Abstract 261: Differential Regulation of Apolipoprotein E Secretion from Primary Human Macrophages by Different Protein Kinase C Isoforms

Abstract

Macrophage-specific apolipoprotein E (apoE) secretion plays an important role in atherosclerosis. Recently, we established that inhibition of protein kinase A, phospholipase C, protein phosphatase 2B and calcium results in a reduction in apoE secretion. Research suggests PKC may have a role in this process, but the isoforms and pathways involved in human macrophages are unknown. Here, we investigate a role for PKC in regulating apoE secretion from primary human monocyte-derived macrophages (HMDM). HMDM treated with pan-PKC inhibitors (Calphostin C, Ro-318220, Bisindolylmaleimide I) that primarily target the classical and novel PKC isoforms result in a dose-dependent decrease in apoE secretion. Further, the specific classical PKC inhibitor, Gö6976, also resulted in a reduction in apoE secretion. Metabolic labelling studies indicate that CalpC and Gö6976 have no effect on apoE synthesis or degradation but directly inhibit the secretion of apoE. This occurred post-golgi as evidenced by normal glycosylation of apoE on 2D electrophoresis. Direct visualisation of apoE-GFP containing vesicles in HMDM by live cell confocal microscopy demonstrated a marked reduction in vesicular speed from 0.42μm/s in control cells to 0.14μm/s or 0.15μm/s by CalpC and Ro-318220 respectively. Finally, siRNA knockdown (∼70%) of the classical PKC isoforms, PKCalpha and PKCbeta, resulted in decreased basal apoE secretion (∼50%), whereas knockdown of PKCdelta (novel PKC isoform) did not. Interestingly, preliminary data demonstrate that treatment of HMDM with the PKCtheta or PKCzeta pseudosubstrate inhibitor increases apoE secretion from HMDM, whereas both the PKCepsilon translocation inhibitory peptide and siRNA knockdown of PKCepsilon did not affect apoE secretion. This suggests that apoE secretion is differentially regulated by different and specific PKC isoforms. Inhibition of PKCalpha and/or PKCbeta decreases apoE secretion by targeting the apoE trafficking pathway in a post-golgi vesicular compartment, whereas inhibition of PKCzeta (atypical PKC isoform) or PKCtheta (novel PKC isoform) increases apoE secretion. Future studies will further confirm and investigate the role of these novel and atypical PKC isoforms in regulating apoE secretion.