Insights into male androgenetic alopecia using comparative transcriptome profiling: hypoxia-inducible factor-1 and Wnt/β-catenin signalling pathways

Abstract

Background: The key pathophysiological changes in androgenetic alopecia (AGA) are limited to hair follicles (HFs) in frontal and vertex regions, sparing the occipital region.

Objectives: To identify biological differences among HF subpopulations.

Methods: Paired vertex and occipital HFs from 10 male donors with AGA were collected for RNA sequencing assay. Furthermore, HF and cell experiments were conducted on the identified key genes to reveal their roles in AGA.

Results: Transcriptome profiles revealed that 506 mRNAs, 55 microRNAs and 127 long noncoding RNAs were differentially expressed in the AGA vertex HFs. Pathway analysis of mRNAs and microRNAs revealed involvement of the hypoxia-inducible factor (HIF)-1, Wnt/β-catenin, and focal adhesion pathways. Differential expression of HIF-1 prolyl hydroxylase enzymes (EGLN1, EGLN3) and Wnt/β-catenin pathway inhibitors (SERPINF1, SFRP2) was experimentally validated. In vitro studies revealed that reduction of EGLN1, EGLN3, SERPINF1 and SFRP2 stimulated proliferation of dermal papilla cells. Ex vivo HF studies showed that downregulation of EGLN1, EGLN3 and SERPINF1 promoted HF growth, postponed HF catagen transition, and prolonged the anagen stage, suggesting that these genes may be potentially utilized as therapeutic targets for AGA.

Conclusions: We characterized key transcriptome changes in male AGA HFs, and found that HIF-1 pathway-related genes (EGLN1, EGLN3) and Wnt pathway inhibitors (SERPINF1, SFRP2) may play important roles in AGA. What is already known about this topic? Multiple differentially expressed genes and signalling pathways have been found between hair follicles (HFs) in the balding area (frontal and vertex regions) and nonbalding area (occipital region) of individuals with androgenetic alopecia (AGA). A whole-transcriptome atlas of the vertex and occipital region is lacking. What does this study add? We identified a number of differentially expressed genes and pathways between balding vertex and nonbalding occipital AGA HFs by using whole-transcriptome analyses. We identified pathways not previously reported in AGA, such as the hypoxia-inducible factor (HIF)-1 signalling pathway. We verified that HIF-1 pathway-related genes (EGLN1, EGLN3) and Wnt pathway inhibitors (PEDF, SFRP2) played important roles in dermal papilla cell activity, hair growth and the hair cycle. What is the translational message? The EGLN1, EGLN3, SERPINF1 and SFRP2 genes may be potentially utilized as therapeutic targets for AGA.

Figure 1

The whole‐genome transcriptional signature of human hair follicles of patients with androgenetic alopecia (AGA). (a) Circle plot showing the results of the integration of transcriptomic data on hair follicle mRNA (red, outer circle), long noncoding RNA (blue, middle circle) and microRNA (yellow, inner circle) with their P‐values from the DESeq2 results. (b, c) Differential expression analyses of (b) mRNAs and (c) microRNAs are shown in the volcano plots. Each dot represents either an expressed mRNA gene or mature microRNA. The red dots represent overexpression in the vertex group and the blue dots indicate downregulation after screening with false discovery rate correction for multiple testing (FDR < 0·05) and log₂ fold change (< −0·5 or > 0·5). (d, e) Hierarchical clustering analysis of differentially expressed (d) mRNAs and (e) microRNAs of 10 patients with AGA. Each column shows the gene expression value of one sample, and each row represents one gene in the model. Orange and blue indicate high and low z‐scored levels, respectively, while red and green on the legend bar represent hair follicles from the vertex and occipital scalp, respectively. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Enrichment of the hypoxia‐inducible factor (HIF)‐1 and Wnt signalling pathways. (a) KEGG pathway enrichment analysis of differentially expressed (DE) mRNAs. The red and blue bars indicate enriched pathways using the upregulated and downregulated genes in the vertex group, respectively. (b–d) Expression levels of HIF‐1 signalling‐related genes, Wnt signalling inhibitor genes and Wnt signalling pathway genes, respectively. The y‐axis represents the row counts of each gene calculated by kallisto and tximport software. P‐values were derived by DESeq2 following false discovery rate correction for multiple testing. Data are shown as the mean and SEM (n = 10) of each group. ***P < 0·001; **P < 0·01; *P < 0·05; ns, not significant. (e) KEGG pathways enriched by each DE microRNA. The hypergeometric test was used to identify the enriched pathways by using the targeted genes of each DE microRNA. The legend bar represents the −log₁₀(P‐value) of the enriched pathway for each DE microRNA. (f) Dot plot showing that the HIF‐1 signalling pathway is significantly enriched by eight DE microRNAs. The dot size represents the number of microRNA target genes belonging to the HIF‐1 signalling pathway. The x‐axis (expected hits) represents the expected number of microRNA target genes belonging to the HIF‐1 signalling pathway by chance. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Validation of the expression of differentially expressed genes involved in the hypoxia‐inducible factor (HIF)‐1 and Wnt signalling pathways. (a) Haematoxylin and eosin staining showed that the hair follicles (HFs) were anagen HFs and the bulb diameter was decreased in vertex HFs compared with occipital HFs. (b) The expression of HIF‐1 pathway‐related genes (EGLN1, EGLN3 and HMOX1) and Wnt signalling pathway‐related genes (PEDF/SERPINF1, SFRP2 and LGR5) was examined in vertex HFs compared with occipital HFs. The mRNA expression of EGLN1, EGLN3, PEDF/SERPINF1 and SFRP2 was significantly higher in the vertex HFs compared by paired‐sample t‐tests. (c) Immunofluorescence staining showed increased protein expression of EGLN1, EGLN3, PEDF or SFRP2 in vertex HFs compared by paired‐sample t‐tests. **P < 0·01, *P < 0·05. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Effects of EGLN1 and EGLN3 on dermal papilla cells (DPCs) and hair follicles (HFs). (a) mRNA expression levels of EGLN1 and EGLN3 were significantly downregulated after corresponding small interfering (si)RNA transfection in cultured DPCs. (b) xCELLigence system detection showed that siEGLN1 or siEGLN3 treatment significantly increased the proliferation of DPCs compared with the negative control (NC) group DPCs. (c) Immunofluorescence staining assay identified decreased protein levels of EGLN1 and EGLN3 after siEGLN1 or siEGLN3 treatment of HFs. (d) EGLN1 or EGLN3 knockdown increased the length of hair shafts compared with the NC after 2 days of culture. (e) Macroscopic images of cultured hair follicles were obtained every other day. The hair cycle stage was macroscopically determined on day 6, and a greater percentage of anagen HFs and a lower percentage of catagen HFs remained in the EGLN1 or EGLN3 siRNA‐treated group compared with the NC group. Data are expressed as the mean (SD) of each group. P‐values were calculated using unpaired‐sample t‐tests. ***P < 0·001, **P < 0·01, *P < 0·05. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Effects of PEDF/SERPINF1 and SFRP2 on dermal papilla cells (DPCs) and hair follicles (HFs). (a) The mRNA expression levels of PEDF/SERPINF1 and SFRP2 were significantly downregulated after corresponding small interfering (si)RNA transfection in cultured DPCs. (b) xCELLigence system detection showed that siPEDF/SERPINF1 or siSFRP2 treatment significantly increased the proliferation of DPCs compared with the negative control (NC) group DPCs. (c) Recombinant protein PEDF or SFRP2 treatment decreased the proliferation of DPCs. (d) Immunofluorescence staining assay identified decreased protein levels of PEDF and SFRP2 after siPEDF or siSFRP2 treatment of HFs. (e) PEDF/SERPINF1 knockdown increased the length of hair shafts, whereas PEDF recombinant protein significantly inhibited HF growth compared with the NC after 2 days of culture. (f) Treatment with siSFRP2 or recombinant SFRP2 protein showed no significant effect on HF growth. (g) Macroscopic images of cultured hair follicles in different groups were obtained every other day. (h, i) Quantification of hair cycle stage from macroscopic images of cultured hair follicles on day 6. A greater percentage of anagen HFs and a lower percentage of catagen HFs remained in the PEDF/SERPINF1 siRNA‐treated group, and PEDF recombinant protein facilitated catagen transition and shortened the anagen stage. By contrast, siSFRP2 and recombinant SFRP2 showed no significant effect on the HF hair cycle. Data are expressed as the mean (SD) of each group. P‐values were calculated using unpaired‐sample t‐tests. **P < 0·01, *P < 0·05. [Colour figure can be viewed at wileyonlinelibrary.com]