Multiple potential roles of thymosin β4 in the growth and development of hair follicles

Abstract The hair follicle (HF) is an important mini‐organ of the skin, composed of many types of cells. Dermal papilla cells are important signalling components that guide the proliferation, upward migration and differentiation of HF stem cell progenitor cells to form other types of HF cells. Thymosin β4 (Tβ4), a major actin‐sequestering protein, is involved in various cellular responses and has recently been shown to play key roles in HF growth and development. Endogenous Tβ4 can activate the mouse HF cycle transition and affect HF growth and development by promoting the migration and differentiation of HF stem cells and their progeny. In addition, exogenous Tβ4 increases the rate of hair growth in mice and promotes cashmere production by increasing the number of secondary HFs (hair follicles) in cashmere goats. However, the molecular mechanisms through which Tβ4 promotes HF growth and development have rarely been reported. Herein, we review the functions and mechanisms of Tβ4 in HF growth and development and describe the endogenous and exogenous actions of Tβ4 in HFs to provide insights into the roles of Tβ4 in HF growth and development.

Previous studies have shown that HF and keratinocyte components may promote hair growth when the amount of DPCs reaches a certain threshold. 14,15 Therefore, the enhancement of the proliferation ability of DPCs is an effective means to promote hair growth.
Currently, three main types of strategies are available to promote hair growth through DPCs. First, the proliferation of DPCs can be promoted by treatment with certain medications. Kang et al 16 found that mackerel-derived fermented fish oil promotes the proliferation of DPCs by activating Wnt/β-catenin signalling for hair growth. Second, the proliferation of DPCs is promoted by the overexpression or inhibition of endogenous genes. Wu et al 17

found that
Wnt10b promotes DPC proliferation in Rex rabbits. In contrast, Yu et al 18 demonstrated that mitotic arrest-deficient protein 2B plays negative roles in T-cell factor 4-induced DPC proliferation in human beings. Finally, hair growth is promoted by increasing the secretion of extracellular vesicles (EVs) by DPCs. 19 Kwack et al 20  Despite significant advances in molecular technologies, the molecular basis of hair growth promotion through DPCs is still not well understood. Hair growth resembles wound healing in that it requires a highly coordinated interplay among cell proliferation, cell differentiation and cell migration. 22,23 The transformation of HF telogen and anagen stages depends on the crosstalk of DPCs and HF stem cells to produce the necessary activators. 24,25 Previous studies have shown that DPCs promote hair growth by secreting Wnt/β-catenin, 26 Notch, 27 bone morphogenetic protein 28 and sonic hedgehog (Shh) 29,30 signalling molecules, which communicate with epithelial cells. 24 In particular, the canonical Wnt/β-catenin signalling pathway plays critical roles in facilitating HF entry into the anagen stage. 31 Similarly, during wound healing in mice with cutaneous injury, Shh levels rise, activating the hedgehog pathway and promoting HF regeneration. Therefore, the overexpression of Shh on the epidermis can lead to extensive HF regeneration in wounds, suggesting that the activation of the Shh signal in Wnt-responsive cells can promote wound healing. 32 These events are driven by compartmentalized cell types and their signalling molecules, which regulate de novo hair formation in embryonic skin and new hair growth cycling in adult skin, switching from dormancy to rapid cell division during cycling. 33 Therefore, the search for key signalling molecules that control these processes has become a major focus of studies of the growth and development of HFs.
Thymosin β4 (Tβ4) is a highly conserved G-actin-sequestering protein that is involved in various biological processes, such as cell migration, angiogenesis and wound healing. Recently, Tβ4 has been shown to play key roles in HF growth and development. Moreover, endogenous Tβ4 can activate the HF cycle transition in mice and affect HF growth and development by promoting the migration and differentiation of HF stem cells and their progeny. 34,35 In addition, exogenous Tβ4 increases the rate of hair growth in mice and promotes cashmere production by increasing the number of secondary HFs in cashmere goats. [36][37][38] However, the molecular mechanisms through which Tβ4 promotes HF growth and development have not been described in detail.
Herein, we review the functions and mechanisms of Tβ4 in HF growth and development and describe the endogenous and exogenous actions of Tβ4 in HFs to provide insights into how Tβ4 mediates HF growth and development.

| THE S TRUC TURE AND D ISCOVERY OF Tβ 4
Thymosins are lymphoid growth factors that were first isolated by Goldstein and White from the thymus of a calf in 1966. 39,40 Based on the isoelectric point (pI), thymosins can be classified into α (pI < 5.0), β (5.0 < pI <7.0) and γ (pI > 7.0) types. 41,42 Among these types, thymosin β, characterized by sequestration of globular actin (G-actin) monomers, is highly conserved at the amino acid sequence level in species ranging from echinoderms to mammals. 43 Currently, 16 F I G U R E 1 Schematic representation and terminology of the structure of the hair follicle. The hair shaft (HS) consists of the medulla (M), hair cortex (Cx) and hair cuticle (Ce). The inner root sheath (IRS) consists of the cuticle of the inner root sheath (Ci), Huxley's layer (Hx) and Henle's layer (He). The companion layer (Cp) is occasionally referred to as a member of the outer root sheath (ORS), which includes the hair matrix (Mx) and dermal papilla (DP) thymosin β members have been reported; among these, Tβ4, thymosin β10 (Tβ10) and thymosin β15 (Tβ15) have been identified and isolated in mammals. Studies have shown that Tβ4, accounting for more than 70% of all thymosins, is found ubiquitously in mammalian body fluids, tissues and cells (except erythrocytes) and can be detected in both the nucleus and cytoplasm. 41 Tβ4 is a water-soluble G-actin-sequestering protein with a molecular weight of 4.9 kDa and a pI of 5.1 (Figure 2). Tβ4 is composed of 43 amino acid residues, the most abundant of which are glutamate and glycine. 44,45 Protein conformation analysis has shown that there is a high helical content at positions 4-15 and 30-40 of the amino acid sequence of Tβ4. Additionally, internal overlap is observed at positions 18-30 and 31-43. N-acetyl-seryl-aspartyl-lysyl-proline is the N-terminal amino acid residue sequence of Tβ4, which can be obtained by direct cleavage of the chemical bond between proline and aspartic acid using prolyl oligopeptidase or Aspn-like protease. 46 Nuclear magnetic resonance analysis showed that Tβ4 mainly contains α helices in aqueous solution, exhibiting a loose structure.
When the pH value of the aqueous solution is 4.5-7.5 and the temperature is between 5 and 40°C, the secondary structure of Tβ4 is difficult to identify. In addition to its binding affinity to G-actin, Tβ4 is able to form complexes with F-actin. 47 In recent years, Tβ4 has been extensively studied, mainly owing to its involvement in various biological processes. In 1995, Grant and colleagues reported that Tβ4 is involved in endothelial cell differentiation. 48 In addition to its ability to induce endothelial cell differentiation, Tβ4 also promotes endothelial cell migration and stimulates microtubule formation and angiogenesis in vitro and in vivo. Moreover, Tβ4 enhances wound healing through various mechanisms, including increased angiogenesis, improved keratinocyte migration, collagen deposition and wound contracture. 49,50 Furthermore, Tβ4 has anti-inflammatory and hair growth improvement effects. 35 Recently, Tβ4 has been shown to play key roles in HF growth and development. Endogenous Tβ4 can activate the HF cycle transition in mice and affects HF growth and development by promoting the migration and differentiation of HF stem cells and their progeny. 34,35 In addition, exogenous Tβ4 increases the rate of hair growth in mice and promotes cashmere production by increasing the number of secondary HFs in cashmere goats. [36][37][38] Taken together, these findings demonstrate that Tβ4 is a pleiotropic protein with important roles in the growth and development of HFs via modulation of HF growth and development through its various functions.

| ROLE S OF ENDOG ENOUS Tβ 4 IN HF G ROW TH AND DE VELOPMENT
According to the literature, few studies have assessed the roles of endogenous Tβ4 in HF growth and development, and most studies on this topic have been published by a single group. 35 The primary

| EFFEC TS OF E XOG ENOUS Tβ ON HF G ROW TH AND DE VELOPMENT
The Philp team first identified the effects of exogenous Tβ4 on HF growth and development by treatment of the skin surface with Tβ4. 34,35 During their study of wound healing in the skin of rats, they unexpectedly observed that, at the histological level, the number of HFs at the wound margins increased after 7 days of topical treatment with Tβ4. In this study, the sides of healthy rats were shaved, and  The Wnt signalling pathway is another possible target of Tβ4 and is involved in regulating the growth and development of HFs. 74,75 Indeed, the Wnt signalling pathway is the main signalling pathway controlling HF growth; β-catenin and lymphoid enhancer-binding factor 1 (LEF-1) are key molecules in this pathway.

| P OSS IB LE S I G NALLING PATHWAYS OF Tβ 4 IN HF G ROW TH AND DE VELOPMENT
In the nucleus, β-catenin directly interacts with LEF-1, activates the keratin gene, promotes hair growth and regulates the HF cycle. [76][77][78][79] In addition, after Wnt signalling is activated, β-catenin can also bind to the TCF/LEF complex to form the β-catenin/TCF/LEF complex. In a study by Liu et al, 37

| CON CLUS IONS
The growth and development of HFs involve many molecules and cells. Therefore, the search for key signalling molecules that control these processes has become a major focus of research. In recent years, the study of Tβ4 in the growth and development of HFs has gradually become a research hotspot in this field, not only because of its involvement in cell differentiation, angiogenesis, wound healing and other biological processes but also because of its potential clinical and production application value. In terms of HF growth and

ACK N OWLED G EM ENTS
We would like to thank Editage (www.edita ge.cn) for English language editing.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.