Increased melanin induces aberrant keratinocyte - melanocyte - basal - fibroblast cell communication and fibrogenesis by inducing iron overload and ferroptosis resistance in keloids

Abstract

Background: Keloid is a typical skin fibrotic disease with unclear mechanisms and limited therapeutic options. Fibroblast-induced fibrogenesis is a crucial cause of KD. However, the types of cells involved in fibroblast fibrogenesis in KD and the specific mechanisms are unclear. This study aimed to investigate the role of melanocyte-secreted melanin in promoting fibroblast fibrogenesis and its mechanism and to evaluate the potential therapeutic effect of intervening melanin in treating keloid.

Methods: The activity of pigmentation-related pathways in KD melanocytes was examined using single-cell RNA-sequence (scRNA-seq) analysis. Masson-Fontana staining or isolated melanin quantification detected the melanin levels and distribution in the skin and cells. Collagen deposition, wounding healing, and proliferation analysis were employed to integratively assess fibroblast fibrogenesis. After melanin treatment, bulk-seq identified fibroblasts' differentially expressed genes (DEGs). The iron levels were detected by Perl's staining or isolated iron quantification. Cell viability, LipidROS, and malondialdehyde assay accessed the ferroptosis levels. The therapeutic potential of ML329 was evaluated in keloid-bearing mice.

Results: We found the enriched skin pigmentation-related pathways in the melanocytes of keloid by single-cell RNA-sequence (scRNA-seq) analysis. We further validated increased melanin levels in keloid patients. Additionally, melanin positively correlated with the Keloid Area and Severity Index in keloid. Furthermore, melanocyte-secreted melanin significantly promoted fibroblast proliferation, migration, and collagen synthesis. Mechanically, melanin increased basal cell permeability and inflammation to facilitate its transfer to the dermis, where it further activated fibroblasts by evoking iron overload and ferroptosis resistance. Consistently, iron overload and ferroptosis resistance were validated in primary fibroblasts and skin tissues of keloid patients. Inhibition of iron overload and ferroptosis resistance effectively diminish melanin-induced fibrogenesis. Interestingly, melanin induced iron overload and ferroptosis resistance in melanocytes in an autocrine manner and further stimulated keratinocytes to take up melanin to deepen skin color by upregulating the F2R-like trypsin receptor 1 (F2RL1). In vivo, the delivery of ML329, a microphthalmia-associated transcription factor (MITF) inhibitor, could suppress melanogenesis and alleviate keloid in human keloid-bearing nude mice. Meanwhile, ML329 decreased the iron content and restored the sensitivities of ferroptosis.

Conclusion: Collectively, melanin-lowing strategies may appear as a potential new therapeutic target for keloid.

Keywords: Ferroptosis resistance; Fibrogenesis; Iron overload; Keloid; Melanin.

Fig. 1

Hyperactive pigmentation in melanocyte cells from KD patients. (A) UMAP of the cell clustering from skin tissues by scRNA-seq analysis. (B) Re-clustering of melanocytes by PMEL, MITF, TYR, and MLANA. (C) The mRNA levels of PMEL, MITF, TYR, and MLANA in KD and KN skin tissues by scRNA-seq analysis. (D-E) Immunofluorescence assay and cell count of MITF-positive melanocytes in epidermis of skins. The dashed white lines represent the epidermis above and the dermis below. N = 12. Scale bar: 100 μm. (F) PMEL, MITF, MLANA, TYRP1, and TYRP2 levels in keloid skin tissues by quantitative Real-time PCR (qPCR) analysis. N = 12. (G) KEGG analysis of the Differentially Expressed Genes (DEGs) of melanocytes between KD and KN from scRNA-seq. (H) Epidermal dermal thickness changes between KD and KN skin tissues. (I-J) Masson-Fontana staining (I) and quantification (J) of melanin contents in skin epidermis. N = 12. 40×, 100×, and 400× means the images are magnified by 40, 100, and 400 times, respectively. Scale bar: 500 μm, 200 μm and 50 μm in 40×, 100× and 400×, respectively. (K) Quantifying isolated melanin from skin tissues by spectrophotometric measurement at 490 nm of absorbance. N = 12. (L) The correlations between KASI and melanin (N = 30). (M-N) The photos of keloid patients. *P < 0.05; **P < 0.001; ***P < 0.001

Fig. 2

Melanin from melanocytes promotes fibroblast growth, migration, and collagen deposition. (A) HE and Masson’s staining of KN and keloid skin tissues. N = 12. Scale bar, 200 μm. (B-C) Dermal thickness and collagen content. (D) Immunofluorescence assay and cell count of S100A4-positive fibroblast cells in keloid skin tissues. N = 6. Scale bar: 50 μm. (E-F) Cell growth and migration of KD fibroblast cells with or without pHEMs medium treatment. (G-I) qPCR and western blot detection of fibrosis-related proteins in KD fibroblast cells with or without pHEMs medium treatment. (J-K) Cell growth and migration of KD fibroblast cells with or without melanin treatment. (L-N) qPCR and western blot detection of fibrosis-related proteins in KD fibroblast cells with or without melanin treatment. (O) Western blot detection of MITF in pHEMs with NC siRNA or si-MITF. (P) Quantifying isolated melanin from pHEMs medium by spectrophotometric measurement at 490 nm of absorbance. (Q) Cell growth of KD fibroblast cells pretreated with NC-transfected pHEMs medium (pHEMs Medium + NC) or si-MITF-transfected pHEMs medium (pHEMs Medium + si-MITF). (R-R1) Cell migration of KD fibroblast cells pretreated with pHEMs medium with NC siRNA or si-MITF. (S-U) qPCR and western blot detection of fibrosis-related proteins in KD fibroblast cells treated with pHEMs Medium + NC or pHEMs Medium + si-MITF. The Scale bar in data F, K, and R were 50 μm. The experiments in E-U were repeated three times independently. *P < 0.05; **P < 0.001; ***P < 0.001

Fig. 3

Melanin transfers to the skin dermis by disrupting the basal membrane. (A) Masson-Fontana staining and quantification of melanin contents in the skin dermis. N = 12. The 40× scale bar is 500 μm and the 200× scale bar is 100 μm, respectively. (B) Quantification of the isolated melanin from the separated dermis. N = 12. (C) The Volcano map of DEGs between KN and KD basal cells. (D) GO and KEGG orthologs of the DEGs in basal cells. (E) Violin plots represent the expression of inflammation, tight junction, and basal marker genes in basal cells from KN and KD skins. (F) qPCR analysis of inflammation and tight junction genes in KN and KD skins. N = 12. (G) ELISA analysis of CCL2 and S100A8/A9 in KN and KD skins. N = 12. (H) qPCR analysis of COL17A1 and KRT15 in KN and KD skins. N = 12. (I) Immunohistochemistry of COL17A1 on skin sections from the KN and KD. N = 12. (J) Protein expression of COL17A1 in KN and KD skins. (K) The schematic diagram of ex vivo explants culture and intraepidermal melanin injection. (L) qPCR analysis of inflammation genes after melanin treatment in skin explants. (M) ELISA analysis of CCL2 and S100A8/A9 after melanin treatment in skin explants. (N-O) mRNA and protein levels of COL17A1 in skin explants after melanin treatment. (P) CCL2 and S100A8/A9 protein expression in TE 354.T basal cells. (Q) The schematic diagram of co-culture of keratinocytes, basal cells, and fibroblasts. (R) Cell permeability assay. (S) Melanin content in cell medium and fibroblast cells with or without melanin treatment. The experiments in P-S were repeated three times independently. *P < 0.05; **P < 0.001; ***P < 0.001

Fig. 4

Melanin induces iron overload to activate fibroblasts. (A-B) The heatmap and Volcano map of DEGs between control and 100 ug/ml melanin-treated fibroblasts. (C-D) GO and KEGG orthologs of the upregulated and downregulated DEGs. (E) The heatmap of the upregulated DEGs involved in mineral absorption and ferroptosis. (F) mRNA expression of FTH1, FTL, TFRC and FPN1 in primary KD fibroblasts. (G-H) FTH1, FTL, TFRC, and FPN1 protein expression in primary KD fibroblasts. (I) Total iron, Fe²⁺, and Fe³⁺ levels in melanin-treated KD fibroblasts. (J-K) Protein expression of α-SMA and Collagen I in primary KD fibroblasts. (L-M) Protein expression of α-SMA and Collagen I in primary KD fibroblasts. (N) Perl’s staining of skin tissues. Scale bar: 500 μm and 50 μm. (O) Total iron, Fe²⁺, and Fe³⁺ levels in KN and KD fibroblasts. (P-Q) Protein expression of FTH1, FTL, and TFRC in primary KD fibroblasts. N = 6. (R-S) Immunofluorescence assay and cell count of FTH1⁺S100A4⁺ fibroblast cells in keloid skin tissues. N = 6. Scale bar: 200 μm. The experiments in A-M were repeated three times independently. *P < 0.05; **P < 0.001; ***P < 0.001

Fig. 5

Melanin inhibits ferroptosis in KD fibroblasts. (A-C) qPCR and western blot detection of ferroptosis-inhibitory proteins SLC3A2/SLC7A11/GPX4 and pro-ferroptotic proteins in KD fibroblast cells with or without melanin stimulation. (D) The mRNA levels of ACSL4, SLC7A11, GPX4, and SLC3A2 in KD and KN skin tissues by scRNA-seq analysis. (E) Evaluation of the effects of melanin on RSL-3-induced LipidROS in KD fibroblast cells. Scale bar: 200 μm. (F) Cell viability of fibroblast cells treated with RSL-3 and melanin by CCK8 assay. (G-H) Relative 4-HNE and MDA levels after fibroblast cell treated withRSL-3 and melanin. (I-J) Western blot analysis of SLC3A2, SLC7A11, GPX4, and ACSL4 protein levels in KN and KD primary fibroblasts. N = 6. (K-L) Immunofluorescence assay and cell count of GPX4⁺S100A4⁺ fibroblast cells in keloid skin tissues. N = 6. Scale bar: 200 μm. The experiments in A-H were repeated three times independently. *P < 0.05; **P < 0.001; ***P < 0.001

Fig. 6

Melanin-induced iron overload and ferroptosis-resistance in melanocytes and benefited keratinocyte cells absorbing melanin. (A-C) The expression of iron metabolism-related genes after 100 ug/ml melanin incubation in pHEMs. (D) Total iron, Fe²⁺, and Fe³⁺ levels in melanin-treated melanocytes. (E) Cell viability of pHEMs treated with RSL-3 and melanin by CCK8 assay. (F-H) qPCR and western blot detection of ferroptosis-inhibitory proteins SLC3A2/SLC7A11/GPX4 and pro-ferroptotic proteins in pHEMs with or without melanin stimulation. (I) Perl’s staining of iron content in epidermal skin tissues. Scale bar: 50 μm. (J) Evaluation of the effects of melanin on RSL-3-induced LipidROS in pHEMs. Scale bar: 200 μm. (K) Melanin contents in HaCaT cells. (L-N) qPCR and western blot analysis of PAR-2 protein levels in HaCaT cells. The experiments were repeated three times independently. *P < 0.05; **P < 0.001; ***P < 0.001

Fig. 7

ML329 improved skin fibrosis in vivo. (A) The keloid-bearing mouse model construction and subcutaneous ML329 injection. (B) Grafts were collected on day 21 and separated into saline and ML329 groups. (C-D) Volume and weight measurement of grafts. (E) HE and Masson stain the collected grafts. Scale bar, 200 μm. (F) Sircol assay showed a decrease in ECM in the ML329 groups. (G-I) Measurements of melanin, total iron, Fe²⁺, Fe³⁺ levels, and MDA levels in KD grafts. (J-M) Immunofluorescence assay of FTH1⁺S100A4⁺ fibroblast cells and GPX4⁺S100A4⁺ fibroblast cells. Scale bar, 200 μm. N = 6. *P < 0.05; **P < 0.001; ***P < 0.001