Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line. Role for endogenous ceramide in mediating the action of exogenous ceramide

Treatment of A549 cells with C(6)-ceramide resulted in a significant increase in the endogenous long chain ceramide levels, which was inhibited by fumonisin B1 (FB1), and not by myriocin (MYR). The biochemical mechanisms of generation of endogenous ceramide were investigated using A549 cells treated with selectively labeled C(6)-ceramides, [sphingosine-3-(3)H]d-erythro-, and N-[N-hexanoyl-1-(14)C]d-erythro-C(6)-ceramide. The results demonstrated that (3)H label was incorporated into newly synthesized long chain ceramides, which was inhibited by FB1 and not by MYR. Interestingly, the (14)C label was not incorporated into long chain ceramides. Taken together, these results show that generation of endogenous ceramide in response to C(6)-ceramide is due to recycling of the sphingosine backbone of C(6)-ceramide via deacylation/reacylation and not due to the elongation of its fatty acid moiety. Moreover, the generation of endogenous long chain ceramide in response to C(6)-ceramide was completely blocked by brefeldin A, which causes Golgi disassembly, suggesting a role for the Golgi in the metabolism of ceramide. In addition, the generation of endogenous ceramide in response to short chain exogenous ceramide was induced by d-erythro- but not l-erythro-C(6)-ceramide, demonstrating the stereospecificity of this process. Interestingly, several key downstream biological activities of ceramide, such as growth inhibition, cell cycle arrest, and modulation of telomerase activity were induced by d-erythro-C(6)-ceramide, and not l-erythro-C(6)-ceramide (and inhibited by FB1) in A549 cells, suggesting a role for endogenous long chain ceramide in the regulation of these responses.     

The role of neutral sphingomyelinase produced ceramide in lipopolysaccharide-mediated expression of inducible nitric oxide synthase

Lipopolysaccharide (LPS) and interferon-gamma (IFN) treatment of C6 rat glioma cells increased the intracellular ceramide level and the expression of the inducible nitric oxide synthase (iNOS) gene. To delineate the possible role of ceramide in the induction of iNOS, we examined the source of intracellular ceramide and associated signal transduction pathway(s) with the use of inhibitors of intracellular ceramide generation. The inhibitor of neutral sphingomyelinase (3-O-methylsphingomyelin, MSM) inhibited the induction of iNOS, whereas inhibitor of acidic sphingomyelinase (SR33557) or that of ceramide de novo synthesis (fumonisin B1) had no effect on the induction of iNOS. MSM-mediated inhibition of iNOS induction was reversed by the supplementation of exogenous C8-ceramide, suggesting that ceramide production by neutral sphingomyelinase (nSMase) is a key mediator in the induction of iNOS. The MSM-mediated inhibition of iNOS gene expression correlated with the decrease in the activity of ras. Inhibition of co-transfected iNOS promoter activity by dominant negative ras supported the role of ras in the nSMase-dependent regulation of iNOS gene. NF-kappaB DNA binding activity and its transactivity were also reduced by MSM pretreatment, and were completely reversed by the supplementation of C8-ceramide. As the dominant negative ras also reduced NF-kappaB transactivity, NF-kappaB activation may be downstream of ras. Our results suggest that ceramide generated by nSMase may be a critical mediator in the regulation of iNOS gene expression via ras-mediated NF-kappaB activation under inflammatory conditions.     

Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells

This study was designed to analyze whether ceramide, a bioeffector of growth suppression, plays a role in the regulation of telomerase activity in A549 cells. Telomerase activity was inhibited significantly by exogenous C(6)-ceramide, but not by the biologically inactive analog dihydro-C(6)-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 microm C(6)-ceramide at 24 h. Moreover, analysis of phosphatidylserine translocation from the inner to the outer plasma membrane by flow cytometry and of poly(ADP-ribose) polymerase degradation by Western blotting showed that ceramide treatment (20 microm for 24 h) had no apoptotic effects. Trypan blue exclusion, [(3)H]thymidine incorporation, and cell cycle analyses, coupled with clonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 microm concentration at 24 h resulted in the cell cycle arrest of the majority of the cell population at G(0)/G(1) with no detectable cell death. These results suggest that the inhibition of telomerase by ceramide is not a consequence of cell death but is correlated with growth arrest. Next, to determine the role of endogenous ceramide in telomerase modulation, A549 cells were transiently transfected with an expression vector containing the full-length bacterial sphingomyelinase cDNA (b-SMase). The overexpression of b-SMase, but not exogenously applied purified b-SMase enzyme, resulted in significantly decreased telomerase activity compared with controls, showing that the increased endogenous ceramide is sufficient for telomerase inhibition. Moreover, treatment of A549 cells with daunorubicin at 1 microm for 6 h resulted in the inhibition of telomerase, which correlated with the elevation of endogenous ceramide levels and growth arrest. Finally, stable overexpression of human glucosylceramide synthase, which attenuates ceramide levels by converting ceramide to glucosylceramide, prevented the inhibitory effects of C(6)-ceramide and daunorubicin on telomerase. Therefore, these results provide novel data showing for the first time that ceramide is a candidate upstream regulator of telomerase.     

Ceramide stimulates a cytosolic protein phosphatase.

A sphingomyelin cycle has been identified whereby the action of certain extracellular agents results in reversible sphingomyelin hydrolysis and the concomitant generation of ceramide. Moreover, a cell-permeable ceramide, C2-ceramide (N-acetylsphingosine), is a potent modulator of cell proliferation and differentiation. We report herein that C2-ceramide, C6-ceramide, and natural ceramides activate a cytosolic serine/threonine protein phosphatase in a dose-dependent manner. Initial activation is observed at concentrations of ceramide as low as 0.1 microM with peak response occurring at 5-10 microM. However, other closely related sphingolipids, sphingosine and sphingomyelin, were largely inactive. Ceramide-stimulated phosphatase was inhibited by okadaic acid, an inhibitor of protein phosphatases, with an IC50 of 0.1-1 nM, depending on the concentration of ceramide. Ceramide-stimulated phosphatase was insensitive to Mg2+ and Mn2+ cations. Using sequential anion exchange chromatography, ceramide-stimulated phosphatase activity could be resolved from ceramide-nonresponsive phosphatases. The activity of partially purified enzyme was stimulated 3.5-fold by ceramide. The identification of a phosphatase as a molecular target for the action of ceramide defines a novel intracellular signaling pathway with potential roles in the regulation of cell proliferation and differentiation.     

R5020 and RU486 act as progesterone receptor agonists to enhance Sp1/Sp4-dependent gene transcription by an indirect mechanism

It has been suggested that ligand-dependent gene activation by the progesterone receptor (PR) can result from recruitment of PR by the promoter bound Sp1. A detailed investigation of the Sp1-dependent agonistic activity of RU486 and R5020 on the folate receptor (FR) type alpha, p27, thymidine kinase 1 and p21 genes reveals a different mechanism. The FR-alpha P4 promoter and the endogenous FR-alpha gene were up-regulated by the PR agonist R5020 through either PR-A or PR-B. The classical antagonist RU486 also activated the promoter but only through PR-B. The most proximal (essential) G/C-rich (Sp1 binding) element and the initiator region constituted the minimal promoter responsive to PR regulation; substitution with a stronger cluster of G/C-rich elements enhanced the magnitude of the PR response. In contrast, substitution of the G/C-rich element with a TATA box resulted in the loss of regulation by PR. Overexpression of Sp1 and Sp4 but not Sp3 enhanced activation of the FR-alpha promoter by PR, knocking down Sp1 decreased the activation in a manner that was reversed by ectopic Sp1 or Sp4. The ligand-dependent action of PR on the promoter was delayed compared with its activation of a classical glucocorticoid response element-driven promoter and activation of both the promoter and the endogenous FR-alpha gene by PR required new protein synthesis. Activation by PR paralleled RNA polymerase II recruitment but was not accompanied by either association of PR or a change in the association of Sp1 with the endogenous FR-alpha P4 promoter. Similar observations were made for PR regulation of the genes encoding p27, thymidine kinase 1, and p21. The results contradict the current view of Sp1-dependent gene regulation by PR and point to the existence of one or more PR target genes whose promoter and cell context(s) must thus be key determinants of the agonistic activity of RU486 on a large group of important Sp1-dependent downstream target genes.     

Ceramide inhibits lipopolysaccharide-mediated nitric oxide synthase and cyclooxygenase-2 induction in macrophages: effects on protein kinases and transcription factors

The goal of this study was to elucidate whether triggering the sphingomyelin pathway modulates LPS-initiated responses. For this purpose we investigated the effects of N-acetylsphingosine (C(2)-ceramide) on LPS-induced production of NO and PGE(2) in murine RAW 264.7 macrophages and explored the signaling pathways involved. We found that within a range of 10-50 microM, C(2)-ceramide inhibited LPS-elicited NO synthase and cyclooxygenase-2 induction accompanied by a reduction in NO and PGE(2) formation. By contrast, a structural analog of C(2)-ceramide that does not elicit functional activity, C(2)-dihydroceramide, did not affect the LPS response. The nuclear translocation and DNA binding study revealed that ceramide can inhibit LPS-induced NF-kappaB and AP-1 activation. The immunocomplex kinase assay indicated that IkappaB kinase activity stimulated by LPS was inhibited by ceramide, which concomitantly reduced the IkappaBalpha degradation caused by LPS within 1-6 h. In concert with the decreased cytosolic p65 protein level, LPS treatment resulted in rapid nuclear accumulation of NF-kappaB subunit p65 and its association with the cAMP-responsive element binding protein. Ceramide coaddition inhibited all the LPS responses. In addition, LPS-induced PKC and p38 mitogen-activated protein kinase activation were overcome by ceramide. In conclusion, we suggest that ceramide inhibition of LPS-mediated induction of inducible NO synthase and cyclooxygenase-2 is due to reduction of the activation of NF-kappaB and AP-1, which might result from ceramide's inhibition of LPS-stimulated IkappaB kinase, p38 mitogen-activated protein kinase, and protein kinase C.