| P311 gene was first found in brain tissue of late embryonic mice in 1993,which was confirmed expressing a small cytoplastic protein about 8 kDa.Because there is a PEST domain at the end of P311protein?s N terminal,the molecule is easy to be degraded by Met-HGF-SF,ubiquitin-protease,metalloprotease and some other pathways,thus makes its short biological half-life.P311 gene was then mainly found in nerve cells,smooth muscle cells,glial cells and chondrocytes.The protein expression of P311 was not found in normal adult tissues generally.Several studies have shown that P311 plays important roles in accelerating nerve tissue repair,wound healing,tumor angiogenesis,embryonic development and maintaining blood pressure balance.However,the molecular weight of P311 protein is small and the expression is extremely unstable,which limits the further study on its structure and function,the futher biological functions of P311 and its regulation are not clear till now.This also makes it impossible to classify it into a specific type of protein family.Different from the other common wounds such as incised wound,the early burn wound bed and the peri-wound exsit ischemia/hypoxia to different degrees.It is well known that ischemia/hypoxia is one of the most important factors leading to the pathological process of skin injury and wound repair.After having been suffered from burns,EpSCs to leaving their niche,migrate,and initiating re-epithelialization are the important and fundmental processes for wound repair.Therefore,it is necessary to investigate the effect of hypoxia on the biological behaviors of Ep SCs.Our preliminary work indicated that P311 highly expressed in the epidermal basal layers and hair follicles of the peri-wound where the Ep SCs reside in.During the early time of a superficial partial thickness model,the migration rate of P311-/-Ep SCs was significantly lower than that of wild-type,which indicates that the enhanced expression of P311 was benefit to the migration of EpSCs.Moreover,the niche of stem cells in the peri-wound which Ep SCs reside in was considered as a hypoxia microenvironment.The expression of most genes are regulated by HIF-1? under hypoxia,which is a hypoxia dependent key transcription factor.At the C terminal of HIF-1?,there was a oxygen dependent degradation domain and two relatively conservative transactivation domains.Therefore,it is Susceptible to be degradated by ubiquitin-proteasome under normoxia,thus makes its? short half-time,HIF-1? is vulnerable to accumulate under anoxic conditions.Under hypoxia,HIF-1? is transferred into the nucleus via CBP/p300(Cyclic AMP response element-binding protein,a co-stimulatory molecules),and by binding to the beta subunit then regulates the transcriptional activation of downstream genes.Hypoxia can induce the expression of HIF-1?,which is highly expressed in chondrocytes,fibroblasts and tumor cells under hypoxic condition s.This is crucial for the activation of downstream genes and strength of tumor metastasis,angiogenesis,embryogenesis,and anaerobic glycolysis.So whether the expression of P311 and the biological activity in EpSC under hypoxia are also regulated by HIF-1??In this study,we simulated a hypoxia environment in vitro,the migration rate of EpSC from wild-type and P311-/-mice at different time points under normoxia and hypoxia(1% O2)were measured,and the influence of HIF-1? on P311 under hypoxia in wild-type EpSCs were detected.The scratch test results showed that P311-/-EpSCs migrated slower than the wild-type mouse Ep SCs under hypoxia at the same time points.Real-time fluorescence quantity RT-PCR showed that the levels of HIF-1 alpha and P311 m RNA in EpSCs were significantly increased at the early time under hypoxia.The protein expression of HIF-1? and P311 were detected by immunoblotting and immunocytochemical staining assay.The results demonstrated that the expression of HIF-1? and P311 were significantly increased at the early stage of hypoxia.On the other hand,after the cell culture system were treated by HIF-1? inhibitor and stabilizer,the m RNA and protein level of P311 was found decreased or increased accordingly.Finally,it was found that the transcriptional activation of P311 may be regulated by HIF-1? via luciferase reporter gene assay.In conclusion,under hypoxia,the expression of P311 in Ep SC may be regulated by the transcriptional activation of HIF-1?,and the interaction between them promote the migration of EpSC.This provides a positive support for the study of the biological behavior of P311 and its biological regulation mechanism further.Methods and materials1.Study on migration rate of mouse EpSCs under different oxygen environment1)The culture of EpSCsNeonatal C57BL/6J mice were anesthetized by inhalation of ether,the skin tissue were separated after stetilized by alcohol,then the Ep SCs were extracted by two step enzymatic digestion and IV collagen rapid adherence.Then the isloated and cultured Ep SCs were identified by flow cytometry.2)The identification of Ep SCsAfter the cells grow to 70% confluence,the FITC labeled anti-mouse-CD71 and PE labeled anti-mouse-CD49 f antibody were used to incubate the EpSCs,then the cells with CD71(-)/CD49f(+)were determined by flow cytometry.3)The effects of HIF-1? and P311 on the migration of EpSCs under hypoxiaThe studies were designed by completely randomized,after having been grouped by SPSS 18.0,the Ep SCs scratch residual width of P311-/-normoxia group,P311-/-hypoxia group,wild-type normoxia group,wild-type hypoxia group,wild-type HIF-1? inhibitor group and wild-type HIF-1? stabilizer group were measured after the cells having been treated for 0,12,24,48 h.The results were presented as mean ±standard deviation,the general differences between all groups were analysed by variance of factorial design analysis and one-ANOVA analysis,and pairwise comparisons was analysed using LSD test(the value of this statistic is automatically deleted).And the variables were corrected by Bonferroni.P<0.05 were considered the differences were statistically significant.2.The effect of HIF-1? on P311 in Ep SCs under hypoxia1)The expression of HIF-1? in EpSC detected by immmunoblottingThe total protein was extracted by the phosphorylated protein extraction kit.The immunoblotting experiment was performed by SDS-PAGE to detect the expression of HIF-1? in wild-type and P311-/-EpSCs after treated under hypoxia at 0,12,24,48 h.Quantitative analysis of the results of gray values of gel imaging system were pcocessed by using Quantity one gel quantitative software.The results were analysed by one-ANOVA analysis,and pairwise comparisons was analysed using LSD test,P<0.05 considered the differences were statistically significant.2)Real-time fluorescence quantitative RT-PCR to detect the effect of HIF-1? on P311 in EpSCsThe grouping method was consistent with 1.3,the total m RNA were extracted by TRNzol extraction method,and UV spectrophotometer was used to detect the concentration and purity,c DNA was reverse transcripted to detect the expression of HIF-1? and P311 in EpSCs by Real-time fluorescence quantitative RT-PCR.The general differences between all groups were analysed by variance of factorial design analysis,and pairwise comparisons was analysed using LSD test.P<0.05 was considered the differences were statistically significant.3)The expression of P311 in Ep SC was detected by immunocytochemistryThe grouping methods refered to 1.2.1.After the cells grew to 50% confluence,the cells were fixed by 4% paraformaldehyde.Then immunocytochemical staining experiments were performed according to the guidelines,the positive expression of P311 was detected by inverted phase contrast microscope.5 perspectives were taken from each picture.The results were analyzed by Image Pro Plus 6 software,and shown with integral abso rbance value.4)the effect of HIF-1? on the transcriptional activation of P311 detected by Luciferase reporter gene assayThe promoter of P311 was amplified by KOD-Plus-Neo High fidelity PCR enzyme,p GL3-basic and the subclonal products of p311 promoter were firstly identified by restriction Kpn I and Nhe I endonuclease.Then the completion of the construction of the P311 expression vector was proved by restriction endonuclease analysis and PCR amplification in vitro.Following confirmation of 90% confluence,the HEK-293 cells were transiently co-transfected with the vectors of p GL3-P311-Luc plasmid.After having been incubated for different time points under normoxia or hypoxia,the level of the luciferase was detected to estimate whether the transcriptional activation of P311 was regulated by HIF-1? under hypoxia.Results1.The effect of HIF-1? and P311 on the migration of Ep SCs1)The purification and identification of epidermal stem cellsThe Ep SCs which showed cobblestone like pattern,and the single cell was small and regular polygon,amd showed different clonal morphology due to the different cell proliferation potential.The proportion of CD71(-)/CD49f(+)cells accounted for about 87%.The results indicated that the Ep SCs pocessed strong stem cell properties and hi gh purity.2)The effect of HIF-1? and P311 on the migration of EpSCs under hypoxiaCompared with the wild-type hypoxia group,the residual width of P311 gene knock-out hypoxia group at 12 and 24 h was wider(P value below 0.05),the residual width of HIF-1 inhibitor group at 12 h was wider(P values all below 0.05),and the residual width of HIF-1? stabilizer group at 12 and 24 h was smaller(P values all below 0.05).There was no obvious difference in the width of 48 h scratch among 7 groups(F=19.02,P>0.05).2.The interaction between HIF-1 and P311 in EpSC under hypoxia1)Immunoblotting to detect the protein level of HIF-1? in Ep SCThe protein level of HIF-1? in wild-type EpSCs at 0,12,24,48 h under hypoxia were 1.02±0.05?2.56±0.09?1.60± 0.17?1.17±0.03 respectively,the general differences were obvious(F=48.30,P<0.05).Compared with 0 h time point under hypoxia,the protein level of HIF-1? at 12 h under hypoxia was markedly enhanced(P<0.01),24 h under hypoxia began to decline but still higher than 0 h under hypoxia(P<0.05),48 h under hypoxia approached to the normoxia level(P>0.05).2)Real-time fluorescence quantitative RT-PCR to detect the effect of HIF-1? on P311 in EpSCsCompared with the wild-type hypoxia group,the expression of P311 was significantly decreased at 12 h time point in HIF-1 inhibitor group(P values all below 0.05),and the HIF-1 stabilizer group at 12 and 24 h under hypoxia were significantly enhanced(P values all below 0.05).Compared with the HIF-1 inhibitor group,the expression of P311 in DMSO group and HIF-1 stabilizer group were markedly enhanced at 12 and 24 h under hypoxia(P values all below 0.05).3)Immunocytochemical staining to detect the protein level of P311 in Ep SCsThe protein expression level of P311 in EpSCs was similar to that of group 2.2.3.The transcriptional activation of P311 regulated by HIF-1?At 0 h under hypoxia,there was no significant difference in the luciferase activity among vector group,wild-type normoxia group,wild-type hypoxia group,wild-type hypoxia+HIF-1 inhibitor group(F=13.33,P>0.05).Compared with the vector group at 12 h under hypoxia,the luciferase activity of P311 hypoxia group was markedly enhanced(P<0.01);compared with wild-type normoxia group,the luciferase activity of P311 hypoxia group higher(P<0.05);compared with wild-type hypoxia group,the luciferase activity of wild-type hypoxia + HIF-1 inhibitor group was significantly lower(P<0.01).Conclusions P311 contributes to HIF-1?-mediated migration of EpSCs at the early time under hypoxia. |
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