We have reported that ceramide mediates binding of atypical protein kinase C (PKC) to its inhibitor protein, PAR-4 (prostate apoptosis response-4), thereby inducing apoptosis in differentiating embryonic stem cells. Using a novel method of lipid vesicle-mediated affinity chromatography, we showed here that endogenous ceramide binds directly to the PKC⅐PAR-4 complex. Ceramide and its analogs activated PKC prior to binding to PAR-4, as determined by increased levels of phosphorylated PKC and glycogen synthase kinase-3␤ and emergence of a PAR-4-to-phosphorylated PKC fluorescence resonance energy transfer signal that co-localizes with ceramide. Elevated expression and activation of PKC increased cell survival, whereas expression of PAR-4 promoted apoptosis. This suggests that PKC counteracts apoptosis, unless its ceramide-induced activation is compromised by binding to PAR-4. A luciferase reporter assay showed that ceramide analogs activate nuclear factor (NF)-B unless PAR-4-dependent inhibition of PKC suppresses NF-B activation. Taken together, our results show that direct physical association with ceramide and PAR-4 regulates the activity of PKC. They also indicate that this interaction regulates the activity of glycogen synthase kinase-3␤ and NF-B.

The formation of stem cell–derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body–derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of β-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

Summary The mechanisms by which tumour cells metastasize and the role cell polarity proteins play in this process are not well understood. We report that Partitioning defective protein 3 (Par3) is dysregulated in metastasis in human breast cancer, and is associated with higher tumour grade and ErbB2-positive status. Downregulation of Par3 cooperated with ErbB2 to induce cell invasion and metastasis in vivo. Interestingly, the metastatic behaviour was not associated with an overt mesenchymal phenotype. However, loss of Par3 inhibited E-cadherin junction stability, disrupted membrane and actin dynamics at cell-cell junctions and decreased cell-cell cohesion in a Tiam1/Rac-GTP pathway dependent manner. Inhibition of this pathway restored E-cadherin junction stability and blocked invasive behaviour of cells lacking Par3 suggesting that loss of Par3 promotes metastatic behaviour of ErbB2-induced tumour epithelial cells by decreasing cell-cell cohesion.

Ceramide is emerging as a key sphingolipid that regulates a variety of cellular processes. To facilitate the study of ceramide localization and its interaction with cellular proteins, we have developed a novel antibody against ceramide. Our results indicate that the antibody (rabbit IgG) specifically recognizes ceramide in lipid overlay assays and detects ceramide species with different fatty acid chain lengths that include C2, C8, C16, C18, C20, and C24. The new antibody was compared with the commercially available anti-ceramide antibody (mouse IgM) in immunocytochemistry experiments to study the localization of ceramide. Although both antibodies stain the same regions on the cell membrane, the rabbit IgG reveals the distribution of ceramide in compartments that are not well identified with the commercially available antibody. In addition to staining of ceramide in protrusions of the plasma membrane, the rabbit IgG also detects ceramide in the Golgi apparatus. Pharmacological depletion or increase of ceramide levels results in a corresponding change in staining intensity, confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine the localization of ceramide and its interaction with proteins by immunocytochemistry.-Krishnamurthy, K., S. Dasgupta, and E. Bieberich. Development and characterization of a novel anti-ceramide antibody. J. Lipid Res. 2007. 48: 968-975.

In mammals, the primitive ectoderm is an epithelium of polarized cells that differentiates into all embryonic tissues. Our study shows that in primitive ectoderm cells, the sphingolipid ceramide was elevated and co-distributed with the small GTPase Cdc42 and cortical F-actin at the apicolateral cell membrane. Pharmacological or RNA interference-mediated inhibition of ceramide biosynthesis enhanced apoptosis and impaired primitive ectoderm formation in embryoid bodies differentiated from mouse embryonic stem cells. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog. Ceramide depletion prevented plasma membrane translocation of PKC/, its interaction with Cdc42, and phosphorylation of GSK-3␤, a substrate of PKC/. Recombinant PKC formed a complex with the polarity protein Par6 and Cdc42 when bound to ceramide containing lipid vesicles. Our data suggest a novel mechanism by which a ceramide-induced, apicolateral polarity complex with PKC/ regulates primitive ectoderm cell polarity and morphogenesis.Our studies have shown that the membrane sphingolipid ceramide regulates apoptosis during embryonic stem (ES) 3 cell differentiation (1-5). This regulation is initiated by direct physical interaction of ceramide with atypical PKC or (5). Homozygous knock-out of PKC in mice results in lethality at embryonic day 9 because of severe defects of post-gastrulation morphology (6). This suggests that atypical PKCs are critical for aspects of early embryonic development, although the degree of functional overlap as well as specific effects and regulation of the two isoforms are not known yet. Our results obtained with ES cells suggest that ceramide-dependent regulation of atypical PKCs contributes to the function of PKC/ during embryonic development.In the pregastrulation embryo, inner cell mass-derived cells give rise to two adjacent epithelia of polarized cells, the primitive endoderm and the underlying primitive ectoderm. The formation of the primitive ectoderm is followed by the removal of cells that are not in contact with the primitive endoderm derived basal lamina. These cells die by apoptosis giving rise to the proamniotic cavity, a process termed cavitation (7). This developmental process is recapitulated in embryoid bodies (EBs) generated from in vitro differentiating ES cells. ES cellderived EBs have been used to determine key regulatory factors of primitive germ layer formation and cavitation, and are a bona fide, in vitro model for early embryo morphogenesis (8 -10). A key experiment to determine the significance of ceramide for cavitation and primitive ectoderm morphogenesis is depleting EBs of ceramide by disruption of ceramide biosynthesis. Ceramide biosynthesis is initiated and regulated by serine palmitoyltransferase (SPT), an enzyme consisting of the two subunits SPT-1 and SPT-2. Homozygous knock-out of SPT-1 or SPT-2 results in embryonic lethality at a stage of development well before E15 indicating the absolute requirement of ceramide biosynthesis for embryo development (...