Valproic acid induces hair regeneration in murine model and activates alkaline phosphatase activity in human dermal papilla cells

Abstract

Background: Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA), a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo.

Methodology/ principal findings: Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP). VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX), a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl(2) also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice.

Conclusions/ significance: Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth.

Figure 1. Effects of VPA and MNX on hair re-growth, hair cycle stage in mouse skin.

The back skin of 7-wk-old male C3H mice was shaved and treated daily with topical application of vehicle, 500 mM valproic acid (VPA), or 100 mM minoxidil (MNX) for 28 d. (A) Gross images showing hair re-growth in C3H mice treated with VPA or MNX for 28 d (upper panel) and H&E staining of the VPA or MNX-treated skin for 28 d (lower panel). (B) Quantitative histomorphometic analyses of skin tissues treated with VPA or MNX for 28 d were performed with 5 mice for each group; Early anagen, anagen I-anagen II; Middle anagen, anagen III-IV; Late anagen, anagen V-anagen VI; Early catagen, catagen I-catagen III; Middle catagen, catagen IV-V; Late catagen, catagen VI-catagen VIII. Asterisks denote significant differences between control and test groups as measured by t-test with one asterisk being p<0.05, two asterisks being p<0.005, and three asterisks being p<0.0001. Original magnification: A, ×100 (H&E staining).

Figure 2. Effects of VPA and MNX on epidermal differentiation markers, ALP activities, and hair follicular stem cell markers.

The back skin of 7-wk-old male C3H mice was shaved and treated daily with topical application of vehicle, 500 mM valproic acid (VPA), or 100 mM minoxidil (MNX) for 7 d or 28 d. (A) Immunohistochemical analysis of epidermis of C3H mice treated with VPA or MNX for 28 d with antibody against β-catenin, filaggrin, loricrin, keratin 14, p-Erk, p-Akt, or PCNA. (B) Immunohistochemical analysis of β-catenin expression in hair follicles (upper panel) and ALP staining (lower panel) of skin treated with vehicle, VPA, or MNX for 7 d. Dark blue region in the dermal papillae of hair follicles (a red arrow in middle panel) represent positivity for ALP activity. (C) Immunohistochemical analysis was performed with anti-keratin 15, or -CD34 antibody. Original magnification: A, ×635; B, ×635 (immunohistochemistry) and ×400 (ALP staining); C, ×635.

Figure 3. Effects of VPA and MNX on the activation status of the Wnt/β-catenin pathway and ALP activity in human dermal papilla cells.

Human dermal papilla cells at passage 11 with minimal ALP activity were used to test the ability of VPA and MNX to recover ALP activity. Cells were grown in DMEM supplemented with 10% heat-inactivated FBS, G418 (100 µg/ml), streptomycin (100 µg/ml), and penicillin G sodium (100 µg/ml) in 5% CO₂ at 37°C, and treated with 1 mM VPA or 100 µM MNX for 72 h. (A) Western blotting for β-catenin, BMP4, ALP and α-tubulin. (B) Immunocytochemical staining with antibody against β-catenin or BMP4. (C) ALP staining. Cell morphology (left panels) was examined under a bright-field microscope. Dark blue staining indicates ALP-expressing cells (right panels). (D) Western blotting for β-catenin, BMP4, ALP, and α-tubulin in human dermal papilla cells treated with VPA or noggin (500 ng/ml). (E) Western blotting for β-catenin, BMP4, ALP, and α-tubulin expression in human dermal papilla cells treated with Wnt3a (200 or 40 ng/ml), BMP4 (100 or 20 ng/ml), or EGF (100 or 20 ng/ml) for 72 h. (F) ALP activity was measured as described in Methods after treatment with Wnt3a (200 ng/ml), BMP4 (100 ng/ml), or EGF (100 ng/ml) for 72 h. Asterisks denote significant differences between control and test group as measured by t-test with one asterisk being p<0.05 and two asterisks being p<0.005. Original magnification: B, ×635; C, ×100.

Figure 4. Effects of Wnt/β-catenin pathway activators on hair re-growth on mouse skin.

The vehicle, 500 mM VPA, 100 mM MNX or derivatives of VPA (500 mM PBA, 500 mM EBA) were topically applied to shaved back skin of C3H mice daily for 28 d. (A) Gross images of hair re-growth (first row panel), immunohistochemistry of the drug-treated skin with antibody against β-catenin (second row panel), and H&E staining of the drug-treated skin (third row panel). (B) Quantitative histomorphometric analyses were performed for 5 mice for each group. Asterisks denote significant differences between control and test group as measured by t-test with one asterisks being p<0.05, two asterisks being p<0.005, and three asterisks being p<0.0001. Original magnification: A, ×100 (H&E staining) and ×635 (immunohistochemistry).

Figure 5. Effects of Wnt/β-catenin pathway activators on the regulation of BMP and alkaline phosphatase activity.

Human dermal papilla cells at passage 11 with minimal ALP activity were used to test the ability to recover ALP activity. (A) Morphology (upper panels) and ALP staining patterns (lower panels) of human dermal papilla cells treated with 1 mM VPA, 1 mM PBA, 1mM EBA. Cells were observed under bright-field. (B) Western blotting for β-catenin, ALP, or α-tubulin expression in extracts prepared from drug-treated human dermal papilla cells. (C) RT-PCR analysis of BMP4, BMP6, ALP, and GAPDH expression using total RNA prepared from drug-treated human dermal papilla cells. (D) ALP activity. Asterisks denote significant differences between control and test group as measured by t-test with one asterisk being p<0.05, two asterisks being p<0.005. Original magnification: B, ×100.

Figure 6. Effects of VPA or MNX on wound-induced hair growth.

Four full-thickness excisions (0.2 cm² circular wounds) of skin were made on the backs of 8-wk-old C3H or TOP-Gal transgenic mice, and 500 mM VPA or 100 mM MNX was topically applied daily to the wounds. (A) Gross images of representative wounded back skin of C3H mice 25 d after drug application. (B) H&E staining of wounded skin treated with VPA or MNX. (C, D) Western blot (C) and Immunohistochemical (D) analyses of β-catenin, filaggrin, loricrin and keratin 14 in the wounds. (E) Wounded skins were treated with vehicle, VPA, or MNX for 14 d, and tissue was subjected to ALP staining assays. (F) Immunohistochemical analysis was performed with anti-keratin 15, or -CD34 antibody. Original magnification: B, ×200; C, ×635; D, ×200; E, ×200; F, ×635.