The effect of abnormal secretion of DKK1 by fibroblasts on melanocytes function in vitiligo

The pathogenesis of vitiligo remains unclear,1 and in recent years, the role of dermal compartment,2 especially fibroblasts,2 has attracted increasing attention. Xu et al.3 found that fibroblasts in different anatomical sites determined different skin autoimmune patterns, which strongly confirmed the importance of mesenchymal components. Yamaguchi et al. reported that DKK1 derived from fibroblasts in different anatomical sites has an effect on the function of human melanocytes,4, 5 followed by studies reporting elevated expression of DKK1 in vitiligo lesions by immunohistochemistry,6 ELISA7 and qRT-PCR,8 respectively. However, there are limited studies on the specific role of fibroblasts-secreted DKK1 in the pathogenesis of vitiligo. This study aims to explore the effect of abnormal expression of DKK1 in fibroblasts on melanocyte melanin synthesis function, further elucidating the potential role of fibroblasts in vitiligo. In this study, skin specimens were taken from the depigmented lesion region of 30 non-segmental trunk vitiligo patients (18 men and 12 women, age range 12–65 years). Primary vitiligo fibroblasts (VF) and normal fibroblasts (NF) were extracted from vitiligo lesions and healthy control, respectively. Skin biopsy was sheared into pieces and digested with 0.1% Collagenase II (Sigma-Aldrich, USA), then cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Absin, China) at 37°C under 5% CO2. We detected the expression of DKK1 in vitiligo lesions and normal tissues, using immunohistochemistry, then followed by the expression levels of DKK1 in VF and NF using qRT-PCR and Western blot. Recombinant adenoviruses Ad-DKK1 and Ad-siDKK1 were used to transfect NF and VF, respectively. Next, we sequenced the transcriptome of VF with silenced DKK1 and conducted KEGG pathway enrichment analysis on differentially expressed genes. Immunocytochemical staining was used to detect senescencing-related markers. We collected cell culture supernatants as conditioned medium (NFDKK1-CM and VFsiDKK1-CM) and assayed the levels of fibroblasts secreted factors using ELISA. Subsequently, human melanocyte (PIG1) and vitiligo melanocyte (PIG3V) were treated with conditioned medium, melanin content, tyrosinase activity and melanogenesis-related proteins were determined. Finally, 3D melanoma skin models were treated with conditioned medium to simulate the in vivo environment and investigate the effects of DKK1 on pigmentation. We found that DKK1 expression was significantly elevated in vitiligo tissues and fibroblasts (Figure 1a,b). Through sequencing, results suggested significant differences in cellular senescence (Figure 1c). The immunocytochemical staining revealed that the overexpression of DKK1 upregulated the expression of HP1, P16 and P21, and conversely, silencing DKK1 expression decreased these markers (data not shown). ELISA analysis revealed that Ad-DKK1 treatment led to a decrease in HGF and NRG-1 secretion, while Ad-siDKK1 treatment upregulated their levels (Figure 1d). Furthermore, PIG1 treated with NFDKK1-CM showed a reduction in melanin content and tyrosinase activity (Figure 1e,f), as well as decrease in the expression of melanin synthesis-related proteins (Figure 2a,c,d). Conversely, treating PIG3V cells with VFsiDKK1-CM yieled the opposite results (Figures 1e,f and 2b–d). In addition, the findings from our experiment on 3D melanin skin models were congruent with those from cellular experiments (Figure 2e). In conclusion, our research findings indicate that the elevated expression of DKK1 in both vitiligo tissues and fibroblasts leads to the fibroblasts senescence, decreases the levels of HGF and NRG-1, affects the cell microenvironment and consequently inhibits melanin synthesis function of melanocytes. These insights into the interplays between dermal and epidermal cells in vitiligo offer valuable leads for the development of innovative therapeutic approaches for the development of vitiligo. However, our research is limited to biological phenomena, and the exploration of the mechanism is still insufficient, which is the direction of our follow-up research. This study was supported by the National Natural Science Foundation of China (grant No. 82073462). None declared. All procedures in this study followed were conducted in accordance with the ethical standards established by the responsible committee on human experimentation whether institutional or regional, and in compliance with the Helsinki Declaration. Data sets analysed or used in the current study can be obtained upon reasonable request from the corresponding authors.