WITHDRAWN: Ceramide kinase regulates the production of TNF{alpha} via inhibition of TNF{alpha}-converting enzyme.

This manuscript was withdrawn by the author.

Ceramide kinase regulates the production of tumor necrosis factor α (TNFα) via inhibition of TNFα-converting enzyme.

Tumor necrosis factor α (TNFα) is a well known cytokine involved in systemic and acute inflammation. In this study, we demonstrate that ceramide 1-phosphate (C1P) produced by ceramide kinase (CERK) is a negative regulator of LPS-induced TNFα secretion. Specifically, bone marrow-derived macrophages isolated from CERK knock-out mice (CERK(-/-)) generated higher levels of TNFα than the wild-type mice (CERK(+/+)) in response to LPS. An increase in basal TNFα secretion was also observed in CERK(-/-) murine embryonic fibroblasts, which was rescued by re-expression of wild-type CERK. This effect was due to increased secretion and not transcription. The secretion of TNFα is regulated by TNFα-converting enzyme (TACE also known as ADAM17), and importantly, the activity of TACE was higher in cell extracts from CERK(-/-) as compared with wild type. In vitro analysis also demonstrated that C1P is a potent inhibitor of this enzyme, in stark contrast to ceramide and sphingosine 1-phosphate. Furthermore, TACE specifically bound C1P with high affinity. Finally, several putative C1P-binding sites were identified via homology throughout the protein sequence of TACE. These results indicate that C1P produced by CERK has a negative effect on the processing/secretion of TNFα via modulation of TACE activity.

Ceramide 1-phosphate is required for the translocation of group IVA cytosolic phospholipase A2 and prostaglandin synthesis.

Little is known about the regulation of eicosanoid synthesis proximal to the activation of cytosolic phospholipase A(2)alpha (cPLA(2)alpha), the initial rate-limiting step. The current view is that cPLA(2)alpha associates with intracellular/phosphatidylcholine-rich membranes strictly via hydrophobic interactions in response to an increase of intracellular calcium. In opposition to this accepted mechanism of two decades, ceramide 1-phosphate (C1P) has been shown to increase the membrane association of cPLA(2)alpha in vitro via a novel site in the cationic beta-groove of the C2 domain (Stahelin, R. V., Subramanian, P., Vora, M., Cho, W., and Chalfant, C. E. (2007) J. Biol. Chem. 282, 20467-204741). In this study we demonstrate that C1P is a proximal and required bioactive lipid for the translocation of cPLA(2)alpha to intracellular membranes in response to inflammatory agonists (e.g. calcium ionophore and ATP). Last, the absolute requirement of the C1P/cPLA(2)alpha interaction was demonstrated for the production of eicosanoids using murine embryonic fibroblasts (cPLA(2)alpha(-/-)) coupled to "rescue" studies. Therefore, this study provides a paradigm shift in how cPLA(2)alpha is activated during inflammation.

Chain length specificity for activation of cPLA2alpha 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 A(2) (cPLA(2)alpha) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of >or=6 carbons efficiently activated cPLA(2)alpha in vitro, whereas C(2)-C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specific activation of cPLA(2)alpha, AA release, and PGE(2) synthesis (EC(50) = 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, C(2)-C1P was shown to be ineffective in the induction of AA release as compared with C(6)-C1P, C(16)-C1P, and C(18:1) C1P. Here, we demonstrate that C1P requires >or=6 carbon acyl-chain to activate cPLA(2)alpha. Thus, published reports on the biological activity of C(2)-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.

Ceramide kinase and ceramide-1-phosphate.

It has been over a decade since the sphingolipid ceramide-1-phosphate (C1P) was described. Until recently, only sparse reports on possible biological functions for this lipid have been published. A large number of reports have now surfaced demonstrating distinct biological mechanisms regulated by C1P produced from ceramide kinase (CERK). In the following methods chapter, the methodologies for examining CERK function in vitro and in cells are outlined in detail. The methodologies for examining C1P levels and the use of exogenous C1P on cells to observe lipid specific effects on a particular biology are also detailed.

Novel organotypic culture model of cholangiocarcinoma progression.

AIM: Recent studies have suggested that increased α-smooth muscle-actin positive myofibroblastic cells (α-SMA positive CAF) in the desmoplastic stroma may relate to a more aggressive cancer and worse survival outcomes for intrahepatic cholangiocarcinoma (ICC) patients. To facilitate investigating cellular and molecular interactions between α-SMA positive CAF and cholangiocarcinoma cells related to ICC progression, we developed a novel 3-D organotypic culture model of cholangiocarcinoma that more accurately mimics the stromal microenvironment, gene expression profile and select pathophysiological characteristics of desmoplastic ICC in vivo. METHODS: This unique model was established by co-culturing within a type I collagen gel matrix, a strain of cholangiocarcinoma cells (derived from an ICC formed in syngeneic rat liver following bile duct inoculation of spontaneously-transformed rat cholangiocytes) with varying numbers of clonal α-SMA positive CAF established from the same tumor type. RESULTS: Cholangiocarcinoma cells and α-SMA positive CAF in monoculture each exhibited cell-specific biomarker gene expression profiles characteristic of stromal myofibroblastic cell versus malignant cholangiocyte cell types. In comparison, the gene expression profile and histopathological characteristics exhibited by the organotypic co-culture closely resembled those of whole tissue samples of the parent orthotopic ICC. We further showed α-SMA positive CAF to significantly enhance cholangiocarcinoma cell "ductal-like" growth and cancer cell migration/invasiveness in vitro, as well as to promote upregulated expression of select genes known to be associated with ICC invasion. CONCLUSION: This novel organotypic model provides an important new resource for studying the effects of microenvironment on cholangiocarcinoma progression in vitro and may have potential as a preclinical model for identifying molecularly targeted therapies.

A ceramide analog inhibits cPLA(2) activity and consequent PGE(2) formation in LPS-stimulated macrophages.

Prostaglandin E(2) (PGE(2)) is an important mediator of the inflammatory response. Phospho-ceramide analogue-1 (PCERA-1), a synthetic phospholipid-like molecule, was previously reported to modulate pro- and anti-inflammatory cytokine production. We show here that PCERA-1 inhibited LPS-stimulated PGE(2) production in RAW264.7 macrophages, without affecting COX-2 expression. Furthermore, PCERA-1 efficiently suppressed arachidonic acid (AA) release in response to LPS. The dephosphorylated derivative of PCERA-1, ceramide analogue-1 (CERA-1), mimicked the inhibitory effect of PCERA-1 on AA release and PGE(2) production in macrophages. Inhibition of PGE(2) production by CERA-1 was completely rescued by addition of exogenous AA. Importantly, PCERA-1 and ceramide-1-phosphate (C1P) stimulated the enzymatic activity of cPLA(2)α in an in vitro assay, whereas CERA-1 and ceramide inhibited both basal and C1P-stimulated cPLA(2)α activity. Collectively, these results indicate that CERA-1 suppresses AA release and subsequent PGE(2) production in LPS-stimulated macrophages by direct interaction with cPLA(2), and suggest that ceramide may similarly counteract C1P effect on cPLA(2) activity in cells. The suppression of PGE(2) production is suggested to contribute to the anti-inflammatory action of PCERA-1.

hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing.

Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non-small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.

Ceramide kinase and the ceramide-1-phosphate/cPLA2alpha interaction as a therapeutic target.

Ceramide kinase (CERK) was discovered more than a decade ago. Since then, numerous reports have been published demonstrating a role for CERK in various signal transduction pathways involved in inflammation, immunity or cancer. In this review, the biosynthesis of ceramide-1-phosphate (C1P) and the various roles of CERK and C1P in biological mechanisms will be overviewed. We will focus on the role of C1P in eicosanoid synthesis, more specifically, in the activation and translocation of cPLA(2)alpha. Furthermore, the possible therapeutic relevance of inhibitors of these mechanisms is discussed.

Ceramide kinase uses ceramide provided by ceramide transport protein: localization to organelles of eicosanoid synthesis.

Ceramide kinase (CERK) is a critical mediator of eicosanoid synthesis, and its product, ceramide-1-phosphate (C1P), is required for the production of prostaglandins in response to several inflammatory agonists. In this study, mass spectrometry analysis disclosed that the main forms of C1P in cells were C(16:0) C1P and C(18:0) C1P, suggesting that CERK uses ceramide transported to the trans-Golgi apparatus by ceramide transport protein (CERT). To this end, downregulation of CERT by RNA interference technology dramatically reduced the levels of newly synthesized C1P (kinase-derived) as well as significantly reduced the total mass levels of C1P in cells. Confocal microscopy, subcellular fractionation, and surface plasmon resonance analysis were used to further localize CERK to the trans-Golgi network, placing the generation of C1P in the proper intracellular location for the recruitment of cytosolic phospholipase A(2)alpha. In conclusion, these results demonstrate that CERK localizes to areas of eicosanoid synthesis and uses a ceramide "pool" transported in an active manner via CERT.

Ceramide-1-phosphate: the "missing" link in eicosanoid biosynthesis and inflammation.

It has been over a decade since ceramide kinase (CERK) and its product, ceramide-1-phosphate (C1P), were first reported. Since itscloning, in 2002, CERK has been the subject of an explosion of publications concerning various signal transduction pathways. The roles of this previously overlooked enzyme, as well as those of its product C1P, continue to expand, and their regulatory functions in the production of eicosanoid inflammatory mediators are proving essential to fundamental signal transduction pathways. In particular, C1P is required for the activation and translocation of cPLA(2)alpha, the initial rate-limiting step of eicosanoid synthesis. The potential for inhibitors of CERK to offer a new generation of anti-inflammatory and anti-cancer therapeutics is especially deserving of further study.