Farwell Sara Lynn N, Kanyi Daniela, Hamel Marianne, Slee Joshua B, Miller Elizabeth A, Cipolle Mark D, Lowe-Krentz Linda J
From the Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015.
From the Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, the Department of Chemistry, Lehigh University, Allentown, Pennsylvania 18103.
J Biol Chem. 2016 Mar 4;291(10):5342-54. doi: 10.1074/jbc.M115.681288. Epub 2016 Jan 14.
Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparin-induced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNFα-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNFα-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.
尽管存在大量肝素和硫酸乙酰肝素结合蛋白，但肝素改变血管细胞生理功能的分子机制仍未完全明确。对血管平滑肌细胞（VSMC）的研究表明，双特异性磷酸酶1（DUSP1）的诱导发挥了作用，它降低了ERK活性并导致细胞增殖减少，这取决于特定的肝素结合。本研究检验了普通肝素通过诱导DUSP1表达来降低内皮细胞（EC）炎症信号转导的假说。此外，还研究了肝素反应是否包括细胞因子诱导的细胞骨架变化减少。通过蛋白质免疫印迹法和免疫荧光显微镜检查发现，用肝素预处理内皮细胞可降低肿瘤坏死因子α（TNFα）诱导的JNK和p38活性以及下游靶点磷酸化。通过基因敲除策略，证实了肝素诱导的DUSP1表达在这些效应中的重要性。定量荧光显微镜检查表明，肝素处理内皮细胞可减少TNFα诱导的应力纤维增加。使用模拟肝素诱导的血管平滑肌细胞变化的单克隆抗体来支持肝素通过与受体相互作用发挥作用的假说。跨膜蛋白184A（TMEM184A）的基因敲除证实了其参与了血管平滑肌细胞中肝素诱导的信号传导。因此，TMEM184A作为肝素受体，介导内皮细胞的抗炎反应，包括降低JNK和p38活性。
