NOTCH activation promotes glycosyltransferase expression in human myeloid leukemia cells

Abstract

NOTCH signaling diversely regulates the growth of acute myeloid leukemia (AML) cells. It is known that glycosylation of NOTCH receptors modulates NOTCH activation. However, little is known about glycosylation of NOTCH in AML cells. We examined the effects of ligand-induced NOTCH activation on the expression of NOTCH-modifying glycosyltransferases in two AML cell lines, THP-1 and TMD7. The cells were stimulated with recombinant NOTCH ligands JAGGED1 and DELTA1, and subjected to immunoblot analysis to evaluate theexpres-sion levels of glycosyltransferases. Ligand stimulation promoted the expression of POFUT1, LFNG, MFNG, RFNG, GXYLT1, GXYLT2, and XXYLT1 in THP-1 cells, and that of RFNG and GXYLT1 in TMD7 cells. We found that NOTCH activation promoted the expression of several glycosyltransferases in AML cells. This suggests that NOTCH activation modulates its sensitivity to NOTCH ligands by increased glycosylation of NOTCH receptors in AML cells. Further investigation is needed to elucidate its biological significance.mmelby stimulation with these ligands.6 NOTCH signaling is activated upon binding of the NOTCH extracellular domain (NECD) to JAGGED and/or DELTA ligands on adjacent cells. The addition of O-fucose moieties to epidermal growth factor-like (EGF) repeats within the NECD (Figure 1) by O-fucosyltransferase-1 (POFUT1) is essential for NOTCH activation. Subsequently, Fringe (FNG) glycosyltransferases mediate the addition of N-acetylglucosamine (GlcNAc) to these O-fucose moieties. Mammals encode three distinct Fringe homologues: lunatic Fringe (LFNG), manic Fringe (MFNG), and radical Fringe (RFNG). While this addition of GlcNAc was reported to promote NOTCH– DELTA binding and reduces NOTCH– JAGGED binding,9-11 the precise roles of GlcNAc have yet to be determined.12,13 The addition of O-glucose moieties to the EGF repeats by O-glucosyltransferase (POG-LUT1) is also essential for NOTCH activation. Conversely, the addition of two xylose residues to these O-glucose molecules byuglu-coside a1–3 xylosyltransferases 1 and 2 (GXYLT1 /2) and xyloside a1-3 xylosyl-transferase 1 (XXYLT1) was reported tommelinhibit NOTCH activity in Drosophila.14 The NOTCH-modifying glycosyltransferase enzymes exhibit aberrant expression in various cancers.13 However, little is known about glycosylation of NOTCH in AML cells. To elucidate the relationship between NOTCH and its glycosylation, we examined the effects of ligand-mediated NOTCH activation on the expression of glycosyltransferases in AML cell lines. Given the technical difficulties associated with measuring glycosylation levels of NOTCH protein, we chose to evaluate glycosyltransferase mRNA and protein expression instead.