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1.
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Approximately 85% of GISTs harbor activating mutations of the KIT or PDGFRA receptor tyrosine kinases. PTEN and SHIP2 are major phosphatases that dephosphorylate PI(3,4,5)P3, one of the intracellular signal pathways downstream of KIT. PTEN is an important tumor suppressor, whereas the involvement of SHIP2 in cancer has been proposed based essentially on cell line studies. We have used a mouse model of GIST, i.e. KitK641E knock-in mice, resulting in the substitution of a Lys by Glu at position 641 of Kit. In homozygous KitK641E mice, PTEN-immunoreactivity (ir) in antrum was found in the hyperplastic Kit-ir layer. The same localization was found for SHIP2. Western blot analysis in antrum showed a large increase in PTEN expression in KitK641E homozygous mice as compared to wild type. In contrast, SHIP2 expression was not affected between the two genotypes. Erk1, but not PKB, phosphorylation appears to be upregulated in KitK641E homozygous mice. In the human GIST882 imatinib sensitive cell line, both PTEN and SHIP2 were expressed and showed, in part, a nuclear localization. The upregulation of PTEN in antrum in KitK641E mice might serve as a feedback mechanism to limit PI 3-kinase activation downstream of Kit in a context of oncogenic mutation.  相似文献   

2.
Gastrointestinal stromal tumors (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the KIT or PDGFRA receptor tyrosine kinases are present in the majority of GIST, leading to ligand-independent activation of the intracellular signal transduction pathways. We previously investigated the gene expression profile in the murine Kit(K641E) GIST model and identified Ntsr1 mRNA, encoding the Neurotensin receptor 1, amongst the upregulated genes. Here we characterized Ntsr1 mRNA and protein expression in the murine Kit(K641E) GIST model and in tissue microarrays of human GIST. Ntsr1 mRNA upregulation in Kit(K641E) animals was confirmed by quantitative PCR. Ntsr1 immunoreactivity was not detected in the Kit positive ICC of WT mice, but was present in the Kit positive hyperplasia of Kit(K641E) mice. In the normal human gut, NTSR1 immunoreactivity was detected in myenteric neurons but not in KIT positive ICC. Two independent tissue microarrays, including a total of 97 GIST, revealed NTSR1 immunoreactivity in all specimens, including the KIT negative GIST with PDGFRA mutation. NTSR1 immunoreactivity exhibited nuclear, cytoplasmic or mixed patterns, which might relate to variable levels of NTSR1 activation. As studies using radio-labeled NTSR1 ligand analogues for whole body tumor imaging and for targeted therapeutic interventions have already been reported, this study opens new perspectives for similar approaches in GIST.  相似文献   

3.

Introduction

Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3Cre/+, devoid of mast cells but with intact Kit signaling.

Design

The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. KitW-sh/W-sh and Cpa3Cre/+ mice, and by use of the mast cell stabilizer cromolyn.

Results

KitW-sh/W-sh mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3Cre/+ mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3Cre/+ mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3+/+). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI.

Conclusions

Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.  相似文献   

4.
Gastrointestinal stromal tumours (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the receptor tyrosine kinase KIT are present in most GIST. KIT K642E was originally identified in sporadic GIST and later found in the germ line of a familial GIST cohort. A mouse model harbouring a germline Kit K641E mutant was created to model familial GIST. The expression profile was investigated in the gastric antrum of the Kit K641E murine GIST model by microarray, quantitative PCR and immunofluorescence. Gja1/Cx43 , Gpc6 , Gpr133 , Pacrg , Pde3a , Prkar2b , Prkcq/Pkce , Rasd2 , Spry4 and Tpbg/5T4 were found to be up-regulated. The proteins encoded by Gja1/Cx43 , Pde3a , Prkcq/Pkce were localized in Kit-ir ICC in wild-type and Kit K641E animals while Spry4 and Tpbg/5T4 were detected in Kit-ir cells only in Kit K641E, but not in Kit WT/WT animals. Most up-regulated genes in this mouse model belong to the gene expression profile of human GIST but also to the profile of normal Kit+ ICC in the mouse small intestine. Spry4 and Tpbg/5T4 may represent candidates for targeted therapeutic approaches in GIST with oncogenic KIT mutations.  相似文献   

5.
This paper aimed at investigating the alterations in interstitial cells of Cajal (ICCs) in the murine small intestine from 0-day to 56-day post-partum (P0–P56) by immunohistochemistry. The Kit+ ICCs, which were situated around myenteric nerve plexus (ICC-MY) formed a loose cellular network at P0 which changed into an intact one before P32. The density of ICC-MY increased from P0 to P12, and then decreased until P32. In contrast, the estimated total amount increased more than 15-fold at P32 than that at P0. Some Kit+/BrdU+ cells were observed at 24 h after one BrdU injection to the different-aged mice, and the number decreased from P2 to P24 and vanished at P32. Actually a few Kit+/BrdU+ cells can be observed at 1 h after one BrdU injection at P10, and the amount doubled at 24 h along with paired Kit+/BrdU+ cells. A number of BrdU+ ICCs were also labeled with CD34, CD44 and insulin-like growth factor I receptor. About 65% ICCs were BrdU+ at P32 after daily BrdU injection from P0. Our results indicate that an age-dependent proliferation is involved in the postnatal development of ICC-MY which increase greatly in cell numbers and proliferative ICCs may originate from ICCs progenitor cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Feng Mei and Jiang Zhu have contributed equally to this work.  相似文献   

6.
Primary graft dysfunction (PGD), as characterized by pulmonary infiltrates and high oxygen requirements shortly after reperfusion, is the major cause of early morbidity and mortality after lung transplantation. Donor, recipient and allograft-handling factors are thought to contribute, although new insights regarding pathogenesis are needed to guide approaches to prevention and therapy. Mast cells have been implicated in ischemic tissue injury in other model systems and in allograft rejection, leading to the hypothesis that mast cell degranulation contributes to lung injury following reperfusion injury.We tested this hypothesis in a mouse model of PGD involving reversible disruption of blood flow to one lung. Metrics of injury included albumin permeability, plasma extravasation, lung histopathology, and mast cell degranulation. Responses were assessed in wild-type (Kit+/+) and mast cell-deficient (KitW-sh/W-sh) mice. Because mouse lungs have few mast cells compared with human lungs, we also tested responses in mice with lung mastocytosis generated by injecting bone marrow-derived cultured mast cells (BMCMC).We found that ischemic lung responses of mast cell-deficient KitW-sh/W-sh mice did not differ from those of Kit+/+ mice, even after priming for injury using LPS. Degranulated mast cells were more abundant in ischemic than in non-ischemic BMCMC-injected KitW-sh/W-sh lungs. However, lung injury in BMCMC-injected KitW-sh/W-sh and Kit+/+ mice did not differ in globally mast cell-deficient, uninjected KitW-sh/W-sh mice or in wild-type Kit+/+ mice relatively deficient in lung mast cells.These findings predict that mast cells, although activated in lungs injured by ischemia and reperfusion, are not necessary for the development of PGD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0095-0) contains supplementary material, which is available to authorized users.  相似文献   

7.
8.
The development and maintenance of interstitial cells of Cajal (ICC) are closely associated with SCF/KIT signal activity. In this study, we evaluate the distribution of ICC in KIT distal kinase domain mutant mice (Wads) and determine whether the loss-of-function mutations in KIT easily lead to gastrointestinal (GI) disorders. ICC were examined by anti-KIT immunohistochemistry and western blotting. The GI microstructure of wild-type (WT) and Wads mice in normal intestines and incomplete intestinal obstruction was evaluated by hematoxylin and eosin staining. The results in Wadsm/m mice were as follows. Myenteric ICC were obviously decreased in the stomach and colon and were totally absent in the small intestine. Intramuscular ICC were nearly absent in the stomach and irregularly distributed in the colon. Moreover, the smooth muscle thickness of the small intestine was increased 1.3-fold in Wadsm/m, compared to WT and Wadsm/+ mice and the diameter of the intestinal lumen was also enlarged in Wadsm/m mice. When constructing an incomplete intestinal obstruction model, the extent of distention involved was greater in Wads mice (1.6-fold in Wadsm/+ mice and 1.8-fold in Wadsm/m mice vs. WT mice). Meanwhile, the intestinal lumen expansion and decrease in ICC were more pronounced in Wads mice than in WT mice. Our results suggest that the KIT distal kinase domain mutation leads to an ICC loss in a subtype and location-specific pattern in Wadsm/m mice. The injury of the KIT signaling in mutant mice results in more serious pathological manifestations after being exposed to pathogenic factors.  相似文献   

9.
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 ~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.  相似文献   

10.
The effect of mutation Kit W-Y found in C57BL/6 mice on fertility, spermatogenesis, and early embryogenesis of mice have been studied. If heterozygotes Kit W /+ are crossed with wild-type mice, fertility decreases by 20%. Homozygotes Kit W-Y /Kit W-Y and compounds Kit W-Y /Kit Ssm are nonviable. The study of spermatogenesis in Kit W /+ mice has demonstrated a negative effect of this mutation on spermatocytes. Histological examination of the testes of mutant males has shown local empty spaces in seminal ducts. Electron microscopic examination of synaptonemal complexes have demonstrated desynapsis disturbance in some nuclei at the diplotene stage of meiotic prophase I. However, these disturbances do not cause a decrease in the number of fertilized oocytes/ova. The decrease in fertility is accounted for disturbances of early embryogenesis. In vivo and in vitro analyses of early embryogenesis have demonstrated that cleavage divisions are asynchronous in Kit W-Y /+ heterozygous embryos. Some of these embryos die before implantation, and others cleave more rapidly than wild-type embryos, which give them selective advantage during the postimplantation period of embryogenesis. The pattern of Kit W-Y expression during spermatogenesis and embryogenesis mimics potential human pathology, which makes these mutants an interesting and valuable object for genetics and developmental biology.  相似文献   

11.

Background

Clinical studies showed the contribution of viral infection to the development of asthma. Although mast cells have multiple roles in the pathogenesis of allergic asthma, their role of in the virus-associated pathogenesis of asthma remains unknown. Most respiratory viruses generate double-stranded (ds) RNA during their replication. dsRNA provokes innate immune responses. We recently showed that an administration of polyinocinic polycytidilic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13.

Methods

The effect of poly IC on allergen-induced airway eosinophilia was investigated for mast cell-conserved Kit+/+ mice and -deficient KitW/KitW-v mice. The outcome of mast cell reconstitution was further investigated.

Results

Airway eosinophilia and IL-13 production were augmented by poly IC in Kit+/+ mice but not in KitW/KitW-v mice. When KitW/KitW-v mice were reconstituted with bone marrow-derived mast cells (BMMCs), the augmentation was restored. The augmentation was not induced in the mice systemically deficient for TIR domain-containing adaptor-inducing IFN-β (TRIF) or interferon regulatory factor (IRF)-3, both mediate dsRNA-triggered innate immune responses. The augmentation was, however, restored in KitW/KitW-v mice reconstituted with TRIF-deficient or IRF-3-deficient BMMCs. Although leukotriene B4 and prostaglandin D2 are major lipid mediators released from activated mast cells, no their contribution was shown to the dsRNA-induced augmentation of airway eosinophilia.

Conclusions

We conclude that mast cells contribute to dsRNA-induced augmentation of allergic airway inflammation without requiring direct activation of mast cells with dsRNA or involvement of leukotriene B4 or prostaglandin D2.  相似文献   

12.
13.
Genetic mutations could cause sperm deficiency, leading to male infertility. Without functional gametes in the testes, patients cannot produce progeny even with assisted reproduction technologies such as in vitro fertilization. It has been a major challenge to restore the fertility of gamete-deficient patients due to genetic mutations. In this study, using a Kitw/Kitwv mouse model, we investigated the feasibility of generating functional sperms from gamete-deficient mice by combining the reprogramming and gene correcting technologies. We derived embryonic stem cells from cloned embryos (ntESCs) that were created by nuclear transfer of Kitw/Kitwv somatic cells. Then we generated gene-corrected ntESCs using TALEN-mediated gene editing. The repaired ntESCs could further differentiate into primordial germ cell-like cells (PGCLCs) in vitro. RFP-labeled PGCLCs from the repaired ntESCs could produce functional sperms in mouse testes. In addition, by co-transplantation with EGFP-labeled testis somatic cells into the testes where spermatogenesis has been chemically damaged or by transplantation into Kitw/Kitwv infertile testes, non-labeled PGCLCs could also produce haploid gametes, supporting full-term mouse development. Our study explores a new path to rescue male infertility caused by genetic mutations.  相似文献   

14.
Sprouty proteins (Sproutys) inhibit receptor tyrosine kinase signaling and control various aspects of branching morphogenesis. In this study, we examined the physiological function of Sproutys in angiogenesis, using gene targeting and short-hairpin RNA (shRNA) knockdown strategies. Sprouty2 and Sprouty4 double knockout (KO) (DKO) mice were embryonic-lethal around E12.5 due to cardiovascular defects. The number of peripheral blood vessels, but not that of lymphatic vessels, was increased in Sprouty4 KO mice compared with wild-type (WT) mice. Sprouty4 KO mice were more resistant to hind limb ischemia and soft tissue ischemia than WT mice were, because Sprouty4 deficiency causes accelerated neovascularization. Moreover, suppression of Sprouty2 and Sprouty4 expression in vivo by shRNA targeting accelerated angiogenesis and has a therapeutic effect in a mouse model of hind limb ischemia. These data suggest that Sproutys are physiologically important negative regulators of angiogenesis in vivo and novel therapeutic targets for treating peripheral ischemic diseases.  相似文献   

15.
Previous studies have reported that only primordial follicles and empty follicles can be found in 7.5 days postparturition (dpp) Sohlh1?/? mouse ovaries and females are infertility. There appears to be a defect in follicle development during the primordial‐to‐primary follicle transition in Sohlh1?/? mouse ovaries. However, detailed analyses of these phenomena have not been performed. In this study, we used Sohlh1?/? transgenic mice to explore the role of Sohlh1 in folliculogenesis. The results showed that only primordial follicles and empty follicles can be observed in Sohlh1?/? ovaries from 0.5 to 23.5 dpp. The expression of Foxo3 and FOXO3 was downregulated; nucleocytoplasmic shuttling of FOXO3 was normal in 7.5‐dpp Sohlh1+/+ but not Sohlh1?/? ovaries; and primordial follicle activation (PFA) was not observed in 7.5‐dpp Sohlh1?/? mice. The expression levels of KIT, AKT, and P308‐AKT were downregulated (p < 0.05), whereas that of P473‐AKT was not significantly changed (p > 0.05). The KIT/PI3K/AKT pathway was inhibited. Furthermore, we conducted a dual luciferase assay and chromatin immunoprecipitation. The results showed that SOHLH1 can upregulate the Kit gene by binding to the ?3698 bp E‐box motif. The absence of Sohlh1 may affect PFA in mouse ovaries via downregulation of Kit and inhibition of the KIT/PI3K/AKT pathway.  相似文献   

16.
In mammals, primordial follicles are generated early in life and remain dormant for prolonged intervals. Their growth resumes via a process known as primordial follicle activation. Recent genetic studies have demonstrated that phosphoinositide 3-kinase (PI3K) is the essential signaling pathway controlling this process throughout life, acting via Akt to regulate nucleocytoplasmic shuttling of Foxo3, which functions as a downstream molecular switch. The receptor tyrosine kinase Kit has been implicated by numerous studies as the critical upstream regulator of primordial follicle activation via PI3K/Akt. Here we present a genetic analysis of the contribution of Kit in regulating primordial follicle activation and early follicle growth, employing a knock-in mutation (KitY719F) that completely abrogates signaling via PI3K. Surprisingly, homozygous KitY719F female mice undergo primordial follicle activation and are fertile, demonstrating that Kit signaling via PI3K is dispensable for this process. However, other abnormalities were identified in KitY719F ovaries, including accelerated primordial follicle depletion and accumulation of morphologically abnormal primary/secondary follicles with persistent nuclear Foxo3 localization. These findings reveal specific roles of Kit in the maintenance of the primordial follicle reserve and in the primary to secondary follicle transition, but argue that Kit is dispensable in primordial follicle activation.  相似文献   

17.
18.
Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-β for early differentiation. To gain pathogenicity, however, Th17 cells require IL-1β and IL-23. The underlying mechanism by which these confer pathogenicity is not well understood. Here we show that Sprouty4, an inhibitor of the PLCγ-ERK pathway, critically regulates inflammatory Th17 (iTh17) cell differentiation. Sprouty4-deficient mice, as well as mice adoptively transferred with Sprouty4-deficient T cells, were resistant to experimental autoimmune encephalitis (EAE) and showed decreased Th17 cell generation in vivo. In vitro, Sprouty4 deficiency did not severely affect TGF-β/IL-6-induced Th17 cell generation but strongly impaired Th17 differentiation induced by IL-1/IL-6/IL-23. Analysis of Th17-related gene expression revealed that Sprouty4-deficient Th17 cells expressed lower levels of IL-1R1 and IL-23R, while RORγt levels were similar. Consistently, overexpression of Sprouty4 or pharmacological inhibition of ERK upregulated IL-1R1 expression in primary T cells. Thus, Sprouty4 and ERK play a critical role in developing iTh17 cells in Th17 cell-driven autoimmune diseases.  相似文献   

19.
20.
The mast/stem cell growth factor receptor KIT has long been assumed to be a specific marker for interstitial cells of Cajal (ICC) in the bladder, with possible druggable perspectives. However, several authors have challenged the presence of KIT+ ICC in recent years. The aim of this study was therefore to attempt to clarify the conflicting reports on KIT expression in the bladder of human beings, rat, mouse and guinea pig and to elucidate the possible role of antibody‐related issues and interspecies differences in this matter. Fresh samples were obtained from human, rat, mouse and guinea pig cystectomies and processed for single/double immunohistochemistry/immunofluorescence. Specific antibodies against KIT, mast cell tryptase (MCT), anoctamin‐1 (ANO1) and vimentin were used to characterize the cell types expressing KIT. Gut (jejunum) tissue was used as an external antibody control. Our results revealed KIT expression on mast cells but not on ICC in human, rat, mouse and guinea pig bladder. Parallel immunohistochemistry showed KIT expression on ICC in human, rat, mouse and guinea pig gut, which confirmed the selectivity of the KIT antibody clones. In conclusion, we have shown that KIT+ cells in human, rat, mouse and guinea pig bladder are mast cells and not ICC. The present report is important as it opposes the idea that KIT+ ICC are present in bladder. In this perspective, functional concepts of KIT+ ICC being involved in sensory and/or motor aspects of bladder physiology should be revised.  相似文献   

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