It is hypothesized that inflammatory cytokines and vasoactive peptides stimulate distinct species of diglycerides that differentially regulate protein kinase C isotypes. In published data, we demonstrated that interleukin-1, in contrast to endothelin, selectively generates ether-linked diglyceride species (alkyl, acyl-and alkenyl, acylglycerols) in rat mesangial cells, a smooth muscle-like pericyte in the glomerulus. We now demonstrate both in intact cell and in cell-free preparations that these interleukin-1 receptor-generated ether-linked diglycerides inhibit immunoprecipitated protein kinase C ␦ and ⑀ but not activity. Neither interleukin-1 nor endothelin affect de novo protein expression of these protein kinase C isotypes. As down-regulation of calciuminsensitive protein kinase C isotypes has been linked to antimitogenic activity, we investigated growth arrest as a functional correlate for IL-1-generated ether-linked diglycerides. Cell-permeable ether-linked diglycerides mimic the effects of interleukin-1 to induce a growtharrested state in both G-protein-linked receptor-and tyrosine kinase receptor-stimulated mesangial cells. This signaling mechanism implicates cytokine receptorinduced ether-linked diglycerides as second messengers that inhibit the bioactivity of calcium-insensitive protein kinase C isotypes resulting in growth arrest.Interleukin-1 (IL-1) 1 -induced activation of rat glomerular mesangial cells (MC) culminates in an inflammatory phenotype often observed in vivo in models of glomerulosclerosis. Our laboratory has been investigating the early, lipid-mediated signal transduction pathways for inflammatory cytokines in MC with particular emphasis on the distinct molecular species of diglycerides (DG) generated and their regulation of protein kinase C (PKC) activity. We have demonstrated previously that the inflammatory cytokine interleukin-1␣ and the vasoconstrictor peptide, endothelin-1 (ET-1) generate distinct species of DG from different membrane-associated phospholipids in MC (1-3). IL-1 receptor activation selectively generates etherlinked species of DG, namely alkyl, acyl-and alkenyl, acylglycerols, whereas ET-1 receptor activation produces predominantly ester-linked diacylglycerols (1). Our laboratory has also demonstrated previously that these IL-1 generated etherlinked DG, in contrast to the PKC-activating diacylglycerols, inhibit total PKC activity as well as inhibit diacylglycerolstimulated PKC ␣ activation (1). In fact, ether-linked DG may competitively inhibit PKC activation induced by diacylglycerol species (4). Additional studies support this signaling mechanism, as ether-linked DG fail to activate total PKC activity in vitro or only activate PKC in the presence of pharmacological concentrations of calcium (5-8).In light of these findings, our interest has now turned to regulation by ether-linked DG of specific PKC isotypes. The PKC family now includes 12 distinct isotypes subdivided into three categories (9). The calcium-sensitive conventional PKCs consist of alpha (␣), beta ( 1 ...
Diglycerides are phospholipid-derived second messengers that serve as cofactors for protein kinase C activation. We have previously shown that, in rat glomerular mesangial cells, the cytokine, interleukin-1␣, and the vasoactive peptide, endothelin, generate diglycerides from unique phospholipid precursors. However, neither the molecular species of these diglycerides nor their biological actions were determined. It is now hypothesized that interleukin-1-and endothelin-treated mesangial cells form distinct molecular species of diglycerides which may serve different roles as intracellular signaling molecules. Diglyceride molecular species were resolved and quantified by TLC and high performance liquid chromatography as diglyceride-[ 14 C]acetate derivatives. Endothelin stimulates predominantly esterlinked species (diacylglycerols) in contrast to interleukin-1 which stimulates only ether-linked species (alkyl, acyl-and alkenyl,acylglycerols). In support of these data, interleukin-1-treated mesangial cells hydrolyze ethanolamine plasmalogens, vinyl ether-linked phospholipids. It has been reported that ether-linked, in contrast to ester-linked, diglyceride species do not activate protein kinase C activity. Thus, we next assessed membrane protein kinase C activity in endothelin-or interleukin-1-treated mesangial cells. Even though interleukin-1 has no effect upon basal protein kinase C activity, this cytokine, through the formation of ether-linked diglyceride second messengers, inhibits endothelin, platelet-activating factor, or arginine vasopressin-stimulated protein kinase C activity. We further demonstrate that ester-linked diacylglycerols but not alkyl,acyl-or alkenyl,acylglycerols substitute for phorbol esters in a cell-free protein kinase C assay. In addition, alkenyl,acylglycerols inhibit diacylglycerol-stimulated immunoprecipitated protein kinase C ␣ activity in vitro and total protein kinase C activity in permeabilized mesangial cells ex vivo. Taken together, these data suggest that interleukin-1-induced formation of ether-linked diglycerides may physiologically serve to down-regulate receptor-mediated protein kinase C activity and that individual molecular species of diglycerides may serve different roles as intracellular signaling molecules.Classical transmembrane signaling theory suggests that receptors are linked to a phosphatidylinositol 4,5-bisphosphate (PtdIns) 1 -specific phospholipase C generating inositol phosphates and diglycerides (DG). However, distinct phospholipase C and phospholipase D activities that hydrolyze ester-and ether-linked phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEth) may also release unique molecular species of DG and phosphatidic acids. We have previously compared and contrasted the early phospholipid-derived second messengers generated by the vasoconstrictor, endothelin-1 (ET), and the cytokine, interleukin-1␣ (IL-1) in rat glomerular mesangial cells (MC). ET maintains the myogenic phenotype of these smooth muscle pericytes inducing MC contraction which leads ...
Human tracheal epithelial (TE) cells selectively incorporate their major lipoxygenase product, 15-hydroxyeicosatetraenoic acid (15-HETE), into the sn-2 position of phosphatidylinositol (PI) (S. E. Alpert and R. W. Walenga. Am. J. Respir. Cell Mol. Biol. 8: 273–281, 1993). Here we investigated whether 15-HETE-PI is a substrate for receptor-mediated generation of 15-HETE-substituted diglycerides (DGs) and whether these 15-HETE-DGs directly activate and/or alter conventional diacylglycerol-induced activation of protein kinase C (PKC) isotypes in these cells. Primary human TE monolayers incubated with 0.5 μM 15-[3H]-HETE or 15-[14C]HETE for 1–2 h were stimulated with 1 nM to 1 μM platelet-activating factor (PAF) for 30 s to 6 min, and the radiolabel in the medium, cellular phospholipids, and neutral lipids was assessed by high-performance liquid and thin-layer chromatography. PAF mobilized radiolabel from PI in a dose-dependent manner (22 ± 5% decrease after 1 μM PAF) without a concomitant release of free intra- or extracellular 15-HETE. 14C-labeled DGs were present in unstimulated TE monolayers incubated with 15-[14C]HETE, and the major 14C band, identified as sn-1,2-15-[14C]HETE-DG, increased transiently in response to PAF. Western blots of freshly isolated and cultured human TE cells revealed PKC isotypes α, βI, βII, δ, ε, and ζ. In vitro, cell-generated sn-1,2-15-[14C]HETE-DG selectively activated immunoprecipitated PKC-α and inhibited diacylglycerol-induced activation of PKC-α, -δ, -βI, and -βII. Our observations indicate that 15-HETE-DGs can modulate the activity of PKC isotypes in human TE cells and suggest an intracellular autocrine role for 15-HETE in human airway epithelia.
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