiquitination, which is necessary for the following proteasomal degradation. In support of this, proteasome inhibitor (AALVS) blocked Mypt1 depletion by GSNO, indicating that Mypt1 was degraded via a ubiquitination-proteasomal degradation pathway. Co-IP with ubiquitin antibody showed that GSNO-induced Mypt1 ubiquitination was blocked also by AALVS. Since PKG phosphorylates Mypt1 at S693, S696 and S854, we mutated these three phosphorylation sites by site-directed mutagenesis and examined whether NO induces degradation of these Mypt1 mutants. Over expression of wild type (WT) or mutant Mypt1 followed by GSNO treatment showed that only WT Mypt1 was degraded by GSNO, suggesting that all three (S693, S696 and S854) are critical to Mypt1 degradation. We established the translational significance of our molecular findings by incubating intact circular muscle strips without the submucosal plexus with 25 μM GSNO for 24 hrs. These strips generate predominantly the GMCs. GSNO treatment significantly increased the contractile response to ACh in these muscle strips. This increase was blocked by PKG inhibitor KT5823 (1 μM). Conclusions: NO released by iNOS during inflammation degrades Mypt1 through a mechanism involving cGMP, PKG, Mypt1 phosphorylation, ubiquitination and proteasomal degradation. The degradation of Mypt1 enhances the generation of GMCs, which is an underlying cause of inflammatory diarrhea.

265 Immunoreactivity for Ano1 Detects Depletion of Interstitial Cells of Cajal in Patients with Slow Transit Constipation Purna C. Kashyap, Pedro J. Gomez-pinilla, Matthew S. Lurken, Robert R. Cima, Eric J. Dozois, David W. Larson, Tamas Ordog, Simon J. Gibbons, Gianrico Farrugia Background: Interstitial cells of Cajal (ICC) play a key role in the control of gastrointestinal motility. Depletion of ICC is associated with motility disorders including diabetic gastroparesis and slow transit constipation. Changes in ICC networks are usually detected by immunolabeling for the receptor tyrosine kinase Kit, which is expressed on mast cells and ICC in the muscle layers of the gastrointestinal tract. Other markers have been identified for ICC e.g. CD44 and Ano1 (DOG1 or TMEM16A) but it has not been determined whether expression of these markers changes in parallel with changes in Kit expression in motility disorders where ICC are depleted. Aim: Our aim was to determine whether Ano1 immunoreactivity can be used as a reliable marker for ICC in tissues where Kit-positive ICC are depleted. Methods: Colon tissue was obtained under IRB approval from 4 control patients undergoing hemicolectomy for non obstructive cancer and from 4 patients with slow transit constipation. Twelve μm sections of fresh frozen tissue were fixed in 25% acetic acid/ 75%ethanol(v/v), and doubly labeled with a mouse monoclonal antibody for Kit (Lab Vision) and a rabbit polyclonal antibody for Ano1 (Abcam). ICC bodies were identified by morphology, location in the muscle layer and the presence of a DAPI-labeled nucleus. Labeled cells were counted from 13 fields in the circular muscle layer from 3 sections for each patient (39 fields total) at 400X magnification. Results: Both the processes and somata of ICC were immunoreactive for Ano1. Mast cells were not positive for Ano1 labeling. There was near complete overlap between Kit and Ano1 labeling. Four out of 1291 cells with ICC morphology were positive for Ano1 and appeared negative for Kit. These cells were identified in tissues from both groups of patients. Tissues from patients with slow transit constipation contained significantly fewer Ano1-positive ICC than control tissues (1.89±0.13 vs 6.43±0.37 cells per field, P <0.05, n = 4, unpaired t test). The numbers of ICC identified by Ano1 and Kit immunoreactivity were nearly identical across the range of ICC numbers from an average of 1.64 to 7.05 cells per field and correlated with an R2 value of 0.99. Conclusion: Ano1 is a reliable and sensitive marker for detecting changes in ICC networks in humans. Depletion of ICC in tissues from patients with slow transit constipation is reproducibly detected by Ano1 immunoreactivity. An advantage of Ano1 is that it does not label Kit-positive mast cells. The small number of Ano1-positive, Kit-negative cells detected in this study may represent labeling of ICC precursor cells or senescent ICC. Supported by DK57061.

268 Proliferation of Interstitial Cells of Cajal Occurs in Adult Mice and Is Dependent On 5HT2B Receptors and Protein Kinase C γ Vivek S. Tharayil, Mira M. Wouters, Jennifer E. Stanich, Michael D. Gershon, Luc Maroteaux, Simon J. Gibbons, Gianrico Farrugia Background: Normal gastrointestinal motility requires intact networks of interstitial cells of Cajal (ICC), the loss of which are associated with several motility disorders. ICC numbers are maintained by a balance between cell loss factors and survival/trophic/growth factors such as stem cell factor, NO and 5HT. We have previously shown that ICC express 5HT2B receptors. Activation of the 5HT2B receptor In Vitro increases numbers of proliferating ICC via protein kinase C γ (PKCγ). Aims: The aims of this study were to investigate whether proliferation occurs in adult ICC networks, whether proliferation and density of ICC networks are altered In Vivo in adult mice lacking 5-HT2B receptors and whether ICC derived from PKCγ knockout (KO) mice are able to proliferate when stimulated by 5HT2B receptor agonists. Methods: Whole mount preparations of the external jejunal muscle layers of 14, 4-week old mice, homozygous for KO of Htr2b gene expression and 14 congenic wild type (WT) controls were studied. ICC and proliferating ICC were identified by their expression, respectively, of Kit and Ki67. Confocal imaging was used to visualize Ki67-positive ICC. Ki67positive nuclei located within Kit-positive ICC were counted from 0.5 μm confocal slices in the deep muscular (DMP) and myenteric plexuses (MP). The volumes of Kit-positive ICC networks were determined by 3D reconstruction and volume rendering using Analyze™. In Vitro proliferation was studied in primary cultures of ICC from 3-5 day old PKCγ KO mice. Results: Proliferation of ICC was detected in ICC networks of adult mice. The numbers of proliferating ICC in the MP region of whole mounts prepared from 5HT2B KO mice were significantly lower than in WT controls (KO; 20.2 ± 3, WT; 33.0 ± 3.1 ICC/mm2, n =14, p<0.01). There was no difference in the DMP region (KO; 8.0 ± 0.9, WT; 7.7 ± 2.5 ICC/ mm2, n =14, p>0.05). The volume of ICC networks from 5HT2B KO mice was 38% decreased when compared to WT mice (KO; 4.1 ± 0.5%, WT; 6.6 ± 0.6% of total image volume, n = 6, p<0.05). In primary culture, ICC derived from PKCγ KO mice did not proliferate in response to the 5HT2B agonist BW723C86 (vehicle 17.9 ± 1.1%, 50 nM BW 15.0 ± 1.1%, n = 4, p>0.05). Conclusions: ICC proliferate in adult mice. Endogenous activation of 5HT2B receptors results in an increase In Vivo in proliferation of ICC-MP but not of ICC-DMP. The decrease in proliferation of ICC due to lack of 5HT2B receptor signaling reduces the density of ICC networks in mature mice. Supported by NIH DK57061 and NS12969.

266 Accelerated Turnover of Interstitial Cells of Cajal (ICC) from Local Progenitors in a Mouse Model of Gastrointestinal Stromal Tumors (GIST) Michael R. Bardsley, Laura Popko, David L. Young, Gianrico Farrugia, Brian P. Rubin, Tamas Ordog Most GISTs originate from the ICC lineage as a result of activating mutations in Kit, the receptor for stem cell factor (Kitl), a key developmental, growth and survival factor for ICC. However, the exact mechanisms leading to aberrant ICC growth remain unclear. Recently we identified gastric KitdimCD44+CD34+ cells as progenitors of ICC and described their requirements for self-renewal and differentiation into mature, Kit+CD44+CD34- ICC (Gastroenterology 2008; 134: 1083-93). The aim of this study was to investigate the role of these cells in ICC hyperplasia in mice homozygous for the oncogenic activating mutation KitK641E (Cancer Res 2005; 65: 6631-9), a model of human familial GIST syndrome. We also studied W/Wv and Sl/Sld mice, which harbor hypomorphic mutations of Kit and Kitl, respectively. ICC and their progenitors in the gastric corpus and antrum were studied by confocal microscopy and quantified by flow cytometry. In homozygous KitK641E:Neo mice, Kit+ myenteric ICC were grossly hyperplastic. In contrast, ICC were very rare in the circular muscle layer and missing from their usual location at the antral submucosal surface. In both wildtype and mutant mice, all ICC were also positive for Ano1, a novel ICC marker. In wildtype mice, ICC progenitors were detected as small, round or oval-shaped cells with short or no processes that co-expressed Kit, Ano1 and CD34 and occurred as rare solitary cells or in small clusters within, or on the submucosal surface of, the circular muscle layer. In homozygous KitK641E:Neo mice there was a marked increase in the number of these cells predominantly in the perivascular space surrounding small blood vessels. Flow cytometry

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showed a significant increase in the absolute number of KitdimCD44+CD34+ progenitors, Kit+CD44+CD34+ late precursors and Kit+CD44+CD34- mature ICC (P<0.03 for all comparisons). The frequency of ICC increased from 5.7[4.7;6.1]% to 19.3[17.4;20.4]% (median[25th;75th percentiles]) of all cells counted (P<0.03). In both W/Wv and Sl/Sld stomachs, KitdimCD44+CD34+ ICC progenitors were unaffected. However, there was a significant decrease in late precursors in Sl/Sld mice (P=0.015) and ICC were reduced in both mutants (P<0.02). In conclusion, the KitK641E oncogenic mutation leads to ICC hyperplasia by increasing the number of KitdimCD44+CD34+ progenitors and stimulating their differentiation into ICC. Consistent with our previous report, these progenitors do not depend on Kit signaling but need membrane-bound Kitl for proper development into Kit+CD44+CD34+ late ICC precursors. W/Wv mutation seems to only affect the final maturation of ICC and, likely, their maintenance. Supported by NIH Grant DK58185.

of GIP. Methods: Immunohistochemistry was used to compare expression patterns in rodents and zebrafish (ZF), the latter one of the earliest vertebrates to possess a distinct extraintestinal pancreas. GIP promoter activity was assessed using luciferase-reporter and gel shift assays. Results: In contrast to the intestinal expression of GIP in rodents, immunohistochemical analysis demonstrated that ZF GIP was expressed principally in the pancreas and, to a lesser extent, in the intestine. Although the proximal 290-bp portion of the GIP promoter is highly conserved among mammals, no significant homology to the ZF GIP promoter was demonstrated. However, a cloned ZF GIP 1072-bp promoter sequence directed a high level of expression in both GTC-1 and βTC-3 cells, mammalian cell lines that express endogenous GIP. Analysis of the ZF GIP promoter identified two potential cis-regulatory elements, which when deleted, reduced promoter activity significantly. The upstream region contained a Nkx2.5 binding motif, which belongs to a transcription factor family implicated in pancreaticspecific gene expression. When this region was mutated, ZF GIP promoter activity was reduced by ~75%. In contrast, no Nkx motif was identified in rodent GIP promoters. The downstream region was identified as a GATA-4 site, which we previously have shown to play a critical role in regulating rat GIP transcriptional activity. When the ZF GATA site was mutated or when GTC-1 cells were transfected with a GATA-4 -specific shRNA, GIP promoter activity was attenuated by ~85%, compared to the full-length promoter. Summary and Conclusion: The ZF GIP promoter was capable of directing gene expression in mammalian cells, indicating the conservation of transcription regulators through evolution. Similar to mammals, GATA-4 appears to be an important transcriptional activator of the ZF GIP promoter. Finally, the importance of Nkx2.5 appears evident by differences in organ-specific expression patterns in the rat and ZF. This divergence in the transcriptional regulation of GIP is consistent with the hypothesis that the enteroinsular axis in ZF has yet to fully evolve.