Ceramide kinase deficiency improves diet-induced obesity and insulin resistance.

Ceramide kinase (CERK) is an enzyme that phosphorylates ceramide to produce ceramide 1-phosphate. Recently, evidence has emerged that CERK has a role in inflammatory signaling of immune cells. Since obesity is accompanied by chronic, low-grade inflammation, we examined whether CERK might be involved using CERK-null mice. We determined that CERK deficiency suppresses diet-induced increases in body weight, and improves glucose intolerance. Furthermore, we demonstrated that CERK deficiency attenuates MCP-1/CCR2 signaling in macrophages infiltrating the adipose tissue, resulting in the suppression of inflammation in adipocytes, which might otherwise lead to obesity and diabetes.

Ceramide kinase is not essential but might act as an Ca2+-sensor for mast cell activation.

Ceramide kinase (CerK) catalyzes the conversion of ceramide to ceramide 1-phosphate (C1P). We previously revealed that CerK is involved in the activation of mast cells. In this study, we performed an advanced investigation into the role of CerK on the activation of mast cells using CERK-/- mice. Although CERK-/- mice were less prone to exhibiting a passive cutaneous anaphylactic shock (PCA)-reaction compared to wild type (WT) mice, the differences were not significant. In bone marrow-derived mast cells (BMMC) activated by cross-linking antigen (Ag)/IgE, not high, but low concentrations of Ag had a reduced effect on degranulation in BMMC from CERK-/- mice compared to effects on BMMC from WT mice. Similarly, when the BMMCs were activated with calcium ionophore to focus on the downstream signaling of Ca(2+)-elevation, only a low concentration of ionophore had a reduced effect on degranulation in the BMMC from CERK-/- mice compared to the effect on BMMC from WT mice. Furthermore, the CerK inhibitor K1 reduced the differences in degranulation observed between the BMMC from CERK-/- and WT mice in a dose-dependent manner, demonstrating a contribution for CerK and its product C1P in degranulation. Although CerK is not essential for activation of mast cells, especially a potent and acute activation such as a PCA reaction, CerK might act as an modulator for mild and chronic activation of mast cells, thus increasing sensitivity to cytoplasmic Ca(2+).

Role of ceramide kinase in peroxisome proliferator-activated receptor beta-induced cell survival of mouse keratinocytes.

Ceramide (Cer) is known to be a lipid mediator in apoptosis and to have an important role in cell fate, via control of intracellular Cer levels. Recently, ceramide kinase (CerK) was identified as an enzyme that converts Cer to ceramide 1-phosphate (C1P). We examined potential functions of CerK in the regulation of keratinocyte survival, and the possible involvement of peroxisome proliferator-activated receptor beta (PPARbeta). PPARbeta is known to be a nuclear receptor acting as a ligand-inducible transcription factor and has been implicated in the control of keratinocyte survival. In the mouse keratinocyte cell line SP1, serum starvation induced cell death and the accumulation of intracellular Cer, an apoptotic event. However, apoptosis was inhibited by activation of PPARbeta. Interestingly, activation of PPARbeta enhanced the mRNA expression of CerK and CerK activity. Furthermore, the cell survival effect of PPARbeta was greatly diminished in keratinocytes isolated from CerK-null mice. Chromatin immunoprecipitation revealed that, in vivo, PPARbeta binds to the CerK gene via a sequence located in the first intron. Electrophoretic mobility-shift assays confirmed that PPARbeta associates with this sequence in vitro. These findings indicated that CerK gene expression was directly regulated by PPARbeta. In conclusion, our results demonstrate that PPARbeta-mediated upregulation of CerK gene expression is necessary for keratinocyte survival against serum starvation-induced apoptosis.

The generation and behavioral analysis of ceramide kinase-null mice, indicating a function in cerebellar Purkinje cells.

The discovery of ceramide kinase (CerK), which phosphorylates ceramide (Cer) to ceramide 1-phisphate (C1P), established a new pathway for Cer metabolism. Among mouse tissues, brain contains the highest CerK activity. In this study, we found that CerK is highly expressed in cerebellar Purkinje cells. Since Purkinje cells are important for motor-related behaviors, we generated CerK-null mice and performed behavioral analyses. The CerK-null mice were healthy, and displayed no histological abnormalities. The mice lost CerK activity completely, suggesting that CerK is the only enzyme that phosphorylate Cer. However, cellular C1P levels were not different between the CerK-null and wild-type mice, indicating the presence of other C1P-producing pathway. The general motor-coordination was not impaired in the CerK-null mice, but emotional behavior was slightly affected. Our findings suggest that CerK is not necessary for survival at an individual level, but might be involved in higher brain function related to emotion.

Homing at an extragenic locus mediated by VDE (PI-SceI) in Saccharomyces cerevisiae.

PI-SceI (VDE), a homing endonuclease with protein splicing activity, is a genomic parasite in the VMA1 gene of Saccharomyces cerevisiae. In a heterozygous diploid of the VDE-less VMA1 allele and a VDE-containing VMA1 allele, VDE specifically cleaves its recognition sequence (VRS) in the VDE-less VMA1 allele at meiosis, followed by 'homing', i.e. a conversion to a VDE-containing allele. We found that upon VDE expression, homing of a marker gene at an extragenic locus occurs only when a 45 bp element containing the VRS is inserted at its allelic site, while mutants of VDE with no endonuclease activity lack authentic extragenic homing activity. Thus, both the VRS and VDE are required for homing. Insertion of the VRS in a homozygous diploid significantly lowered the spore germination ability, indicating that a template for gene repair at its allelic locus is essential for efficient homing and survival of yeast cells.

Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization.

Ceramide-1-phosphate is a sphingolipid metabolite that has been implicated in membrane fusion of brain synaptic vesicles and neutrophil phagolysosome formation. Ceramide-1-phosphate can be produced by ATP-dependent ceramide kinase activity, although little is known of this enzyme because it has not yet been highly purified or cloned. Based on sequence homology to sphingosine kinase type 1, we have now cloned a related lipid kinase, human ceramide kinase (hCERK). hCERK encodes a protein of 537 amino acids that has a catalytic region with a high degree of similarity to the diacylglycerol kinase catalytic domain. hCERK also has a putative N-myristoylation site on its NH(2) terminus followed by a pleckstrin homology domain. Membrane but not cytosolic fractions from HEK293 cells transiently transfected with a hCERK expression vector readily phosphorylated ceramide but not sphingosine or other sphingoid bases, diacylglycerol or phosphatidylinositol. This activity was clearly distinguished from those of bacterial or human diacylglycerol kinases. With natural ceramide as a substrate, the enzyme had a pH optimum of 6.0-7.5 and showed Michaelis-Menten kinetics, with K(m) values of 187 and 32 microm for ceramide and ATP, respectively. Northern blot analysis revealed that hCERK mRNA expression was high in the brain, heart, skeletal muscle, kidney, and liver. A BLAST search analysis using the hCERK sequence revealed that putative ceramide kinases (CERKs) exist widely in diverse multicellular organisms including plants, nematodes, insects, and vertebrates. Phylogenetic analysis revealed that CERKs are a new class of lipid kinases that are clearly distinct from sphingosine and diacylglycerol kinases. Cloning of CERK should provide new molecular tools to investigate the physiological functions of ceramide-1-phosphate.