Dina Durotimi O, Maiellaro Miriam, Camera Emanuela, Connelly John T
Centre for Cell Biology and Cutaneous Research, Faculty of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
Laboratory of Cutaneous Physiopathology and Integrated Center of Metabolomics Research, San Gallicano Dermatological Institute, IRCCS, Rome, Italy.
JID Innov. 2025 May 14;5(4):100383. doi: 10.1016/j.xjidi.2025.100383. eCollection 2025 Jul.
The lipid composition of the epidermis plays a critical role in the skin's barrier function, and defects in lipid synthesis or assembly can cause a spectrum of skin diseases, ranging from dry skin to severe ichthyoses. The aim of this study was to develop an in vitro model of human skin with tunable inhibition of lipid synthesis. Human N/TERT keratinocytes were engineered to express doxycycline-inducible short hairpin RNAs targeting ceramide synthase 3, which is essential for synthesis of ultralong-chain ceramides and skin barrier function. We show that 3-dimensional human skin equivalents with induced knockdown of ceramide synthase 3 display normal stratification and terminal differentiation but have reduced Nile red staining for polar lipids. Further analysis of the lipidome by mass spectrometry confirmed a significant reduction in specific classes of ceramides and ceramide chain length in the ceramide synthase 3-depleted human skin equivalents. We also show that ceramide synthase 3 knockdown is reversible upon removal of doxycycline and can be used to study recovery and repair of epidermal lipids. Together, these findings provide an overall strategy for genetically regulating the lipid composition within human skin models and establish a tunable in vitro model of ceramide deficiency.
表皮的脂质组成在皮肤屏障功能中起着关键作用，脂质合成或组装缺陷可导致一系列皮肤疾病，从干性皮肤到严重鱼鳞病。本研究的目的是建立一种可调节脂质合成抑制的人皮肤体外模型。对人N/TERT角质形成细胞进行工程改造，使其表达靶向神经酰胺合酶3的强力霉素诱导型短发夹RNA，神经酰胺合酶3对超长链神经酰胺的合成和皮肤屏障功能至关重要。我们发现，神经酰胺合酶3诱导敲低的三维人皮肤等效物表现出正常的分层和终末分化，但极性脂质的尼罗红染色减少。通过质谱对脂质组进行的进一步分析证实，在神经酰胺合酶3缺失的人皮肤等效物中，特定类别的神经酰胺和神经酰胺链长度显著减少。我们还表明，去除强力霉素后，神经酰胺合酶3的敲低是可逆的，可用于研究表皮脂质的恢复和修复。总之，这些发现为在人皮肤模型中基因调控脂质组成提供了一个总体策略，并建立了一种可调节的神经酰胺缺乏体外模型。
