Abstract：
BACKGROUND: Cadmium exposure induces dermatotoxicity and epidermal barrier disruption and leads to the development of various pathologies. HaCaT cells are immortalized human keratinocytes that are widely used as alternatives to primary human keratinocytes, particularly for evaluating cadmium toxicity. HaCaT cells bear two gain-of-function (GOF) mutations in the TP53 gene, which strongly affect p53 function. Mutant forms of p53 are known to correlate with increased resistance to various stimuli, including exposure to cytotoxic substances. In addition, keratin 17 (KRT17) was recently shown to be highly expressed in HaCaT cells in response to genotoxic stress. Moreover, p53 is a direct transcriptional repressor of KRT17. However, the impact of TP53 mutations in HaCaT cells on the regulation of cell death and keratin 17 expression is unclear. In this study, we aimed to evaluate the impact of p53 on the response to Cd-induced cytotoxicity.
RESULTS: Employing the MTT assay and Annexin V/propidium iodide staining, we demonstrated that knockout of TP53 leads to a decrease in the sensitivity of HaCaT cells to the cytotoxic effects of cadmium. Specifically, HaCaT cells with TP53 knockout (TP53 KO HaCaT) exhibited cell death at a cadmium concentration of 10 μM or higher, whereas wild-type cells displayed cell death at a concentration of 30 μM. Furthermore, apoptotic cells were consistently detected in TP53 KO HaCaT cells upon exposure to low concentrations of cadmium (10 and 20 μM) but not in wild-type cells. Our findings also indicate that cadmium cytotoxicity is mediated by reactive oxygen species (ROS), which were significantly increased only in TP53 knockout cells treated with 30 μM cadmium. An examination of proteomic data revealed that TP53 knockout in HaCaT cells resulted in the upregulation of proteins involved in the regulation of apoptosis, redox systems, and DNA repair. Moreover, RT‒qPCR and immunoblotting showed that cadmium toxicity leads to dose-dependent induction of keratin 17 in p53-deficient cells but not in wild-type cells.
CONCLUSIONS: The connection between mutant p53 in HaCaT keratinocytes and increased resistance to cadmium toxicity was demonstrated for the first time. Proteomic profiling revealed that TP53 knockout in HaCaT cells led to the activation of apoptosis regulatory circuits, redox systems, and DNA repair. In addition, our data support the involvement of keratin 17 in the regulation of DNA repair and cell death. Apparently, the induction of keratin 17 is p53-independent but may be inhibited by mutant p53.
RESULTS: Employing the MTT assay and Annexin V/propidium iodide staining, we demonstrated that knockout of TP53 leads to a decrease in the sensitivity of HaCaT cells to the cytotoxic effects of cadmium. Specifically, HaCaT cells with TP53 knockout (TP53 KO HaCaT) exhibited cell death at a cadmium concentration of 10 μM or higher, whereas wild-type cells displayed cell death at a concentration of 30 μM. Furthermore, apoptotic cells were consistently detected in TP53 KO HaCaT cells upon exposure to low concentrations of cadmium (10 and 20 μM) but not in wild-type cells. Our findings also indicate that cadmium cytotoxicity is mediated by reactive oxygen species (ROS), which were significantly increased only in TP53 knockout cells treated with 30 μM cadmium. An examination of proteomic data revealed that TP53 knockout in HaCaT cells resulted in the upregulation of proteins involved in the regulation of apoptosis, redox systems, and DNA repair. Moreover, RT‒qPCR and immunoblotting showed that cadmium toxicity leads to dose-dependent induction of keratin 17 in p53-deficient cells but not in wild-type cells.
CONCLUSIONS: The connection between mutant p53 in HaCaT keratinocytes and increased resistance to cadmium toxicity was demonstrated for the first time. Proteomic profiling revealed that TP53 knockout in HaCaT cells led to the activation of apoptosis regulatory circuits, redox systems, and DNA repair. In addition, our data support the involvement of keratin 17 in the regulation of DNA repair and cell death. Apparently, the induction of keratin 17 is p53-independent but may be inhibited by mutant p53.
摘要：
背景：镉暴露会引起皮肤毒性和表皮屏障破坏，并导致各种病理的发展。HaCaT细胞是永生化的人角质形成细胞，被广泛用作原代人角质形成细胞的替代品。特别是评估镉的毒性。HaCaT细胞在TP53基因中具有两个功能获得（GOF）突变，强烈影响p53功能。已知p53的突变形式与对各种刺激的抗性增加相关。包括接触细胞毒性物质。此外,最近显示角蛋白17（KRT17）在HaCaT细胞中高度表达，以响应基因毒性应激。此外，p53是KRT17的直接转录阻遏物。然而,HaCaT细胞中TP53突变对细胞死亡和角蛋白17表达调节的影响尚不清楚.在这项研究中,我们旨在评估p53对Cd诱导的细胞毒性反应的影响。
结果：采用MTT法和膜联蛋白V/碘化丙啶染色，我们证明敲除TP53导致HaCaT细胞对镉细胞毒作用的敏感性降低.具体来说,具有TP53敲除（TP53KOHaCaT）的HaCaT细胞在10μM或更高的镉浓度下表现出细胞死亡，而野生型细胞在30μM的浓度下显示细胞死亡。此外，暴露于低浓度镉（10和20μM）后，在TP53KOHaCaT细胞中始终检测到凋亡细胞，但在野生型细胞中未检测到。我们的发现还表明，镉的细胞毒性是由活性氧（ROS）介导的，仅在用30μM镉处理的TP53敲除细胞中显着增加。对蛋白质组数据的检查显示，在HaCaT细胞中敲除TP53导致参与细胞凋亡调节的蛋白质上调，氧化还原系统,DNA修复此外，RT-qPCR和免疫印迹表明，镉毒性导致p53缺陷细胞中角蛋白17的剂量依赖性诱导，而野生型细胞中则没有。
结论：首次证明了HaCaT角质形成细胞中突变型p53与镉毒性抗性增加之间的联系。蛋白质组学分析显示，在HaCaT细胞中敲除TP53导致凋亡调节回路的激活，氧化还原系统,DNA修复此外,我们的数据支持角蛋白17参与DNA修复和细胞死亡的调节.显然,角蛋白17的诱导是p53非依赖性的，但可能被突变型p53抑制。
结果：采用MTT法和膜联蛋白V/碘化丙啶染色，我们证明敲除TP53导致HaCaT细胞对镉细胞毒作用的敏感性降低.具体来说,具有TP53敲除（TP53KOHaCaT）的HaCaT细胞在10μM或更高的镉浓度下表现出细胞死亡，而野生型细胞在30μM的浓度下显示细胞死亡。此外，暴露于低浓度镉（10和20μM）后，在TP53KOHaCaT细胞中始终检测到凋亡细胞，但在野生型细胞中未检测到。我们的发现还表明，镉的细胞毒性是由活性氧（ROS）介导的，仅在用30μM镉处理的TP53敲除细胞中显着增加。对蛋白质组数据的检查显示，在HaCaT细胞中敲除TP53导致参与细胞凋亡调节的蛋白质上调，氧化还原系统,DNA修复此外，RT-qPCR和免疫印迹表明，镉毒性导致p53缺陷细胞中角蛋白17的剂量依赖性诱导，而野生型细胞中则没有。
结论：首次证明了HaCaT角质形成细胞中突变型p53与镉毒性抗性增加之间的联系。蛋白质组学分析显示，在HaCaT细胞中敲除TP53导致凋亡调节回路的激活，氧化还原系统,DNA修复此外,我们的数据支持角蛋白17参与DNA修复和细胞死亡的调节.显然,角蛋白17的诱导是p53非依赖性的，但可能被突变型p53抑制。
