Protein kinase C β and δ isoenzymes mediate cholesterol accumulation in PMA-activated macrophages

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Abstract

Previously, we showed that PMA activation of human monocyte-derived macrophages stimulates macropinocytosis (i.e., fluid-phase endocytosis) of LDL and transforms these macrophages into foam cells. The current study aimed to learn which PKC isoenzymes mediate cholesterol accumulation in PMA-activated human macrophages incubated with LDL. Cholesterol accumulation by PMA-activated macrophages incubated with LDL was nearly completely inhibited (>85%) by the pan PKC inhibitors Go6850, Go6983, and RO 32-0432, but only was inhibited about 50% by the classical group PKC inhibitor, Go6976. This indicated that cholesterol accumulation was mediated by both a classical group and some other PKC isoenzyme. PKC β was determined to be the classical group isoenzyme that mediated PMA-stimulated cholesterol accumulation. A pseudosubstrate myristoylated peptide inhibitor of PKC α and β showed partial inhibition (≅50%) of cholesterol accumulation. However, a small molecule inhibitor of PKC α, HBDDE, show minimal inhibition of cholesterol accumulation while a small molecule inhibitor of PKC β, LY333513, could completely account for the inhibition of cholesterol accumulation by the classical group PKC isoenzyme. Thus, our findings show that β and some other PKC isoenzyme, most likely δ, mediate cholesterol accumulation when macropinocytosis of LDL is stimulated in PMA-activated human monocyte-derived macrophages.

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Materials and methods

PKC inhibitors. PKC pseudosubstrate myristoylated peptide inhibitors for α + β (P-205) and ε (P-223), HBDDE, RO 32-0432, Go6976, and Go6983 were obtained from Biomol. PKC pseudosubstrate myristoylated peptide inhibitors for theta (#539636) and eta (#539602) and Go6850 were obtained from Calbiochem. LY333531 was obtained from Alexis. The non-PKC inhibitors genistein and H89 were obtained from Biomol.

Culture of human monocyte-derived macrophages. Human monocytes were purified with counterflow

Kinase dependency of PMA-stimulated macrophage cholesterol accumulation

Previously we showed that PMA-stimulated macrophage cholesterol accumulation during incubation with native LDL could be inhibited with broad specificity PKC inhibitors [6]. To learn whether other protein kinase classes functioned in PMA-stimulated cholesterol accumulation we tested the effects of genistein, a pan tyrosine kinase inhibitor, and HA89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, on this process. Neither of these protein kinase inhibitors significantly decreased

Discussion

Macrophage foam cell formation has been thought to occur only by receptor-mediated uptake of modified LDL. We recently showed that PMA-activated macrophages take up native LDL through receptor-independent fluid-phase macropinocytosis and form foam cells due to massive accumulation of LDL-derived cholesterol [6], [7]. In this report, we have identified β and some additional PKC isoenzyme, most likely δ, as mediators of PMA-stimulated macrophage cholesterol accumulation. These two PKC isoenzymes

Acknowledgment

We thank Rani Rao for technical assistance, and the Department of Transfusion Medicine, Clinical Center, National Institutes of Health, for providing elutriated monocytes. This research was supported in part by the Intramural Research Program of the NIH, NHLBI.

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