Roles of Melatonin in Goat Hair Follicle Stem Cell Proliferation and Pluripotency Through Regulating the Wnt Signaling Pathway

Abstract

Emerging studies show that melatonin promotes cashmere development through hypodermic implantation. However, the impact and underlying mechanisms are currently unknown. In vitro study has previously demonstrated that melatonin induces cashmere growth by regulating the proliferation of goat secondary hair follicle stem cells (gsHFSCs), but there is limited information concerning the effects of melatonin on cell pluripotency. It is also known that Wnt signaling may actively participate in regulating cell proliferation and stem cell pluripotency. Therefore, in the current investigation, goat hair follicle stem cells were exposed to multiple concentrations of melatonin and different culture times to reveal the relationship between melatonin and the activation of Wnt signaling. A proportionally high Catenin beta-1 (CTNNB1) response was induced by 500 ng/L of melatonin, but it was then suppressed with the dosages over 1,000 ng/L. Greater amounts of CTNNB1 entered the cell nuclei by extending the exposure time to 72 h, which activated transcription factor 4/lymphoid enhancer-binding factor 1 and promoted the expression of the proliferation-related genes C-MYC, C-JUN, and CYCLIND1. Moreover, nuclear receptor ROR-alpha (RORα) and bone morphogenetic protein 4 (BMP4) were employed to analyze the underlying mechanism. RORα presented a sluggish concentration/time-dependent rise, but BMP4 was increased dramatically by melatonin exposure, which revealed that melatonin might participate in regulating the pluripotency of hair follicle stem cells. Interestingly, NOGGIN, which is a BMP antagonist and highly relevant to cell stemness, was also stimulated by melatonin. These findings demonstrated that melatonin exposure and/or NOGGIN overexpression in hair follicle stem cells might promote the expression of pluripotency markers Homeobox protein NANOG, Organic cation/carnitine transporter 4, and Hematopoietic progenitor cell antigen CD34. Our findings here provided a comprehensive view of Wnt signaling in melatonin stimulated cells and melatonin mediated stemness of gsHFSCs by regulating NOGGIN, which demonstrates a regulatory mechanism of melatonin enhancement on the growth of cashmere.

Keywords: CTNNB1; Wnt pathway; cashmere; hair follicle stem cells; melatonin.

FIGURE 1

Melatonin activated Wnt signaling pathway in gsHFSCs. (A) CTNNB1 detected by immunofluorescence staining after stimulation with different concentrations of melatonin. Scale bar, 50 μm. (B) Relative mRNA level of CTNNB1 by RT-qPCR; *p < 0.05, **p < 0.01. (C) CTNNB1 marked by immunofluorescence in gsHFSCs. Scale bar, 20 μm (white circles circumscribe the position of CTNNB1 in the nuclei), and relative immunofluorescence intensity of CTNNB1 in the nuclei; *p < 0.05. (D) CTNNB1 in nuclei/cytoplasm as detected by Western blotting. (E) Fluorescence density analysis of CTNNB1 in the nuclei of gsHFSCs.

FIGURE 2

Melatonin maintained the continuous activation of Wnt signaling in gsHFSCs. (A) CTNNB1, as detected by immunofluorescence staining after stimulation by different periods of melatonin exposure. Scale bar, 50 μm. (B) Relative mRNA levels of CTNNB1 (Control: green; melatonin: pink); *p < 0.05, **p < 0.01. (C) CTNNB1 marked by immunofluorescence in gsHFSCs. Scale bar, 20 μm (white circles circumscribe the position of CTNNB1 in the nuclei), and relative immunofluorescence intensity of CTNNB1 in nuclei; ***p < 0.001. (D) Protein levels of CTNNB1 between 36 and 72 h in treated gsHFSCs as shown by Western blotting. (E) Fluorescence density analysis of CTNNB1 in the nuclei of gsHFSCs after stimulation with different periods of melatonin exposure.

FIGURE 3

Melatonin promoted the expression of Wnt downstream factors in gsHFSCs. (A) Relative mRNA level of TCF4 and LEF1 as detected by RT-qPCR; **p < 0.01, *p < 0.05 (Control: green; 36 h melatonin: orange; 72 h melatonin: blue). (B) TCF4 and LEF1 tested by immunofluorescence staining. Scale bar, 50 μm. (C) TCF4 and LEF1 were compared between melatonin exposed and control cells using Western blotting.

FIGURE 4

Melatonin promoted the proliferation and cell cycle of gsHFSCs by promoting Wnt downstream factor expression, ^∗∗∗p < 0.001. (A) MTT analysis of melatonin on gsHFSCs proliferation following 72 h of exposure. (B) Cell cycle analysis of gsHFSCs after melatonin exposure at 72 h. (C) Relative mRNA level of C-JUN, C-MYC, CYCLIND1, and CDK6 as detected by RT-qPCR after melatonin exposure for 72 h; ^∗∗p < 0.01, ^∗p < 0.05 (Control: green; 72 h melatonin: blue). (D) Proteins tested by immunofluorescence staining. Scale bar, 50 μm.

FIGURE 5

Melatonin mediated the expression of RORα, BMP4, and NOGGIN. (A) Relative mRNA levels of RORα, BMP4, and NOGGIN in gsHFSCs before/after melatonin exposure (Control: green; 36 h melatonin: orange; 72 h melatonin: blue); ^∗∗p < 0.01. (B) Protein levels of RORα, BMP4, and NOGGIN in gsHFSCs as revealed by immunofluorescence staining. Scale bar, 50 μm. (C) Protein levels of RORα, BMP4, and NOGGIN in gsHFSCs as shown by Western blotting. (D) Fluorescence intensity analysis of RORα in melatonin exposed gsHFSCs. (E) Fluorescence intensity analysis of BMP4 in melatonin exposed gsHFSCs. (F) Fluorescence intensity of NOGGIN in melatonin exposed gsHFSCs.

FIGURE 6

Protein levels of CTNNB1, BMP4, NOGGIN, NANOG, OCT4, and CD34 were detected after exposure to 500 ng/L melatonin with or without NOGGIN overexpression as shown by immunofluorescence staining at 72 h. Scale bar, 50 μm.

FIGURE 7

Model summarizing the main findings of this study.