CD271 Combined With TrkA Promoted The Proliferation And Migration Of Epidermal Stem Cells To Support Cutaneous Wound Healing

BackgroundBurn wound can significantly reduce quality of patient's life in physiology and psychology,even threaten their lives.Approximately 11 million people suffering from burns receive medical treatment each year.Many treatments were proposed,including stem cells therapy,but there is still no effective method to cure it.Therefore,the development of a safe,efficient,easy-to-use and inexpensive wound treatment is becoming increasingly imperative.Epidermal stem cells(eSCs),located in the basal epidermal layer,which are proliferating and differentiating during migration to the suprabasal layer,cause the closure of the wound epithelium inducing wound healing.eSCs are characterized by expression of cytokeratin(CK)19 and the cell adhesion molecule ?1(?1).In contrast,differentiated keratinocytes(KCs)are marked by CK10.CD271,also called p75 neurotrophin receptor,is expressed in eSCs.It has been reported that CD271 has a significant role in the stem cells growth and renewal.CD271 may promote deep partial burn wounds healing process in mice.However,the role of CD271 in skin wound healing is not clear.Moreover,TrkA is another neurotrophin receptor.It plays a role in various stem cells and neuron cells.In the nervous system,CD271 receptor plays different roles based on the expression of TrkA.Although CD271 and TrkA receptor interaction is clearly involved in human epidermal homeostasis,a few studies have shown the role of TrkA combined with CD271 in wound-healing process.This research aims to highlight the role of CD271 with or not TrkA promotes migration and proliferation in eSCs during the skin wounds healing.ObjectiveThe role of CD271 in skin wounds via activating eSCs,and the potential roles of TrkA mediated with CD271 promoting eSCs migration and proliferation during the wound healing process were detected.Methods1.In vitro cell study(1)Isolation and culture of eSCseSCs were isolated according to the protocols previously established.Skin was then carved into 0.5 square centimetre and digested in a 0.05%separation enzymatic solution for 10 min at 37?.The digestion was terminated by DMEM/10%FBS.The cells were filtrated and seeded into plates pre-covered with collagen type ?.The markers of eSCs were detected by western blot after eSCs were incubated in serum-free medium(KSFM,Gibco)at 370C under 5%CO2.The eSCs passage 4(P4)were used for cellular analyses.Colony formation assay was used to measure cell renewal capability,in which eSCs(P4)were plated into six-well plates and incubated for 10 days.The colonies were fixed using methyl alcohol and stained with haematoxylin for 10 min.(2)Lentivirus TransfectionPrimary eSCs were transfected with CD271 carrying lentivirus,CD271-shRNA lentivirus or empty puro lentivirus separately.Lentivirus particles were obtained from the Genome Ditech Company.After 8 hr,the medium was replaced by fresh medium.At day 3 after transfection,cells were observed under an inverted fluorescence microscope to calculate the transfection efficiency.(3)Cell cycle analysisCell cycle was tested by flow cytometry.The eSCs P4 were separately collected and washed three times with cold phosphate buffer saline(PBS),then fixed with cold 70%ethanol at 4?.The 500 ?L of Propidium Iodide(PI)was added and incubated at 37?in the dark for another 30 min according to the manufacturer's protocol.The data were collected and analysed using flow cytometry.(4)Cell Proliferation studyCell Counting Kit-8(CCK-8)was employed to test cell proliferation.eSCs(P4)were seeded into 96-well plates and cultured.The CCK-8 solution was added to the wells after 24 hr,48 hr,72 hr,and 96 hr of culture.The absorbance was measured by a Multimode Reader at a wave length of 450 nm.(5)Cell migration assayNon-coated and pre-coated with diluted Matrigel Transwell membranes(8.0 ?m pore size)were used to test cell migration ability.The eSCs(P4)(approximately 5*105 cells)suspended in 200 ?L of epidermal growth factor(EGFR)-free medium were seeded into the upper chamber.The growth factor medium containing EGFR was added to the lower chamber as a chemo-attractant.The cells were incubated for 24 hr for migration assay and for 36 hr for the metastasis assay.Migrated cells on the membrane were fixed in methanol and stained with haematoxylin.(6)Cell apoptosis analysiseSCs(1*106/well)were collected and washed with PBS.These eSCs(P4)were then stained with 5 ?L of PE-7-AAD and Annexin-V-FITC at room temperature in the dark for 15 min.The cellular apoptosis was analysed using flow cytometry.The rate of Q2 means the rate of apoptosis.The rate of Q4 means the rate of death.Hoechst 33342/PI double staining was also used to analyse cell apoptosis.The eSCs(P4)were seeded into a 24-well plate 24 hr before the test.Hoechst33342 and PI(2 ?g/ml)were added into the culture medium and incubated for 15 min.Cells were then washed and observed under an inverted fluorescence microscope to count the number of dead and alive cell.(7)Inhibition of TrkA by K252aThe three groups of infected eSCs at passage 4(P4)were cultured and plated at a density of 1.0×105 cells/cm2 in KSFM and then pre-treated for 24 hr with 100 nM K252a for the inhibitor assays.2.In vivo animal study(1)Generation of burn wounds in miceC57BL/6 mice(6 weeks old,male,20-25 g)were purchased from the Shandong University Laboratory Animal Centre.The back hairs of mice were shaved and wiped the skin with 75%alcohol after anesthetized.Copper billets were heated in boiling water then placed on the expos.ed backs skin of mice(n=5)for 4 sec to create burn wound.The mice were intraperitoneally injected 5 ml lactated Ringer's liquid immediately.At days 7,14,and 21 after burn wounds,the mice were sacrificed and full-thickness skin around the wound was collected for western blot and immuno-staining analyses.(2)Generation of full-thickness wounds in miceC57BL/6 mice(6 weeks old,male,20-25 g)were purchased from the Shandong University Laboratory Animal Centre.The mice were randomly divided into four groups(n=5).The mice were anesthetized,shaved and wiped as welist before.Full-thickness skin wounds on mouse back measuring 0.5 cm in diameter were cut to create the wound model.(3)Generation of TrkA-deficit in miceC57BL/6 mice(male,20-25 g,6 weeks old)were purchased and divided into four groups randomly(n=5).In control and CD271 group,intraperitoneal injections of vehicle(saline containing 2%DMSO)were given,and an intra-peritoneal k252a(5?g/kg)dissolved in saline containing 2%DMSO was injected to the k252a group and CD271+k252a group[39].The absence of TrkA expression in k252a group and CD271+k252a group was confirmed by western blot(WB)at protein levels.(4)Injection of eSCs to wounds4.1 C57BL/6 mice(6 weeks old,male,20-25 g)were randomly divided into four groups(n=5))and burn wounds were generated as described above.An equal amount(1*107)of control eSCs(P4)and CD271-vo eSCs(P4)re-suspennded in 150 ?L of PBS was injected into the dermis layer around the skin wounds at days 0,3 and 5 after wounding and the healing rate was compared to PBS injected wounds.The area of the wound was measured and photographed at days 0,7,14,17,19 and 21 after wounding.The size of wound area was measured by using a metric ruler.At day 21,full-thickness skin biopsies around wounds were taken to detect the expression of biomarkers.Wound healing rates(%)at specific time points were evaluated by calculation of(wound area/original wound area)*100.4.2 For the control group,150 ?L of PBS was injected into the dermis layer around the skin wound.The other groups of mice were injected with an equal amount(1*107)of CD271-vo eSCs,CD271-vo eSCs+K252a,and control eSCs+K252a suspended in 150 ?L of PBS.All groups were injected at days 0,3 and 5,and the areas of the wound were measured by tracing the wound at days 0,3,and 7 post-injury.The wound area in each of the seven groups at days 3 and 7 was compared with the original wound area in that group at 0 day by measuring with a metric ruler:(wound area rate at days(3,7))%=(the wound area at days(3,7))/(the wound area at 0 day).(5)Immunohistochemistry(IHC)stainingDeparaffinized slides in turpentine for 30 min and rehydrate slides in 100%,90%,80%,70%alcohol and PBS.The slides were then blocked by an endogenous peroxidase-blocking reagent,and incubated at 4? overnight with the monoclonal antibody specific for ?1,CD271,CK19,or CK10.The secondary antibodies and the streptavidin-biotin complex were added and incubated at 37? for 30 min.The slides were conterstained with Diaminobenzidine and haematoxylin.The staining of the slides were observed and photographed under a microscope.The cultured cells were similarly stained as the slides.The cells were fixed for 20 min with 4%paraformaldehyde,and incubated with primary antibodies against CD271 and TrkA at 4? overnight.(6)Protein isolation and western blotting(WB)Proteins were extracted using radio immune precipitation assay buffer containing 1%phenyl methanesulfonyl fluoride following the manufacturer's protocol.The protein concentration was determined with a bicinchoninic acid protein assay kit.Protein(40?g)was added and separated on a 6%or 10%SDS polyacrylamide gel electrophoresis.The separated protein bands were then transferred to a nitrocellulose membrane.After blocked with 5%powdered milk,the blots were incubated with primary antibodies:CD271,? 1,CK10,CK19,TrkA,phosphorylated ERK,phospho p44/42,JNK,phospho JNK,Akt,and phospho Akt,glyceraldehydes-3-phosphate dehydrogenase(GAPDH)at 4? overnight.The blots were incubated with the corresponding secondary antibodies.3.Statistical analysisData were expressed as means ± standard deviation.Student's t-test is used for statistical analysis between two mean values of unpaired data.The one-way analysis of variance(ANOVA)was performedand an analysis of variance was used to compare multiple groups for determining the significance values using Statistical Product and Service Solutions software(SPSS,IL,USA)17.0.AP value<0.05 was accepted as statistically significant difference.Result1.Cell experiments(1)Differentiation of eSCs mediated by CD271 in vitroThe differentiation of eSCs plays an important role in re-epithelialization of epidermis during wound healing.Therefore,the role of CD271 in eSCs differentiation in vitro culture system was evaluated.The eSCs colonies were cultured in collagen coated plates,and they were expanded in serum free medium.CD271 was over-expressed by lentiviras carrying CD271(CD271-vo),or silenced by the lentivirus having shRNA to target CD271(CD271-kd).The Control eSCs were infected by control virus.The virus infection was confirmed by detecting green fluorescence as these viruses carry green fluorescence protein gene.The efficiency of blockage(CD271-kd eSCs(P4))or over-expression of CD271(CD271-vo eSCs(P4))was evaluated by western blot and immune-staining respectively.The protein level of CD271 increased in CD271-vo eSCs but decreased in CD271-kd eSCs shown by protein bands and by immune-staining patterns.The impact of CD271 manipulation on differentiation was determined by measuring the level of differentiation marker CK10.The levels of stem cell markers CK19 and ? 1 were also evaluated.When CD271 was over-expressed,the level of CK10 increased but CK19 decreased.In contrast,when CD271 was silenced,level of CK10 decreased but CK19 and ? 1 increased in CD271-kd eSCs.These results demonstrated that expression of CD271 was efficiently manipulated by the lentivirus and CD271 promotes the differentiation of eSCs culture system.(2)Proliferation and migration of eSCs mediated by CD271 in vitroNext the impacts of CD271 manipulation on proliferation and migration of eSCs in vitro were investigated.CD271 increased the cell populations in G2 phase(CD271-vo eSCs)compared to the control as shown by flow cytometry.However,silencing of CD271 showed opposite effect(CD271-kd eSCs)compared to overexpression(CD271-vo eSCs).Over-expression of CD271 increased colony forming efficiency but silencing decreased it.CD271 also increased cell proliferation during eSCs culture shown by cell numbers evaluated by cell counting kit-8.The role of CD271 on cell migration of eSCs was also evaluated by using Matrigel Transmembrane assay.CD271 increased the number of migrated cells detected on the membrane(CD271-vo eSCs),however CD271 silencing decreased it.In contrast,CD271 silencing substantially increased dead cells as shown by PI staining and flow cytometric analyses(Q2+Q4).These data demonstrated that CD271 promotes proliferation and migration of eSCs,although removing CD271 drives cells to apoptosis.(3)The level of TrkA regulated by CD271 in vitroTrkA is another neurotrophins receptor which binds to Ngf.The relative ratio of CD271 and TrkA may affect the cell proliferation or cell survival mediated by Ngf.Therefore TrkA in CD271-vo eSCs(P4)and CD271-kd eSCs was detected by using IHC and western blot analyses.When CD271 was over-expressed,the expression of TrkA dramatically decreased as shown IHC and western blot.In contrast,CD271 silenced cells decreased the level of TrkA.(4)CD271 inhibited apoptosis of eSCs by interaction with TrkATo further verify the role of TrkA in impacting the effect of CD271 on the anti-apoptosis in eSCs,cell apoptosis and death rates were examined.CD271-vo eSCs decreased the rates of apoptotic/dead cells(TUNEL staining cells)compared with the control eSCs,as exhibited by TUNEL/DAPI staining.Moreover,compared withK252a eSCs,eSCs apoptosis increased along with inhibiting TrkA,even CD271over-expressed.Similarly,CD271 promoted the apoptosis of eSCs in absence of TrkA detected by the rates of Q2+Q4(apoptotic/dead rate)in FCM analysis.(5)CD271 promoted proliferation of eSCs by interaction with TrkAThe results of colony formation assay showed that CD271 inhibited the proliferation of eSCs in absence of TrkA.In addition,the G2 phase cells are the cells in or will in the phase of division on behalf of the proliferation ability of cells.Over-expression of CD271 increased the number of the G2 phase cells as shown by cell cycle analysis to detect the proliferation of eSCs.A significant decrease in proliferation was observed in eSCs with TrkA inhibition,even when CD271 was over-expressed,as indicated by numbers of cells in the G2 phase.(6)CD271 promoted eSCs migration by interaction with TrkARe-epithelialization of the epidermis was influenced by CD271 and TrkA mediated eSCs migration.So we further investigated whether changes in CD271 and TrkA complex affected eSCs migration.To elucidate the role of TrkA combined with CD271 in CD271-vo eSCs,K252a,a tyrosine inhibitor of TrkA,was used to block the expression of TrkA.The expressions of TrkA and CD271 in control eSCs,CD271-vo eSCs,K252a eSCs,and CD271-vo eSCs+K252a were detected by WB.The result showed a significant decrease of TrkA expression in CD271-vo eSCs.Compared with control eSCs,the protein levels of TrkA were nearly close to zero in K252a eSCs and CD271-vo eSCs+K252a.Compared with control eSCs,CD271 expression was high in CD271-vo eSCs,K252a eSCs,and CD271-vo eSCs+K252a measured by WB and FCM.The Matrigel transmembrane assay showed that the migration ability of CD271-vo eSCs was higher than that of control eSCs.In the absence of TrkA,over-expression of CD271 had the lowest migratory ability,indicating that TrkA deficiency had important effect on the migration of eSCs participating in wound closure.(7)ERK and Akt were involved in the interaction of CD271 and TrkA to promoteeSCs biological activityTo explore the potential underlying mechanism,proteins were extracted from control eSCs,CD271-vo eSCs,K252a eSCs,and CD271-vo eSCs+K252a,the levels of pERK,pAkt and pJNK were examined.Compared with control eSCs,the protein level of pERK increased in CD271-vo eSCs.Furthermore,we examined the role of CD271 without TrkA in pERK signaling,and found that pERK was significantly down-regulated in the CD271-vo eSCs+K252a,which compared to K252a eSCs and CD271-vo eSCs.Further analysis indicated that pAkt level remained up-regulation when elevated CD271 and resulted in significant decrease in CD271-vo eSCs+K252a in parallel with the level of pERK.Moreover,we observed a significant decrease in pJNK protein expression in CD271-vo eSCs compared with control eSCs.In the absence of TrkA,pJNK level in K252a and CD271-vo eSCs+K252a significantly increased.2.Animal experiments(1)Expression of CD271 increased during the burn wound healingThe expression levels of CD271 were assessed during burn wound healing.The deep partial thickness burn wounds were generated by pushing heated cupper billets on mouse skin for 4 sec.The wound extended through the epidermis and into the dermis,and the damage reached to reticular region of the dermis,which is classified as a deep partial-thickness burn.The expression levels of CD271,eSCs markers CK19 and 0 1,and differentiation marker CK10 were measured at different stages of healing.The wounds were healed by reducing of wounded area at day 7 and 14 through re-epithelialization and completely closed at day 21 after wounding.The whole skin wounds were harvested at different stages and analysed by both western blot and immunohistochemistry(IHC).Protein level of CD271 decreased at day 7,but increased at mid-late stages of day 14 and 21,which is consistent to the increases in CK19 and ? 1.However,CK10 decreased at day 7 and 14 but increased at day 21.The IHC studies indicated that CD271 and CK19 were mainly expressed in thickened epidermis at day 21.The ? 1 also increased,which mainly expressed in the basement membrane.The level of CK10 in cornified layer of the epidermis was reduced at day 7 but increased drastically at day 21.(2)The burn wound healing accelerated by injection of CD271 over-expressing eSCsThe role of CD271 in burn wound healing was investigated by injecting eSCs over-expressing CD271.The same deep partial thickness burn wounds were generated in mouse skin.The CD271 over-expressing eSCs or the control eSCs were injected into dermis around burn wounds.The healing rates of eSCs injected wounds were compared to non-injected control wounds.The wound healing was evaluated by measuring wound areas at different stages.Injection of the both the control eSCs and CD271-vo eSCs substantially accelerated the wound healing rates at day 14 and 17 compared to non-injected control during wounds were re-epithelialized.However,these injections did not affect the wound closure rate at early stage of day 7.In addition,injection of CD271-vo eSCs showed higher healing rate compared to control eSCs group with statistical significance difference although changes were small.These results indicated that injection of eSCs or CD271 expressing eSCs accelerated the burn wound healing at the mid to late stages of burn wound healing when wounds were re-epithelized.(3)CD271 promoted wound healing in mice by interaction with TrkAIn order to identify the potential the role of TrkA mediated with CD271 in eSCs on wound healing,full-thickness skin mouse wound closure model(5 mm diameter)was used and the ability of CD271 over-expressed and TrkA-deficient to heal wounds was compared with their corresponding control mice.CD271-vo eSCs were over-expressed by a lentivirus carrying CD271 and control eSCs were infected with a puro lentivirus as previously described.The wound area was measured by tracing the wound at days 0,3 and 7 post-injury.Before the experiments,the absence of TrkA expression in K252a-intraperitoneal injection(K252a and CD271+K252a)group and the presence of TrkA expression in DMSO-intraperitoneal injection(control and CD271)group were confirmed at protein levels.Compared with the control group(eSCs group),CD271 eSCs group exhibited enhanced wound healing from day 3.Wound healing of mice in CD271+K252a group was delayed at days 3 and 7 macroscopically than those of mice in K252a group and CD271 group.The CD271+K252a group had the largest wound area in all groups.The reepithelialization stage and the proliferation and migration of eSCs were observed and evaluated by HIC and WB at days 7 during wound healing.The expressions of eSCs markers(?1 and CK19)were analyzed.?1 and CK19 were mainly expressed in basal layer,and their protein levels increased in CD271 group.Compared with K252a group,?1 and CK19 at protein levels both decreased significantly in CD271+K252a group.The least eSCs gathered around the wound edge in CD271+K252a group.In addition,the PCNA staining and TUNEL staining were examined for assessing the proliferation and apoptosis ability at days 7 after wound.In the wound sites,PCNA staining revealed that PCNA labeling indices were the highest in CD271 group and the lowest in CD271+K252a group,and the TUNEL labeling indices were opposite.These suggested that CD271 promoted wound healing via eSCs but inhibited it with the absence of TrkA.ConclusionOur studies showed CD271 promoted cutaneous burn wound healing.especially at the re-epithelialization stage,probably by facilitating proliferation,migration and differentiation of eSCs.The present findings laid the foundation to introduce CD271 as the promising molecular target to accelerate the regeneration of epidermis after deep partial thickness burn wound.Moreover,the CD271/TrkA interaction accelerates the migration and proliferation of eSCs to facilitate wound healing via activation of the ERK and Akt signaling pathways and that CD271 is a potential target gene for mediating the functional role of TrkA,CD271 mediated TrkA could be a key complex involved in reconstruction of the epidermal structure in wound healing and a new therapeutic target.