Expression of protein kinase C isozymes in guinea pig epidermis: selective inhibition of PKC-beta activity by 13-hydroxyoctadecadienoic acid-containing diacylglycerol.
Prompted by the reversal of skin hyperproliferation to normal by 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid, we investigated a possible mechanism for this antiproliferative action. To address this we first demonstrated that 13-HODE is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns4,5-P2) and released as 13-HODE-containing diacylglycerol by epidermal phospholipase C. Secondly, we tested the possibility whether this putative 13-HODE-containing DAG (13HODE-DAG) could exert a modulatory effect on epidermal protein kinase C (PKC) activity which previously has been associated with skin hyperproliferation. Unlabeled 13HODE-DAG was generated from 13-HODE-containing phosphatidylcholine after phospholipase C hydrolytic cleavage. The effects of the 13HODE-DAG were determined on: i) total epidermal PKC activity; ii) diolein-activated PKC activity; and iii) the two identified epidermal PKC-isozymes (PKC-beta and PKC-alpha). Our data revealed over a twofold activation of total basal PKC activity by diolein. In contrast, replacement of diolein (1,2-dioleoylglycerol) with 13HODE-DAG (1-palmitoyl,2-13HODE-glycerol) in the incubation mixture exerted no effect on total basal PKC activity. In an another experiment, 13HODE-DAG inhibited diolein-activated PKC activity in a dose-dependent manner. To determine whether the effects of 13HODE-DAG are selective, we tested its effects on DEAE-Sephacel-purified and Western blot-confirmed PKC isozymes. Our data revealed that 13HODE-DAG selectively inhibited the activity of PKC-beta isozyme, while exerting negligible effect on the PKC-alpha isozyme. This selective inhibitory effect of 13HODE-DAG on a major epidermal PKC isozyme activity suggests that 13HODE-containing DAG seemingly can modulate epidermal PKC activity, which purportedly is associated with epidermal hyperproliferation.
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Department of Dermatology, School of Medicine, University of California, Davis 95616.
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D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation; D004817:Epidermis / Q000201:enzymology*; D006168:Guinea Pigs; D007527:Isoenzymes / Q000096:biosynthesis*; D008041:Linoleic Acids / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D064546:Protein Kinase C beta; D011869:Radioligand Assay; D013110:Sphingosine / Q000494:pharmacology
D004075:Diglycerides; D007527:Isoenzymes; D008041:Linoleic Acids; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D013110:Sphingosine; C013965:diolein