Study On The Function And Mechanism Of ERK/MAPK Signal During Intestinal Homeostasis Maintenance

Adult stem cells are critical for tissue homeostasis and regeneration.As one of the excellent system for studying adult stem cell function,the intestinal epithelium undergoes rapid cell turnover,well-controlled cell differentiation and maturation.The maintenance of self-renew of epithelium is supported by two populations of the intestinal stem cells(ISCs)which are Lgr5-positive fast-cycling crypt base columnar(CBC)stem cells wedged between Paneth cells and +4 quiescent "reserve stem cells".ISCs also constantly generate transit-amplifying(TA)cells which are further differentiated into postmitotic absorptive lineage(enterocytes)and secretory lineage(goblet cells,Paneth cells,enteroendocrine cells,Tuft cells,etc.)in small intestine.Stem cell hierarchies in the small intestine are rigorously controlled by the orchestration of reciprocal niche signals from neighboring Paneth cells and underlying mesenchymal cells.Wnt signaling is the essential driving force to fuel the ISCs and maintains the crypts as well as inducing Paneth cells maturation.Cooperatively with Wnt signaling,Notch and EGF pathways promote crypt expansion and regulate cell differentiation.While BMP and Hedgehog pathways negatively regulate epithelial Wnt signaling to promote cell differentiation.Moreover,epithelial cell produced Hedgehog(Shh and Ihh)factors induced mesenchymal BMP secretion is critical for epithelial-mesenchymal interactions and villus patterning.However,it is still obscure what is the upstream signaling which regulates Hedgehog activity.The mitogen-activated protein kinases(MAPK)pathway regulates intestinal epithelial cell growth,survival and Paneth versus goblet cell choice,but the underline mechanism is still discrepant.As one of the major direct targets of Ras-Raf-MEK1/2 cascade,the extracellular signal-regulated kinases 1 and 2(ERK1/2)reciprocally regulate AKT/mTOR pathway through numerous negative feedback commonly observed in human tumours,but whether and how the feedback regulation functions during development is still under exploration.To this end,we used the Villin-cre tool mice to induce the Erkl/2 gene inactivation in the intestinal epithelium specifically(hereinafter abbreviated Erk?IEC mice)to carry out this subject.First,ERK1/2 inactivation in the intestinal epithelium severely damaged the epithelial structure and resulted in about half of the mice died two weeks after birth.Traditionally,Erk1/2 is a proliferation-promoting signal,whereas,cell proliferation in the intestinal epithelium of Erkl/2-deficient mice increased greatly.Cell migration increased and cell differentiation was abnormal,Paneth cells differentiation earlier and increased the number of goblet cells decreased,the number of tuft cells reduced and it had no effect on the differentiation of endocrine cells.The effect of Erk1/2 deletion on the differentiation of two major secretory cells was consistent with that of epithelial-inactivated APC gene which leads to activation of Wnt signaling pathways.Wnt signaling pathway was detected by immunohistochemistry and western blotting to detect the target genes Sox9,C-myc and Cyclindl which indicated the over-activated Wnt signaling pathway.At the same time,RNA-seq data analysis also showed excessive activation of Wnt signal.At the same time,the number of Lgr5-positive stem cells in Erk?IEC mice decreased 14 days after birth and may be associated with Wnt signal activity above threshold.Besides the normal behavior of epithelial cells was destroyed,we also found that the arrangement of intestinal epithelium-villus axis was destroyed.Interstitial cells play an important role in the development of intestinal villi during embryonic development.A-sma positive cells reduced and distribution was abnormal.A-sma positive cells were mainly smooth muscle cells and myofibroblasts underneath intestinal epithelial,and the proliferation of stromal cells was also decreased in Erk?IEC mice.Further investigation revealed that the activity of Hedgehog-BMP signal pathway in Erk?IEC mice was decreased.Hedgehog-BMP signal abnormalities affect the epithelial and mesenchymal normal interaction so that their development was destroyed.Erk?IEC mice abnormal structure disorder of epithelial and stromal is similar to the morphological characteristics of hamartoma.In humans,hamartomatous polyposis is associated with inactivation mutation of BMP signaling and activation mutation of AKT/mTOR signaling.Most of these mutations associated with this disease have been verified by genetically modified animal models.Our data showed that AKT/mTOR signaling in the intestinal tissue of Erk?IEC mice was over-activated.Knockout of TSC1 to further activate the mTOR signal based on inactivation of Erk1/2 gene in intestinal epithelium will lead to more severe intestinal epithelial cell differentiation and tissue morphology defects.Inhibition of mTOR signaling pathways by rapamycin intraperitoneal injection Erk?IEC mice,the mice tissue morphology,cell differentiation and abnormal signal activity were partially restored,thereby improving the survival rate of mice.These positive and negative experimental evidences strongly suggested that AKT/mTOR signaling played an important role in the development of intestinal defects in Erk?IEC mice.In cancer,the MAPK signaling pathway can adjust AKT/mTOR signaling pathways through a variety of negative feedback mechanism.We found that Erk1/2 gene inactivation can cause upstream of the RAS-RAF-MEK feedback cascade activation,leading to the activation of AKT/mTOR.While others possible feedback molecular activity did not change,such as EGFR,MEK1 protein,showing the feedback mechanism in intestinal development stage had certain specificity.