Increased Expression of Zyxin and Its Potential Function in Androgenetic Alopecia

Abstract

Androgenetic alopecia (AGA) is the most common progressive form of hair loss, occurring in more than half of men aged > 50 years. Hair follicle (HF) miniaturization is a feature of AGA, and dermal papillae (DP) play key roles in hair growth and regeneration by regulating follicular cell activity. Previous studies have revealed that adhesion signals are important factors in AGA development. Zyxin (ZYX) is an actin-interacting protein that is essential for cell adhesion and migration. The aim of this research was to investigate the expression and potential role of ZYX in AGA. Real-time polymerase chain reaction (RT-PCR) analysis revealed that ZYX expression was elevated in the affected frontal HF of individuals with AGA compared to unaffected occipital HF. Moreover, increased ZYX expression was also observed within DP using immunofluorescence staining. Our in vivo results revealed that ZYX knockout mice showed enhanced hair growth and anagen entry compared to wild-type mice. Reducing ZYX expression in ex vivo cultured HFs by siRNA resulted in the enhanced hair shaft production, delayed hair follicle catagen entry, increased the proliferation of dermal papilla cells (DPCs), and upregulated expression of stem cell-related proteins. These results were further validated in cultured DPCs in vitro. To further reveal the mechanism by which ZYX contributes to AGA, RNA-seq analysis was conducted to identify gene signatures upon ZYX siRNA treatment in cultured hair follicles. Multiple pathways, including focal adhesion and HIF-1 signaling pathways, were found to be involved. Collectively, we discovered the elevated expression of ZYX in the affected frontal hair follicles of AGA patients and revealed the effects of ZYX downregulation on in vivo mice, ex vivo hair follicles, and in vitro DPC. These findings suggest that ZYX plays important roles in the pathogenesis of AGA and stem cell properties of DPC and may potentially be used as a therapeutic target in AGA.

Keywords: RNA-seq; Zyxin; androgenetic alopecia; dermal papilla cell; hair follicle.

Figure 1

Detection of ZYX in AGA hair follicles and dermal papilla cells. (A) HE staining of hair follicles. Scale bar: 5 μm. (B,C) mRNA levels of ZYX in hair follicles. N = 20. (D) Immunofluorescence examination of ZYX in hair follicle tissues. Scale bar: 5 μm. (E) Cell counting of ZYX-positive dermal papilla cells. N ≥ 3; *P < 0.05, ***P < 0.001 Pregnant group vs. Alendronate group. Control bars represent the mean ± SD.

Figure 2

Effect of ZYX on hair growth in mice. (A) Size of hair covered skin area in Zyx-/- and WT mice. Different morphology of hair shaft (B) and hair follicles (C) in Zyx-/- and WT mice. (D) HF percentage and cycling score in Zyx-/- and WT mice. Scale bar: 5 μm, N =6 per group; *P < 0.05, ***P < 0.001. Control bars represent the mean ± SD.

Figure 3

The ratio of hair follicle growth after ZYX knockdown in vitro. (A) Measurement of the length of hair shaft in ZYX knockdown and negative control groups. (B) Distribution of hair follicle cycle in ZYX knockdown and control groups. (C,D) Bulb diameter and cell counting of Ki67-positive cells in si-ZYX HFs and NC HFs. (E) Evaluation of apoptosis using the TUNEL method after ZYX knockdown. (F) Inductivity marker expression in si-ZYX HFs. *P < 0.05, **P < 0.01, ***P < 0.001. Control bars represent the mean ± SD.

Figure 4

Proliferation ability and inductivity properties in ZYX-deficient DP cells. (A) ZYX levels in DPCs after NC or si-ZYX treatment. (B,C) RTCA assay of si-ZYX- and NC-treated DP cells. (D,E) Cell cycle distribution in si-ZYX- and NC-treated DP cells. (F) Inductivity marker expression in si-ZYX HFs using qPCR assay. (G) Inductivity ability assay in si-ZYX or control DPCs. *P < 0.05, **P < 0.01, ***P < 0.001. Control bars represent the mean ± SD.

Figure 5

Transcriptomics changes in ZYX-deficient DP cells. (A) Heat map of DEGs between ZYX knockdown and control DP cells. (B) List of the upregulated and downregulated DEGs. (C,D) KEGG analysis of the DEGs. N = 3 per group.