Secretion of Apolipoprotein E From Macrophages Occurs via a Protein Kinase A– and Calcium-Dependent Pathway Along the Microtubule Network

Abstract

Abstract-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 (Ca 2ϩ ) 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% (PϽ0.01). 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 ([Ca 2ϩ ] i ) with 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetate-acetoxymethyl ester (BAPTA-AM) inhibited apoE secretion by 77.2% (PϽ0.01). Injection of c57Bl6 apoE ϩ/ϩ bone marrow-derived macrophages into the peritoneum of apoE Ϫ/Ϫ 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.

Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages

“…Human apoAI, acetylated LDL, and lipoprotein-deficient serum were all prepared as described previously (49). The apoE-green fluorescent protein (GFP) construct was generated as described previously (16).…”

“…For inhibitor experiments, HMDMs were incubated with the indicated concentrations of PKC inhibitors or corresponding vehicle (DMSO) control in RPMI medium containing 0.1% (w/v) BSA. Levels of secreted apoE in the medium were measured by ELISA and/or Western blot, and cellular apoE protein levels were analyzed by Western blot, as described previously (16). Western blots were quantified using ImageJ version 1.42b (21).…”

“…It is known that newly synthesized apoE follows the classical secretory pathway, whereby it is trafficked into the endoplasmic reticulum, post-translationally glycosylated and sialylated in the Golgi network, and transported in vesicles to the plasma membrane for secretion (1,8,15,16) or reinternalization and resecretion or degradation (2,(17)(18)(19). ApoE secretion is a constitutive process controlled by the ATP-binding cassette transporter, ABCA1 (20,21).…”

“…Recently, our laboratory has demonstrated roles for phospholipase C, protein kinase A (PKA), intracellular calcium (Ca 2ϩ ), and protein phosphatase 2B (PP2B) 2 in regulating apoE secretion from macrophages (16,27). In macrophages, apoEcontaining vesicles are associated with the microtubular network, and perturbing this network inhibits apoE secretion (16).…”

“…In macrophages, apoEcontaining vesicles are associated with the microtubular network, and perturbing this network inhibits apoE secretion (16). Stimulation of PKA activity did not enhance apoE secretion, suggesting that additional pathways were active.…”