Enhanced expression of uridine diphosphate-N-acetylglucosaminyl transferase (OGT) in a stable, tetracycline-inducible HeLa cell line using histone deacetylase inhibitors: kinetics of cytosolic OGT accumulation and nuclear translocation.
We have created a stable, tetracycline-inducible HeLa cell line that overexpresses murine uridine diphosphate-N-acetylglucosaminyl transferase (OGT). Tetracycline increased cytosolic OGT activity about 4-fold in a dose-dependent manner (ED(50)=0.03 microg/ml) with enhanced activity observable at 8h and maximal activity observable by 40h. Enhanced OGT activity was due to overexpression of OGT protein as determined by Western analysis. Trichostatin A (TSA), a potent and specific histone deacetylase inhibitor (HDI), markedly enhanced tetracycline-induced OGT gene expression, resulting in a >10-fold increase in OGT activity (>50-fold compared to that of uninduced cells). Other HDIs such as butyrate (ED(50)=1.6mM) and propionate (ED(50)=8mM) were similarly effective, but less potent than TSA (ED(50)=120 nM). We next examined the appearance of recombinant OGT in cytosol and nucleosol at various times (10 min to 6h) after inducing OGT gene. Within 2h, recombinant OGT was detected by Western analysis in both cytosol and nucleosol. This indicates rapid biosynthesis and accumulation of recombinant OGT in the cytosol and subsequent nuclear translocation. Entry of OGT into the nucleus was closely correlated with enhanced O-linked glycosylation of nuclear proteins, indicating that recombinant OGT was enzymatically active. The ability to rapidly induce OGT expression in a stable cell line provides an excellent model system to study the mechanism(s) underlying OGT nuclear translocation and a useful system to elucidate the cascade of signaling events related to O-linked glycosylation.
Marshall
Stephen
S
Hexos, Inc., Woodinville, WA 98072, USA. Hexos@comcast.net
Duong
Trung
T
Wu
Tong
T
Hering
Michelle A
MA
Yada
Jason
J
Higgins
Sarah
S
Orbus
Ryan J
RJ
Yan
Zhong-Hua
ZH
Rumberger
John M
JM
D000818:Animals; D001692:Biological Transport; D002087:Butyrates / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005227:Fatty Acids / Q000737:chemistry / Q000494:pharmacology; D005690:Galactose / Q000031:analogs & derivatives; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D002087:Butyrates; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D006023:Glycoproteins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D011994:Recombinant Proteins; C012589:trichostatin A; D014537:Uridine Diphosphate N-Acetylglucosamine; D013752:Tetracycline; D005690:Galactose