The Mechanism Of T?4 Regulating On The Hair Growth Through JNK And Notch Signaling Pathways

Hair can protect against skin damage,infection,dehydration,and regulate body temperature.Hair develops from hair follicles,which are derivatives of the skin.?thymosin belongs to a family of polar protein molecules.Up to now,there are 15 kinds of ? thymosin have been found.Some studies have shown that T?4 can stimulate the growth of hair follicles.Notch signaling pathway regulates the late stage formation of follicles in embryo and maintains the hair follicles growth balance after birth.If there are some key factors defects in Notch signaling pathway,they will cause thin,short and curve hair in the rats.The JNK signaling pathway regulates the growth direction and cycle of hair follicles.the JNK and Notch signaling pathway have important roles in regulating the growth of hair follicles.In present study,we will investigate whether T?4 regulate the hair follicles growth through JNK and Notch signaling pathway.1.Expression of T?4 in the hair follicle cycleWe harvested the skin of mice of 2d,6d,15 d and 30 d after birth.The mRNA levels were detected by real-time quantitative PCR.the mRNA levels of 15 d and 30 dmice were higher than that in the control group,which was 26.9 times and 48.3 times of the control group respectively.The results showed that T?4 expressed higher in the catagen and telogen phase in the first hair follicles cycle.2.The relationship between T?4,Notch and JNK signaling pathwayIn this part,the skin of wild-type mice and T?4 gene knockout mice were used as experimental samples.We detected Grb2,MKK7,Hes5 and Notch1 genes transcription by real-time quantitative PCR.These genes were relevant to JNK and Notch signaling pathways.In T?4 knockout mice,the mRNA expression level of Grb2 and MKK7 which were relevant to JNK pathway were 0.17 times and 0.84 times of that in control group.Hes5 and Notch1 mRNA expression level which were relevant to Notch pathway were 0.97 times and 0.72 times of that in control group.We also detected these four genes by Western blot detection.The results showed that the expression of these four genes were 0.47 times,0.10 times,0.53 times,0.9 times of that in the control group respectively.These results indicated that the JNK signaling pathway was regulated by T?4 gene.Notch1,which is an upstream gene in Notch signaling pathway was not regulated by T?4.However,the downstream gene Hes5 was regulated by T?4.3.The relationship between Notch and JNK signaling pathwayWe further explored T?4 acting point on the network of Notch and JNK signaling pathway in this part.DAPT and sp600125 were used to inhibit Notch and JNK pathway in WT murine fetal fibroblasts.The fibroblasts were harvested after treated by the inhibitor(1?mol/L)for 48 h.Grb2,MKK7,Hes5 and Notch1 were detected by real-time quantitative PCR in both signaling pathway.The mRNA expression level of these four genes in DAPT treated cells were 0.13 times,0.23 times,0.08 times,0.59 times lower than that in the control group.In protein level,these four genes expression were 0.2 times,0.14 times,0.36 times,0.08 times respectively of that in control group.The mRNA expression of these four genes in sp600125 treated cells were 0.20 times,0.49 times,0.09 times,1.02 respectively ofthat in control group.And were 0.2 times,0.14 times,0.37 times,1.04 times respectively of that in control group in the protein level.The results showed that the two genes of the JNK pathway were located between Notch1 and Hes5 in the Notch signaling pathway.Therefore,we can deduce that the acting site of T?4 was located between Notch1 and MKK7.The above experimental results confirmed that T?4 had highest expression in the catagen and telogen phase in the first hair follicles development period,which indicated that T?4 had most significant regulation effect on hair follicles during the catagen and the telogen period.T?4 knock-out gene had a direct inhibitive effect on the expression of JNK signaling pathway.The upstream Notch1 gene regulated the expression of T?4 in Notch signaling pathway.The T?4 knockout gene down-regulate the expression of downstream gene Hes5 in Notch pathway.In conclusion,the regulating relationship between T?4 and these four genes in JNK and Notch pathways was as Notch1?T?4?MKK7?Grb2?Hes5.The present study confirmed that T?4 was relevant to JNK and Notch signaling pathways in regulating hair follicles growth.The acting site of T?4 in these two signaling pathways was Notch1?T?4?MKK7?Grb2?Hes5.These results will provide experimental basis for further investigation on T?4 in regulating the hair follicles growth.