REPOSITORY > RESULTS

Abstract

Glucosamine (GlcN) is a naturally occurring derivative of glucose and an over-the-counter food additive. However, the mechanism underlying GlcN action on cells is unknown. In this doctoral dissertation, we investigated the effect of GlcN on natural killer (NK) cells. We showed that GlcN decreases NK cell cytotoxicity in a dose-dependent manner and that this inhibition is reversible. We demonstrated that GlcN affects NK-92 cell cytotoxicity by altering the distribution of cathepsin C, a cysteine protease required for granzyme processing in cytotoxic granules. The relocation of cathepsin C due to GlcN was shown to be accompanied by a decrease in the intracellular enzyme activity and its extracellular secretion. Similarly, the relocation of endosomal aspartic cathepsin E was observed. Furthermore, we elucidated that repositioning of cathepsin C is a consequence of altered signaling pathways of cytotoxic granule movement. The inhibition of phosphorylation upstream and downstream of ERK by GlcN disturbed the polarized release of cytotoxic vesicles. Considerable changes in the ERK phosphorylation dynamics, but not in those of p38 kinase or JNK, were observed in the IL2-activated NK-92 cells. GlcN causes prolonged phosphorylation of ERK kinase, as well as its nuclear translocation, which changes its substrates. The prolonged ERK phosphorylation is a consequence of decreased phosphorylation of the transcription factor FOXO1, an upstream negative regulator of ERK phosphorylation. Similarly, a protein downstream of the ERK phosphorylation cascade, paxillin, was less phosphorylated, resulting in a diffuse distribution of cytotoxic granules. Additionally, we showed that TNFα, a cytokine released by NK cells, has no effect on NK cell cytotoxicity, neither on the viability of NK, K562, nor U937 cells. Furthermore, TNFα effect U937 only in combination with CHX, an inhibitor of the proteasome. Apoptosis of K562 cells by TNFα/CHX causes the release of cathepsins from lysosomes and stepwise degradation of endogenous cysteine cathepsin inhibitor stefin B. Taken together, our results suggest that dietary GlcN affects signaling pathway activation of NK-92 immune cells and that TNFα has no effect on NK cell cytotoxicity.