Pro-pigmentary Action of 5-Fluorouracil Through the Stimulated Secretion of CXCL12 by Dermal Fibroblasts
Vitiligo is a common acquired pigmentary disorder affecting approximately 1% of the global population, characterized by depigmented skin patches due to the immune-mediated destruction of melanocytes in the epidermis. Current treatments, particularly UVB-based phototherapy, have shown efficacy in stimulating repigmentation. However, a significant proportion of patients exhibit poor responses to these therapies, highlighting the need for novel strategies to enhance skin repigmentation, especially in UVB-resistant cases.
Recent clinical observations have suggested that intradermal injections of 5-fluorouracil (5-FU) can achieve significant repigmentation in localized vitiligo patients who are unresponsive to UVB phototherapy. This pro-pigmentary effect of 5-FU has prompted investigations into its potential extra-genotoxic activities that may favor melanocyte recruitment. This study explores the mechanisms underlying the pro-pigmentary action of 5-FU, focusing on its ability to stimulate the secretion of CXCL12 by dermal fibroblasts, thereby promoting melanocyte migration.
To investigate the effects of 5-FU on melanocyte mobilization and migration, the study utilized a full-thickness excisional skin wound model in Dct-LacZ transgenic mice. These mice carry the LacZ reporter under the control of the Dct promoter, allowing for the dynamic visualization of melanocytes during the wound-healing process. Wounds were created on the dorsal and tail skins of the mice and treated topically with 5% 5-FU or normal saline as a control. Skin specimens were harvested from the wound margins on the third, seventh, and twenty-first days post-surgery and subjected to whole-mount and cryosection X-Gal staining.
The results revealed that LacZ-positive melanocytes were significantly more abundant in the margins of wounds treated with 5-FU compared to the untreated controls. Specifically, on the seventh day post-surgery, the number of LacZ-positive cells in the dorsal skin wound margins treated with 5-FU was notably higher than in the control group. Similarly, in the tail skin, 5-FU treatment resulted in a marked increase in LacZ-positive melanocytes. Cryosection X-Gal staining further confirmed that LacZ-positive cells were present in the outer root sheath (ORS) and interfollicular epidermis of the dorsal skin wound margins treated with 5-FU, whereas they were scarcely observed in the untreated controls.
The study also examined the in-situ expression of CXCL12 in the wound margins using immunofluorescence staining. The results showed that CXCL12 immunostaining was significantly increased in the dermal compartments of wounds treated with 5-FU compared to the untreated controls. This upregulation of CXCL12 suggests a potential role for this chemokine in recruiting melanocytes to the wound site.
To further investigate the relationship between 5-FU and CXCL12, primary mouse dermal fibroblasts were cultured and treated with 50 μmol/L 5-FU for 24 hours. The expression levels of CXCL12 mRNA and protein were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyses. The results demonstrated that 5-FU significantly upregulated the expression of both CXCL12 mRNA and protein in the cultured fibroblasts. Specifically, the relative expression levels of CXCL12 mRNA in 5-FU-treated fibroblasts were 1.54 ± 0.06 compared to 1.00 ± 0.08 in the control group (P < 0.05). Similarly, the protein levels of CXCL12 were 2.93 ± 0.10 in 5-FU-treated fibroblasts versus 1.00 ± 0.06 in the controls (P < 0.05).
The role of the CXCL12/CXCR4 axis in 5-FU-induced melanocyte migration was further corroborated through siRNA-mediated knockdown of CXCL12 in murine fibroblasts and the use of a CXCR4 antagonist (AMD3100). Fibroblasts transfected with CXCL12 siRNA showed a significant reduction in CXCL12 expression, as confirmed by qRT-PCR and Western blotting. Transwell migration assays revealed that the chemotactic migration of melanocytes towards CXCL12-silenced fibroblasts was markedly reduced. Additionally, pretreatment of melanocytes with AMD3100, a specific CXCR4 antagonist, inhibited their migration towards 5-FU-treated fibroblasts. The F-actin staining pattern in melanocytes treated with the conditioned medium from 5-FU-treated fibroblasts showed a coarse actin filament pattern throughout the cytoplasm, indicative of a motile phenotype. In contrast, melanocytes treated with the conditioned medium from CXCL12-silenced fibroblasts exhibited a diffuse or fine network-like F-actin distribution.
These findings collectively suggest that 5-FU stimulates dermal fibroblasts to secrete CXCL12, which in turn activates the CXCL12/CXCR4 axis to drive the chemotactic migration of melanocytes. This pro-pigmentary activity of 5-FU may offer a novel therapeutic approach for enhancing skin repigmentation in vitiligo patients, particularly those who are unresponsive to conventional treatments.
The study also highlights the potential of 5-FU to modulate the microenvironment to favor melanocyte recruitment. Previous research has primarily focused on the antimitotic activity of 5-FU in the treatment of skin cancer, but this study reveals its extra-genotoxic actions in promoting melanocyte migration. The selective cytotoxicity of 5-FU, which spares melanocytes while affecting other epidermal cells, may contribute to its pro-pigmentary effects. This selective action could be harnessed to develop targeted therapies for vitiligo.
In conclusion, this study provides evidence that 5-FU possesses a pro-pigmentary activity through the activation of the CXCL12/CXCR4 axis, which drives the chemotactic migration of melanocytes. The enhanced secretion of CXCL12 by dermal fibroblasts in response to 5-FU treatment represents a novel mechanism that could be exploited to improve repigmentation in vitiligo patients. Future research should explore the potential of 5-FU in combination with other therapeutic modalities to further enhance its efficacy in treating vitiligo.
doi.org/10.1097/CM9.0000000000001689
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