Kockx Maaike, Guo Dongni Lily, Traini Mathew, Gaus Katharina, Kay Jason, Wimmer-Kleikamp Sabine, Rentero Carles, Burnett John R, Le Goff Wilfried, Van Eck Miranda, Stow Jennifer L, Jessup Wendy, Kritharides Leonard
Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia.
J Biol Chem. 2009 Sep 4;284(36):24144-54. doi: 10.1074/jbc.M109.032615. Epub 2009 Jul 9.
Cyclosporin A (CsA) is an immunosuppressant that inhibits protein phosphatase 2B (PP2B/calcineurin) and is associated with hyperlipidemia, decreased cholesterol efflux via ATP-binding cassette transporter A1 (ABCA1), and increased risk of atherosclerosis. Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, the secretion of which from human macrophages is regulated by the serine/threonine protein kinase A (PKA) and intracellular calcium (Ca(2+)) (Kockx, M., Guo, D. L., Huby, T., Lesnik, P., Kay, J., Sabaretnam, T., Jary, E., Hill, M., Gaus, K., Chapman, J., Stow, J. L., Jessup, W., and Kritharides, L. (2007) Circ. Res. 101, 607-616). As PP2B is Ca(2+)-dependent and has been linked to PKA-dependent processes, we investigated whether CsA modulated apoE secretion. CsA dose- and time-dependently inhibited secretion of apoE from primary human macrophages and from Chinese hamster ovary cells stably transfected with human apoE and increased cellular apoE levels without affecting apoE mRNA. [(35)S]Met kinetic modeling studies showed that CsA inhibited both secretion and degradation of apoE, increasing the half-life of cellular apoE 2-fold. CsA also inhibited secretion from primary human Tangier disease macrophages and from mouse macrophages deficient in ABCA1, indicating that the effect is independent of the known inhibition of ABCA1 by CsA. The role of PP2B in mediating apoE secretion was confirmed using additional peptide and chemical inhibitors of PP2B. Importantly, kinetic modeling, live-cell imaging, and confocal microscopy all indicated that CsA inhibited apoE secretion by mechanisms quite distinct from those of PKA inhibition, most likely inducing accumulation of apoE in the endoplasmic reticulum compartment. Taken together, these results establish a novel mechanism for the pro-atherosclerotic effects of CsA, and establish for the first time a role for PP2B in regulating the intracellular transport and secretion of apoE.
环孢素A（CsA）是一种免疫抑制剂，可抑制蛋白磷酸酶2B（PP2B/钙调神经磷酸酶），与高脂血症、通过ATP结合盒转运蛋白A1（ABCA1）的胆固醇流出减少以及动脉粥样硬化风险增加有关。载脂蛋白E（apoE）是脂质代谢和动脉粥样硬化的重要调节因子，人巨噬细胞中apoE的分泌受丝氨酸/苏氨酸蛋白激酶A（PKA）和细胞内钙（Ca(2+)）调节（Kockx，M.，Guo，D. L.，Huby，T.，Lesnik，P.，Kay，J.，Sabaretnam，T.，Jary，E.，Hill，M.，Gaus，K.，Chapman，J.，Stow，J. L.，Jessup，W.，和Kritharides，L.（2007年）《循环研究》101，607 - 616）。由于PP2B依赖于Ca(2+)且与PKA依赖的过程相关，我们研究了CsA是否调节apoE的分泌。CsA剂量和时间依赖性地抑制原代人巨噬细胞以及稳定转染人apoE的中国仓鼠卵巢细胞中apoE的分泌，并增加细胞内apoE水平，而不影响apoE mRNA。[(35)S]甲硫氨酸动力学建模研究表明，CsA抑制apoE的分泌和降解，使细胞内apoE的半衰期延长2倍。CsA还抑制原发性人类丹吉尔病巨噬细胞以及缺乏ABCA1的小鼠巨噬细胞的分泌，表明该效应独立于CsA对ABCA1的已知抑制作用。使用PP2B的其他肽类和化学抑制剂证实了PP2B在介导apoE分泌中的作用。重要的是，动力学建模、活细胞成像和共聚焦显微镜均表明，CsA抑制apoE分泌的机制与抑制PKA的机制截然不同，很可能是诱导apoE在内质网区室中积累。综上所述，这些结果确立了CsA促动脉粥样硬化作用的新机制，并首次确立了PP2B在调节apoE的细胞内转运和分泌中的作用。
