We have previously demonstrated that hexanoyl-D-erythrosphingosine (C 6 -ceramide), an anti-mitogenic cell-permeable lipid metabolite, limited vascular smooth muscle growth by abrogating trauma-induced Akt activity in a stretch injury model of neointimal hyperplasia. Furthermore, ceramide selectively and directly activated protein kinase C (PKC) to suppress Akt-dependent mitogenesis. To further analyze the interaction between ceramide and PKC, the ability of ceramide to localize within highly structured lipid microdomains (rafts) and activate PKC was investigated. Using rat aorta vascular smooth muscle cells (A7r5), we now demonstrate that C 6 -ceramide treatment results in an increased localization and phosphorylation of PKC within caveolin-enriched lipid microdomians to inactivate Akt. In addition, ceramide specifically reduced the association of PKC with 14-3-3, a scaffold protein localized to less structured regions within membranes. Pharmacological disruption of highly structured lipid microdomains resulted in abrogation of ceramide-activated, PKC-dependent Akt inactivation, whereas molecular strategies suggest that ceramide-dependent PKC phosphorylation of Akt3 at Ser 34 was necessary for ceramide-induced vascular smooth muscle cell growth arrest. Taken together, these data demonstrate that structured membrane microdomains are necessary for ceramide-induced activation of PKC and resultant diminished Akt activity, leading to vascular smooth muscle cell growth arrest.

Many forms of cellular stress cause an elevation of endogenous ceramide levels leading to growth arrest or apoptosis. Ceramidases (CDase) play a critical role in regulating apoptosis by hydrolyzing ceramide into sphingosine, a precursor for promitogenic sphingosine-1-phosphate. Growth factor induction of neutral CDase (nCDase) has been shown to have a cytoprotective effect against cytokine-induced increases in ceramide levels. To further define the physiological regulation of nCDase, we identified a 200 bp promoter region and demonstrated that serum activated this proximal promoter, which correlated with a serum-induced increase in human nCDase mRNA expression. Computational analysis revealed a putative cis-element for AP-1, a transcription factor activated by serum. Electrophoretic mobility shift assays demonstrated that the identified transcriptional response element binds to AP-1 transcription factors. RNA interference-mediated knockdown of the AP-1 subunit, c-Jun, inhibited the activity of the human nCDase proximal promoter, whereas, c-Jun overexpression increased promoter activity, which directly correlated with human nCDase mRNA transcription, decreased ceramide mass, and protection against caspase 3/7-dependent apoptosis. Taken together, our findings suggest that c-Jun/AP-1 signaling may, in part, regulate serum-induced human nCDase gene transcription.

Houck KL, Fox TE, Sandirasegarane L, Kester M. Etherlinked diglycerides inhibit vascular smooth muscle cell growth via decreased MAPK and PI3K/Akt signaling. Am J Physiol Heart Circ Physiol 295: H1657-H1668, 2008. First published August 22, 2008 doi:10.1152/ajpheart.00141.2008 are phospholipid-derived second messengers that regulate PKC-dependent signaling pathways. Distinct species of DGs are generated from inflammatory cytokines and growth factors. Growth factors increase diacyl-but not ether-linked DG species, whereas inflammatory cytokines predominately generate alkyl, acyl-and alkenyl, acyl-linked DG species in rat mesenchymal cells. These DG species have been shown to differentially regulate protein kinase C (PKC) isotypes. Esterlinked diacylglycerols activate PKC-ε and cellular proliferation in contrast to ether-linked DGs, which lead to growth arrest through the inactivation of PKC-ε. It is now hypothesized that ether-linked DGs inhibit mitogenesis through the inactivation of ERK and/or Akt signaling cascades. We demonstrate that cell-permeable ether-linked DGs reduce vascular smooth muscle cell growth by inhibiting platelet-derived growth factor-stimulated ERK in a PKC-ε-dependent manner. This inhibition is specific to the ERK pathway, since ether-linked DGs do not affect growth factor-induced activation of other family members of the MAPKs, including p38 MAPK and c-Jun NH2-terminal kinases. We also demonstrate that ether-linked DGs reduce prosurvival phosphatidylinositol 3-kinase (PI3K)/Akt signaling, independent of PKC-ε, by diminishing an interaction between the subunits of PI3K and not by affecting protein phosphatase 2A or lipid (phosphatase and tensin homologue deleted in chromosome 10) phosphatases. Taken together, our studies identify ether-linked DGs as potential adjuvant therapies to limit vascular smooth muscle migration and mitogenesis in atherosclerotic and restenotic models. cell migration; proliferation; bioactive lipids; mitogen-activated protein kinase; phosphatidylinositol 3-kinase DIGLYCERIDES (DGs) are bioactive phospholipid-derived second messengers. Our laboratory and others have shown that there are distinct DG species produced endogenously in mammalian cells (20,23,27,32). It has been demonstrated that growth factors, such as platelet-derived growth factor, lead to the formation of ester-linked diacylglycerols (DAGs), whereas cytokines, such as interleukin-1, generate ether-linked DGs, which contain either an alkyl-or alkenyl-chain, via hydrolysis of ether-linked phosphatidylethanolamine (32). DAGs are linked to vascular smooth muscle (VSM) cell mitogenesis (27).In contrast, we have demonstrated that ether-linked DGs inhibit mesenchymal cell mitogenesis (27). In fact, these novel ether-linked, phospholipid-derived, second messengers mimic the effect of interleukin-1 to inhibit growth factor-induced cellular proliferation (27).