Transcriptional Mechanism of the Mouse ß4-Galactosyltransferase 6 Gene in Mouse Neuroblastoma Cell Line Neuro-2a.

Lactosylceramide (Lac-Cer) constitutes the backbone structure of various gangliosides whose abnormal expression is associated with malignancy of neuroblastoma. The understanding of the regulatory mechanism of Lac-Cer contributes to the development of neuroblastoma therapy. In this study, the transcriptional mechanism of mouse ß4-galactosyltransferase (ß4GalT) 6, which is one of Lac-Cer synthase, was analyzed using mouse neuroblastoma cell line Neuro-2a. The -226 to -13 region relative to the most downstream transcriptional start site was determined to be the promoter region by luciferase assay using the 5'-deletion constructs. The mutation into the activating protein (AP) 4-binding site -110/-101 drastically decreased the promoter activity, indicating that this site is mainly implicated in the transcription. Furthermore, the mutation into the GATA-binding site -210/-201 or another AP4-binding site -202/-193 partially decreased the promoter activity. The study suggests that the mouse ß4GalT6 gene is transcriptionally regulated by AP4 in cooperation with GATA family transcription factor in neuroblastoma.

Phosphorylation of Specificity Protein 3 Is Critical for Activation of ß4-Galactosyltransferase 3 Gene Promoter in SH-SY5Y Human Neuroblastoma Cell Line.

Elevated expression of ß4-galactosyltransferase (ß4GalT) 3 is correlated with poor clinical outcome of neuroblastoma patients. Our recent study has revealed that the transcription of the ß4GalT3 gene is activated by Specificity protein (Sp) 3 in SH-SY5Y human neuroblastoma cell line. Here we report the biological significance of the Sp3 phosphorylation in the transcriptional activation of the ß4GalT3 gene. The treatment of SH-SY5Y cells with 10% fetal bovine serum (FBS) increased the mitogen-activated protein kinase (MAPK) signaling and the promoter activity of the ß4GalT3 gene. Meanwhile, the treatment with U0126, an inhibitor for MAPK kinase, decreased the MAPK signaling and the promoter activity. These findings indicate that the transcriptional activation of the ß4GalT3 gene is mediated by the MAPK signaling. In SH-SY5Y cells cultured in the medium containing 10% FBS, the serine (Ser) residues in Sp3 were phosphorylated. Human Sp3 contains four Ser residues, Ser73, Ser563, Ser566, and Ser646, as the putative phosphorylation sites. Sp3 mutant with the mutation of Ser73 did not decrease the promoter activation of the ß4GalT3 gene, indicating that Ser73 is uninvolved in the promoter activation of the ß4GalT3 gene by Sp3. In contrast, Sp3 mutants with the mutations of Ser563, Ser566, and Ser646 significantly reduced the promoter activation by Sp3. The results suggest that the phosphorylation of these Ser residues is implicated in the promoter activation by Sp3. This study demonstrates that the phosphorylation of Sp3 plays important roles in the transcriptional activation of the ß4GalT3 gene in human neuroblastoma.

Suppression of Malignant Potentials of A549 Human Lung Cancer Cell Line by Downregulation of the ß4-Galactosyltransferase 1 Gene Expression.

Our previous study demonstrated that downregulation of transcription factor Specificity protein (Sp) 1 suppresses the malignant potentials of A549 human lung cancer cell line with the reduced ß4-galactosylation of highly branched N-glycans on cell surface glycoproteins. The reduced ß4-galactosylation was brought about by the decreased expression of the ß4-galactosyltransferase 1 (ß4GalT1) gene. Herein, we examined whether the reduced ß4-galactosylation by decreasing the ß4GalT1 gene expression suppresses the malignant potentials of A549 cells. In the ß4GalT1-downregulated cells, the ß4-galactosylation of highly branched N-glycans was reduced in several glycoproteins such as lysosome-associated membrane protein-1 and E-cadherin. The anchorage-independent growth and migratory ability of the ß4GalT1-downregulated cells decreased when compared with the control cells. Furthermore, the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) decreased in the ß4GalT1-downregulated cells. These results indicate that downregulation of the ß4GalT1 gene decreases the ß4-galactosylation of highly branched N-glycans and the phosphorylation of p44/42 MAPK, and suppresses the malignant potentials of A549 cells.

Transcription of human ß4-galactosyltransferase 3 is regulated by differential DNA binding of Sp1/Sp3 in SH-SY5Y human neuroblastoma and A549 human lung cancer cell lines.

Poor prognosis of neuroblastoma patients has been shown to be associated with increased expression of ß4-galactosyltransferase (ß4GalT) 3. To address the underlying mechanism of the increased expression of ß4GalT3, the transcriptional regulation of the human ß4GalT3 gene was investigated in SH-SY5Y human neuroblastoma cell line comparing with A549 human lung cancer cell line, in which the ß4GalT3 gene expression was the lowest among four cancer cell lines examined. The core promoter region was identified between nucleotides -69 and -6 relative to the transcriptional start site, and the same region was utilized in both cell lines. The promoter region contained two Specificity protein (Sp)1/3-binding sites at nucleotide positions -39/-30 and -19/-10, and the sites were crucial for the promoter activity. Although the gene expression of Sp family transcription factors Sp1 and Sp3 was comparable in each cell line, Sp3 bound to the promoter region in SH-SY5Y cells whereas Sp1 bound to the region in A549 cells. The promoter activities were enhanced by Sp1 and Sp3 in SH-SY5Y cells. In contrast, the promoter activities were enhanced by Sp1 but reduced by Sp3 in A549 cells. Furthermore, the function of each Sp1/3-binding site differed between SH-SY5Y and A549 cells due to the differential binding of Sp1/Sp3. These findings suggest that the transcription of the ß4GalT3 gene is regulated by differential DNA binding of Sp3 and Sp1 in neuroblastoma and lung cancer. The increased expression of ß4GalT3 in neuroblastoma may be ascribed to the enhanced expression of Sp3, which is observed for various cancers.

Mechanism for Decreased Gene Expression of ß4-Galactosyltransferase 5 upon Differentiation of 3T3-L1 Mouse Preadipocytes to Adipocytes.

Upon differentiation of cells, remarkable changes in the structures of glycans linked to lipids on cell surface have been observed. Lactosylceramide (Lac-Cer) serves as a common precursor for a series of glycosphingolipids with diverse structures. In the present study, we examined the underlying mechanism for the biosynthesis of Lac-Cer upon differentiation of 3T3-L1 mouse preadipocytes to adipocytes. TLC analysis showed that the amounts of Lac-Cer decrease in 3T3-L1 adipocytes compared to 3T3-L1 preadipocytes. In accordance with this change, the gene expression level of ß4-galactosyltransferase (ß4GalT) 5, which was identified as Lac-Cer synthase, decreased drastically upon differentiation of 3T3-L1 preadipocytes. The analysis of the transcriptional mechanism of the ß4GalT5 gene demonstrated that the core promoter region is identified between nucleotides -299 and -1 relative to the translational start site. During adipocyte differentiation, the expression levels and promoter activities of the ß4GalT5 gene decreased dramatically. Since the Specificity protein 1 (Sp1)-binding sites in the promoter region were critical for the promoter activity, it is suggested that Sp1 plays an important role for the expression of the ß4GalT5 gene in 3T3-L1 cells. The gene and protein expression of Sp1 decreased significantly upon differentiation of 3T3-L1 preadipocytes. Taken together, the present study suggest that the expression of the ß4GalT5 gene decreases through reduced expression of the Sp1 gene and protein upon differentiation of 3T3-L1 peradipocytes to adipocytes, which may lead to the decreased amounts of Lac-Cer in 3T3-L1 adipocytes.

Screening Method for Anti-Colon Cancer Drugs Using Two Sensor Cell Lines with Human ß4-Galactosyltransferase 4 Gene Promoters.

The increased expression of ß4-galactosyltransferase (ß4GalT) 4 is closely associated with poor prognosis of colon cancer. Recently, we showed that the expression of the ß4GalT4 gene is regulated by the 0.17 kb core promoter region containing one binding site for Specificity protein 1 (Sp1). To develop a screening method for anti-colon cancer drugs, two sensor cell lines having the luciferase gene under the control of two ß4GalT4 gene promoters that differed in length were established from SW480 human colon cancer cells. The hGT4-0.17-sensor cells possessed the luciferase reporter driven by the 0.17 kb promoter, while the hGT4-0.3-sensor cells possessed the luciferase reporter driven by the 0.3 kb promoter containing one binding site each for colon cancer-related transcription factors including activator protein 2, E2F, caudal-related homeobox transcription factors, and Runt-related transcription factors besides Sp1. Upon treatment with mitogen-activated protein kinase signaling inhibitor U0126, the promoter activities of the hGT4-0.3-sensor cells decreased significantly, while those of the hGT4-0.17-sensor cells remained unchanged. These results suggest that the responsiveness to U0126 differs between two sensor cell lines due to the different regulation of the luciferase reporters. This study provides the screening method for anti-colon cancer drugs by the combination of two sensor cell lines.

Downregulation of Transcription Factor Sp1 Suppresses Malignant Properties of A549 Human Lung Cancer Cell Line with Decreased ß4-Galactosylation of Highly Branched N-Glycans.

Dramatic changes in the glycan structures of cell surface proteins have been observed upon malignant transformation of cells as induced by the altered expression levels of glycosyltransferases. Such changes are closely associated with the malignant properties of cancer cells. Transcription factor Sp1 regulates the gene expression of various molecules including glycosyltransferases. Herein, we investigated whether or not Sp1-downregulation affects to N-glycosylation of glycoproteins and malignant properties of A549 human lung cancer cell line. We established a stable clone whose Sp1-expression level was reduced to 50% of a control clone by RNA interference. Lectin blotting revealed that the ß4-galactosylation of highly branched N-glycans decreases mainly in cell adhesion molecule, E-cadherin. The analysis of underlying mechanism for decreased ß4-galactosylation of N-glycans showed that the gene expression level of ß4-galactosyltransferase (ß4GalT) 1 decreases dramatically by downregulation of Sp1 without changes in those of ß4GalT2 and N-acetylglucosaminyltransferase V. Mutations in the Sp1-binding sites of the ß4GalT1 gene promoter showed that the promoter activity decreases significantly, indicating that the gene expression is regulated by Sp1. These results indicate that the ß4-galactosylation of highly branched N-glycans decreases by downregulation of Sp1 through the reduced expression of the ß4GalT1 gene. Furthermore, the Sp1-downregulated cells showed the suppression of the anchorage-independent growth in soft agar and migratory activity when compared to the control cells. The present study demonstrates that downregulation of Sp1 suppresses the malignant properties of A549 cells through the decreased ß4-galactosylation of highly branched N-glycans.

Transcriptional Mechanism of the ß4-Galactosyltransferase 4 Gene in SW480 Human Colon Cancer Cell Line.

Increased expression of ß4-galactosyltransferase (ß4GalT) 4 has been shown to be associated with metastatic ability and poor prognosis of colon cancer cells. To solve the up-regulation of ß4GalT4 in colon cancer cells at transcriptional level, we examined the transcriptional mechanism of the ß4GalT4 gene in SW480 human colon cancer cell line. Luciferase assay using the deletion constructs revealed that the promoter activity of the ß4GalT4 gene is associated with the region between nucleotides -122 and -55 relative to the transcriptional start site, which contained one Specificity protein 1 (Sp1)-binding site. The mutation into the Sp1-binding site resulted in dramatic decreased promoter activity. Meanwhile, ectopic Sp1 expression stimulated the promoter activity significantly. The present study suggests that the expression of the ß4GalT4 gene is controlled by Sp1, and Sp1 plays a key role in the activation of the ß4GalT4 gene in colon cancer cells.

Gene expression levels of ß4-galactosyltransferase 5 correlate with the tumorigenic potentials of B16-F10 mouse melanoma cells.

Our previous studies showed that mouse ß4-galactosyltransferase 5 (ß4GalT5) is a lactosylceramide (Lac-Cer) synthase, and that its gene expression increases by 2- to 3-fold upon malignant transformation of cells. In the present study, we examined whether or not the tumorigenic and metastatic potentials of B16-F10 mouse melanoma cells can be suppressed by reducing the expression of the ß4GalT5 gene. We isolated a stable clone named E5 whose ß4GalT5 gene expression level was reduced to 35% that of a control clone C1 by transfection of its antisense cDNA. Thin-layer chromatography analysis of glycosphingolipids showed that the amounts of Lac-Cer and ganglioside GM3 are significantly less in clone E5 than in clone C1. Clone C1 and E5 cells were each transplanted subcutaneously or injected intravenously into C57BL/6 mice, and the sizes of tumors and numbers of colonies formed in the lungs were determined. The average tumor size and average number of colonies formed with clone E5 were decreased to 44 and 49%, respectively, of those formed with clone C1. Furthermore, the numbers and sizes of colonies formed in the soft agarose gels, and the volumes of tumors formed in athymic mice with fibroblasts from wild type, heterozygous and homozygous ß4GalT5-knockout mouse embryos upon transformation with the polyoma virus oncogene correlated with the ß4GalT5 gene dosage. These results strongly indicate that the amounts of Lac-Cer synthesized by ß4GalT5 correlate with the tumorigenic potentials of malignantly transformed cells.

ß4GalT6 is involved in the synthesis of lactosylceramide with less intensity than ß4GalT5.

Glycosphingolipids are expressed on the cell membrane and act as important factors in various events that occur across the plasma membrane. Lactosylceramide (LacCer) is synthesized from glucosylceramide and is a common precursor of various glycosphingolipids existing in whole body. Based on the enzyme purification, ß1,4-galactosyltransferase 6 (B4galt6) cDNA was isolated as a LacCer synthase-coding gene in the rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of ß4GalT6. B4galt6 KO mice were born and grew up apparently normal. LacCer synthase activity and the composition of acidic glycosphingolipids in the brain were almost equivalent or minimally different between wild-type and KO mice. Studies by mouse embryonic fibroblasts (MEFs) revealed that the silencing of B4galt5 gene resulted in the marked reduction in LacCer synthase activity and this reduction was more severe in MEFs derived from B4galt6 KO mice than those from wild-type mice. These results suggested that ß4GalT6 plays a role as a LacCer synthase, whereas ß4GalT5 acts as a main enzyme for LacCer biosynthesis in these tissues and cells.