| The enzyme (1,4-galactosyltransferase ((1,4GT; EC 2.4.1.38) is a constitutively expressed type II membrane-bound glycoprotein. It resides in two distinct subcellular compartments, the trans-Golgi network, where it is one of the key enzymes involved in the biosynthesis of complex-type oligosaccharide, and the plasma membrane, where it serves as an adhesion molecule and participates in a number of cellular interactions. Since the promoter region upstream of the 4.1 kb (1,4GT1 start-site is occupied mainly by ubiquitous transcription factor Sp1, it is considered as a housekeeping gene for a long time. However, accumulated evidences have shown that its expression could be regulated by a variety of stimuli. Transfection of p16, a cyclin-dependent-kinase inhibitor, into A549 human lung cancer cells led to down-regulation of (1,4GT1. Inhibition of another cell cycle related protein, TGF-(, in SMMC-7721 cells showed the same result. Since the study on murine (1,4-GT1 promoter region couldn't explain the transcriptional regulation of (1,4-GT1, we cloned the human (1,4-GT1 promoter region and examined the promoter activity during the change of cell cycle, metastasis and apoptosis.HeLa cells were transfected with the (1,4GT1/Luc constructs and kept quiescent by serum starvation, serum-restimulation resulted in a sharp increase in luciferase reporter activity. The change in reporter activities was much greater when cells were restimulated by EGF, indicating that EGF is one of the most important growth factors that regulate (1,4GT1 transcription. Consistent with that, Northern blot analysis indicated an obvious increase in (1,4GT1 mRNA after stimulated by EGF. Cotransfection of p16 or antisense TGF-( expression plasmid and (1,4GT1/Luc constructs showed that certain transcription factors between -318 and +52 mediated its downregulation.(1,4GT1 activity is upregulated on cell surface of highly metastatic cells. In seven of eight human adrenal carcinoma cell lines, the level of (1,4-GT1 correlates with their relative degree of in vitro invasiveness. Additionally, in two B16 murine melanoma sublines with distinct in vivo metastatic abilities, cell surface (1,4-GT1 activity is elevated on the highly metastatic variant. Moreover, the degree of metastasis is, in fact, influenced by the relative expression of surface (1,4-GT1. However, the mechanism by which (1,4GT1 is upregulated in highly metastatic cellsremains inknown. PGLH7 and PGBE1 cells, isolated from metastatic human lung giant cell carcinoma (PG), were two cell sublines with different spontaneous metastatic potentials. In our study, it was found that highly metastatic PGBE1 cells have higher (1,4GT1 mRNA levels than low metastatic PGLH7 cells. It was also shown by northern and western blot analysis that E1AF/PEA3, a member of the Ets transcription factor family that confers invasive phenotype in non-small-cell lung cancers, was elevated in highly metastatic PGBE1 cells. Co-transfection of (1,4GT1/Luc and E1AF/PEA3 expression plasmid increased luciferase activity in a dose dependent manner and this activation could be blocked by over-expressing antisense to E1AF/PEA3. Stable transfection of E1AF/PEA3 expression plasmid into PGLH7 cells resulted in elevated (1,4GT1 mRNA expression. The region between -318(-98, which contributed to basal promoter activity, is sufficient for activation by E1AF/PEA3. All these results suggested that elevated (1,4GT1 in highly metastatic lung cells was mediated by increased E1AF/PEA3 expression. The effect of the association of these two metastatic related proteins is under investigation. Since no Ets binding element was found in this region by searching the trancription factor database, we suggested E1AF/PEA3 might activate (1,4GT1 by protein-protein interaction, a mechanism called protein-tethered tansactivation, instead of direct transactivation.In our previous study, we first reported that (1,4GT1 was associated with cycloheximide-induced apoptosis in human hepatocarcinoma cells. The mRNA level of (1,4GT1 increased greatly dur...
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