title: Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner.
abstract: A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity.
authors.affiliation: From the Departments of Microbiology and Immunology.
authors.forename: Tejas S
authors.initials: TS
authors.lastname: Tirodkar
authors.affiliation: From the Departments of Microbiology and Immunology.
authors.forename: Ping
authors.initials: P
authors.lastname: Lu
authors.affiliation: Biochemistry and Molecular Biology, and.
authors.forename: Aiping
authors.initials: A
authors.lastname: Bai
authors.affiliation: From the Departments of Microbiology and Immunology.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Scheffel
authors.affiliation: Biochemistry and Molecular Biology, and the Department of Molecular Biology and Genetics, 34662 Istanbul, Turkey.
authors.forename: Salih
authors.initials: S
authors.lastname: Gencer
authors.affiliation: Public Health, Medical University of South Carolina, Charleston South Carolina 29425 and.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Garrett-Mayer
authors.affiliation: Biochemistry and Molecular Biology, and.
authors.forename: Alicja
authors.initials: A
authors.lastname: Bielawska
authors.affiliation: Biochemistry and Molecular Biology, and.
authors.forename: Besim
authors.initials: B
authors.lastname: Ogretmen
authors.affiliation: From the Departments of Microbiology and Immunology, johnsocv@musc.edu.
authors.forename: Christina
authors.initials: C
authors.lastname: Voelkel-Johnson
mesh_terms: D055573:Acid Ceramidase / Q000235:genetics / Q000378:metabolism*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002518:Ceramides / Q000494:pharmacology*; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013107:Sphingolipids / Q000378:metabolism; D051049:Sphingosine N-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D002518:Ceramides; D008565:Membrane Proteins; D012333:RNA, Messenger; D013107:Sphingolipids; C506123:CERS6 protein, human; D051049:Sphingosine N-Acyltransferase; D048031:JNK Mitogen-Activated Protein Kinases; D055573:Acid Ceramidase; D005472:Fluorouracil
keywords: acid ceramidase; c-Jun transcription factor; cell death; ceramide synthase; colon cancer; sphingolipid
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