Effects Of KLF4 On Hair Follicle Formation And Melanocytes

Melanocytes originated from melanocytes derived from the neural crest,migrated and differentiated in the skin epidermis,and then participate in the formation of hair.Therefore,pigment deposition is a complex process.Some studies have shown that KLF4 is involved in the production of pigment,but its mechanism still unknown.And Then,the development of hair follicles and hair cycle also related to the production of pigment.Therefore,this study revealed the role of KLF4 in hair follicle development,hair follicle cycle and melanin deposition mechanism through a series of experiments including immunofluorescence,q PCR,western blot,immunohistochemistry,immunofluorescence,cell cycle,cell apoptosis,cell migration,transcriptome sequencing,et al.The experiment contents and results are as followed:(1)Real time PCR,western blotting,immunofluorescence and immunohistochemistry were used to detect the expression of KLF4 at different stages of hair follicle development.It was found that,compared with E13.5 days,E15.5 KLF4 m RNA significantly increased in embryo(P<0.01),KLF4 protein was significantly increased at E15.5(P<0.05).After birth,P8 KLF4 m RNA was significantly higher than P2 and P4(P<0.01),while KLF4 protein showed a decreasing trend.From E13.5 to E19.5,KLF4 was expressed in undifferentiated epidermis,hair bud and hair peg.On days P2~P8,KLF4 was expressed in epidermis,hair bulb and outer root sheath,while on days P8,KLF4 was less expressed in hair bulb.Through immunofluorescence,we found that KLF4 and MITF co-expression in each stage of hair follicle development,and we observed that KLF4 and MITF were co-located in the hair bulb and outer root sheath from day P2 to day P8.Thus,KLF4 is involved in hair follicle development.(2)Real time PCR,western blotting and immunohistochemistry were used to detect the expression of KLF4 in different stages of the hair follicle cycle.It was found that the expression levels of KLF4 m RNA and protein in the hair cycle ofcatagen were significantly higher than those in the telogen and anagen(P<0.01).In catagen,KLF4 protein was expressed in the outer root sheath of hair follicles,hair papilla and epidermis.At thetelogen,KLF4 proteins are located in the sebaceous glands and epidermis as well as the dermis.At the anagen,KLF4 protein is expressed in the hairy papilla,hair matrix,outer root sheath,sebaceous glands,epidermis and dermis.After skin detection of hair follicle synchronization period,compared with d3 days,the expression levels of KLF4 m RNA in d8,d13,d16,d20 and d25 were significantly increased in the whole hair growth period(P<0.01),the KLF4 proteinslevelsin d5,d8 and d10 increased significantly(P<0.05),the KLF4 proteins levels in d16 increased significantly(P<0.01).The expression level of d20 KLF4 protein was significantly higher than that of d3(P<0.05).Meanwhile,at d3,positive staining of KLF4 protein was found in the hair papilla,sebaceous glands,and epidermis.At d5,the positive staining of KLF4 protein was located in the bulb,sebaceous glands,and epidermis.At d8,the positive staining of KLF4 protein was located in the inner root sheath,sebaceous glands,and epidermis.At d10,d13 and d16 days,the positive staining of KLF4 protein was located in the inner and outer root sheath,sebaceous glands and epidermis.At d20 days,positive staining of KLF4 protein was found in the outer root sheath,sebaceous glands,and epidermis.At d25 days,KLF4 protein was located in the bulb,outer root sheath,sebaceous glands,and epidermis.Thus KLF4 is involved in the hair follicle cycle.(3)Successfully constructed the mouse melanocytes that overexpression of KLF4.The MITF,TYR m RNA(P<0.001)expression significantly increased,the MITFprotein expression was significantly increased(P<0.01),the TYRprotein expression was increased(P<0.05).Compared with control,the content of melanin in pigment cells was significantly higher(P<0.01),the number of melanocyte dendrites increased significantly(P<0.001),significantly increased the expression levels of Rac1 and Rac3 m RNA(P<0.05).In order to find more melanin synthesis and transport mechanism,melanocytesoverexpressing KLF4 were deeply sequenced,we screened out two genes which related to pigment production,such as Kitl,E-cadherin,Col6a3,S100a6,S100a4.The enrichment results of GO function showed that the different genes were related to vesicle targeting and vesicle fusion.Thus,GO result distribution suggests that KLF4 may be involved in melanin transport.In the KEGG enrichment results,it was found that the up-regulated genes wwre enriched in the "PI3K-Akt signaling pathway",which may regulate the expression of MITF through the PI3K-Akt signaling pathway.(4)The expression of KLF4 in different hair colors was detected by real time PCR,western blotting,immunofluorescence and immunohistochemistry.Compared with white sheep skin,the relative expression of KLF4 m RNA was extremely significantly in black sheep skin(P<0.01),and significantly in brown sheep skin(P<0.05).The expression level of KLF4 protein in black skin was significantly higher than that in white skin sheep(P<0.05).Immunohistochemistry showed that KLF4 protein was expressed in hair follicles,mainly in the outer root sheath of the hair follicles,epidermis and hair bulb.After transfection with KLF4 into sheep melanocytes,it showed that KLF4 inhibited the proliferation of melanocytes and this decrease in cell proliferation was coupled with both an induction of the S cell cycle arrest and with an increased number of early apoptotic cells.In vitro cell migration assays showed that KLF4 suppresses cell migration.Melanin contents and pigment-related gene expression revealed that KLF4 significantly promoted melanogenesis of sheep melanocytes.Collectively,our findings showed that KLF4 has an important role in the regulation of melanin production and melanocyte homeostasis.