Valproic acid-mediated transcriptional regulation of human GM3 synthase (hST3Gal V) in SK-N-BE(2)-C human neuroblastoma cells¹

Abstract

Key wordsvalproic acid; human GM3 synthase; human n e u r o b l a s t o m a ; C R E B ; t r a n s c r i p t i o n a l regulation 1 This work was supported by a grant (M1061 9010001-08N1901-00110) from the Korea Science and Engineering Foundation (KOSEF). 6 Correspondence to Prof Young-choon LEE. AbstractAim: To investigate whether valproic acid (VPA) modulates human GM3 synthase (hST3Gal V) mRNA expression, as a part of ganglioside GM3 biosynthesis, in human neuroblastoma cells. Methods: Using RT-PCR and immunofluorescent confocal microscopy, we examined hST3Gal V mRNA and GM3 levels during VPA-induced differentiation of human neuroblastoma SK-N-BE(2)-C cells. We characterized the VPA-inducible promoter region within the hST3-Gal V gene using luciferase constructs carrying 5'-deletions of the hST3Gal V promoter. Results: RT-PCR indicated that VPA-mediated hST3Gal V induction is transcriptionally regulated. Functional analysis of the 5'-flanking region of the hST3Gal V gene demonstrated that the -177 to -83 region, which contains a cAMP-responsive element (CRE) at -143, functions as the VPA-inducible promoter by actively binding CRE binding protein (CREB). In addition, sitedirected mutagenesis and electrophoretic mobility shift assay indicated that the CRE at -143 is crucial for the VPA-induced expression of hST3Gal V in SK-N-BE(2)-C cells. Conclusion: Our results isolated the core promoter region in the hST3Gal V promoter, a CRE at -143, and demonstrated that it is essential for transcriptional activation of hST3Gal V in VPA-induced SK-N-BE(2)-C cells. Subsequent CREB binding to this CRE mediates VPA-dependent upregulation of hST3Gal V gene expression.

Upregulation of Human ST8Sia VI (α2,8-Sialyltransferase) Gene Expression by Physcion in SK-N-BE(2)-C Human Neuroblastoma Cells

“…Cell viability assay was performed as described previously [7,8,9,10,11]. The amount of formazan produced was quantified by measuring the absorbance at 490 nm with an ELISA plate reader (Bio-Rad, Hercules, CA, USA).…”

“…Recently we studied the transcriptional regulation mechanism of human sialyltransferase genes that are specifically expressed at high levels in cell lines treated with natural compounds [7,8,9,10,11]. In the current study, we investigated the effect of physcion isolated from the Polygonum multiflorum roots on the human sialyltransferase gene expression in SK-N-BE(2)-C human neuroblastoma cells.…”

Transcriptional activation of human GM3 synthase (hST3Gal V) gene by valproic acid in ARPE-19 human retinal pigment epithelial cells

“…Ganglioside GM3 expression levels were analyzed by the same procedure as described previously (6,7). Mouse anti-GM3 monoclonal antibody (mouse IgM, Kappa-chain, clone: GMR6; Seigakagu; Japan) and FITC-conjugated goat-antimouse IgM (Sigma; St. Louis, MO, USA) as the secondary antibody were used to visualize GM3 expression in ARPE-19 cells induced by VPA.…”

“…These changes reflect the differential regulation of ganglioside biosynthesis catalyzed by a family of glycosyltransferases in the Golgi apparatus. Recently, we demonstrated that VPA modulates the mRNA expression of human GM3 synthase (hST3Gal V) and GD3 synthase (hST8Sia I) in human neuroblastoma cells (6,7).…”

Isolation and functional analysis of the human glioblastoma-specific promoter region of the human GD3 synthase (<italic>hST8Sia I</italic>) gene

“…Analysis of ganglioside GD3 levels on the cell membrane was carried out as described previously [16]. Briefly, SK-N-BE(2)-C, U-87MG and T98G cells were seeded on round cover slips ( pretreated with 2% of 3-aminopropyl-triethoxysilan) in an eight-well multiplate.…”