Transition of autophagy and apoptosis in fibroblasts depends on dominant expression of HIF-1α or p53

Abstract

It has been revealed that hypoxia is dynamic in hypertrophic scars; therefore, we considered that it may have different effects on hypoxia-inducible factor-1α (HIF-1α) and p53 expression. Herein, we aimed to confirm the presence of a teeterboard-like conversion between HIF-1α and p53, which is correlated with scar formation and regression. Thus, we obtained samples of normal skin and hypertrophic scars to identify the differences in HIF-1α and autophagy using immunohistochemistry and transmission electron microscopy. In addition, we used moderate hypoxia in vitro to simulate the proliferative scar, and silenced HIF-1α or p53 gene expression or triggered overexpression to investigate the changes of HIF-1α and p53 expression, autophagy, apoptosis, and cell proliferation under this condition. HIF-1α, p53, and autophagy-related proteins were assayed using western blotting and immunofluorescence, whereas apoptosis was detected using flow cytometry analysis, and cell proliferation was detected using cell counting kit-8 (CCK-8) and 5-bromo-2′-deoxyuridine (BrdU) staining. Furthermore, immunoprecipitation was performed to verify the binding of HIF-1α and p53 to transcription cofactor p300. Our results demonstrated that, in scar tissue, HIF-1α expression increased in parallel with autophagosome formation. Under hypoxia, HIF-1α expression and autophagy were upregulated, whereas p53 expression and apoptosis were downregulated in vitro. HIF-1α knockdown downregulated autophagy, proliferation, and p300-bound HIF-1α, and upregulated p53 expression, apoptosis, and p300-bound p53. Meanwhile, p53 knockdown induced the opposite effects and enhanced HIF-1α, whereas p53 overexpression resulted in the same effects and reduced HIF-1α. Our results suggest a teeterboard-like conversion between HIF-1α and p53, which is linked with scar hyperplasia and regression.

摘要

目的

在增生性瘢痕中缺氧是动态变化的, 因此我们认为它可能对缺氧诱导因子1α(HIF-1α)和肿瘤抑制蛋白p53表达有不同的影响。在本研究中, 我们旨在确认HIF-1α和p53之间存在类似跷跷板的转换, 并与瘢痕的形成和消退相关, 进一步探讨HIF-1α/p53介导细胞自噬和凋亡功能变化在增生性瘢痕形成中的作用。

创新点

(1)通过体外中度缺氧模拟增生性瘢痕增生期环境, 分别沉默HIF-1α或p53基因表达或过表达p53, 研究HIF-1α和p53表达的变化, 验证HIF-1α和p53之间存在类似跷跷板的转化;(2)发现成纤维细胞自噬和凋亡的转变依赖于HIF-1α或p53的显性表达, 细胞增殖和胶原蛋白的产生也随之发生变化;(3)发现HIF-1α和p53不仅在蛋白质水平上相互调节, 而且还竞争性结合转录辅因子p300。

方法

首先, 通过免疫组织化学和透射电镜观察增生期增生性瘢痕相对于正常皮肤中HIF-1α的表达与自噬的变化;其次, 在体外模拟的中度缺氧环境中培养人皮肤成纤维细胞(HDFs), 通过western blot和免疫荧光染色检测细胞自噬相关蛋白, 利用流式细胞技术检测细胞凋亡率, 采用细胞计数试剂盒8(CCK-8)和5-溴脱氧尿嘧啶核苷(BrdU)染色检测细胞增殖活力;再则使用慢病毒载体转染HDFs, 分别敲低HIF-1α、敲低p53、过表达p53, 检测基因表达规律及相应细胞自噬和凋亡等变化;最后, 应用免疫沉淀验证HIF-1α和p53竞争性结合p300。

结论

在中度缺氧的环境中, 细胞通过增强HIF-1α表达提高自噬, 降低凋亡水平。HIF-1α和p53之间存在此消彼长的转换关系, 影响细胞自噬和凋亡水平, 并在作为转录因子发挥调节作用时与转录辅因子p300竞争性结合。两者水平转换可能在瘢痕增生和消退机制中起重要作用。靶向HIF-1α/p53可以减少过度的成纤维细胞增殖和随之而来的胶原蛋白沉积, 并为开发新型增生性瘢痕疗法提供理论基础。