Abstract
N-acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. Our previous studies have shown that modification of N-glycans by GnT-III affects a number of intracellular signaling pathways. In this study, the effects of GnT-III on the cellular response to reactive oxygen species (ROS) were examined. We found that an overexpression of GnT-III suppresses H(2)O(2)-induced apoptosis in HeLaS3 cells. In the case of GnT-III transfectants, activation of Jun N-terminal kinase (JNK) following H(2)O(2) treatment was markedly reduced compared with control cells. Either the depletion of protein kinase C (PKC) by prolonged treatment with phorbol 12-myristate 13-acetate or the inhibition of PKC by the specific inhibitor H7 attenuated the H(2)O(2)-induced activation of JNK1 and apoptosis in control cells but not in the GnT-III transfectants. Furthermore, we found that H(2)O(2)-induced phosphorylation of PKC delta was markedly suppressed in GnT-III transfectants. Rottlerin, a specific inhibitor of PKC delta, significantly inhibited H(2)O(2)-induced activation of JNK1 in control cells, indicating that PKC delta is involved in the pathway. These findings suggest that the overexpression of GnT-III suppresses H(2)O(2)-induced activation of PKC delta-JNK1 pathway, resulting in inhibition of apoptosis.
Highlights
It is generally thought that oligosaccharides attached to glycoproteins play crucial roles in the folding, stability, and sorting of the protein [1]
When cells were treated with 300 M H2O2 for 18 h, the nuclei consistently appeared condensed and fragmented by DAPI staining in control cells (Fig. 1C, panel b), and chromatin condensation was strongly suppressed in the GnTIII transfectants (Fig. 1C, panel d)
When cells were pretreated with caspase3-specific inhibitor, DEVD-CHO, to prevent apoptotic cell death, 300 M H2O2-induced cell death in control cells was markedly reduced to the same level as in GnT-III transfectants (Fig. 1D)
Summary
GnT-III, N-acetylglucosaminyltransferase III; ROS, reactive oxygen species; MTT, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide; JNK, Jun N-terminal kinase; SEK1, stress-activated protein kinase/extracellular signalregulated kinase kinase 1; ERK, extracellular signal-regulated protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; MAPK, mitogen-activated protein kinase; DAPI, 4,6-diamidino-2phenylindole; EGF, epidermal growth factor. The metastatic potential of B16 mouse melanoma cells is decreased by the introduction of the GnT-III gene [6]. The overexpression of the GnT-III gene affects nerve growth factor signaling by suppressing the dimerization of TrkA in PC12 cells [8]. We reported that GnT-III gene induction resulted in changes in N-glycan structures and enhanced epidermal growth factor (EGF)-induced Erk phosphorylation in HeLaS3 cells, and we found that this up-regulation of Erk phosphorylation is due to the enhancement of receptor internalization [9]. We demonstrate that GnT-III overexpression in HeLaS3 cells suppresses H2O2-induced apoptosis and the activity of two stressregulated MAPKs, JNK and p38, but not Erk 1/2. The results indicate that the suppression of PKC␦ activity had an inhibitory effect on JNK activity and apoptosis in GnT-III transfectants
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