Knockdown of FOXA2 Impairs Hair-Inductive Activity of Cultured Human Follicular Keratinocytes

Abstract

Reciprocal interactions between hair-inductive dermal cells and epidermal cells are essential for de novo genesis of hair follicles. Recent studies have shown that outer root sheath (ORS) follicular keratinocytes can be expanded in vitro, but the cultured cells often lose receptivity to hair-inducing dermal signals. In this study, we first investigated whether the hair-inductive activity (trichogenicity) of cultured human ORS follicular keratinocytes was correlated with the cultivation period. ORS follicular keratinocytes from the scalp were cultured for 3, 4, 5, or 6 weeks and were then implanted into nude mice along with freshly isolated neonatal mouse dermal cells. We observed that the trichogenicity of the implanted ORS cells was inversely correlated with their cultivation period. These initial findings prompted us to investigate the differentially expressed genes between the short-term (20 days) and long-term (42 days) cultured ORS cells, trichogenic and non-trichogenic, respectively, by microarray analysis. We found that forkhead box protein A2 (FOXA2) was the most up-regulated transcription factor in the trichogenic ORS cells. Thus, we investigated whether the trichogenicity of the cells was affected by FOXA2 expression. We found a significant decrease in the number of induced hair follicles when the ORS cells were transfected with a FOXA2 small interfering RNA versus control small interfering RNA. Taken together, our data strongly suggest that FOXA2 significantly influences the trichogenicity of human ORS cells.

Keywords: forkhead box protein A2; hair induction; outer root sheath; transcription factor; trichogenicity.

FIGURE 1

Diagram of human outer root sheath (ORS) follicular keratinocytes cultivation. (A) Human ORS cells were cultured as described in the Materials and Methods section. Representative images from a patient (male, age 37 years) are shown. (B) Cells in various culture periods (3, 4, 5, and 6 weeks) from a patient (male, age 28 years) were immunostained with cytokeratin 8 antibody.

FIGURE 2

Inverse correlation of hair-inductive activity of ORS cells with their cultivation period. (A) Diagram of a “patch” hair reconstitution assay used in this study. Cultured human ORS cells (1 × 10⁶ cells) were combined with freshly isolated neonatal mouse dermal cells (1 × 10⁶ cells) and then subcutaneously implanted into the skin on the backs of 7-week-old female nude mice. (B) Hair follicle (HF) formation was examined from the back skin of the mice after 2 weeks and representative pictures are shown. High power images of boxed regions are shown in Supplementary Figure S2. (C) Total induced HFs were quantified and the data are the means ± standard deviation of four determinations using same batch ORS cells from a patient (male, age 26 years). *P < 0.05, **P < 0.01.

FIGURE 3

Comparison of the gene expression profiles between the short-term and long-term cultured ORS cells. (A) Diagram of twofold up- and down-regulated genes in short-term cultured ORS cells. Two independent cell lines (#1 and #2) from two male patients (ages 50 and 64 years) were cultured for 20 days and 42 days, and microarray analysis was performed with transcripts derived from those cells. The number of genes with a more than twofold increase and decrease in the short-term cultured ORS cells are shown on the left and right, respectively. A total of 144 genes had a more than twofold increase in both short-term cultured ORS cells (listed in Supplementary Table S1), and 278 genes had a more than twofold decrease in both short-term cultured ORS cells (listed in Supplementary Table S2). (B) Heat map of differentially expressed genes (DEGs). The up-regulated genes were shown in red color and down-regulated genes in green color. (C) List of genes related to the skin signaling pathways from twofold up-regulated (2.0×) and down-regulated (0.5×) genes. Genes were selected based on gene-set enrichment analysis (GSEA) information in each pathway (HH, FGF, VEGF, Notch, BMP, and TGF-b). (C’) List of up-regulated Wnts and their receptor (FZD) among WNT signaling pathway. (D) Relative fold changes of six up-regulated transcription factors. (E) Relative fold changes of four down-regulated transcription factors. (F) Relative levels of FOXA2 expression of ORS cells in various cultivation periods (3, 4, 5, and 6 weeks). Data are expressed as means ± standard deviation of three different ORS cell lines from three male patients (ages 33, 38, and 67 years).

FIGURE 4

Impairment of hair-inductive activity of ORS cells by knockdown of FOXA2. (A) A schematic illustration of the experimental procedure. (B) The relative expression of FOXA2 in ORS cells transfected with control siRNA or FOXA2 siRNA was measured by qPCR. (C) Representative pictures of induced hair follicles from nude mice skin are shown. (D) The total number of induced hair follicles was counted at each injection site. Data are expressed as means ± standard deviation of eight determinations using three different ORS cell lines from three male patients (ages 38, 50, and 58 years). *P < 0.05.