- 學位論文
角質蛋白-1/CD32/LOX-1為C反應蛋白受器對介導C反應蛋白/氧化低密度脂蛋白在人類主動脈內皮細胞功能之影響
KRT1 as a CRP Receptor with CD32 and LOX-1 Mediates the Effect of CRP/OxLDL on Functions of Human Aortic Endothelial Cells
摘要
動脈粥狀硬化是動脈慢性發炎的疾病，C反應蛋白 (C reactive protein; CRP)與氧化低密脂蛋白 (oxidized low density lipoprotein; oxLDL)扮演促發炎的反應，CRP可以與許多分子結合，包括oxLDL，也可能與CD32、LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1)以外的受器結合。而CRP與oxLDL如何形成複合體並藉由哪一相對應的受器與人內主動脈內皮細胞作用並不清楚，過去我們利用親和性管柱實驗及液相層析串聯質譜分析證實Keratin 1 (KRT1)會與CRP結合，以抗體阻斷KRT1會降低內皮細胞對於CRP的攝取及回復因CRP作用而降低的一氧化氮 (nitric oxide; NO)釋放量，CRP誘導KRT1表現的結果相似於氧化壓力誘導CD32與LOX-1表現，另外，將pcDNA3.1-KRT1質體DNA轉染至內皮細胞，增加KRT1蛋白表現量，另一方面，利用小髮夾RNAs (small hairpin RNAs)降低KRT1蛋白表現也會使因CRP降低的NO釋放量回升。另一方面我們發現CRP與oxLDL形成複合體對內皮細胞NO的釋放與CRP/oxLDL濃度比例有關，當CRP/oxLDL濃度比例為1:20時明顯降低NO的產生，利用阻斷CD32、LOX-1及KRT1受器可以提升NO的釋放，另外我們也發現CD32、LOX-1及KRT1介導細胞對CRP/oxLDL複合體的攝取，實驗發現KRT1與LOX-1在內皮細胞有共位現象，CRP/oxLDL對內皮細胞的作用或許提供了對於心血管治療的新機制。
並列摘要
Atherosclerosis is a chronic inflammatory disease of the arteries. C-reactive protein (CRP) and oxidized low-density lipoprotein (oxLDL) play a significant role in the pro-inflammatory response observed in various types of vascular disease. CRP is capable of binding to various molecules including oxLDL and may bind to the receptor other than CD32 and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). However, how CRP/oxLDL complexes are formed, as well as their effect and corresponding receptors on endothelial cells, remains unknown. Here, we identified keratin 1 (KRT1) on endothelial cells as a novel protein for CRP binding by affinity chromatography and mass spectrometry. The interaction between KRT1 and CRP was also confirmed by the antibody blockade of KRT1 that decreased the CRP uptake and CRP-mediated reduction in nitric oxide (NO) release in human aortic endothelial cells (HAECs). Similar to CD32 and LOX-1 induced by oxdative stress, KRT1 was induced by CRP. Additionally, an increased KRT1 expression by transfection of plasmid pcDNA3.1-KRT1 enhanced but a reduced KRT1 expression by KRT1 small hairpin RNAs reversed the CRP antagonism in NO release. On the other hand, we found that the induction of NO release by CRP and oxLDL co-incubates was dependent on the ratio of CRP and oxLDL. Specifically, the concentration ratio of ~1:20 (CRP/oxLDL-1:20) resulted in the greatest inhibition of endothelial NO secretion. Pretreatment of neutralizing antibodies for CD32, LOX-1, or KRT1 abolished the effect of CRP/oxLDL-1:20 on NO production. Furthermore, we found that CD32, LOX-1, and KRT1 mediated the cellular uptake of CRP/oxLDL complexes, in which LOX-1 co-localized with KRT1, but not CD32. These results provide a novel mechanism by which the multifaceted effects of CRP/oxLDL complexes on endothelial cells can be targeted for the treatment of vasculature diseases.