Lactoferrin promotes hair growth in mice and increases dermal papilla cell proliferation through Erk/Akt and Wnt signaling pathways

Abstract

Hair loss affects men and women of all ages. Dermal papilla (DP) plays a crucial role in regulating the growth and cycling of hair follicles. Lactoferrin (LF) exhibits a wide range of biological functions, including antimicrobial activity and growth regulation. However, its effect on DP and its role in hair growth remain unknown. In this study, we found that bovine LF (bLF) promoted the proliferation of DP cells and enhanced the phosphorylation of Erk and Akt. The bLF-mediated proliferation was significantly blocked by the Erk phosphorylation inhibitor PD98059 or the Akt phosphorylation inhibitor LY294002. Moreover, biotin-labeled bLF could bind to DP cells, and the binding was independent of lipoprotein receptor-related protein 1, a known LF receptor. Importantly, bLF stimulated hair growth in both young and aged mice. Moreover, we also found that bLF significantly induced the expression of Wnt signaling-related proteins, including Wnt3a, Wnt7a, Lef1, and β-catenin. The bLF-mediated DP cell proliferation could be significantly reversed by the Wnt pathway inhibitor XAV939. Our findings suggest that bLF promotes hair growth in mice and stimulates proliferation of DP cells through Erk/Akt and Wnt signaling pathways. This study highlights a great potential of the use of bLF in developing drugs to treat hair loss.

Keywords: Alopecia; Dermal papilla; Hair; Lactoferrin.

Fig. 1

bLF increases cell proliferation in DP cells. a DP cells were treated with 50 μg/mL bLF for 0–5 days. b Cell growth at different concentrations of bLF at day 5. Cell proliferation was analyzed by the MTT assay. Data are presented as the mean values ± SD from three independent experiments. ***p < 0.001

Fig. 2

bLF increases phosphorylation of Erk and Akt in DP cells. a DP cells were serum-starved for 1 h and subsequently treated with 50 μg/mL of bLF for 1 h. Signals of phosphorylated proteins on Western blot analyses were quantified and normalized to their total protein levels. b DP cells treated with 50-μg/mL bLF and 5-μM LY294002, 5-μM PD98059, or a combined treatment of LY294002 and PD 98059 for 3 days. Cell proliferation was analyzed by the MTT assay. Data are presented as the mean values ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 3

Binding of bLF to DP cells. a DP cells were treated with 0.1-μΜ biotin-labeled bLF or biotin-labeled BSA in the presence of 100-fold molar excess of unlabeled bLF or BSA for 4 h at 4 °C. The binding of biotin-labeled bLF to DP cells was detected by incubating them with HRP-conjugated avidin, followed by adding the OPD substrate reagent and measuring the absorbance at 492 nm. b Following methanol fixation and permeabilization, cells treated with biotin-labeled bLF or biotin-labeled BSA were stained for streptavidin-FITC Ab (green), and their nuclei were stained with DAPI (blue). Fluorescent microscopy showing DP cells treated with 0.1–1 μΜ of biotin-labeled BSA and biotin-labeled bLF for 1 h. The fluorescent levels of cells bound by biotin-bLF were quantified using AxioVision Software and normalized to the levels of biotin-BSA control. Scale bars, 50 μm. Data are presented as the mean values ± SD from three independent experiments. ***p < 0.001

Fig. 4

RAP does not compete with bLF for binding to DP cells. a The RT-PCR products of β-actin (lanes 1), LRP1 (lanes 2) and intelectin-1 (lane 3) were separated by an agarose gel. Intelectin-1 is a lactoferrin receptor expressed by the small intestine. b DP cells were plated onto 96-well tissue culture plates (1 × 10⁴ cells/well) and incubated with biotin-labeled bLF or biotin-labeled BSA in the presence of the indicated concentrations of RAP (0.1–0.4 μM) for 4 h at 4 °C. The binding of biotin-labeled bLF to DP cells was detected by incubation with HRP-conjugated avidin, which was followed by adding the OPD substrate reagent and measuring the absorbance at 492 nm. Data are presented as the mean values ± SD from three independent experiments. ***p < 0.001

Fig. 5

bLF increases hair growth in both young and aged mice. a Young mice treated with 200-mg/mL bLF or ddH₂O. b Hemotoxylin and eosin staining of the back skin from the young mice. Longitudinal sections of hair follicles (arrows). Scale bar = 200 μm. c Aged mice treated with 40-mg/mL bLF or ddH₂O control for 7 days. Hair growth was quantified using ImageQuant TL software and normalized to their levels at day 0. *p < 0.05

Fig. 6

bLF increases the expression of Wnt signaling-related genes in DP cells. The mRNA expression levels of Wnt3a, Wnt7a, and Lef1 in DP cells treated with 0.5–2.5 μM of bLF or 1–2.5 μM of minoxidil was analyzed by real-time RT-PCR. Data are presented as the mean values ± SD from three independent experiments. *p < 0.05; ***p < 0.001

Fig. 7

bLF increases the Wnt pathway-related proteins in DP cells. a DP cells were treated with 50 μg/mL of bLF and/or 5-μM XAV939 for 48 h (left) or 72 h (right). Signals of proteins on Western blots were quantified and normalized to their total protein levels. b Cell viability of DP cells treated with 50-μg/mL bLF and/or 5-μM XAV939 for 5 days. Cell viability was analyzed using the MTT assay. Data are presented as the mean values ± SD from three independent experiments. *p < 0.05; **p < 0.01

Fig. 8

A schematic depicting the potential mechanisms involved in bLF-induced hair growth. bLF bovine lactoferrin, Erk extracellular signal‐regulated kinase, Akt protein kinase B