Human glycolipid transfer protein gene (GLTP) expression is regulated by Sp1 and Sp3: involvement of the bioactive sphingolipid ceramide.
Glycolipid transfer protein (GLTP) accelerates glycolipid intermembrane transfer via a unique lipid transfer/binding fold (GLTP fold) that defines the GLTP superfamily and is the prototype for functional GLTP-like domains in larger proteins, i.e. FAPP2. Human GLTP is encoded by the single-copy GLTP gene on chromosome 12 (12q24.11 locus), but regulation of GLTP gene expression remains completely unexplored. Herein, the ability of glycosphingolipids (and their sphingolipid metabolites) to regulate the transcriptional expression of GLTP via its promoter has been evaluated. Using luciferase and GFP reporters in concert with deletion mutants, the constitutive and basal (225 bp; ∼78% G+C) human GLTP promoters have been defined along with adjacent regulatory elements. Despite high G+C content, translational regulation was not evident by the mammalian target of rapamycin pathway. Four GC-boxes were shown to be functional Sp1/Sp3 transcription factor binding sites. Mutation of one GC-box was particularly detrimental to GLTP transcriptional activity. Sp1/Sp3 RNA silencing and mithramycin A treatment significantly inhibited GLTP promoter activity. Among tested sphingolipid analogs of glucosylceramide, sulfatide, ganglioside GM1, ceramide 1-phosphate, sphingosine 1-phosphate, dihydroceramide, sphingosine, only ceramide, a nonglycosylated precursor metabolite unable to bind to GLTP protein, induced GLTP promoter activity and raised transcript levels in vivo. Ceramide treatment partially blocked promoter activity decreases induced by Sp1/Sp3 knockdown. Ceramide treatment also altered the in vivo binding affinity of Sp1 and Sp3 for the GLTP promoter and decreased Sp3 acetylation. This study represents the first characterization of any Gltp gene promoter and links human GLTP expression to sphingolipid homeostasis through ceramide.
Zou
Xianqiong
X
The Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.
Gao
Yongguang
Y
Ruvolo
Vivian R
VR
Gardner
Tawnya L
TL
Ruvolo
Peter P
PP
Brown
Rhoderick E
RE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001943:Breast Neoplasms; D002352:Carrier Proteins / Q000235:genetics*; D002518:Ceramides / Q000378:metabolism*; D005260:Female; D020868:Gene Silencing; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D024363:Transcription Initiation Site / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology
D002352:Carrier Proteins; D002518:Ceramides; C511922:GLTP protein, human; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases