Secretion of apolipoprotein E from macrophages occurs via a protein kinase A and calcium-dependent pathway along the microtubule network.
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 PKI(14-22)) 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.
Macrophage Biology Group, Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Australia.
Maaike
M
Kockx
Dongni Lily
DL
Guo
Thierry
T
Huby
Philippe
P
Lesnik
Jason
J
Kay
Tharani
T
Sabaretnam
Eve
E
Jary
Michael
M
Hill
Katharina
K
Gaus
John
J
Chapman
Jennifer L
JL
Stow
Wendy
W
Jessup
Leonard
L
Kritharides
D000199:Actins / Q000378:metabolism; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism; D001057:Apolipoproteins E / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002227:Carbazoles / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology; D002460:Cell Line; D017690:Cell Transplantation; D002614:Chelating Agents / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000637:transplantation; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011758:Pyrroles / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D050257:Tubulin Modulators / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D000199:Actins; D016632:Apolipoprotein A-I; D001057:Apolipoproteins E; D002227:Carbazoles; D002352:Carrier Proteins; D002614:Chelating Agents; D007211:Indoles; D007546:Isoquinolines; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D013449:Sulfonamides; D050257:Tubulin Modulators; C477818:myristoylated protein kinase A inhibitor amide 14-22; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; C057416:KT 5720; D014747:Vinblastine; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D003078:Colchicine