Expression And Significance Of Notch Signaling In The Proliferation And Differentiation Of Rat Tracheal Stem Cells

IntroductionAdult stem cells have the capacity to self-renew and generate functionally differentiated cells that replenish lost cells in the organ throughout the lifespan of the organism.It has been demonstrated that tracheal stem cells exist in tracheal epithelium, they are scarce but crucial for they can maintain tracheal epithelium homeostasis and they are indispensable for damage repair of tracheal epithelium and disease recovery of upper airway.However,the research progress of tracheal stem cells is very slow because it is quite difficult to construct tracheal stem cell line for the complicated structure and microenviroument of tracheal epithelium.We constructed a tracheal regeneration model induced by fluorouracil(5-FU)ex vivo which revived the process of proliferation and differentiation of tracheal stem cells successfully and made it possible to explore the mechanism involved in the regulation of this process.Under the treatment of 5-FU,the tracheal epithelia in proliferating phase desquamated and only a few cells in Go phase remained in the basal membrane. Thereafter,the tracheal epithelia turned flat then cuboidal and restored to its original mode at about 48h after removal of 5-FU.It is obviously that those cells in Go phase immediately following the removal of 5-FU contained tracheal stem cells and it is just though the proliferation and differentiation of tracheal stem cells that the tracheal epithelium restored,to its original mode of pseudostratified ciliated columnar epithelium which has been demonstrated before.However,the precise molecular mechanisms involved in the regulation of this process has been elusive. Notch comprises a family of highly conserved receptors,whose activation is induced by their specific ligands through cell--cell interactions.Notch signaling is involved in various aspects of cellular regulation,including stem cell maintenance and its components' expression and its role is not always the same in different tissues.The components' expression of Notch signal in tracheal epithelium and its role in regulating stem cell fate remains unclear.In this study,we demonstrate the role of Notch signaling in the maintenance of tracheal stem cells and provide a theoretical evidence for advanced study.Materials and methods1.Preparation of Tracheal Epithelium Regeneration Model and DAPT TreatmentTracheas were excised sterilely from male and female Wistar rats(～200 g)and cultured in DMEM/F12 containing 120 mg/ml 5-FU and 10%FBS for 12h at 37℃. Following removal of 5-FU,tracheas were cultured in DMEM/F12 containing 10% FBS with or without 5μM DAPT or 40μ.M Jag-1.Tracheas were removed at 0,3,6,9, 12,24,and 48 h after removing 5-FU and analyzed by one of several methods.Another group of tracheas were firstly cultured in DMEM/F12 containing 10%FBS with or without 5μM DAPT or 0μM Jag-1 for 12h and cultured for another 12h in DMEM/F12 containing 10%FBS with 5-FU,then the tracheas were cultured in DMEM/F12 containing 10%FBS for 48h.Tracheas were removed after DAPT or Jag-1 treatment, after 5-FU treatment and at 0,3,6,9,12,24,and 48 h after removing 5-FU respectively. For RT-PCR and western blot analyses,tracheal epithelia were digested and stored at -70℃until use.For immunofluorescent analysis,tracheas were fixed in 4% paraformaldehyde,and prepared as paraffin-embedded tissue sections for hematoxylin-eosin(HE)stain and immunofluorescent staining.Untreated tracheas were used as controls.2.RT-PCR analysis RT-PCR was performed with the TaKaRa RNA PCR Kit(AMV)version 3.0, according to the manufacturer's protocol.β-actin was used as an endogenous control. PCR conditions were as follows:94℃for 2 rain,94℃for 30 s,variable temperature for 40 s,and 72℃for 1 min,for 35 cycles.Reverse transcription reactions lacking reverse transcriptase served as negative controls.PCR products were visualized by ethidium bromide staining on 2%agarose gels on a gel scanner.3.Western blot analysisTotal cell homogenates were prepared by lysing cells in NP40 lysis buffer.Total protein was subjected to SDS-PAGE,followed by blotting to PVDF.Membranes were blocked with nonfat dried milk in PBS,incubated with primary antibody in overnight at 4℃with shaking,then incubated with secondary antibodies for 2 hours at room temperature.Membranes were washed again and then incubated with DAB at room temperature.When bands reached the desired intensity,membranes were washed in water,followed by PBS.Finally,membranes were dried and photographed and scanned. After scanning,the densitometric analysis was performed using Image J 1.33 software.4.Indirect ImmunofluorescenceIndirect immunofluorescence staining was performed to the serial tissue sections from tracheas during the recovery from injury with Notch3,Jagged1,ABCG2,CK19 and PCNA antibodies respectively.Briefly,rabbit anti-Notch-3,rabbit anti-CK19,goat anti-Jagged-1,goat anti-ABCG2 and goat anti-PCNA(dilution 1:100)were used as primary antibodies.Fluorescein isothiocyanate(FITC)-conjugated goat antirabbit immunoglobulin G(IgG)and Rhodamine isothiocyanate(TRITC)-conjugated rabbit antigoat IgG(dilution 1:100)were used as secondary antibodies.Both antibodies were diluted with 1%bovine serum albumin-PBS.After sections were treated with the secondary antibody,they were incubated with DAPI for nuclear counterstaining. Specimens were examined with an epi-illumination fluorescence microscope BX50. For serum controls,1%bovine serum albumin-PBS was used instead of the primary antibody as a negative control. 5.Statistical analysisData from at least three independent experiments were used for statistical analysis by SPSS 11.5.All values were expressed as mean±standard deviation(SD).Statistical analyses were preformed by one way ANOVA,where p＜0.05 was considered significant.Results1.Expression levels of ABCG2,CK19,and PCNA proteins in rat tracheal epithelium during recovery from injury induced by 5-FUIn untreated rat tracheal epithelia,almost no ABCG2 and PCNA was detected, while levels of CK19 were very high relative to levels in 5-FU treated tracheal epithelia. Immediately following removal of 5-FU,levels of ABCG2 and PCNA increased together and reached peak levels at 6h after removal of 5-FU,and both decreased gradually,returning to normal levels about 48h after removal of 5-FU.Expression of CK19 was lowest at Oh and increased slowly until 6h,thereafter the expression of CK19 increased sharply and nearly returned to normal level about 48h after removal of 5-FU.2.Expression of Notch signaling pathway components in the rat tracheal epitheliumRT-PCR showed that levels of Notchl,Notch3,Jagged1,D114,Hes1,Hey1,and Hey2 were very low in the untreated rat tracheal epithelium.Following treatment with 5-FU,expression levels of Notchl,D114,Hes1,and Hey2 did not change obviously, while Notch3,Jagged1,and Hey1 levels changed markedly.Expression of Notch2, Notch4,Jagged2,D111,D113,Hes3,and Hes5 was not detected in the rat tracheal epithelium during this process.3.Expression of N3ICD,Jagged-1,and Hey1 in the rat tracheal epithelium during injury recovery Western blot showed that expression levels of N3ICD,Jagged1,and Hey1 were very low in the normal rat tracheal epithelium.After treatment with 5-FU,expression levels increased and reached peak levels at 6h,6h,and 12h,respectively,and decreased gradually thereafter to return to normal levels by about 48h.4.Immunofluorescence of anti-Jagged-1,anti-Notch-3,anti-ABCG2 and anti-CK19Immunofluorescence showed that after treatment with 5-FU,most of the cells remaining in the basement membrane exhibiting nuclear Notch3 increased.By about 6h of treatment,the proportion of cells with nuclear Notch3 reached peak levels,and decreased thereafter.By 48h,the rate returned to baseline levels.Immediately following removal of 5-FU,very few Jagged1-positive cells were detected. Subsequently,the number of Jaggedl-positive cells increased gradually and reached peak levels at about 6h,at which point expression began to decrease.Most Jagged1 positive cells were interspersed with nuclear Notch3 positive cells,and these were distributed within similar regions within the tracheal epithelium.Both ABCG2 and CK19 were expressed on the cell membranes and the change trends of them were consisted with the results from western blot analysis.Most of the ABCG2 positive cells were Notch3 nuclear positive.5.The effects of disruption or persistent activation of Notch signaling on the injury recovery of rat tracheal epithelium.5μM DAPT could reduce Notch signaling within 6h of treatment,and block Notch signaling within 24h of treatment.In DAPT treated group,expression of ABCG2 increased more slowly,and peaked at a lower level compared with the control group at 3h.From 3h and on,expression of ABCG2 ceased to increase,and decreased sharply from 6h.The expression of CK19 increased rapidly and maintained a high level from 12h,and then peaked at a higher level compared with non-DAPT treated samples. PCNA expression increased slightly after 5-FU treatment.24h later,PCNA expression was not detected.The expression of PCNA decreased compared with non-DAPT treated samples.In the Jag-1 treated group,the expression of ABCG2 and PCNA increased significantly from 6h;the expression of CK19 decreased significantly from 6h compared with the control group.HE staining showed that in the DAPT-treated group,morphological differences with the control group appeared beginning around 6h after treated with DAPT. DAPT-treated tracheal epithelial cells became cuboidal around 6h earlier than the control group.By 12h,cilia had already appeared on the surface of the tracheal epithelium,earlier than in the control group.From 12h to 48h,there was no obvious increase in the number of epithelial cells in the DAPT-treated group.In the Jag-1 treated group,the ceil number increased rapidly and no callia was observed on the surface of the epithelium.48h later,tracheal epithelium didn't convert to its original appearance.6.The effects of DAPT or Jag-1 pretreatment on the injury recovery of rat tracheal epitheliumHE stain showed that immediately after DAPT pretreatment,the cell number in tracheal epithelium decreased,after 5-FU treatment,almost no cells remained within the basement membrane and no recovery trend was observed.After Jag-1 pretreatment, cillia cells decreased obviosly.After treated with 5-FU,more cells remained in the basement membranes and 24h later,the tracheal epithelium almost revert to its original appearance.Immunofluorescence with anti-ABCG2 showed that ABCG2 positive cell was not detectable after DAPT treatment in the group pretreated with DAPT,while after Jag-1 pretreatment,ABCG2 positive cells increased.Conclusions1.48h after 5-FU treatment,the morphology and the proliferation and differentiation state of tracheal epithelium can revert to its original appearance; 2.Notch-1,Notch-3,Jagged-1,Hes1,Hey1 and Hey2 may be involved in the maintenance of normal rat tracheal epithelium;3.Notch-3,Jagged-1 and Hey1 may play a role in the proliferation and differentiation of rat tracheal stem cells;4.Notch signaling pathway plays a casual role in the maintenance and proliferation of tracheal stem cells and is indispensable for the injury recovery of tracheal epithelium.