Published online before print July 27, 2007, doi: 10.1161/CIRCRESAHA.107.157198
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Submitted on August 24, 2006
Revised on June 4, 2007
Accepted on July 17, 2007
Secretion of Apolipoprotein E From Macrophages Occurs via a Protein Kinase A– and Calcium-Dependent Pathway Along the Microtubule Network
Maaike Kockx ;
From the Macrophage Biology Group (M.K., D.L.G., K.G., W.J., L.K.), Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Australia; INSERM U551 (T.H., P.L., J.C.), Dyslipoproteinemia and Atherosclerosis Research Unit, Paris, France; UMR S551 (T.H., P.L., J.C.), Université Pierre et Marie Curie-Paris6, Paris, France; Institute for Molecular Bioscience (J.K., J.L.S.), The University of Queensland, Brisbane, Australia; The Anzac Research Institute (T.S.), University of Sydney; Department of Physiology and Pharmacology (E.J., M.H.), University of New South Wales, Australia; and Department of Cardiology (L.K.), Concord Repatriation General Hospital, University of Sydney, Australia.
* To whom correspondence should be addressed. E-mail: l.kritharides{at}unsw.edu.au.
Macrophage-specific expression of apolipoprotein (apo)E protects against atherosclerosis; however, the signaling and trafficking pathways regulating secretion of apoE are unknown. We investigated the roles of the actin skeleton, microtubules, protein kinase A (PKA) and calcium (Ca2+) in regulating apoE secretion from macrophages. Disrupting microtubules with vinblastine or colchicine inhibited basal secretion of apoE substantially, whereas disruption of the actin skeleton had no effect. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14–22) all decreased basal secretion of apoE by between 50% to 80% (P<0.01). Pulse-chase experiments demonstrated that inhibition of PKA reduced the rate of apoE secretion without affecting its degradation. Confocal microscopy and live cell imaging of apoE–green fluorescent protein–transfected RAW macrophages identified apoE–green fluorescent protein in vesicles colocalized with the microtubular network, and inhibition of PKA markedly inhibited vesicular movement. Chelation of intracellular calcium ([Ca2+]i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM) inhibited apoE secretion by 77.2% (P<0.01). Injection of c57Bl6 apoE+/+ bone marrow–derived macrophages into the peritoneum of apoE-/- C57Bl6 mice resulted in time-dependent secretion of apoE into plasma, which was significantly inhibited by transient exposure of macrophages to BAPTA-AM and colchicine and less effectively inhibited by H89. We conclude that macrophage secretion of apoE occurs via a PKA- and calcium-dependent pathway along the microtubule network.
Key words: apolipoprotein E • atherosclerosis • macrophages • signaling pathways
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