STAT3 Signaling Pathway in Keloid Pathogenesis Implicated by Preliminary Transcriptome and ATAC Analyses

Keloids are wounding-induced fibroproliferative human tumor–like skin scars of complex genetic makeup and poorly defined pathogenesis. Fibroblasts are the principal mediator of fibroproliferative disorders. To reveal dynamic epigenetic and transcriptome changes of keloid fibroblasts, a vertical study from RNA-seq and ATAC-seq analyses followed by in vivo confirmation of candidate molecule expression and subsequent functional testing was carried out using an early passage, freshly isolated keloid fibroblast cell strain and its paired normal control. These keloid fibroblasts produce keloid-like scars in a plasma clot-based skin equivalent humanized keloid animal model. RNA-seq analysis reveals that Hepatic fibrosis is the most significant pathway followed by Wnt–b-catenin signaling, TGF-b signaling, regulation of the EMT pathway, the STAT3 pathway, and adherens junction signaling. ATAC-seq analysis shows that STAT3 signaling is the most active pathway in keloid fibroblasts, followed by Wnt signaling (Wnt5) and regulation of the EMT pathway. Immunohistochemistry confirms that activated STAT3, (Tyr705 phospho-STAT3) and/or b-catenin are upregulated in dermal fibroblasts of keloid clinical specimens and mature keloid skin equivalent implants from the humanized mouse model compared to the normal control. The effect of STAT3 signaling on keloid fibroblast collagen expression was further tested in plasma clot-based skin equivalents using Cucurbitacin I, a selective JAK2/STAT3 inhibitor. A non-linear dose response of Cucurbitacin I was observed in collagen type I expression indicating a likely role of STAT3 signaling pathway in keloid pathogenesis. This work also demonstrates the utility of the recently established humanized keloid mouse model in exploring the mechanism of keloid formation. Control and keloid samples, each in duplicate瘢痕疙瘩（Keloids）是一类创伤诱导的、遗传构成复杂且发病机制尚不明确的纤维增殖性人源肿瘤样皮肤瘢痕。成纤维细胞（Fibroblast）是纤维增殖性疾病的核心介导因子。为揭示瘢痕疙瘩成纤维细胞的动态表观基因组与转录组变化，本研究采用早期传代、新鲜分离的瘢痕疙瘩成纤维细胞株及其配对正常对照，通过RNA测序（RNA-seq）与转座酶可及性测序（ATAC-seq）开展纵向研究，随后对候选分子表达进行体内验证，并完成后续功能实验。该瘢痕疙瘩成纤维细胞可在基于血浆凝块的皮肤等效物人源化瘢痕疙瘩动物模型中形成瘢痕疙瘩样瘢痕。RNA-seq分析显示，富集程度最高的通路为肝纤维化（Hepatic fibrosis），其次为Wnt-β-连环蛋白信号通路、转化生长因子-β（TGF-β）信号通路、上皮间质转化（EMT）通路调控、信号转导与转录激活因子3（STAT3）信号通路及黏着连接信号通路。ATAC-seq分析表明，瘢痕疙瘩成纤维细胞中活性最强的通路为STAT3信号通路，其次为Wnt信号通路（Wnt5）及EMT通路调控。免疫组织化学结果证实，相较于正常对照，瘢痕疙瘩临床标本及人源化小鼠模型中成熟瘢痕疙瘩皮肤等效物移植物的真皮成纤维细胞内，激活型STAT3（Tyr705位点磷酸化STAT3）及/或β-连环蛋白的表达显著上调。本研究进一步利用选择性JAK2/STAT3抑制剂葫芦素I（Cucurbitacin I），在基于血浆凝块的皮肤等效物体系中检测了STAT3信号通路对瘢痕疙瘩成纤维细胞胶原表达的调控作用。实验观察到葫芦素I对I型胶原表达的调控呈现非线性剂量效应，提示STAT3信号通路可能参与瘢痕疙瘩的发病过程。本研究同时验证了新近建立的人源化瘢痕疙瘩小鼠模型在探究瘢痕疙瘩形成机制中的应用价值。本研究的对照样本与瘢痕疙瘩样本均设置双重复。