Abstract
Our previous studies have shown that human native low density lipoprotein (LDL) can be oxidized by activated human monocytes. In this process, both activation of protein kinase C (PKC) and induction of superoxide anion (O-2) production are required. PKC is a family of isoenzymes, and the functional roles of individual PKC isoenzymes are believed to differ based on subcellular location and distinct responses to regulatory signals. We have shown that the PKC isoenzyme that is required for both monocyte O-2 production and oxidation of LDL is a member of the conventional PKC group of PKC isoenzymes (Li, Q., and Cathcart, M. K. (1994) J. Biol. Chem. 269, 17508-17515). The conventional PKC group includes PKCalpha, PKCbetaI, PKCbetaII, and PKCgamma. With the exception of PKCgamma, each of these isoenzymes was detected in human monocytes. In these studies, we investigated the requirement for select PKC isoenzymes in the process of monocyte-mediated LDL lipid oxidation. Our data indicate that PKC activity was rapidly induced upon monocyte activation with the majority of the activity residing in the membrane/particulate fraction. This enhanced PKC activity was sustained for up to 24 h after activation. PKCalpha, PKCbetaI, and PKCbetaII protein levels were induced upon monocyte activation, and PKCalpha and PKCbetaII substantially shifted their location from the cytosol to the particulate/membrane fraction. To distinguish between these isoenzymes for regulating monocyte O-2 production and LDL oxidation, PKCalpha or PKCbeta isoenzyme-specific antisense oligonucleotides were used to selectively suppress isoenzyme expression. We found that suppression of PKCalpha expression inhibited both monocyte-mediated O-2 production and LDL lipid oxidation by activated human monocytes. In contrast, inhibition of PKCbeta expression (including both PKCbetaI and PKCbetaII) did not affect O-2 production or LDL lipid oxidation. Further studies demonstrated that the respiratory burst oxidase responsible for O-2 production remained functionally intact in monocytes with depressed levels of PKCalpha because O-2 production could be restored by treating the monocytes with arachidonic acid. Taken together, our data reveal that PKCalpha, and not PKCbetaI or PKCbetaII, is the predominant isoenzyme required for O-2 production and maximal oxidation of LDL by activated human monocytes.
Highlights
It has been suggested that monocyte-mediated low density lipoprotein (LDL)1 oxidation may play an important role in atherogenesis and inflammatory tissue injury
Because our previous studies indicated that cPKC activity was required for monocyte-mediated LDL lipid oxidation (7), in further studies we focused on the members of the cPKC group of isoenzymes that are detectable in these cells, namely protein kinase C (PKC)␣, I, and II
Our laboratory published studies indicating that the activity of a member of the cPKC isoenzyme family was required for human monocyte-mediated LDL lipid oxidation and for the production of superoxide anion (7)
Summary
Materials—Cytochrome c and superoxide dismutase (SOD) from bovine erythrocytes were purchased from Sigma. Cell-mediated LDL lipid oxidation was determined by a modified thiobarbituric acid (TBA) assay described by Schuh et al (13). Cell lysates were prepared by sonication in hypotonic buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgSO4, 0.5 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 20 g/ml leupeptin) as described previously (14). For experiments using ODN treatment, human monocytes (1.0 –2.5 ϫ 106 cells/ml) were cultured in Dulbecco’s modified Eagle’s medium with 10% bovine serum albumin in the presence or absence of sense or antisense oligomers for 20 h. Human monocytes (1 ϫ 106 cells/ml) and cytochrome c (320 M; Sigma) were incubated in the presence or absence of 150 unit/ml SOD (from bovine erythrocytes; Sigma) in 96well cell culture plates in RPMI 1640 without phenol red (Whittaker, Walkersville, MD). Data points were considered significantly different if the p value was Ͻ0.05
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