Tissue Inhibitor of Metalloproteinase–3 (TIMP-3) induces FAS dependent apoptosis in human vascular smooth muscle cells

Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain.

在血管平滑肌细胞（vascular smooth muscle cells, VSMCs）中过表达金属蛋白酶组织抑制剂-3（Tissue Inhibitor of Metalloproteinases-3, TIMP-3）可诱导细胞凋亡，并减少隐静脉间置移植术或冠状动脉支架置入术后的新生内膜形成。本研究为阐明TIMP-3诱导人血管平滑肌细胞凋亡的分子机制，通过实验发现：TIMP-3可增强半胱氨酸天冬氨酸蛋白酶-8（caspase-8）的激活，而细胞因子应答修饰物A（Cytokine response modifier A, CrmA）与显性失活型Fas相关死亡结构域蛋白（FADD）的过表达可抑制该凋亡过程。TIMP-3诱导的细胞凋亡并未引发线粒体膜电位去极化，也未增强半胱氨酸天冬氨酸蛋白酶-9（caspase-9）的激活，且B细胞淋巴瘤因子2（B-cell Lymphoma 2, Bcl2）的过表达无法抑制该凋亡过程，提示该通路不依赖线粒体，属于I型死亡受体通路。TIMP-3可上调死亡受体Fas（First Apoptosis Signal receptor, FAS）的表达水平，采用短发夹RNA（shRNA）敲低Fas的表达后，TIMP-3诱导的细胞凋亡受到抑制，表明该凋亡过程依赖Fas通路。免疫共沉淀与免疫荧光实验结果显示，TIMP-3可诱导死亡诱导信号复合物（Death-Inducing Signalling Complex, DISC）的形成。在未表达TIMP-3的细胞中，细胞型Fas相关死亡结构域蛋白样白细胞介素-1β转换酶抑制蛋白（Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein, c-FLIP）与Fas共定位于细胞外周；而当细胞表达TIMP-3后，该共定位现象消失，c-FLIP转而定位于细胞核周围区域。尽管TIMP-3可抑制Fas的胞外域脱落，但这并未提升Fas在细胞表面的总表达水平，反而使Fas在细胞表面的局部区域出现富集。解整合素金属蛋白酶17（A Disintegrin And Metalloproteinase 17, ADAM17）可被TIMP-3抑制，采用shRNA敲低ADAM17的表达后，Fas的胞外域脱落水平显著降低。但敲低ADAM17既无法诱导细胞凋亡，也无法模拟TIMP-3引发的细胞表面Fas局部聚集效应。当ADAM17敲低的细胞表达亚凋亡水平的TIMP-3时，仍可激活半胱氨酸天冬氨酸蛋白酶-3（caspase-3），这表明ADAM17介导的Fas胞外域脱落仅在TIMP-3诱导的细胞凋亡过程中发挥部分作用。综上，本研究证实，TIMP-3诱导的血管平滑肌细胞凋亡高度依赖Fas通路，且与Fas及c-FLIP的定位改变密切相关，但并非完全依赖Fas的胞外域脱落。