Abstract

Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/β and not PKCδ, -ε, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages.

Highlights

  • Macrophage-specific apolipoprotein E secretion has been implicated as protective in atherosclerosis

  • Distinct Pan-protein kinase C (PKC) Inhibitors Inhibit Basal ApoE Secretion from human monocyte-derived macrophages (HMDMs)—To investigate whether PKC plays a role in apolipoprotein E (apoE) secretion from HMDMs, the effect of several panPKC inhibitors with differing sites of action was investigated

  • We identified six other proteins that were consistently secreted by HMDMs from multiple independent donors: matrix metalloproteinase 9 (MMP9) [73], chitinase-3-like-1 protein (CHI3L1; known as HC-gp-39) [74, 75], lysozyme [76], fibronectin [76, 77], cyclophilin A (CypA) [78], and heat shock protein (HSP) 90 [79]

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Summary

Background

Macrophage-specific apolipoprotein E (apoE) secretion has been implicated as protective in atherosclerosis. Results: Protein kinase C (PKC) inhibition or knockdown in macrophages inhibits apoE secretion at a post-Golgi, vesicular transport level and is mediated by MARCKS. Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKC␣/␤ and not PKC␦, -⑀, -␪, or -␫/␨ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Because PKA and protein kinase C (PKC) interact in various cell signaling networks [28, 29] and PKC is known to be activated downstream of phospholipase C [30], it is likely that PKC plays a role in apoE secretion. We identify for the first time likely roles for the classical PKC isoforms in this process, establish that PKC acts independently of ABCA1, and report a likely role for MARCKS as a downstream mediator of this process

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