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.

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.