A Study On The Regulation Of TGF-β,BMP2, And Sex Hormone Related Proteins On Yak Hair Follicle Cycle

The yak（Bos grunniens）lives in the plateau region at an altitude of 3,000⁵,000 meters and is one of the important livestock species in the region.According to reports,annual average temperature of the plateau region is in the range of-3^-5°C;compared with the annual average temperature at sea level（16.5²3.1°C）,the plateau region shows low temperature characteristics.The skin is an organ that’s in direct contact with the external environment,and its accessory hair follicles（HFs）produce plush in cold seasons,and the strong moisture resistance of hair fiber are beneficial for yak lives in the plateau region.In order to explore the regulation characteristics of Transforming growth factor-β（TGF-β）,Bone morphogenetic protein2（BMP2）,and sex hormone related proteins on the HF cycle of yak,adult male yak was used as the research object,and skin samples were collected at 5 different growth phases of HFs during telogen,pro-anagen,mid-anagen,late-anagen,and catagen.The following three aspects were studied:（1）Both TGF-βand heat shock protein 70（HSP70）are important factors for regulating the transition from anagen to catagen of HFs.In this study,immunohistochemistry,immunofluorescence,real-time quantitative polymerase chain reaction（qRT-PCR）,and western blotting were used to detect the expression of TGF-β,TGF-βRⅡ,HSP70,and casepase-3 in the skin of HF mid-anagen,late-anagen,and catagen.The results showed that TGF-β2 mRNA level was significantly higher than transcripts of TGF-β1 and TGF-β3.Meanwhile,the expressions of TGF-β2,TGF-βRⅡ,and caspase-3 were higher in the catagen than in mid-anagen,and the results of immunofluorescence double staining showed that some TGF-βRⅡ-positive cells in the catagen HFs were TUNEL-positive.The expression of HSP70 in mid-anagen was higher than those in late-anagen and catagen,and the lowest and highest expression levels of HSP70 were opposite to those of Caspase-3.These results indicated that TGF-β2 plays an important role in the induction of catagen HFs,and it may induce apoptosis of HF epithelial cells through the TGF-βRII-mediated pathway.In contrast,HSP70 may regulate the transition of HFs to catagen by protecting cells from apoptosis.In summary,TGF-β2 and HSP70 play an important regulatory role in the development of yak HF catagen.（2）BMP2,BMP receptor-ⅠA（BMPR-ⅠA）,and Noggin are important proteins involved in regulating the transition from telogen to anagen of HF.In order to explore the expression profiles of BMP2,BMPR-ⅠA,and Noggin in the yak skin at different phases of HFs,we collected yak skin in the telogen,pro-anagen,and mid-anagen of HFs and evaluated gene and protein expression of BMP2,BMPR-ⅠA,and Noggin by qRT-PCR,western blotting and immunohistochemistry.Both qRT-PCR and western blotting results showed that the expression levels of BMP2 and BMPR-ⅠA were highest in the telogen phase,while the highest expression of Noggin was observed in the pro-anagen.Moreover,from the telogen to pro-anagen phase,the expression of BMP2 and BMPR-ⅠA were down-regulated,while Noggin was up-regulated.Immunohistochemistry results showed that BMP2,BMPR-ⅠA,and Noggin were expressed in the skin epidermis,HF outer root sheath,and hair matrix.In summary,the characteristic expression of BMP2,BMPR-ⅠA,and Noggin suggested that BMP2and BMPR-IA have important regulatory effects on the maintenance of yak telogen HFs,whereas Noggin may plays an important role in regulating the growth of yak HFs,which mainly by affecting proliferation and differentiation of skin epithelial cells.（3）Dihydrotestosterone（DHT）and 17β-estradiol（E₂）are sex hormones that regulate human HFs growth and are produced from testosterone（T）by 5αreductase（5α-red）and aromatase respectively.In order to determine whether yak HFs have sex hormone synthase and receptor expression,in this study,immunohistochemistry,qRT-PCR and western blotting were used to detect and observe the expression of DHT and E₂ synthesis-related proteins and their receptors in yak skin during different HF stages（telogen,anagen,and catagen）.The results showed that 5α-red1,5α-red2,and androgen receptor（AR）were expressed in epithelial cells and AR was also expressed in the hair papilla.At the same time,the transcription level of 5α-red1mRNA at different HF stages was significantly higher than that of 5α-red2 mRNA;5α-red1 and 5α-red2 proteins peaked during the anagen and telogen phase respectively;whereas,AR protein was only expressed in the skin during the anagen phase.In addition,the aromatase and estrogen receptors（ERαand ERβ）were expressed in the cutaneous epithelial cells,both ERαand ERβwere also expressed in the dermal papilla;the transcription level of ERαmRNA in HFs at each stage was much higher than that of ERβmRNA.From the catagen to telogen phase,aromatase and ERβprotein expression was down-regulated,while the ERαprotein expression was up-regulated.The characteristic expression of DHT-related proteins may indicate that the skin cells have a DHT synthesis pathway and mechanism of action,and DHT may have an important regulatory effect on the growth of yak HFs;the expression trend of E₂-related proteins suggests that yak skin cells may exist a E₂ synthesis pathway and its receptor mechanism,and E₂ may play an important regulatory role in the catagen of yak HFs.In conclusion,（1）TGF-β2 may be involved in the regulation of the regression of yak HFs through the mediated pathway of TGF-βRII;while HSP70 may affect the growth of yak HFs by participating in protecting cells from apoptosis.（2）BMP2 and BMPR-IA can participate in the regulation of the telogen maintenance of yak HFs by affecting the proliferation and differentiation of yak skin epithelial cells,while Noggin may play an important role in the regulation of HFs transition from telogen to anagen by affecting the proliferation of yak skin epithelial cells.（3）The skin cells of yak exist a DHT synthesis pathway and mechanism of action,and DHT may have an important regulatory effect on the growth of yak HFs.The yak skin cells also have a synthesis pathway and its receptor mechanism of E₂,and E₂ may play an important regulatory role in the catagen of yak HFs.These provides a theoretical basis for further study on the role of these factors in the growth cycle of yak HFs and its mechanism of action,and also provides a strong support for further revealing the adaptation mechanism of yak to the special environment of the plateau.