Maria H. Granado

Control of metabolism and signaling of simple bioactive sphingolipids: Implications in disease

Simple bioactive sphingolipids include ceramide, sphingosine and their phosphorylated forms sphingosine 1-phosphate and ceramide 1-phosphate. These molecules are crucial regulators of cell functions. In particular, they play important roles in the regulation of angiogenesis, apoptosis, cell proliferation, differentiation, migration, and inflammation. Decoding the mechanisms by which these cellular functions are regulated requires detailed understanding of the signaling pathways that are implicated in these processes. Most importantly, the development of inhibitors of the enzymes involved in their metabolism may be crucial for establishing new therapeutic strategies for treatment of disease.

Ceramide 1-phosphate (C1P) promotes cell migration: Involvement of a specific C1P receptor

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid that is implicated in the regulation of cell homeostasis and the control of inflammation. It is mitogenic for fibroblasts and macrophages, and has been described as potent inhibitor of apoptosis. Using RAW 264.7 macrophages we have now discovered a new biological activity of C1P: stimulation of cell migration. This novel action can only be observed when C1P is applied exogenously to the cells in culture, and not by increasing the intracellular levels of C1P. This fact led to identify a specific receptor through which C1P stimulates cell migration. The receptor is coupled to G(i) proteins and causes phosphorylation of extracellularly regulated kinases 1 and 2, and protein kinase B (also known as Akt) upon ligation with C1P. Inhibition of either of these pathways completely abolished C1P-stimulated macrophage migration. In addition, C1P stimulated the DNA binding activity of nuclear factor kappa B, and blockade of this transcription factor resulted in complete inhibition of macrophage migration. This newly identified receptor could be an important drug target for treatment of illnesses that are associated to inflammatory processes, or to diseases in which cell migration is a major cause of pathology, as it occurs in metastatic tumors.

Ceramide 1-phosphate stimulates macrophage proliferation through activation of the PI3-kinase/PKB, JNK and ERK1/2 pathways

Ceramide 1-phosphate (C1P) was first shown to be mitogenic for fibroblasts, but the mechanisms whereby it stimulated cell proliferation have remained largely unknown. Here we demonstrate that C1P stimulates DNA synthesis and cell division in murine bone marrow-derived macrophages. C1P caused rapid phosphorylation of protein kinase B (PKB, also known as Akt), a downstream target of phosphatidylinositol 3-kinase (PI3-K). Selective inhibition of PI3-K blocked both DNA synthesis and cell growth. C1P induced phosphorylation of GSK-3beta, which is a major target of PKB, and this effect was also abolished by inhibition of PI3-K. In addition, C1P upregulated the expression of cyclin D1 and c-Myc, two major targets of GSK-3beta, which are important regulators of cell proliferation. C1P stimulated the activity of NF-kappaB, and inhibitors of this transcription factor completely blocked macrophage proliferation. Lastly, C1P induced phosphorylation of the mitogen activated protein kinases (MAPK) extracellularly regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK). Inhibition of ERK1/2 and JNK also blocked C1P-induced macrophage proliferation. It can be concluded that C1P stimulates macrophage proliferation through activation of the PI3-K/PKB, ERK and JNK pathways, and that GSK-3beta, c-Myc, cyclin D1, and NF-kappaB are important downstream effectors in this action.

Ceramide 1-phosphate inhibits serine palmitoyltransferase and blocks apoptosis in alveolar macrophages

We previously reported that incubation of bone-marrow derived macrophages in the absence of macrophage-colony stimulating factor (M-CSF), a cytokine that is essential for their growth and survival, resulted in stimulation of acid sphingomyelinase, accumulation of ceramides, and induction of apoptosis [A. Gomez-Munoz et al. 2004. Ceramide 1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages. J Lipid Res 45: 99-105]. Here, we show that alveolar NR8383 macrophages, which are not dependent on M-CSF for viability, undergo apoptosis when they are incubated in the absence of serum. NR8383 cells showed increased levels of ceramides under apoptotic conditions, but in contrast to bone marrow macrophage acid and neutral sphingomyelinases were only slightly activated. We found that the major mechanism for ceramide generation in NR8383 macrophages was stimulation of their synthesis de novo. This action involved activation of serine palmitoyltransferase (SPT), the key regulatory enzyme of this pathway. A relevant finding was that ceramide 1-phosphate (C1P) inhibited SPT activity and ceramide accumulation leading to inhibition of apoptosis. Furthermore, C1P enhanced the activity of antiapoptotic protein kinase B and its downstream effector nuclear factor kappa B. These observations add a new dimension to the understanding of the pro-survival actions of C1P in mammalian cells.

Chain length specificity for activation of cPLA2α by C1P: use of the dodecane delivery system to determine lipid-specific effects

Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifically activated group IVA cytosolic phospholipase A2 (cPLA2&agr;) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of ≥6 carbons efficiently activated cPLA2&agr; in vitro, whereas C2-C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specific activation of cPLA2&agr;, AA release, and PGE2 synthesis (EC50 = 400 nM) when compared to structurally similar lipids. C1P delivered as vesicles in water also induced a lipid-specific increase in AA release. Mass spectrometric analysis demonstrated that C1P delivered via ethanol/dodecane induced a 3-fold increase in endogenous C1P with little metabolism to ceramide. C1P was also more efficiently delivered (>3-fold) to internal membranes by ethanol/dodecane as compared to vesiculated C1P. Using this now established delivery method for lipids, C2-C1P was shown to be ineffective in the induction of AA release as compared with C6-C1P, C16-C1P, and C18:1 C1P. Here, we demonstrate that C1P requires ≥6 carbon acyl-chain to activate cPLA2&agr;. Thus, published reports on the biological activity of C2-C1P are not via eicosanoid synthesis. Furthermore, this study demonstrates that the alcohol/dodecane system can be used to efficiently deliver exogenous phospholipids to cells for the examination of specific biological effects.—Wijesinghe, D. S., P. Subramanian, N. F. Lamour, L. B. Gentile, M. H. Granado, A. Bielawska, Z. Szulc, A. Gomez-Munoz, and C. E. Chalfant. Chain length specificity for activation of cPLA2&agr; by C1P: use of the dodecane delivery system to determine lipid-specific effects.

Activation of protein kinase C-α is essential for stimulation of cell proliferation by ceramide 1-phosphate

We previously demonstrated that ceramide‐1‐phosphate (C1P) stimulates fibroblast and macrophage proliferation, but the mechanisms involved in this action have only been partially described. Here we demonstrate that C1P induces translocation of protein kinase C‐alpha (PKC‐α) from the soluble to the membrane fraction of bone marrow‐derived macrophages. Translocation of this enzyme was accompanied by its phosphorylation on Ser 657 residue. Activation of PKC‐α was independent of prior stimulation of phosphatidylinositol‐dependent or phosphatidylcholine‐dependent phospholipase C activities, but required activation of sphingomyelin synthesis. Inhibition of PKC‐α activation also blocked C1P‐stimulated macrophage proliferation indicating that this enzyme is essential for the mitogenic effect of C1P.

Ceramide-1-Phosphate in Cell Survival and Inflammatory Signaling

An important metabolite of ceramide is ceramide-1-phosphate (C1P). This lipid second messenger was first demonstrated to be mitogenic for fibroblasts and macrophages and later shown to have antiapoptotic properties. C1P is also an important mediator of the inflammatory response, by stimulating the release of arachidonic acid through activation of group IVA cytosolic phospholipase A2, the initial rate-limiting step of eicosanoid biosynthesis. C1P is formed from ceramide by the action of a specific ceramide kinase (CerK), which is distinct from the sphingosine kinases that synthesize sphingosine-1-phosphate. CerK is specific for natural ceramides with the erythro configuration in the base component and esterified to long-chain fatty acids. CerK can be activated by different agonists, including interleukin 1-beta, macrophage colony stimulating factor, or calcium ions. Most of the effects of C1P so far described seem to take place in intracellular compartments; however, the recent observation that C1P stimulates cell migration implicates a specific plasma membrane receptor that is coupled to a G(i) protein. Therefore, C1P has a dual regulatory capacity acting as an intracellular second messenger to regulate cell survival, or as extracellular receptor ligand to stimulate chemotaxis.

Activation of mTOR and RhoA is a major mechanism by which ceramide 1-phosphate stimulates macrophage proliferation

This study tested the hypothesis that Ceramide 1-phosphate (C1P) stimulates macrophage proliferation through activation of the mammalian target of rapamycin (mTOR). We first reported that C1P is mitogenic for fibroblasts and macrophages, but the mechanisms whereby it stimulates cell proliferation are incompletely understood. Here we demonstrate that C1P causes phosphorylation of mTOR in primary (bone marrow-derived) macrophages. Activation of this kinase was tested my measuring the phosphorylation state of its downstream target p70S6K after treatment with C1P. These actions were dependent upon prior activation of phosphoinositide 3 kinase (PI3-K), as selective inhibition of this kinase blocked mTOR phosphorylation and activation. In addition, C1P caused phosphorylation of PRAS40, a component of the mTOR complex 1 (mTORC1) that is absent in mTORC2. Furthermore, inhibition of the small G protein Ras homolog enriched in brain (Rheb), which is also a specific component of mTORC1, with FTI277, completely blocked C1P-stimulated mTOR phosphorylation, DNA synthesis and macrophage growth. In addition, C1P caused phosphorylation of another Ras homolog gene family member, RhoA, which is also involved in cell proliferation. Interestingly, inhibition of the RhoA downstream effector RhoA-associated kinase (ROCK) also blocked C1P-stimulated mTOR and cell proliferation. It can be concluded that mTORC1, and RhoA/ROCK are essential components of the mechanism whereby C1P stimulates macrophage proliferation.

Involvement of nitric oxide in the promotion of cell survival by ceramide 1‐phosphate

Macrophages play vital roles in inflammatory responses, and their number at sites of inflammation is strictly regulated by cell death and division. Here, we demonstrate that production of nitric oxide (NO) is a major mechanism whereby ceramide‐1‐phosphate (C1P) blocks apoptosis in macrophages. However, NO failed to stimulate macrophage proliferation. The prosurvival effect of C1P was blocked by inhibitors of inducible NO synthase. The antiapoptotic effect of C1P was also blocked by phosphatidylinositol 3‐kinase or nuclear factor‐kappa B inhibitors. Moreover, NO reversed the inhibitory effect of C1P on acid sphingomyelinase, but the prosurvival effect of C1P was independent of this action.

New signalling pathway involved in the anti-proliferative action of vitamin D3 and its analogues in human neuroblastoma cells. A role for ceramide kinase

1α,25-Dihydroxyvitamin D3 (1,25(OH)₂D₃), a crucial regulator of calcium/phosphorus homeostasis, has important physiological effects on growth and differentiation in a variety of malignant and non-malignant cells. Synthetic structural hormone analogues, with lower hypercalcemic side effects, are currently under clinical investigation. Sphingolipids appear to be crucial bioactive factors in the control of the cell fate: the phosphorylated forms, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are mitogenic factors, whereas sphingosine and ceramide (Cer) usually act as pro-apoptotic agents. Although many studies correlate S1P function to impaired cell growth, the relevance of C1P/Cer system and its involvement in neuroblastoma cells remain to be clarified. Here, we demonstrated the anti-proliferative effect of 1,25(OH)₂D₃ as well as of its structural analogues, ZK156979 and ZK191784, in human SH-SY5Y cells, as judged by [³H]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for C1P synthesis, by specific gene silencing or pharmacological inhibition, drastically reduced cell proliferation. 1,25(OH)₂D₃ and ZK191784 treatment induced a significant decrease in CerK expression and C1P content, and an increase of Cer. Notably, the treatment of SH-SY5Y cells with ZK159222, antagonist of 1,25(OH)₂D₃ receptor, trichostatin A, inhibitor of histone deacetylases, and COUP-TFI-siRNA prevented the decrease of CerK expression elicited by 1,25(OH)₂D₃ supporting the involvement of VDR/COUP-TFI/histone deacetylase complex in CerK regulation. Altogether, these findings provide the first evidence that CerK/C1P axis acts as molecular effector of the anti-proliferative action of 1,25(OH)₂D₃ and its analogues, thereby representing a new possible target for anti-cancer therapy of human neuroblastoma.