Proliferation Of Interstitial Cells Of Cajal Research Articles

Knockdown of miR-19a suppresses gastrointestinal dysmotility diarrhea after TBI by regulating VIP expression.

Traumatic brain injury (TBI) is the main cause of death and can lead to a variety of physiological complications, including gastrointestinal dysfunction. The present study aimed to confirm the miR-19a-mediated suppression of diarrhea after TBI through the regulation of VIP expression. A rat model of TBI induced by controlled cortical injury was used to observe gastrointestinal morphology by opening the abdomen after TBI. After 72h of injury, the fecal water content of the rats was measured. The end ileal segments were removed, and HE staining was used to observe the histopathological changes in the intestine. The levels of serum miR-19a and VIP mRNA were detected by qRT-PCR. ELISA was performed to detect VIP levels in serum. Immunohistochemistry was used to detect the level of VIP in ileal tissues, and immunofluorescence was used to detect c-kit expression in ileal tissue. CCK-8 assay was used to detect the cell viability of interstitial cells of Cajal (ICCs), and TUNEL assay was used to detect apoptosis of ICCs. miR-19a and VIP were highly expressed in the serum of TBI rats, and the knockdown of miR-19a alleviated TBI-induced diarrhea. In addition, the overexpression of miR-19a or VIP inhibited the proliferation of ICCs, promoted apoptosis, and suppressed intracellular Ca2+ levels, whereas miR-19a suppression had the opposite effects. A nonselective nitric oxide synthase inhibitor (L-NA), PKG inhibitors (KT-5823 and RP-8CPT-cGMPS), and a guanylate cyclase inhibitor (ODQ) restored the inhibitory effects of VIP on ICC proliferation, anti-apoptosis effects, and Ca2+ concentrations. Knockdown of miR-19a inhibits activation of the VIP-NO-cGMP-PKG pathway through suppression of VIP expression, which in turn inhibits diarrhea after TBI.

Effects of Banxia Xiexin Decoction on apoptosis of interstitial cells of Cajal via regulation of MiR-451-5p: An in vivo and in vitro study

Ethnopharmacological relevanceBanxia Xiexin Decoction (BXD) is a traditional Chinese medical formula applied to gastrointestinal (GI) motility disorders. Previous studies showed that miR-451-5p was down-regulated in rats with GI motility disorders induced by gastric electrical dysrhythmia. Interstitial cells of Cajal (ICCs) are pacemakers for GI motility, while loss of ICCs is responsible for GI motility disturbance. Thus, the underlying interaction mechanisms for BXD regulating ICCs apoptosis via miR-451-5p remain to be explored. Aim of the studyIn this work, the main objectives were to examine the efficacy of BXD on ICCs via miR-451-5p both in GI motility disorders rats model and in vitro, as well as the potential contributions of SCF/c-kit signaling. Materials and methodsRats with gastric electrical dysrhythmia were established in male SD rats by using a single-day diet and a double fasting method (drinking diluted hydrochloric acid water during the period) for 4 weeks. The gastric slow wave (GSW) recording, RT-qPCR, and western blot were performed to examine the effects of BXD on ICCs apoptosis in rats with GED and miR-451-5p expression. In vitro assays included CCK-8, flow cytometry analysis, RT-qPCR, and western blot were applied to investigate the potential molecular mechanism of BXD on ICCs apoptosis via miR-451-5p. ResultsBXD promoted gastric motility, reduced ICCs apoptosis, and elevated miR-451-5p in GED rats. In addition, miR-451-5p was significantly up-regulated in ICCs after BXD treatment compared with that in ICCs with miR-451-5p inhibitor transfection. Meanwhile, high miR-451-5p expression with either BXD treatment or miRNA mimics enhanced ICCs proliferation and inhibit apoptosis. Moreover, overexpression of miR-451-5p can reverse G0/G1 arrest in ICCs by BXD treatment. Further, SCF and c-kit protein levels were detected to demonstrate that modulation of miR-451-5p by BXD treatment was involved in this signaling. ConclusionsThrough this study, we demonstrated that BXD could promote ICCs proliferation and inhibit apoptosis via miR-451-5p and may involve the modulations of SCF/c-kit signaling, thus suggesting a new therapy basis for GI motility dysfunction from the perspective of modulation of ICCs apoptosis by targeting miR-451-5p.

Gastric Electrical Stimulation Increases the Proliferation of Interstitial Cells of Cajal and Alters the Enteric Nervous System in Diabetic Rats

BackgroundLack of interstitial cells of Cajal (ICC) and neuropathy were the most possible pathological mechanisms of diabetic gastroparesis. Gastric electrical stimulation (GES) is a promising way to treat gastroparesis. This study aimed to explore the impact of GES on ICC together with enteric neurons in diabetic rats and the possible mechanisms involved. Materials and MethodsA total of 60 rats were randomized into six groups, including the normal control group, diabetic group (DM), diabetic with sham GES group (DM + SGES), and three groups of diabetic rats with GES (DM + GES1, DM + GES2, and DM + GES3). The proliferation of ICC and expressions of 5-hydroxytryptamine (serotonin) receptor 2B (5-HT2B), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (CHAT), protein gene product 9.5, and glia cell line–derived neurotrophic factor (GDNF) in the antrum of the stomach were evaluated by immunofluorescence staining or Western blot. The levels of 5-HT in blood and tissue were determined by enzyme-linked immunosorbent assay. ResultsThe proliferation of ICC was significantly reduced in the DM group, together with the DM + SGES group, but increased in the three DM + GES groups. The expression of 5-HT2B was decreased in the DM group and enhanced in the DM + GES groups. Similarly, the levels of 5-HT in the blood and distal stomach tissue were increased in the DM + GES groups. Both nNOS labeled neurons and CHAT-positive neurons were reduced in the myenteric plexus of the DM group, whereas these neurons were dramatically increased the in DM + GES groups. The expression of GDNF protein in the diabetic rats was down-regulated, whereas GES increased the expression of GDNF. ConclusionsGES improves the proliferation of ICC possibly related with the 5-HT/5-HT2B signal pathway and alters the enteric nervous system partly though the GDNF expression.

SCF/c-kit signaling pathway participates in ICC damage in neurogenic bladder

ABSTRACT Neurogenic bladder (NB) is a type of double renal dysfunction caused by nerve lesions. The interstitial cells of Cajal (ICC) damage are involved in bladder dysfunction. The aim of this study is to investigate the effect of stem cell factor (SCF)/c-kit signaling pathway on ICC damage in NB model rats. Maximum cystometric capacity (MCC), bladder leak point pressures (BLPP), and bladder compliance (BC) were measured in sham-operated and NB model rats. Immunofluorescent staining for c-kit was performed to determine ICC count in rat bladder trigone. The morphology and ultrastructure changes of ICCs were observed under an electron microscope. The mRNA levels of c-kit and SCF in bladder tissues were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein levels of c-kit, SCF, p-JAK, p-STAT1, and p-STAT3 in bladder tissues were determined by western blot. ICC proliferation was detected by CCK-8 assay. NB resulted in changes in ultrastructure changes of ICCs and a decrease in the number of ICCs and in expression of c-kit, SCF, p-JAK, p-STAT1, and p-STAT3 in NB tissues. Inhibition of SCF/c-kit signaling pathway suppressed ICC proliferation by inhibiting JAK/STAT3 pathway. Moreover, inhibition of SCF/c-kit signaling pathway impaired the SCF-induced attenuation of ICC damage in NB model rats. Collectively, our data indicate that SCF/c-kit signaling pathway participates in ICC damage in NB.

EAVK segment “c” sequence confers Ca2+-dependent changes to the kinetics of full-length human Ano1

Anoctamin1 (Ano1 and TMEM16A) is a calcium-activated chloride channel specifically expressed in the interstitial cells of Cajal (ICC) of the gastrointestinal tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full-length human Ano1 sequence includes an additional exon, exon "0," at the NH2 terminus. Ano1 with exon 0 [Ano1(0)] had a lower EC50 for intracellular calcium ([Ca2+]i) and faster chloride current (ICl) kinetics. The Ano1 alternative splice variant with segment "c" encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alter ICl at low [Ca2+]i Exon 13 is expressed in 75-100% of Ano1 transcripts in most human tissues but only 25% in the human stomach. Our aim was to determine the effect of EAVK deletion on Ano1(0)ICl parameters. By voltage-clamp electrophysiology, we examined ICl in HEK293 cells transiently expressing Ano1(0) with or without the EAVK sequence [Ano1(0)ΔEAVK]. The EC50 values of activating and deactivating ICl for [Ca2+]i were 438 ± 7 and 493 ± 9 nM for Ano1(0) but higher for Ano1(0)ΔEAVK at 746 ± 47 and 761 ± 26 nM, respectively. Meanwhile, the EC50 values for the ratio of instantaneous to steady-state ICl were not different between variants. Congruently, the time constant of activation was slower for Ano1(0)ΔEAVK than Ano1(0) currents at intermediate [Ca2+]i These results suggest that EAVK decreases the calcium sensitivity of Ano1(0) current activation and deactivation by slowing activation kinetics. Differential expression of EAVK in the human stomach may function as a switch to increase sensitivity to [Ca2+]i via faster gating of Ano1.NEW & NOTEWORTHY Calcium-activated chloride channel anoctamin1 (Ano1) is necessary for normal slow waves in the gastrointestinal interstitial cells of Cajal. Exon 0 encodes the NH2 terminus of full-length human Ano1 [Ano1(0)], while exon 13 encodes residues EAVK on its first intracellular loop. Splice variants lack EAVK more often in the stomach than other tissues. Ano1(0) without EAVK [Ano1(0)ΔEAVK] has reduced sensitivity for intracellular calcium, attributable to slower kinetics. Differential expression of EAVK may function as a calcium-sensitive switch in the human stomach.

Stem Cell Factor/Kit Signal Insufficiency Contributes to Hypoxia-Induced Intestinal Motility Dysfunctions in Neonatal Mice.

Gastrointestinal (GI) motility disorders represent a group of problems that more constantly encountered in preterm infants. However, whether hypoxia exposure contributes to the GI dysfunctions is still unclear. Newborn mice were exposed to hypoxia (10%) from P1 to P7. Intestinal motilities were examined by a strain gauge transducer. The proliferation of ICCs was detected by using immunostaining for BrdU, Ki67, Kit, Ano1, and insulin-like growth factor 1 receptor (IGF-1R+). Smooth muscle cells and enteric neurons were revealed by immunostaining for α-SMA and NF200, respectively. Apoptosis was assessed by TUNEL assay. Kit signal pathway was examined by western blot and qPCR. Intestinal motilities were found weakened significantly in the hypoxic small intestines as compared to controls on P8. Kit+ or Ano1+ interstitial cells of Cajal (ICCs) were found obviously decreased in the myenteric ICCs (ICC-MY) of neonatal mice after exposed to hypoxia. A large number of ICC progenitors (IGF-1R+) were found highly mitotic (BrdU+ Ki67+) to populate ICC during early postnatal development in the normoxic mice. We found the ICC proliferation was significantly inhibited upon hypoxia exposure, without increasing apoptosis (TUNEL+). We next identified that Kit phosphorylation was inhibited 3days after hypoxia exposure. The inhibition of Kit signaling was largely due to decreased the expression of the ligand of Kit receptor, stem cell factor (SCF), in the intestinal walls. Exposure to imatinib, a Kit receptor inhibitor, for 3days from P4 phenocopied the effect of hypoxia on the neonatal pups that resulted in inhibited intestinal motilities and decreased Kit+ ICC numbers. All together, our findings indicate the SCF/Kit signaling insufficiency may contribute to the underdevelopment of ICCs and intestinal motility dysfunction upon hypoxia exposure. The decease in ICC density is likely due to the cell cycle arrest of ICC progenitor cells.

Role of stem cell factor in the regulation of ICC proliferation and detrusor contraction in rats with an underactive bladder.

Stem cell factor (SCF) is critical in regulating the proliferation, differentiation and function of the interstitial cells of Cajal (ICCs), which are closely associated with smooth muscle dysfunction. The present study aimed to examine the effect of SCF on ICC proliferation and detrusor contraction in rats with an underactive bladder. Sprague-Dawley rats were divided into four groups comprising control, control+SCF, detrusor underactivity (DU), and DU+SCF groups. The ICC count was determined using immunofluorescence; serum levels of SCF were determined using an enzyme-linked immunosorbent assay; mRNA and protein levels of c-kit and SCF in tissues were assessed using reverse transcription-quantitative polymerase chain reaction and western blot analyses, respectively. Detrusor contractility was determined using muscle strips, based on the contraction amplitude and frequency determined in each specimen. Significantly fewer ICCs were observed in the DU group, in addition to decreased expression levels of SCF and c-kit, compared with the control group. In addition, the detrusor contraction frequency and amplitude were markedly reduced. However, the administration of SCF significantly increased the number of ICCs, and the levels of SCF and c-kit in animals with DU, and resulted in markedly amplified detrusor contraction frequency and amplitude. Similarly, the number of ICCs and levels of SCF and c-kit were higher in the control+SCF group, compared with the control group. Overall, these findings suggested that exogenous SCF improved the organ dysfunction caused by reduced ICC number, providing a novel approach for organ repair.

Effects of Exogenous Interleukin-9 on the Growth, Proliferation and Activity of Interstitial Cells of Cajal Cultured in vitro

Objectives: To evaluate the role of exogenous interleukin-9 (IL-9) in the growth, proliferation and activity of interstitial cells of Cajal (ICCs) cultured in vitro, and to assess its role in maintaining the functions of ICCs. Methods: ICCs of murine gastric antrum were isolated and cultured in vitro. ICCs were identified with c-Kit and ANO1 immunofluorescent antibodies. Both fluorescence microscope and confocal microscopy were used to observe the effects of IL-9 on the growth, proliferation and activity of ICCs in cultured in vitro. ICCs were loaded with fluorescence probe Fluo-3/AM and the fluorescence of intracellular calcium concentration ([Ca²⁺]_i) was measured by confocal microscopy. The effects of exogenous IL-9 on the sulfated cholecystokinin octapeptide (CCK-8S)-evoked [Ca²⁺]_i elevation were observed by confocal microscopy. Results: Immunofluorescence results confirmed the successful separation and culture of ICCs in vitro. IL-9 in concentrations ranging from 0.02 to 1 μg/ml promoted the growth, proliferation and activity of ICCs in culture, and ICCs grew best with 0.5 mg/ml of IL-9. The presence of IL-9 could significantly increase the CCK-8S-evoked [Ca²⁺]_i oscillation, which is probably caused by facilitating the maintenance of the functions of ICCs under suitable conditions for culture. Conclusion: IL-9 could promote the growth, proliferation and activity of ICCs, reinforce the CCK-8S-induced [Ca²⁺]_i increment in ICCs, and facilitate the maintenance of the functions of ICCs under suitable culture condition.

Su2023 IL-4 via STAT6 Regulates a Promoter for the Human Ano1 Gene

Background: Ano1 is a Ca2+ activated Clchannel expressed in interstitial cells of Cajal (ICC) in the smooth muscle layers of the GI tract. Ano1 has been shown to play an important role in the electrical activity of ICC, where it is recognized as the primary channel responsible for the endogenous Ca2+-activated Clcurrent, and also plays a role in ICC proliferation. Multiple isoforms have been described for Ano1 with diverse electrophysiological properties. Relative expression levels of isoform are altered in diabetic gastroparesis thus potentially affecting the function of the channel, however the mechanisms underlying the differential expression of Ano1 are not known. Recently, we identified a region with promoter activity adjacent to a new exon of human Ano1 upstream of the published exon 1. Aim: The aim of the present study was to characterize the Ano1 promoter to gain insight into the regulatory mechanisms governing the expression of Ano1. Methods: Consensus sequences for core promoter and potential regulatory elements were identified in silico. A luciferase reporter assay was used for the evaluation of the activity of the wild-type and mutant promoters and chromatin immunoprecipitation (ChIP) assays were used to characterize the recruitment of RNA polymerase II (PolII) and transcription factors of interest to Ano1 promoter. Results: When expressed in HEK293 cells, the putative promoter region representing 1.8 kb upstream of the novel transcription start site (TSS) drove a significant increase in the luciferase activity under basal conditions (6.7±2.1 fold). IL-4, a known regulator of Ano1 expression, further increased promoter activity (1.6 ± 0.02 fold over vehicle control). Using deletion mutant constructs we identified a 405 bp region as the minimal functional promoter. Bioinformatics analyses identified core promoter elements in this region. No TATA box was found but there was an initiator element (INR) 30 bp upstream of the TSS, as well as a CpG island. ChIP assay using antibodies against the activated form of PolII, phosphorylated on serine 5, confirmed that the TSS identified by 5' RACE was functional and located 202 bp upstream of the translational start codon. The promoter region contains putative binding sites for multiple transcription factors including STAT6, a downstream effector of IL-4 signaling. A combination of luciferase experiments, site directed mutagenesis, siRNA knockdown and ChIP confirmed the involvement of STAT6 in the IL-4 regulation of the Ano1 gene. Conclusions: In conclusion, a promoter for the human Ano1 gene identified upstream of a newly described exon drives Ano1 expression and is modulated by IL4 via STAT6. Identification of the human Ano1 promoter and its regulation allows further understanding of the regulation of Ano1 and changes in expression associated with disease. Supported by NIH grant DK57061.

789 A Novel Exon of the Human Calcium-Activated Chloride Channel Ano1 Imparts Greater Sensitivity of Cl-Current to Intracellular CA2+

INTRODUCTION: Anoctamin 1 (Ano1, also known as TMEM16A) is a Ca 2+-activated Cl channel expressed on interstitial cells of Cajal (ICC). The mechanisms by which Ca 2+ regulates Ano1 are still controversial and not well understood. Ano1 is required for normal slow wave activity in the gastrointestinal tract and involved in regulating cell proliferation of ICC. Splice variants of Ano1 have varying electrophysiological properties and altered expression in diabetic gastroparesis. Recently, we identified a transcript for Ano1 with a novel exon (exon 0) upstream of exon 1 with a transcriptional start (TSS) site that is in frame with the previously assumed TSS of human Ano1. The electrophysiological properties of the resulting full length Ano1 are unknown. AIM: Determine the functional contribution of the newly identified exon to the electrophysiological properties of Ano1. METHODS: Constructs with (+0) or without (-0) the newly identified exon of Ano1 were conjugated to red fluorescent protein (RFP) and transfected into HEK293 cells. Voltage clamp electrophysiology was used to record whole cell Clcurrents, elicited by +20 mV steps to -100 through +100 mV from the holding potential of -100 mV. Baseline Clcurrent densities of non-fluorescent HEK cells did not exceed 10 pA/pF or -2 pA/pF during activation or deactivation, respectively. The extracellular solution contained (in mM): 150 Cl -, 150 NMDG + or 150 Cs +, 10 HEPES, 5.5 glucose, and 2.5 Ca. The intracellular solution contained (in mM): 150 Cl-, 150 Cs+, and 10 HEPES. Intracellular Ca2+ concentrations ([Ca ]i, 10-500 nM) were obtained by buffering with EGTA (2 mM) based on calculations using Maxchelator software (http://www.stanford.edu/~cpatton/). RESULTS: Constructs containing the newly identified exon generated Cl currents that were more sensitive to regulation by free intracellular Ca 2+. At 500 nM [Ca ]i, the whole cell Cl current densities recorded during a +100 mV activation step from cells expressing Ano1 (+0) were larger than currents from cells expressing Ano1(-0) (412±82 vs. 118±24 pA/pF, respectively; n=5, P 0.05). CONCLUSION: A novel exon upstream of the previously assumed transcriptional start site of Ano1 imparts greater sensitivity of Cl current to intracellular Ca 2+ concentrations. Supported by NIH grant DK57061 and the Mayo Clinic Center for Cell Signaling in Gastroenterology (NIDDK P30DK084567).

Roles of interleukin-9 in the growth and cholecystokinin-induced intracellular calcium signaling of cultured interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal (GI) tract and loss of ICC is associated with many GI motility disorders. Previous studies have shown that ICC have the capacity to regenerate or restore, and several growth factors are critical to their growth, maintenance or regeneration. The present study aimed to investigate the roles of interleukin-9 (IL-9) in the growth, maintenance and pacemaker functions of cultured ICC. Here, we report that IL-9 promotes proliferation of ICC, and culturing ICC with IL-9 enhances cholecystokinin-8-induced Ca2+ transients, which is probably caused by facilitating maintenance of ICC functions under culture condition. We also show co-localizations of cholecystokinin-1 receptor and IL-9 receptor with c-kit by double-immunohistochemical labeling. In conclusion, IL-9 can promote ICC growth and help maintain ICC functions; IL-9 probably performs its functions via IL-9 receptors on ICC.

Electroacupuncture at ST36 Ameliorates Gastric Emptying and Rescues Networks of Interstitial Cells of Cajal in the Stomach of Diabetic Rats

Depletion of interstitial cells of Cajal (ICC) is certified in the stomach of diabetic patients. Though electroacupuncture (EA) at ST36 is an effective therapy to regulate gastric motility, the mechanisms of EA at ST36 on gastric emptying and networks of ICC remain to be elucidated. The aims of this study were to investigate the effects of EA on gastric emptying and on the alterations of ICC networks. Rats were randomized into the control, diabetic rats (DM), diabetic rats with sham EA (DM+SEA), diabetic rats with low frequency EA (DM+LEA) and diabetic rats with high frequency EA groups (DM+HEA). The expression of c-kit in each layer of gastric wall was assessed by western blotting. The proliferation of ICC was identified by immunolabeling of c-kit and Ki67 as the apoptosis of ICC was examined by TUNEL staining. The results were as follows: (1) Gastric emptying was severely delayed in the DM group, but accelerated in the LEA and HEA group, especially in the LEA group. (2) The expression of c-kit in each layer was reduced apparently in the DM group, but also up-regulated in the LEA and HEA group. (3) Plentiful proliferated ICC (c-kit+/Ki67+) forming bushy networks with c-kit+ cells were observed in the LEA and HEA group, while the apoptotic cells (c-kit+/TUNEL+) were hardly captured in the LEA and HEA group. Collectively, low and high frequency EA at ST36 rescue the damaged networks of ICC by inhibiting the apoptosis and enhancing the proliferation in the stomach of diabetic rats, resulting in an improved gastric emptying.

Electroacupuncture Regulates Apoptosis/Proliferation of Intramuscular Interstitial Cells of Cajal and Restores Colonic Motility in Diabetic Constipation Rats

Injury of interstitial cells of Cajal (ICC) is associated with gut dysmotility in diabetic rats. We have shown an acceleration of the colonic contractility by electroacupuncture stimulation (EAS). However, little is known about potential roles of EAS on colonic transit and ICC. In this study, we evaluate the effect of EAS on colonic transit and investigate whether apoptosis/proliferation of ICC was involved in regulative effect of EAS on colonic transit. Rats were randomly assigned to normal, diabetic, diabetic-plus-sham stimulation, diabetic-plus-low-frequency stimulation, and diabetic-plus-high-frequency stimulation groups. Bead expulsion test was used for measuring the distal colonic transit. The Kit (ICC marker) was detected by western blot. Apoptotic ICC was detected by terminal dUTP nucleotide end labeling. Proliferating ICC was identified by Kit/Ki67 double immunofluorescent staining on whole mount preparations. Ultrastructure changes of ICC were studied using electron microscopy. Results showed that high-frequency stimulation significantly promoted colonic transit. Low- and high-frequency stimulation markedly rescued intramuscular ICC from apoptosis. Abundant proliferating intramuscular ICC was found in low- and high-frequency stimulation groups. Our results indicate that high-frequency EAS has stimulatory effect on the distal colonic transit, which may be mediated by downregulation of the apoptosis and upregulation of the proliferation of intramuscular ICC.

Inhibition of cell proliferation by a selective inhibitor of the Ca2+-activated Cl− channel, Ano1

BackgroundIon channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca2+-activated Cl− channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16Ainh-A01 as a specific inhibitor of Ano1. AimTo investigate the effect of the T16Ainh-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1. MethodsInhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16Ainh-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively. ResultsT16Ainh-A01 inhibited Ca2+-activated Cl− currents by 60% at 10μM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16Ainh-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16Ainh-A01. In organotypic cultures of smooth muscle strips from mouse jejunum, the proliferation of ICC was reduced but the total number of proliferating cells/confocal stack was not affected, suggesting that the inhibitory effect was specific for ICC. ConclusionsThe selective Ano1 inhibitor T16Ainh-A01 inhibited Ca2+-activated Cl− currents, reduced the number of proliferating ICC in culture and inhibited proliferation in the pancreatic cancer cell line CFPAC-1. These data support the notion that chloride channels in general and Ano1 in particular are involved in the regulation of proliferation.

Late Embryonic and Postnatal Development of Interstitial Cells of Cajal in Mouse Esophagus: Distribution, Proliferation and Kit Dependence

This paper investigates alterations in interstitial cells of Cajal (ICC) in the esophagus of mice from embryonic day 13.5 (E13.5) to 36 days postpartum (P0–P36) using immunohistochemistry. At E13.5, Kit+ cells presented in clusters and differentiated into spindle-like cells with biopolar processes within the outer (longitudinal) and inner (circular) muscle layers at E17.5. These Kit+ ICC with long processes were also Ano1+ and prominent at birth. The density of ICC gradually decreased, and at P36 it became about one twentieth of that at birth. Kit ligand (stem cell factor) expression is lower in striated muscle cells than that in smooth muscle cells. The ICC number was higher in the distal (close to the cardia) than in the proximal esophagus (close to the pharynx). Some Kit+/Ki67+ and Kit+/bromodeoxyuridine+ cells were observed within the muscle layers, and proliferation persisted from birth through adulthood (P28) with a gradually decreasing cell number. At 24 h, Kit+ ICC were dramatically decreased and almost missing 48 h after administration of imatinib (a Kit inhibitor). Our results indicate that ICC proliferation is age dependent and persists throughout the postnatal period. There is a dramatic decrease in the ICC number from P0 to adult life. The Kit signal is essential for the postnatal development of ICC in the esophagus.

Ano1 as a regulator of proliferation

Ano1 is a Ca2+ activated Cl− channel expressed in several proliferating cell types including various epithelia, gastrointestinal stromal tumors, interstitial cells of Cajal (ICC) and CFPAC‐1 cells. The aim of this study was to determine if Ano1 regulates proliferation. ICC from Ano1(−/−) and Ano1(+/+) mice were cultured with Cl− channel inhibitors or in low Cl− media. CFPAC‐1 cells grown in low Cl− media were pulsed with EdU for 1 h. Incorporation was measured by flow cytometry. For immunoblots on mouse jejunum, antibodies to pRb, phospho(Ser773)‐pRb and GAPDH were used.Fewer proliferating ICC were detected in Ano1(−/−) cultures (15 ± 3.5% Ki67+ ICC vs 25 ± 4% in Ano1(+/+), mean ± SEM, n = 4, p < 0.05). Cl− channel inhibitors DIDS (10 μM), niflumate (10 μM) and tamoxifen (1 μM) decreased ICC proliferation by 53, 53 and 59%, respectively (p < 0.05). This effect was reduced in cultures from Ano1(−/−) mice (29% and 19 % decrease, 22% increase, p > 0.05). Fewer proliferating ICC and CFPAC‐1 cells were detected in low Cl− media (% EdU+ ICC: 120 mM; 19.8 ± 5.3, 12 mM; 8.5 ± 3.2, n = 4, and % EdU+ CFPAC‐1: 120 mM; 29.5, 12 mM; 14.3 p < 0.05). Proportionally less Ser773‐phosphorylated pRb was detected in jejunum from Ano1(−/−) mice (Ano1(+/+); 5.98 ± 0.78, Ano1(−/−); 3.60 ± 0.49, mean ± SEM, n = 7, p < 0.05). In conclusion, Ano1 regulates proliferation possibly by promoting the G1/S transition of the cell cycle. Support: DK57061 and DK68055.

Exogenous stem cell factor improves interstitial cells of Cajal restoration after blockade of c-kit signaling pathway

Objective. Interstitial cells of Cajal (ICC) have been endowed with considerable intrinsic plasticity. Blockade of the c-kit signaling pathway results in the shift of ICC towards a smooth muscle-like phenotype. Little is known about stem cell factor (SCF), the ligand of c-kit, and the role it plays in the process of restoration. The aim of this study was to determine whether exogenous SCF can promote ICC replenishment following the blockade of c-kit signaling. Material and methods. Neutralizing anti-c-kit monoclonal antibody (ACK2) was administered to mice for 8 days after birth. Jejunal muscle strips were cultured up to 7 days. Electrical rhythmic changes were monitored and ICC were examined by immunohistochemistry. Expression of c-kit mRNA was detected by reverse transcriptase-polymerase chain reaction, and expression of Kit protein was detected by Western blot. Results. When c-kit receptors were blocked, ICC nearly disappeared from the jejunum accompanied by the loss of electrical slow waves. By day 7, after in vitro culture with SCF (100 ng/ml), the amplitude of muscle strip slow waves was restored to 0.19 ± 0.07 mV (p < 0.05), whereas the frequency recovered to 13.7 ± 3.32/min (p < 0.01). Furthermore, labeling for c-kit+ cells in the myenteric plexus increased and c-kit mRNA and protein expression were up-regulated compared to that of non-treatment with SCF. Conclusions. The c-kit signaling pathway, activated by SCF, is the critical pathway associated with the control of ICC survival and proliferation. The restoration of ICC number and jejunal electrical rhythm, resulting from blockade of the c-kit signaling pathway, could be facilitated by local SCF administration.

Hydrogen Sulfide, a Gaseous Transmitter, Stimulates Proliferation of Interstitial Cells of Cajal via Phosphorylation of AKT Protein Kinase

Interstitial cells of Cajal (ICC) are distributed throughout the gastrointestinal (GI) tract and have important functions in the control of GI motility. Loss of ICC is associated with several GI motility disorders; yet, the mechanisms modulating ICC survival and proliferation are not fully understood. Hydrogen sulfide (H(2)S) has been reported to be a gaseous transmitter that regulates cellular proliferation. This study aims to establish whether H(2)S participates in regulation of ICC proliferation. The effect of H(2)S was studied in primary cultures of ICC, prepared from the mouse small intestine. To determine the extent of ICC proliferation, we used immunofluorescent staining to study alterations in the number of cells expressing c-Kit(+) and CD44(+), markers for mature ICC. Phosphorylation of Akt was measured by Western blot analysis. Treatment with low concentrations of NaHS (H(2)S donor, 1-30 microM) showed no apparent toxicity, as judged from cell numbers. Importantly, treatment with NaHS (15 microM) for 24 hours increased the numbers of c-Kit(+)/CD44(+) ICC by 23.3 +/- 1.4% (P < 0.05). Moreover, NaHS increased Akt phosphorylation, which was prevented with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (5 microM). LY294002 also blocked the NaHS-mediated increase in the number of ICC. In addition, H(2)S enhanced the proliferation of mature ICC in the in vitro culture system used here in a concentration-dependent manner. The present study suggests that H(2)S may be a critical factor in maintaining ICC numbers and may have a novel, Akt-dependent role in proliferation of mature ICC.

Lack of serotonin 5-HT2Breceptor alters proliferation and network volume of interstitial cells of Cajalin vivo

Normal gastrointestinal motility requires intact networks of interstitial cells of Cajal (ICC). Interstitial cells of Cajal numbers are maintained by a balance between cell loss factors and survival/trophic/growth factors. Activation of 5-HT(2B) receptors expressed on ICC increases ICC proliferation in vitro. It is not known whether 5-HT(2B) receptors on ICC are activated in vivo. The aims of this study were to investigate if adult ICC proliferate, whether the proliferation of ICC in vivo is affected by knocking out the 5-HT(2B) receptor, and if alterations in proliferation affect ICC networks. Proliferating ICC were identified by immunoreactivity for Ki67 in both the myenteric and deep muscular plexus regions of the jejunum in mice with a targeted insertion of a neomycin resistance cassette into the second coding exon of the htr2b receptor gene. Adult ICC do proliferate. The number of proliferating ICC was lower in the myenteric plexus region of Htr2b(-/-) compared to Htr2b(+/+) mice. The volume of Kit-positive ICC was 30% lower in the myenteric plexus region and 40% lower in the deep muscular plexus region in Htr2b(-/-) mice where the number of ICC was also reduced. Interstitial cells of Cajal proliferate in adult mice and activation of 5-HT(2B) receptors results in increased proliferation of ICC in vivo. Furthermore, lack of 5-HT(2B) receptor signaling reduces the density of ICC networks in mature mice. These data suggest that 5-HT(2B) receptor signaling is required for maintenance of ICC networks, adding 5-HT to the growing number of factors shown to regulate ICC networks.

Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life

This paper aimed at investigating the alterations in interstitial cells of Cajal (ICC) in the proximal, middle and distal colon of mice from 0-day to 56-day post-partum (P0-P56) by immunohistochemistry. The Kit(+) ICC, which situated around myenteric nerve plexus (ICC-MY) were prominent at birth, meanwhile those cells within the smooth muscle layers (ICC-IM) and in the connective tissue beneath serosa (ICC-SS) began to appear. ICC-SM, which located at the submucosal border of circular muscle layer emerged at P6 in the proximal colon and subsequently in the distal colon at P8, and ICC in the oral side of colon revealed an earlier development in morphology and a higher density than that in the anal side. The density of ICC altered obviously during postnatal period, and the estimated total amount of ICC increased approximately 30 folds at P56 than that at P0. Some Kit(+)/Ki67(+) and Kit(+)/BrdU(+) cells were observed in ICC-MY, ICC-IM and ICC-SS, but not in ICC-SM from P0 to P24. Our result indicates a proximal to distal and transmural gradient development of ICC in the postnatal colon along with a dramatic increase of ICC cell number from neonatal to adult life, and an age-dependent proliferation of ICC is also involved.