ERK / C/EBP-β信号通路调控角蛋白6c在砷致皮肤角化中的作用及机制

Hyperkeratosis of the skin plays an important role in the diagnosis, health assessment, and cancer warning of arsenic poisoning, but the mechanism is unknown. For a long time, the abnormal expression of keratin has been the focus of study on arsenic-induced skin keratosis, and there is a difference between acute and chronic exposure. Recent studies have shown that Krt6c activation is involved in arsenic-induced skin keratosis, and pre-experimental study found that arsenic can upregulate the transcriptional expression of Krt6c by inhibiting C/EBP-β. This project intends to compare the changes of the expression of ERK / C/EBP-β signaling pathway-related proteins and Krt6c in the skin of the control group and the arsenic poisoning patients with skin lesions (pigment change, hyperkeratosis, carcinogenesis), to clarify the role of ERK / C/EBP-β signaling pathway, Krt6c in arsenic induced skin keratosis; Then, using short-term and repeated arsenic treatment cell experiments, Krt6c, cell proliferation, and other related biomarkers for skin keratosis were detected, to study the regulation of ERK / C/EBP-β signaling pathway on the above biomarkers from the positive and negative; Finally, Chip and target gene validation experiments were used to confirm the target regulatory effect of transcription factor C / EBP-β on Krt6c, and the possible binding sites, for the study is expected to provide a new direction and scientific basis for the explanation of the pathogenesis and the selection of possible interventional target, in skin keratosis of endemic arsenic poisoning.

皮肤角化过度在砷中毒诊断、健康评估、癌症预警中发挥重要作用，但机制未明。长期以来，角蛋白的异常表达一直是砷致皮肤角化研究的焦点，且存在急、慢性暴露差异。最近的研究表明，Krt6c活化参与了砷引起的皮肤角化，且预实验研究发现，砷可通过抑制C/EBP-β上调Krt6c转录表达。本项目拟通过比较砷中毒皮肤病变（色素改变、角化过度、癌变）患者及对照人群皮肤中ERK/C/EBP-β信号通路相关蛋白及Krt6c表达变化，初步明确ERK/C/EBP-β信号通路、Krt6c在砷致皮肤角化中的作用；然后利用短期和重复染毒细胞实验，检测Krt6c、细胞增殖等皮肤角化相关标志，从正、反向探讨ERK/C/EBP-β信号通路对上述标志的调控；最后利用Chip和靶基因验证实验，明确转录因子C/EBP-β对Krt6c的靶向调控作用及可能结合位点，为砷中毒皮肤角化发病机制的阐释和可能干预靶点的选择提供新的方向和科学依据。

皮肤角化过度在砷中毒诊断、健康评估、癌症预警中发挥重要作用，但机制未明。本项目在前期研究基础上，从人群、细胞两个层次侧重探讨了细胞外调节蛋白激酶（Extracellular regulated protein kinases，ERK）/CCAAT 增强子结合蛋白β（CCAAT enhancer binding protein beta，CEBPB）信号通路在砷致皮肤角化中的作用及机制；同时，应用具有区域特色的刺梨活性成分刺梨苷进行干预探索。经过四年的项目实施，获得了一系列研究成果：① 从人群层面初步明确ERK/CEBPB信号通路、Krt6c在砷致皮肤角化中的关键作用。发现燃煤砷可诱发多种皮肤病变；燃煤砷可活化ERK/CEBPB信号通路，促进Krt6c的表达，进而引发人群皮肤的过度角化；ERK/CEBPB信号通路的活化与Krt6c的表达存在明显的相关关系。② 从细胞层面探索并验证了ERK/CEBPB信号通路调控Krt6c在砷致皮肤角化中的作用机制。发现短期、高剂量砷暴露可活化ERK/CEBPB信号通路，促进人永生化角质形成细胞（Human immortalized keratinocytes，HaCaT）中反映皮肤角化相关指标Krt6c的表达；长期、低剂量砷暴露可诱导ERK/CEBPB信号通路的持续活化和Krt6c的表达增高，进而抑制细胞凋亡，促进细胞迁移；敲低CEBPB可通过抑制CEBPB的表达进而降低砷诱导的HaCaT细胞 Krt6c的表达，反之亦然；敲低ERK1/2可通过抑制ERK/CEBPB信号通路的活化进而降低砷诱导的HaCaT细胞Krt6c的表达，反之亦然；双荧光素酶报告基因实验显示转录因子CEBPB可正向调控Krt6c的表达。③ 从细胞层面探讨刺梨苷在砷致皮肤角化中的潜在应用价值。发现刺梨苷可抑制ERK/CEBPB信号通路的活化，减轻砷所致的皮肤细胞损伤。本项目的系列研究成果较系统阐释了地方性砷中毒皮肤角化的发病机制，为新时期地方性砷中毒皮肤角化可能干预靶点的选择和优化新的干预策略提供实验依据。