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

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. 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. 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. Collectively, melanin-lowing strategies may appear as a potential new therapeutic target for keloid. Current treatments for keloid are ineffective. Our research demonstrates that melanin levels increase in keloid patients and play a significant role in keloid progression by mediating aberrant keratinocyte − melanocyte − basal − fibroblast cell crosstalk. Importantly, we found that pharmacological inhibition of melanogenesis using an MITF inhibitor, ML329, shows promise in effectively alleviating keloid, offering a potential breakthrough in keloid treatment. The melanin synthesis pathway is abnormally activated in KD melanocytes. Melanin destroys the basal membrane barrier by triggering inflammation and translocates to the dermal layers of the skin in a paracrine manner to induce fibroblast overgrowth, migration, and ECM deposition by inducing iron overload and ferroptosis resistance. Melanin maintains melanocytes' hyperproliferative and non-immortal properties in an autocrine manner. It further enhances the keratocyte PAR-2 to promote transit to the superficial epidermis skin layers, which may be related to the deepening of skin color. Inhibition of melanin synthesis by ML329 alleviates KD in mice.