The Effect And Mechanism Of Action Of Thymosin Beta 4 On Hair Growth

Thymosin beta 4 (Tβ4) is known to induce hair growth and hair follicle development; however, its mechanism is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in hair follicle development and explore the mechanism of Tβ4 on hair growth. To construct Tβ4 knockout mice by TALEN, a suitable targeting site and DNA recognition sequences were designed. T04 global knockout mice and mice that overexpressed Tβ4 in the epidermis were generated by pronucleus injection with TALENs and Tβ4 overexpressing plasmid. The speed of hair growth in three groups was observed after depilation. Skin tissue samples in the three experimental groups were collected and cut into 7μm sections, which were stained with immunofluorescence and H&E to observe the distribution of Tβ4 and the pattern of hair follicles and count the number of hair shafts. The mRNA and protein levels of Tβ4, vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and E-cadherin (E-cad) were detected by real-time PCR and western blotting. The activity of Wnt, MAPK and PI3K signaling pathway were also examined by western blotting to explore the mechanism of Tβ4 on hair growth.1. The construction of knockout plasmids by TALEN and Tβ4 overexpressing plasmid in the epidermis.According to the coding sequence (CDs) of Tβ4, a suitable targeting site and DNA recognition sequences were designed. Six pairs of TALEN plasmids were respectively transfected into the mouse NIH3T3 cells to choose a pair whose the targeting efficiency was the highest for the subsequent experiments. The targeting efficiency in L1×R3 group was 93%. The fragment including Krt14 promoter, the CDs of Tβ4 and polyA sequence in pODsRed-KTP1 was cut and purified to generate mice that overexpressed Tβ4 in the epidermis by pronucleus injection.2. The generation of knockout mice and mice that overexpressed Tβ4 in the epidermis.127 injected zygotes were transplanted into 5 receptors and 25 mice were born in which 8 mice were detected to be knockout mice. The mutation rate was 32%. Meanwhile 225 injected zygotes were transplanted into 7 receptors to generate 40 mice in which 3 mice were examined to be mice that overexpressed Tβ4 in the epidermis. The positive rate was 7.5%.3. The effect of Tβ4 on hair growth in mice.The control and experimental mice aged 8-10 weeks were depilated by a mixture of rosin and paraffin wax on dorsal skin to activate hair growth started at the same time. The conditions of hair growth in all mice were monitored and took photos during 11 days after depilation. Skin tissue samples in the three experimental groups were collected during the same hair growth conditions (wild type mice:13 days; transgenic mice: 11 day; knockout mice:16 days) after depilation and cut into 7μm sections, which were stained with the immunofluorescence and H&E to observe the distribution of T04 and the pattern of hair follicles and count the number of hair shafts. Results of depilation indicated that comparing with the control mice the speed of hair re-growth was faster and the hair was thicker and longer in T04 over-expressing mice, while the speed of hair re-growth was slower and there was no hair on the depilated skin in knockout mice. Histological examination revealed that hair shafts of the control mice were separate and T04 protein was only distributed in the cells around hair follicles. Tβ4 overexpressing mice had a higher number of hair follicles and hair shafts clustered together to form groups, when Tβ4 protein was distributed in the skin. Hair shafts of knockout mice were separate and hair follicles were significantly reduced compared with control mice, at the same time there was no T04 distribution in the cells and tissues around hair follicles.4. The mechanism of Tβ4 on hair growth.With the increase of the Tβ4 levels, compared to control mice the mRNA and protein levels of MMP-2 and VEGF were increased in T(34 overexpressing mice (P<0.05), while the levels of E-cadherin remained stable (P>0.05). Further, in Tβ4 global knockout mice compared to control mice the mRNA and protein levels of MMP-2 and VEGF were decreased dramatically (P<0.05) and the levels of E-cadherin were also stable (P>0.05). The changes in the protein levels of P-catenin and LEF-1, two key molecules in the Wnt signaling pathway, were similar to the changes in T04 expression in control and experimental mice. The Tβ4 overexpressing mice showed increased protein level and phosphorylation of P38, ERK1/2 and AKT, whereas knockout mice had decreased the protein levels and phosphorylation of these proteins, especially the phosphorylation of ERK1/2 (P<0.05).Based on our results, in the skin of adult mice Tβ4 regulates the levels of VEGF and MMP-2 via the Wnt/p-catenin/LEF-1 signaling pathway. This interaction between two proteins further affects the activity of MAPK/P38, MAPK/ERK1/2, PI3K/AKT signaling to control the speed of hair re-growth, the number of hair shafts and the pattern of hair follicles, and thus ultimately affects hair follicle development. The far-reaching effect of T(34 on hair growth and hair follicle development and its process should be confirmed in future studies and the interaction between Tβ4 and other proteins and its mechanism should be actively explored.