Publications

• Upregulation of the human alkaline ceramidase 1 and acid ceramidase mediates calcium-induced differentiation of epidermal keratinocytes.

  Sun W., Xu R., Hu W., Jin J., Crellin H.A., Bielawski J., Szulc Z.M., Thiers B.H., Obeid L.M., Mao C.

  Extracellular calcium (Ca2+(o)) potently induces the growth arrest and differentiation of human epidermal keratinocytes (HEKs). We report that Ca2+(o) markedly upregulates the human alkaline ceramidase 1 (haCER1) in HEKs; and its upregulation mediates the Ca2+(o)-induced growth arrest and differen ... >> More

  Extracellular calcium (Ca2+(o)) potently induces the growth arrest and differentiation of human epidermal keratinocytes (HEKs). We report that Ca2+(o) markedly upregulates the human alkaline ceramidase 1 (haCER1) in HEKs; and its upregulation mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs. haCER1 is the human ortholog of mouse alkaline ceramidase 1 that we previously identified. haCER1 catalyzed the hydrolysis of very long-chain ceramides to generate sphingosine (SPH). This in vitro activity required Ca2+. Ectopic expression of haCER1 in HEKs decreased the levels of D-e-C(24:1)-ceramide and D-e-C(24:0)-ceramide but elevated the levels of both SPH and its phosphate (S1P), whereas RNA interference-mediated knockdown of haCER1 caused the opposite effects on the levels of these sphingolipids in HEKs. Similar to haCER1 overexpression, Ca2+(o) increased the levels of SPH and S1P, and this was attenuated by haCER1 knockdown. haCER1 knockdown also inhibited the Ca2+(o)-induced growth arrest of HEKs and the Ca2+(o)-induced expression of keratin 1 and involucrin in HEKs. In addition, the acid ceramidase (AC) was also upregulated by Ca2+(o); and its knockdown attenuated the Ca2+(o)-induced expression of keratin 1 and involucrin in HEKs. These results strongly suggest that upregulation of haCER1 and AC mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs by generating SPH and S1P. << Less

  J. Invest. Dermatol. 128:389-397(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase.

  El Bawab S., Birbes H., Roddy P., Szulc Z.M., Bielawska A., Hannun Y.A.

  We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this stu ... >> More

  We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited in vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity with d-erythro-sphingosine (Km of 0.16 mol %, and Vmax of 0.3 micromol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Km of 1.7 mol % and a Vmax of 0.63 micromol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified, l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid. << Less

  J. Biol. Chem. 276:16758-16766(2001) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Expression, purification, and characterization of a recombinant neutral ceramidase from Mycobacterium tuberculosis.

  Okino N., Ikeda R., Ito M.

  Ceramidase (CDase) catalyzes the hydrolysis of ceramide (Cer) to sphingosine (Sph) and fatty acid. We have reported the molecular cloning and preliminary characterization of the Mycobacterium CDase (MtCDase) (J. Biol. Chem., 274, 36616-36622 (1999)). To determine its function further, MtCDase was ... >> More

  Ceramidase (CDase) catalyzes the hydrolysis of ceramide (Cer) to sphingosine (Sph) and fatty acid. We have reported the molecular cloning and preliminary characterization of the Mycobacterium CDase (MtCDase) (J. Biol. Chem., 274, 36616-36622 (1999)). To determine its function further, MtCDase was expressed in Escherichia coli and purified by Ni-Sepharose and gelfiltration. The purified recombinant enzyme showed a single band and a molecular weight estimated to be 71 kDa on SDS-PAGE. It had a pH optimum at 8.0-9.0 and quite broad specificity for various Cers. Of the Cers of different fatty acid moieties tested, those composed of C6-C24 fatty acids were well hydrolyzed, and Cers with mono unsaturated fatty acids were much more hydrolyzed than those with saturated fatty acids. Using N-dodecanoyl-7-nitrobenz-2-oxa-1,3-4-diazole (NBD)-D-erythro-sphingosine (C12-NBD-Cer) as substrates, the reaction followed normal Michaelis-Menten kinetics. The apparent Km and Vmax values for C12-NBD-Cer were 98.7 muM and 21.1 pmol/min respectively. The purified enzyme also catalyzed the synthesis of Cer in vitro, using NBD-labeled dodecanoic acid and Sph as substrates. << Less

  Biosci. Biotechnol. Biochem. 74:316-321(2010) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Golgi alkaline ceramidase regulates cell proliferation and survival by controlling levels of sphingosine and S1P.

  Xu R., Jin J., Hu W., Sun W., Bielawski J., Szulc Z., Taha T., Obeid L.M., Mao C.

  Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, promotes cell proliferation and survival whereas its precursor, sphingosine, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of bot ... >> More

  Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, promotes cell proliferation and survival whereas its precursor, sphingosine, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of both sphingosine and S1P by controlling the hydrolysis of ceramides. haCER2 is localized to the Golgi complex and is highly expressed in the placenta. High ectopic expression of haCER2 caused fragmentation of the Golgi complex and growth arrest in HeLa cells due to sphingosine accumulation. Low ectopic expression of haCER2 increased S1P without sphingosine accumulation, promoting cell proliferation in serum-free medium. This proliferative effect was suppressed by dimethylsphingosine, an inhibitor of the S1P formation, or by the RNA interference (RNAi) -mediated inhibition of S1P(1,) a G-protein-coupled receptor for S1P. The RNAi-mediated down-regulation of haCER2 enhanced the serum deprivation-induced growth arrest and apoptosis of HeLa cells, which was inhibited by addition of exogenous S1P. Serum deprivation up-regulated both haCER2 mRNA and activity in HeLa cells. haCER2 mRNA is also up-regulated in some tumors. Taken together, these results suggest that haCER2 is important for the generation of S1P and S1P-mediated cell proliferation and survival, but that its overexpression may cause cell growth arrest due to an accumulation of sphingosine. << Less

  FASEB J. 20:1813-1825(2006) [PubMed] [EuropePMC]