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Thymosin Beta-4 Induces Mouse Hair Growth.

Gao X, Liang H, Hou F, Zhang Z, Nuo M, Guo X, Liu D - PLoS ONE (2015)

Bottom Line: Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown.Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting.Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

View Article: PubMed Central - PubMed

Affiliation: National Research Center for Animal Transgenic Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.

ABSTRACT
Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action 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 HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

No MeSH data available.


Related in: MedlinePlus

Construct design and strategies for Tβ4 knockout mice by TALEN and an analysis of the transcriptional and protein expression levels of Tβ4.(A) A representative schematic of the TALEN is indicated, showing the left and right TALEN DNA recognition sequences (underlined) and the spacer region fused to the FokI endonuclease catalytic domain. (B) The red box indicates the gap in the genomic DNA of KO mouse. (C) This graphical representation demonstrates the pattern of Tβ4 mRNA and protein levels between the dorsal skin samples of the three groups obtained from real-time PCR and western blotting analysis. Gapdh in real-time PCR and α-tubulin in western blotting were used as internal controls. Samples from the WT group were normalized to 1. Each bar represents the mean ± SEM (n = 3). Different superscripts on the bar indicate statistical difference (P < 0.01). (D) Protein expression of Tβ4 was detected by western blotting. α-Tubulin was used as the loading control. The molecular mass of Tβ4 and α-tubulin were approximately 4.9 kDa and 50 kDa, respectively. The results of one representative image of three independent experiments are presented.
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pone.0130040.g001: Construct design and strategies for Tβ4 knockout mice by TALEN and an analysis of the transcriptional and protein expression levels of Tβ4.(A) A representative schematic of the TALEN is indicated, showing the left and right TALEN DNA recognition sequences (underlined) and the spacer region fused to the FokI endonuclease catalytic domain. (B) The red box indicates the gap in the genomic DNA of KO mouse. (C) This graphical representation demonstrates the pattern of Tβ4 mRNA and protein levels between the dorsal skin samples of the three groups obtained from real-time PCR and western blotting analysis. Gapdh in real-time PCR and α-tubulin in western blotting were used as internal controls. Samples from the WT group were normalized to 1. Each bar represents the mean ± SEM (n = 3). Different superscripts on the bar indicate statistical difference (P < 0.01). (D) Protein expression of Tβ4 was detected by western blotting. α-Tubulin was used as the loading control. The molecular mass of Tβ4 and α-tubulin were approximately 4.9 kDa and 50 kDa, respectively. The results of one representative image of three independent experiments are presented.

Mentions: We used TALENs to disrupt the Tβ4 gene, designing a spacer nearby the ATG start codon of Tβ4 to generate a frame shift mutation and loss of Tβ4 function. The appropriate TALEN targeting site was designed using the online TAL Effector Nucleotide Targeter 2.0 software program (https://tale-nt.cac.cornell.edu/node/add/talen-old) (Fig 1A). The targeted sequence was within the second exon of Tβ4 (accession number: NC_000086), on chromosome X. DNA recognized sequences were as follows: left: 5’-GTCTGACAAACCCGATA-3’, right: 5’-TCAACTTCGACTTATCG-3’. The construction of the Tβ4 targeted TALENs was conducted according to the protocol of the FastTALETM TALEN Kit (Sidansai Biotechnology, Shanghai, China). The Tβ4-targeted TALENs RNA mixture was microinjected into zygotes to obtain Tβ4 KO mice, as described below.


Thymosin Beta-4 Induces Mouse Hair Growth.

Gao X, Liang H, Hou F, Zhang Z, Nuo M, Guo X, Liu D - PLoS ONE (2015)

Construct design and strategies for Tβ4 knockout mice by TALEN and an analysis of the transcriptional and protein expression levels of Tβ4.(A) A representative schematic of the TALEN is indicated, showing the left and right TALEN DNA recognition sequences (underlined) and the spacer region fused to the FokI endonuclease catalytic domain. (B) The red box indicates the gap in the genomic DNA of KO mouse. (C) This graphical representation demonstrates the pattern of Tβ4 mRNA and protein levels between the dorsal skin samples of the three groups obtained from real-time PCR and western blotting analysis. Gapdh in real-time PCR and α-tubulin in western blotting were used as internal controls. Samples from the WT group were normalized to 1. Each bar represents the mean ± SEM (n = 3). Different superscripts on the bar indicate statistical difference (P < 0.01). (D) Protein expression of Tβ4 was detected by western blotting. α-Tubulin was used as the loading control. The molecular mass of Tβ4 and α-tubulin were approximately 4.9 kDa and 50 kDa, respectively. The results of one representative image of three independent experiments are presented.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4470810&req=5

pone.0130040.g001: Construct design and strategies for Tβ4 knockout mice by TALEN and an analysis of the transcriptional and protein expression levels of Tβ4.(A) A representative schematic of the TALEN is indicated, showing the left and right TALEN DNA recognition sequences (underlined) and the spacer region fused to the FokI endonuclease catalytic domain. (B) The red box indicates the gap in the genomic DNA of KO mouse. (C) This graphical representation demonstrates the pattern of Tβ4 mRNA and protein levels between the dorsal skin samples of the three groups obtained from real-time PCR and western blotting analysis. Gapdh in real-time PCR and α-tubulin in western blotting were used as internal controls. Samples from the WT group were normalized to 1. Each bar represents the mean ± SEM (n = 3). Different superscripts on the bar indicate statistical difference (P < 0.01). (D) Protein expression of Tβ4 was detected by western blotting. α-Tubulin was used as the loading control. The molecular mass of Tβ4 and α-tubulin were approximately 4.9 kDa and 50 kDa, respectively. The results of one representative image of three independent experiments are presented.
Mentions: We used TALENs to disrupt the Tβ4 gene, designing a spacer nearby the ATG start codon of Tβ4 to generate a frame shift mutation and loss of Tβ4 function. The appropriate TALEN targeting site was designed using the online TAL Effector Nucleotide Targeter 2.0 software program (https://tale-nt.cac.cornell.edu/node/add/talen-old) (Fig 1A). The targeted sequence was within the second exon of Tβ4 (accession number: NC_000086), on chromosome X. DNA recognized sequences were as follows: left: 5’-GTCTGACAAACCCGATA-3’, right: 5’-TCAACTTCGACTTATCG-3’. The construction of the Tβ4 targeted TALENs was conducted according to the protocol of the FastTALETM TALEN Kit (Sidansai Biotechnology, Shanghai, China). The Tβ4-targeted TALENs RNA mixture was microinjected into zygotes to obtain Tβ4 KO mice, as described below.

Bottom Line: Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown.Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting.Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

View Article: PubMed Central - PubMed

Affiliation: National Research Center for Animal Transgenic Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.

ABSTRACT
Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action 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 HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

No MeSH data available.


Related in: MedlinePlus