Dickkopf-related Protein 2 Promotes Hair Growth by Upregulating the Wnt/β-catenin Signaling Pathway in Human Dermal Papilla Cells

Abstract

Background: The Wnt/β-catenin signaling pathway is crucial for the development, initiation, and growth of hair follicles (HFs). The Dickkopf-related protein (DKK) gene family encodes secreted proteins modulating the Wnt/β-catenin signaling pathway. Studies have reported that DKK1 promotes the regression of HFs and serves as a pathogenic mediator in male pattern baldness. However, the role of DKK2 on human hair growth has not yet been explored.

Objective: This study investigates direct effect of DKK2 on hair growth using human dermal papilla cell (DPC) cultures and ex vivo human HF organ cultures.

Methods: To elucidate the effect of DKK2 on hair growth, we examined the effect of recombinant human DKK2 (rhDKK2) treatment on cell viability, expression of mRNA and protein related to the Wnt/β-catenin signaling pathway, and cell growth in cultured human DPCs. We also performed ex vivo organ culture of HFs with rhDKK2 and measured changes in hair shaft length for 8 days.

Results: Treatment with rhDKK2 led to a dose-dependent rise in the proliferation of human DPCs (p<0.05), reaching levels comparable to those induced by 1 μM minoxidil. Moreover, rhDKK2 increased the expression of Wnt/β-catenin target genes, phosphorylated extracellular signal-regulated kinase, and cyclin-D1; it also increased the BAX-to-Bcl-2 ratio and downregulated the bone morphogenetic protein 2 gene. In human HF organ cultures, relative to the control treatment, rhDKK2 treatment significantly increased hair shaft elongation (p<0.01).

Conclusion: Our results indicate that rhDKK2 could promote hair growth by facilitating the proliferation of human DPCs through activation of the Wnt/β-catenin signaling pathway.

Keywords: Beta catenin; Dickkopf 2 protein, human; Hair follicle; Wnt signaling pathway.

Fig. 1. rhDKK2 induced the proliferation of human DPCs analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. (A) Relative to controls treated with phosphate-buffered saline, rhDKK2 was found to induce proliferation of human DPCs in a dose-dependent manner: 113% at 25 ng/ml, 120% at 50 ng/ml, and 123% at 100 ng/ml of rhDKK2. (B) The cells were also cultured with combination treatment with rhDKK2 (50 μM) and DHT (20 μM), and the viability did not change. Data are presented as percentages of the non-treated control, and values are expressed as mean ± standard deviation.

DHT: 5α-dihydrotestosterone, rhDKK: recombinant human Dickkopf-related protein, DPC: dermal papilla cell. ^*p<0.05, ^**p<0.01, ^***p<0.001.

Fig. 2. Treatment with rhDKK2 upregulated the expression of Wnt/β-catenin target genes. Expression levels of target genes were determined using real-time polymerase chain reaction assays. Data are presented as percentages of the non-treated control, and values are expressed as mean ± standard deviation.

DHT: 5α-dihydrotestosterone, rhDKK: recombinant human Dickkopf-related protein, Wnt3: wingless type MMTV integration site family, member 3, Wnt5a: wingless type MMTV integration site family, member 5a, Wnt7a: wingless type MMTV integration site family, member 7a, Wnt10b: wingless type MMTV integration site family, member 10b, Lef1: lymphoid enhancer-binding factor-1, AP1: activator protein 1, Axin2: axin-related protein 2. ^*p<0.05, ^**p<0.01, ^***p<0.001.

Fig. 3. rhDKK2 modulated BMP signaling pathway and regulated Bcl-2/BAX and ERK/cyclin-D1 in human DPCs. (A) Western blot assay findings revealed that rhDKK2 increased the phosphorylation of ERK in human DPCs. (B) The ratio of Bcl-2-to-BAX and the expression level of β-catenin in groups treated with rhDKK2 were increased relative to those of the non-treated control group. (C) Treatment with rhDKK2 downregulated the expression of BMP2 gene. Expression levels of target genes were determined using real-time polymerase chain reaction. (D) The expression level of cyclin-D1 was also increased after treatment with rhDKK2. Data are presented as percentages of the non-treated control, and values are expressed as mean ± standard deviation.

DHT: 5α-dihydrotestosterone, rhDKK: recombinant human Dickkopf-related protein, ERK: extracellular signal-regulated kinase, BMP: bone morphogenetic protein, DPC: dermal papilla cell. ^*p<0.05, ^**p<0.01.

Fig. 4. rhDKK2 promoted hair shaft elongation in ex vivo hair organ culture. (A-E) The photographs show representative hair follicles from each group at days 0, 2, 4, 6, and 8 of culture, respectively. (F) Changes in hair shaft length were measured using a stereomicroscope at 2, 4, 6, and 8 days of culture. Values are expressed as mean ± standard deviation.

PBS: phosphate-buffered saline, DHT: 5α-dihydrotestosterone, rhDKK: recombinant human Dickkopf-related protein. ^*p<0.05, ^**p<0.01.