Kit Signaling Regulates The Development And Proliferation Of ICCs In Neonatal Mouse Colon

Interstitial cells of Cajal (ICCs) are a kind of speical interstial cells, and distribute throughout the entire gastrointestinal tract. They play a very important role in the regulation of gastrointestinal(GI) motility by generating and propagating spontaneous electric slow-wave activity and mediating nitrergic and cholinergic neurotransmission. ICCs can also mediate responses to stretch and serve as mechanosensors. Recent clinical studies have indicated that ICCs are associated with many GI motility disorders such as Infantile hypert- rophic pyloric stenosis(IHPS), Hirschsprung's disease(HD), neonatal pseudo-obstruction and temporary post-operational entero-paralysis. Although the etiological factor and pathogenesis of these diseases have not been kown, they all showed up reduction of ICCs in numbers and impaired of the cell network, which indicated that the prolonged or abnormal of development of ICCs must be one of the reasons for GI motility dysfunctions. Clarification of the development of ICCs will help us to understand the etiology and pathophysiology of disorders characterized by a deficiency of ICCs, and provides us with new insight into the treatment of relevant disease.ICC in the colon of adult animals are divided into four subtypes according to their distinct locations and morphologic features: 1) At the level of Auerbach's plexus (ICC-MY) ; 2) At the border between circular muscle layer and submucosa (ICC-SM); 3) Within the longitudinal and circular smooth muscle layers (ICC-IM) and 4) In the connective tissue beneath serosa (ICC-SS). Different subtype of ICCs has different function and developmental process, for instance ICC-SM are pacemaker cells but ICC-SS act as mechanosensors of the colon, so it is necessary to understand the developmental process of all subtypes of ICCs. Moreover, our previous study has indicated that the number of ICCs in mouse small intestine increased 30 folds from newborns to adults, and these increased cells might derived from proliferation of insulin-like growth factor 1 receptor(IGF-1R)positive ICCs. Whether proliferation is also involved in the colon? Are there being local progenitors? Both are our concerned questions.ICCs express typeâ…¢tyrosine kinase receptor Kit protein, the gene product of c-kit proto-oncogene. Stem cell factor(SCF)is the natural ligand for Kit protein, which can activate the receptor and then regulates the development, survival, proliferation and phenotypic maintained of ICCs. Development of ICCs will be affected under conditions of c-kit mutation in the W/W~V mice and W/W~ S rats models and Sl/Sl~d mice models harbored Sl mutant; or neutralization antibody ACK2 and inhibitor of Kit receptor block Kit signaling in late embryo stage and early neonatal stage; or add neutralization antibody ACK2 and remove exogenous SCF from the cultural system. The number of ICCs decrease sharply and accompany with a set of symptoms of GI motility dysfunction such as gastric retention, reflux esophagitis, regurgitation of duodemun content. Our recent studies on mice and Guinea-pigs have also indicated that Kit signaling palyed an important role in survival and motor function maintained of mature ICCs. Recently, researchers have found that there are ICCs progenitors without ability of generating spontaneous electric slow-wave in postnatal mouse GI. When the number of ICCs decreased under some pathological factors, insulin/IGF-IR signaling and Kit/SCF signaling would stimulate proliferate of these progenitors and differentiate into mature ICCs. Whether Kit signaling can regulate the development and survival of different subtype of colonic ICCs? Response of these ICCs? How to regulate the proliferation of ICCs by Kit /SCF signaling and insulin/IGF-IR signaling? It is eager to elucidate these questions.This investigation is divided into three parts:Part 1: Development of neonatal mouse colon.In order to investigate the alterations of distribution, morphology and cell numbers of ICC in each segment of mouse colon over the period extending from neonatal (P0) to adult life (P56), immunofluorescent staining of Kit protein was utilized. The results are as followings:1. ICC-MY were prominent at birth while ICC-IM and ICC-SS began to appear, and ICC-SM emerged at P6, indicated that time difference was existed between ICCs subtypes during the developmental process.2. ICC-SM emerged at P6 in the proximal colon and subsequently in the distal colon at P8, and ICC in the oral end of colon revealed a better development in morphology and a higher density than that in the anal side. Which indicated that a proximal to distal and transural gradient of ICC distribution in the postnatal development of colon.3. The estimated total cell numbers of ICCs increased from birth to adulthood along with the lengthening and extending of mouse colon.Part 2: The proliferation of ICCs in neonatal mouse colon.BrdU incorporation and anti-Ki67 multi-immunofluorescent staining were utilized to investigate the proliferative potential and characteristics of ICCs in postnatal mouse colon. The results are as followings:1. ICC-MY, ICC-IM and ICC-SS had proliferative potential, and presented an age-dependent characteristics.2. There were some Kit/IGF-IR/Ki67 multi-labeled ICCs progenitors in neonatal mouse colon, which might be the source of increased ICCs.3. The proliferative characteristics of ICC-SM is different from other subtypes, the increased of estimated total cell numbers might rely on proliferation of Kit negative progenitors and subsequently differentiate into mature ICCs.Part 3: Kit signaling regulates the development and proliferation of ICCs in neonatal mouse colon.Mice with Imatinib administration or PPP intraperitoneal injection were used as animal models, immunofluorescent staining and Western blot methods were utilized to investigate the alteration of survival and proliferation of colonic ICCs at neonatal stage after blockade of Kit signaling and IGF-I signaling. Further realized the proliferative mode of postnatal mouse colon. The results are as followings:1. Imatinib was a potent inhibitor of Kit signaling in vivo.2. Each subtype of ICCs except ICC-SM reduced in numbers and incompleted of cell network after blockade of Kit signaling, indicated that Kit signaling was essential for survival of neonatal ICCs except ICC-SM.3. The proliferation of Kit positive ICCs or/and progenitors was inhibited completely by blocking Kit signaling. However, only a half of proliferative ICCs were disappeared after blockade of IGF-IR signaling. Which demonstrated that proliferation of ICCs depent on Kit signaling but was regulated by IGF-IR signaling in part.4. Number of ICCs could recover up to normal rapidly but few of proliferative Kit positive ICCs emerged at later stage followed the withdrawal of Imatinib. So we presumed that Kit negative progenitors might be one of the sources of increased ICCs.The third part revealed that Kit signaling played a key role in survival and proliferation of ICCs in neonatal mouse colon. Kit negative progenitors could be expanded substantially when reduction of Kit positive ICCs under some pathology factors in order to maintain the normal physiology function of GI. IGF-IR signaling participated in regulating proliferation of ICCs.To sum up, this study demonstrated that the further development of ICCs along with the constant improvement of colonic structure from newborn to adult. Proliferative potential of ICC-MY, ICC-IM and ICC-SS characterized by age-dependence. Primary source of proliferative ICCs might be Kit~+/CD44~+/CD34~+/IGF-IR~+ multi-positive progenitors. Kit singnaling regulated survival and proliferation of ICCs except for ICC-SM.