Single‑cell RNA sequencing reveals fibroblast heterogeneity and identifies CLOCK as a key regulator in fibrotic skin diseases

Abstract Fibrosis, characterized by excessive extracellular matrix (ECM) accumulation and fibroblast proliferation, significantly contributes to global morbidity and mortality, affecting millions worldwide. Despite its prevalence, the mechanisms underlying fibrotic skin diseases remain poorly understood, and effective treatments are scarce. This study leverages single-cell RNA sequencing (scRNA-seq) to unravel the heterogeneity of fibroblasts in fibrotic skin diseases, including normal skin, scar, keloid, and scleroderma. Through comprehensive analysis of scRNA-seq data from public repositories, we identified distinct fibroblast subpopulations specific to each fibrotic condition. Notably, pivotal regulators for each sub-fibroblast cluster were discovered: IRF4 for scar, CLOCK for keloid, RUNX3 for scleroderma, and HOXC4 for normal skin. Further, CLOCK was found to be predominantly expressed in keloid tissues, with its upregulation enhancing fibroblast proliferation and migration in vitro. Analysis of The Cancer Genome Atlas (TCGA) data revealed that CLOCK and its regulon genes were upregulated in skin cutaneous melanoma and even more so in metastatic tumors. Our findings underscore the utility of scRNA-seq in dissecting the cellular complexity of fibrotic skin diseases and highlight potential therapeutic targets. This study not only advances our understanding of fibroblast heterogeneity in fibrosis but also opens avenues for targeted therapeutic strategies, moving closer to personalized medicine for fibrotic diseases.