TWEAK regulates the functions of hair follicle stem cells via the Fn14-Wnt/β-catenin-CXCR4 signalling axis

Abstract

Hair follicle stem cells (HFSCs) are crucial for maintaining cutaneous functions under various pathological conditions, including wounds. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) interacts with its receptor, fibroblast growth factor-inducible 14 (Fn14), and plays a role in the development and tissue repair of skin diseases. This study aims to elucidate the effects of TWEAK/Fn14 signalling on HFSCs and the associated mechanisms. The expressions of HFSC markers, including K19, integrin β1 and K15, were analysed via immunohistochemistry in normal and Fn14-deficient mouse skin. Primary HFSCs were cultured in vitro and then treated with TWEAK or a chemokine (CXC motif) (CXCR) 4 inhibitor. The phenotype markers and secreted cytokines of HFSCs were assessed via immunofluorescence analysis, Western blotting and real-time polymerase chain reaction. Our results showed that both Fn14 and CXCR4 were highly expressed in hair follicles. Fn14 deficiency led to a decrease in the expression levels of K19 and CD34. Exogenous TWEAK enhanced the expression of K15, K19, integrin β1, tumour necrosis factor receptor type 2 and CXCR4 in cultured HFSCs. Additionally, TWEAK induced the proliferation, migration and cytokine production in HFSCs. Furthermore, the Wnt/β-catenin signalling pathway was upregulated in HFSCs upon TWEAK stimulation, and inhibitors of β-catenin or CXCR4 suppressed the effects of TWEAK on the differentiation and secretory functions of HFSCs. In conclusion, TWEAK/Fn14 interaction regulates the expression of differentiation markers and secretory functions of HFSCs in vitro. Wnt/β-catenin signalling or CXCR4 activation mediates the effects of TWEAK on HFSCs. Targeting the Fn14-Wnt/β-catenin-CXCR4 signalling axis may offer a potential approach for managing HFSC-related skin diseases, such as wounds.

Keywords: CXCR4; Fn14; TNFR; TWEAK; hair follicle stem cell.

FIGURE 1

TWEAK/Fn14 signalling upregulates the expressions of HFSC markers. (A) Immunofluorescence analysis of Fn14 and CXCR4 expression in mouse hair follicles. The white arrows indicate the Fn14‐ or CXCR4‐positive cells; however, these positive cells were significantly diminished and even became indistinct in the Fn14‐deficient specimen. Scale bar = 20 μm. (B) Histochemical analysis of Fn14, K19 and CD34 expression in mouse hair follicles. Scale bar = 20 μm. (C) Primary human HFSCs cultured in vitro. (D) Immunofluorescence analysis of β1 integrin and K15 expression in HFSCs stimulated with TWEAK (10 ng/mL). Scale bar = 10 μm. (e, f) Western blotting of K19, integrin β1 and K15 proteins. Data represent mean ± SEM from three independent experiments. *p < 0.05, compared with the 0 ng/mL TWEAK group. CXCR4, chemokine (C—X—C motif) receptor 4; Fn14, fibroblast growth factor‐inducible 14; HFSC, hair follicle stem cells; K15/K19, keratins 15/19; TWEAK, tumour necrosis factor‐like weak inducer of apoptosis.

FIGURE 2

TWEAK induces proliferation, migration and cytokine production in HFSCs. Human HFSCs were cultured in vitro and stimulated with TWEAK (0–250 ng/mL). (A) Proliferation of HFSCs analysed via flow cytometry. n = 5 per group. (B) Cell migration assessed via scratch analysis. n = 5 per group. (C) mRNA expression levels of EGF, BFGF, TGF‐β, NGF and VEGF measured via qRT‐PCR. n = 3 per group. (D) Cytokine levels in the supernatants determined via ELISA. n = 3 per group. Data represent mean ± SEM from three to five independent experiments. *p < 0.05 compared with the 0 ng/mL group. #p < 0.05 compared with the 10 ng/mL group. HFSC, hair follicle stem cells; qRT‐PCR, quantitative real‐time PCR; SEM, standard error of the mean; TWEAK, tumour necrosis factor‐like weak inducer of apoptosis.

FIGURE 3

TWEAK upregulates the expressions of HFSC phenotype markers. Human HFSCs were cultured in vitro and stimulated with TWEAK (0–250 ng/mL). (A) Fn14 and TNFR2 expressions detected via immunofluorescence. (B) mRNA expression levels of FN14, TNFR2, IGFR and CXCR4 analysed via qRT‐PCR. (C, D) Protein expressions of these markers determined via Western blotting, with quantification using ImageJ software. Data represent mean ± SEM from three independent experiments. *p < 0.05 compared with the 0 ng/mL group. #p < 0.05 compared with the 10 ng/mL group. Δp < 0.05 compared with the 50 ng/mL group. HFSC, hair follicle stem cells; qRT‐PCR, quantitative real‐time PCR; SEM, standard error of the mean; TWEAK, tumour necrosis factor‐like weak inducer of apoptosis.

FIGURE 4

TWEAK activates Wnt/β‐catenin signalling in HFSCs. Human HFSCs were cultured in vitro and stimulated with TWEAK (10 ng/mL). (A) mRNA expression levels of WNT5A, CTNNB and GSK3B analysed via qRT‐PCR. (B, C) Protein expressions of these markers detected via Western blotting, with quantification using ImageJ software. (D) mRNA expression levels of TNFR2, IGFR and CXCR4 analysed in cells pretreated with the β‐catenin inhibitor XAV‐939. (E, F) Protein expressions of these markers detected via Western blotting, with quantification using ImageJ software. Data represent mean ± SEM from three independent experiments. In (A, C), *p < 0.05 compared with the blank group. In (D, F), *p < 0.05 compared with the PBS alone group; #p < 0.05 compared with the XAV‐939 group. Δp < 0.05 compared with the TWEAK alone group. HFSC, hair follicle stem cells; qRT‐PCR, quantitative real‐time PCR; TWEAK, tumour necrosis factor‐like weak inducer of apoptosis.

FIGURE 5

CXCR4 inhibitor suppresses the effect of TWEAK on HFSCs. Human HFSCs were cultured in vitro and treated with TWEAK (10 ng/mL) in the presence or absence of the CXCR4 inhibitor EPI‐X4. (A) Proliferation of HFSCs assessed via flow cytometry. n = 5 per group. (B, C) Protein expressions of differentiation markers detected via Western blotting and quantitated using ImageJ software. n = 3 per group. (D). Cytokines synthesised by HFSCs and measured via ELISA in the culture supernatants. n = 3 per group. Data represent mean ± SEM from three to five independent experiments. *p < 0.05 compared with the blank group. #p < 0.05 compared with the TWEAK alone group. CXCR4, chemokine (C—X—C motif) receptor 4; HFSC, hair follicle stem cells; TWEAK, tumour necrosis factor‐like weak inducer of apoptosis.