Institute of Physiology, Charité - Universitätsmedizin, Berlin, Germany.
Institute of Pathology, University Medicine Mainz, Mainz 55131, Germany.
Clin Sci (Lond). 2021 Apr 16;135(7):887-905. doi: 10.1042/CS20201399.
Promyelocytic leukemia protein (PML) is a constitutive component of PML nuclear bodies (PML-NBs), which function as stress-regulated SUMOylation factories. Since PML can also act as a regulator of the inflammatory and fibroproliferative responses characteristic of atherosclerosis, we investigated whether PML is implicated in this disease. Immunoblotting, ELISA and immunohistochemistry showed a stronger expression of PML in segments of human atherosclerotic coronary arteries and sections compared with non-atherosclerotic ones. In particular, PML was concentrated in PML-NBs from α-smooth muscle actin (α-SMA)-immunoreactive cells in plaque areas. To identify possible functional consequences of PML-accumulation in this cell type, differentiated human coronary artery smooth muscle cells (dHCASMCs) were transfected with a vector containing the intact PML-gene. These PML-transfected dHCASMCs showed higher levels of small ubiquitin-like modifier (SUMO)-1-dependent SUMOylated proteins, but lower levels of markers for smooth muscle cell (SMC) differentiation and revealed more proliferation and migration activities than dHCASMCs transfected with the vector lacking a specific gene insert or with the vector containing a mutated PML-gene coding for a PML-form without SUMOylation activity. When dHCASMCs were incubated with different cytokines, higher PML-levels were observed only after interferon γ (IFN-γ) stimulation, while the expression of differentiation markers was lower. However, these phenotypic changes were not observed in dHCASMCs treated with small interfering RNA (siRNA) suppressing PML-expression prior to IFN-γ stimulation. Taken together, our results imply that PML is a previously unknown functional factor in the molecular cascades associated with the pathogenesis of atherosclerosis and is positioned in vascular SMCs (VSMCs) between upstream IFN-γ activation and downstream SUMOylation.
早幼粒细胞白血病蛋白 (PML) 是 PML 核体 (PML-NBs) 的组成成分，其作为应激调节 SUMO 化工厂发挥作用。由于 PML 还可以作为动脉粥样硬化特征性炎症和纤维增生反应的调节剂，我们研究了 PML 是否与该疾病有关。免疫印迹、ELISA 和免疫组织化学显示，与人非动脉粥样硬化冠状动脉段相比，动脉粥样硬化冠状动脉段的 PML 表达更强。特别是，PML 集中在斑块区域的 α-平滑肌肌动蛋白 (α-SMA) 免疫反应性细胞的 PML-NBs 中。为了确定 PML 在这种细胞类型中积累的可能功能后果，用含有完整 PML 基因的载体转染分化的人冠状动脉平滑肌细胞 (dHCASMCs)。与转染缺乏特定基因插入的载体或转染编码无 SUMO 化活性的 PML 形式的突变 PML 基因的载体的 dHCASMCs 相比，这些 PML 转染的 dHCASMCs 显示出更高水平的小泛素样修饰物 (SUMO)-1 依赖性 SUMO 化蛋白，但平滑肌细胞 (SMC) 分化标记物的水平更低，并且显示出比 dHCASMCs 更高的增殖和迁移活性。当用不同的细胞因子孵育 dHCASMCs 时，仅在干扰素 γ (IFN-γ) 刺激后观察到更高的 PML 水平，而分化标记物的表达水平较低。然而，在用干扰素 γ 刺激之前用小干扰 RNA (siRNA) 抑制 PML 表达处理的 dHCASMCs 中未观察到这些表型变化。总之，我们的结果表明，PML 是动脉粥样硬化发病机制相关分子级联反应中以前未知的功能因素，位于血管平滑肌细胞 (VSMCs) 中，位于上游 IFN-γ 激活和下游 SUMO 化之间。
