Recent studies suggest that androgen-driven alteration to the autocrine and paracrine factors produced by scalp dermal papilla (DP) cells may be a key to androgen-potentiated balding. Here, we screened dihydrotestosterone (DHT)-regulated genes in balding DP cells and found that dickkopf 1 (DKK-1) is one of the most upregulated genes. DKK-1 messenger RNA is upregulated in 3-6 hours after 50-100 nM DHT treatment and ELISA showed that DKK-1 is secreted from DP cells in response to DHT. A co-culture system using outer root sheath (ORS) keratinocytes and DP cells showed that DHT inhibits the growth of ORS cells, and neutralizing antibody against DKK-1 significantly reversed the growth inhibition of ORS cells. Analysis of co-cultured ORS cells showed a significant increment of sub-G1 apoptotic cells in response to DHT. Also, recombinant human DKK-1 inhibited the growth of ORS cells and triggered apoptotic cell death. In addition, DHT-induced epithelial cell death in cultured hair follicles was reversed by neutralizing DKK-1 antibody. Moreover, immunoblotting showed that the DKK-1 level is up in the bald scalp compared with the haired scalp of patients with androgenetic alopecia. Altogether, our data strongly suggest that DHT-inducible DKK-1 is involved in DHT-driven balding.

Recently, we suggested that Dickkopf 1 (DKK-1) is a pathogenic mediator involved in male pattern baldness. As premature catagen onset is a key characteristic of male pattern baldness, in this study, we evaluated whether DKK-1 has a role as a catagen inducer in hair cycling. Herein, we report that recombinant human DKK-1 (rhDKK-1) injection into the hypodermis of mice during anagen caused premature onset of catagen, whereas neutralizing DKK-1 antibody delayed anagen-to-catagen transition in mice. Moreover, treatment with rhDKK-1 led to a decrease in final hair follicle length, whereas DKK-1 antibody led to an increase compared with control animals. In addition, DKK-1 and DKK-1 messenger RNA expression is most upregulated in follicular keratinocytes of late anagen in depilation-induced hair cycle progression. Moreover, we observed that rhDKK-1 blocks canonical Wnt-mediated activation of b-catenin signaling and induces the proapoptotic protein Bax, resulting in apoptosis in outer root sheath keratinocytes. Taken together, our data strongly suggest that DKK-1 is involved in anagento-catagen transition in the hair cycle by regulating the activity of follicular keratinocytes.

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Autocrine and paracrine factors are produced by balding dermal papilla (DP) cells following dihydrotestosterone (DHT)-driven alterations and are believed to be key factors involved in male pattern baldness. Herein we report that the IL-6 is upregulated in balding DP cells compared with non-balding DP cells. IL-6 was upregulated 3  hours after 10-100  nM DHT treatment, and ELISA showed that IL-6 was secreted from balding DP cells in response to DHT. IL-6 receptor (IL-6R) and glycoprotein 130 (gp130) were expressed in follicular keratinocytes, including matrix cells. Recombinant human IL-6 (rhIL-6) inhibited hair shaft elongation and suppressed proliferation of matrix cells in cultured human hair follicles. Moreover, rhIL-6 injection into the hypodermis of mice during anagen caused premature onset of catagen. Taken together, our data strongly suggest that DHT-inducible IL-6 inhibits hair growth as a paracrine mediator from the DP.

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Dermal papillae (DP) play key roles in hair growth and regeneration by regulating follicular cell activity. Owing to the established roles of exosomes (Exos) in the regulation of cell functions, we investigated whether DP‐derived Exos, especially those from three‐dimensional (3D)‐cultured DP cells, affect hair growth, cycling and regeneration. Exos derived from 3D DP (3D DP‐Exos) promoted the proliferation of DP cells and outer root sheath (ORS) cells and increased the expression of growth factors (IGF‐1, KGF and HGF) in DP cells. 3D DP‐Exo treatment also increased hair shaft elongation in cultured human hair follicles. In addition, local injections of 3D DP‐Exos induced anagen from telogen and also prolonged anagen in mice. Moreover, Exo treatment in human DP spheres augmented hair follicle neogenesis when the DP spheres were implanted with mouse epidermal cells. Similar results were obtained using Exos derived from 2D‐cultured DP cells (2D DP‐Exo). Collectively, our data strongly suggest that Exos derived from DP cells promote hair growth and hair regeneration by regulating the activity of follicular dermal and epidermal cells; accordingly, these findings have implications for the development of therapeutic strategies for hair loss.

Hair loss is a common medical problem affecting both males and females. Dermal papilla (DP) cells are the ultimate reservoir of cells with the potential of hair regeneration in hair loss patients. Here, we analyzed the role of macrophage-derived Wnts (3a and 7b) and macrophage extracellular vesicles (MAC-EVs) in promoting hair growth. We studied the proliferation, migration, and expression of growth factors of human-DP cells in the presence or absence of MAC-EVs. Additionally, we tested the effect of MAC-EV treatment on hair growth in a mouse model and human hair follicles. Data from western blot and flow cytometry showed that MAC-EVs were enriched with Wnt3a and Wnt7b, and more than 95% were associated with their membrane. The results suggest that Wnt proteins in MAC-EVs activate the Wnt/β-catenin signaling pathways, which leads to activation of transcription factors (Axin2 and Lef1). The MAC-EVs significantly enhanced the proliferation, migration, and levels of hair-inductive markers of DP cells. Additionally, MAC-EVs phosphorylated AKT and increased the levels of the survival protein Bcl-2. The DP cells treated with MAC-EVs showed increased expression of vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF). Treatment of Balb/c mice with MAC-EVs promoted hair follicle (HF) growth in vivo and also increased hair shaft size in a short period in human HFs. Our findings suggest that MAC-EV treatment could be clinically used as a promising novel anagen inducer in the treatment of hair loss.

Hypoxia induces the survival and regenerative potential of adipose-derived stem cells (ASCs), but there are tremendous needs to find alternative methods for ASC preconditioning. Therefore, this work investigated: (1) the ability of low-dose ultraviolet B (UVB) radiation to stimulate the survival, migration, and tube-forming activity of ASCs in vitro; (2) the ability of UVB preconditioning to enhance the hair growth-promoting capacity of ASCs in vivo; and (3) the mechanism of action for ASC stimulation by UVB. Although high-dose UVB decreased the proliferation of ASCs, low-dose (10 or 20 mJ/cm 2 ) treatment increased their survival, migration, and tube-forming activity. In addition, low-dose UVB upregulated the expression of ASC-derived growth factors, and a culture medium conditioned by UVB-irradiated ASCs increased the proliferation of dermal papilla and outer root sheet cells. Notably, injection of UVB-preconditioned ASCs into C 3 H/HeN mice significantly induced the telogen-to-anagen transition and increased new hair weight in vivo. UVB treatment significantly increased the generation of reactive oxygen species (ROS) in cultured ASCs, and inhibition of ROS generation by diphenyleneiodonium chloride (DPI) significantly attenuated UVB-induced ASC stimulation. Furthermore, NADPH oxidase 4 (Nox4) expression was induced in ASCs by UVB irradiation, and Nox4 silencing by small interfering RNA, like DPI, significantly reduced UVB-induced ROS generation. These results suggest that the primary involvement of ROS generation in UVB-mediated ASC stimulation occurs via the Nox4 enzyme. This is the first indication that a low dose of UVB radiation and/or the control of ROS generation could potentially be incorporated into a novel ASC preconditioning method for hair regeneration.