Abstract
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.
Highlights
HMDM were incubated in methionine/cysteine-free Dulbecco’s modified Eagle’s medium with 250 Ci/ml [35S]methionine/cysteine for 3 h and received a preincubation of 10 M CsA during the last hour
At the indicated times 35S-labeled apoE was immunoprecipitated from media and cell lysates, separated by SDS-PAGE, and quantified by phosphorimaging as described under “Experimental Procedures.” k1 and k2 represent the secretion and degradation rate constants, respectively
Es represents the percent of cellular apoE in the stable pool; ERF represents the error function of the fit of modeling parameters to experimental data, indicating error Ͻ0.01 for both control and CsA
Summary
CsA, cyclosporin A; ABCA1, ATP-binding cassette transporter A1; PKA, protein kinase A; PP2B, protein phosphatase 2B; ER, endoplasmic reticulum; LDL, low density lipoprotein; AcLDL, acetylated LDL; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; HMDM, human monocyte-derived macrophage; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GFP, green fluorescent protein; BMDM, bone marrow-derived macrophages; TD, Tangier disease. We demonstrate for the first time that CsA and other PP2B inhibitors markedly inhibit apoE secretion from human macrophages and that this effect does not occur via suppression of ABCA1 activity
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