Ceramide 1-phosphate mediates endothelial cell invasion via the annexin a2-p11 heterotetrameric protein complex.

The bioactive sphingolipid, ceramide 1-phosphate (C-1-P), has been implicated as an extracellular chemotactic agent directing cellular migration in hematopoietic stem/progenitor cells and macrophages. However, interacting proteins that could mediate these actions of C-1-P have, thus far, eluded identification. We have now identified and characterized interactions between ceramide 1-phosphate and the annexin a2-p11 heterotetramer constituents. This C-1-P-receptor complex is capable of facilitating cellular invasion. Herein, we demonstrate in both coronary artery macrovascular endothelial cells and retinal microvascular endothelial cells that C-1-P induces invasion through an extracellular matrix barrier. By employing surface plasmon resonance, lipid-binding ELISA, and mass spectrometry technologies, we have demonstrated that the heterotetramer constituents bind to C-1-P. Although the annexin a2-p11 heterotetramer constituents do not bind the lipid C-1-P exclusively, other structurally similar lipids, such as phosphatidylserine, sphingosine 1-phosphate, and phosphatidic acid, could not elicit the potent chemotactic stimulation observed with C-1-P. Further, we show that siRNA-mediated knockdown of either annexin a2 or p11 protein significantly inhibits C-1-P-directed invasion, indicating that the heterotetrameric complex is required for C-1-P-mediated chemotaxis. These results imply that extracellular C-1-P, acting through the extracellular annexin a2-p11 heterotetrameric protein, can mediate vascular endothelial cell invasion.

The therapeutic potential of nanoscale sphingolipid technologies.

Nanotechnologies, while small in size, widen the scope of drug delivery options for compounds with problematic pharmacokinetics, such as bioactive sphingolipids. We describe the development of historical sphingolipid nanotechnologies, such as nanoliposomes, and project future uses for a broad repertoire of nanoscale sphingolipid therapy formulations. In particular, we describe sphingo-nanotherapies for treatment of cancer, inflammatory disease, and cardiovascular disease. We conclude with a discussion of the challenges associated with regulatory approval, scale-up, and development of these nanotechnology therapies for clinical applications.

Ceramide kinase regulates TNFα-stimulated NADPH oxidase activity and eicosanoid biosynthesis in neuroblastoma cells.

A persistent inflammatory reaction is a hallmark of chronic and acute pathologies in the central nervous system (CNS) and greatly exacerbates neuronal degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNFα) plays a pivotal role in the initiation and progression of inflammatory processes provoking oxidative stress, eicosanoid biosynthesis, and the production of bioactive lipids. We established in neuronal cells that TNFα exposure dramatically increased Mg(2+)-dependent neutral sphingomyelinase (nSMase) activity thus generating the bioactive lipid mediator ceramide essential for subsequent NADPH oxidase (NOX) activation and oxidative stress. Since many of the pleiotropic effects of ceramide are attributable to its metabolites, we examined whether ceramide kinase (CerK), converting ceramide to ceramide-1-phosphate, is implicated both in NOX activation and enhanced eicosanoid production in neuronal cells. In the present study, we demonstrated that TNFα exposure of human SH-SY5Y neuroblastoma caused a profound increase in CerK activity. Depleting CerK activity using either siRNA or pharmacology completely negated NOX activation and eicosanoid biosynthesis yet, more importantly, rescued neuronal viability in the presence of TNFα. These findings provided evidence for a critical function of ceramide-1-phospate and thus CerK activity in directly linking sphingolipid metabolism to oxidative stress. This vital role of CerK in CNS inflammation could provide a novel therapeutic approach to intervene with the adverse consequences of a progressive CNS inflammation.

Exogenous ceramide-1-phosphate reduces lipopolysaccharide (LPS)-mediated cytokine expression.

Toll-like receptor 4 (TLR4) is a component of the innate immune system that recognizes a diverse group of molecular structures, such as lipopolysaccharide (LPS) from Gram-negative bacteria. TLR4 signaling ultimately leads to activation of the transcription factor, nuclear factor κB (NF-κB), and the production of cytokines. Ceramide is a bioactive sphingolipid that has been suggested to regulate TLR4-induced NF-κB signaling, although reports on the role of ceramide in TLR4 activation conflict. We investigated the possibility that ceramide metabolites, such as ceramide-1-phosphate (C-1-P), may explain these discrepancies. We now report that exogenous C-1-P, but not ceramide, reduces NF-κB-mediated gene transcription in HEK 293 cells stably transfected with human TLR4, CD14, and MD-2. We demonstrate that inhibition of NF-κB by exogenous C-1-P is dose-dependent and specific to TLR4 in a reporter assay. We further demonstrate a requirement for both the phosphate moiety and the sphingoid backbone to inhibit LPS-activated NF-κB transcription. Specifically, C-1-P prevents the degradation of IκB, the phosphorylation of the p65 subunit of NF-κB, and LPS-stimulated MAPK activation. The functional consequence of C-1-P inhibition of NF-κB is a reduction in LPS-mediated cytokine release from HEK 293 TLR4-expressing cells and human peripheral blood mononuclear cells. Taken together, these data demonstrate that C-1-P may function as an anti-inflammatory lipid mediator of immune response.

Overexpression of the opioid growth factor receptor downregulates cell proliferation of human squamous carcinoma cells of the head and neck.

The opioid growth factor (OGF) is a constitutively expressed negative growth regulator whose action is mediated by the OGF receptor (OGFr). The OGF-OGFr axis tonically regulates the growth of human squamous cell carcinoma of the head and neck (SCCHN). To examine the repercussions of amplifying OGFr in SCCHN, constructs were prepared to overexpress OGFr in SCC-1 cells; six clonal lines were examined. OGFr binding assays of clonal cells revealed a 2.4- to 8.4-fold increase in binding capacity compared to wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. OGFr protein expression, as measured by quantitative immunohistochemistry and Western blotting, was increased in clonal cell lines compared to controls. Under standard growth conditions the cell number of the OGFr clonal lines was reduced by 11 to 68% from the WT group, and doubling times were 7 to 67% longer. Addition of exogenous OGF further reduced (8 to 37%) cell growth of the clonal lines. Depletion of endogenous OGF with antibodies to this peptide increased growth 2-fold in cells amplifying OGFr relative to increases of 32 and 34% for the WT and EV groups, respectively. DNA synthesis of cells overexpressing OGFr was reduced from the WT group by 46 to 75%. These data indicate that the OGF receptor is integral to cell replication of SCCHN, and support treatment modalities that amplify OGFr in order to decrease the growth of these neoplasias.