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Effects of stem cell factor on hypoxia-inducible factor 1 alpha accumulation in human acute myeloid leukaemia and LAD2 mast cells.

Gibbs BF, Yasinska IM, Oniku AE, Sumbayev VV - PLoS ONE (2011)

Bottom Line: However, the mechanisms of this pathophysiological effect remain unclear.We demonstrated that LAD2 cells have a more robust glutathione (GSH)-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner.BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD) activity in THP-1 cells and to near attenuation of it in LAD2 cells.

View Article: PubMed Central - PubMed

Affiliation: Medway School of Pharmacy, University of Kent, Kent, United Kingdom. B.F.Gibbs@kent.ac.uk

ABSTRACT
Stem cell factor (SCF) is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1) in hematopoietic cells--a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH)-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD) activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation--an important stage of the myeloid leukaemia cell life cycle.

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Several pathways impact SCF-dependent HIF-1α accumulation via mTOR in THP-1 human myeloid cells.THP-1 cells were pre-treated for one hour with the indicated concentrations of the outlined inhibitors. In one case cells were transfected with dominant-negative form of ASK1 (ASK1-KM) as indicated in Materials and methods. THP-1 cells were then exposed for 4 h to 100 ng/ml SCF followed by Western blot analysis of HIF-1α and mTOR accumulation as well as S2448 phosphorylation. LAD2 cells were cultured for 24 h in the absence or presence of 100 ng/ml SCF. All Western blot data are from one experiment representative of three that gave similar results. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. a – differences are significant when comparing two indicated values (P<0.01).
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pone-0022502-g006: Several pathways impact SCF-dependent HIF-1α accumulation via mTOR in THP-1 human myeloid cells.THP-1 cells were pre-treated for one hour with the indicated concentrations of the outlined inhibitors. In one case cells were transfected with dominant-negative form of ASK1 (ASK1-KM) as indicated in Materials and methods. THP-1 cells were then exposed for 4 h to 100 ng/ml SCF followed by Western blot analysis of HIF-1α and mTOR accumulation as well as S2448 phosphorylation. LAD2 cells were cultured for 24 h in the absence or presence of 100 ng/ml SCF. All Western blot data are from one experiment representative of three that gave similar results. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. a – differences are significant when comparing two indicated values (P<0.01).

Mentions: Finally, we investigated the impact of ASK1 (as a pro-apoptotic negative regulator of protein synthesis), PKC-δ and XOD on mTOR accumulation and S2448 phosphorylation (XOD contributes to a crucial step of purine nucleotide catabolism, possibly influencing biochemical events associated with the whole transcription/translation system). In these experiments we pre-treated THP-1 cells with 10 µM rapamycin, 100 µM rottlerin or 250 µg/ml allopurinol for 1 h followed by 4 h of exposure to 100 ng/ml SCF. Some of the THP-1 cells were also transfected with the dominant-negative isoform of ASK1 and then exposed for 4 h to 100 ng/ml SCF. The accumulation of mTOR and HIF-1α protein were then analysed. We found that SCF clearly upregulated mTOR accumulation/S2448 phosphorylation in THP-1 cells. These processes were attenuated by rapamycin, rottlerin and allopurinol showing that both PKC-δ and XOD contribute to SCF-induced mTOR activation (Figure 6). Downregulation of ASK1 induced by its dominant-negative form led to a further non-significant increase in intracellular mTOR and its S2448 phosphorylation levels in THP-1 cells exposed to SCF. The effects of the above agents on the mTOR accumulation/S2448-phosphorylation correlated with those observed for HIF-1α (Figure 6). Interestingly, in LAD2 cells, mTOR was constitutively expressed and phosphorylated when they were cultured in the presence of 100 ng/ml SCF or kept for 24 h in the absence of this cytokine, which is consistent with absence in changes in HIF-1α accumulation (Figure 6).


Effects of stem cell factor on hypoxia-inducible factor 1 alpha accumulation in human acute myeloid leukaemia and LAD2 mast cells.

Gibbs BF, Yasinska IM, Oniku AE, Sumbayev VV - PLoS ONE (2011)

Several pathways impact SCF-dependent HIF-1α accumulation via mTOR in THP-1 human myeloid cells.THP-1 cells were pre-treated for one hour with the indicated concentrations of the outlined inhibitors. In one case cells were transfected with dominant-negative form of ASK1 (ASK1-KM) as indicated in Materials and methods. THP-1 cells were then exposed for 4 h to 100 ng/ml SCF followed by Western blot analysis of HIF-1α and mTOR accumulation as well as S2448 phosphorylation. LAD2 cells were cultured for 24 h in the absence or presence of 100 ng/ml SCF. All Western blot data are from one experiment representative of three that gave similar results. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. a – differences are significant when comparing two indicated values (P<0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140519&req=5

pone-0022502-g006: Several pathways impact SCF-dependent HIF-1α accumulation via mTOR in THP-1 human myeloid cells.THP-1 cells were pre-treated for one hour with the indicated concentrations of the outlined inhibitors. In one case cells were transfected with dominant-negative form of ASK1 (ASK1-KM) as indicated in Materials and methods. THP-1 cells were then exposed for 4 h to 100 ng/ml SCF followed by Western blot analysis of HIF-1α and mTOR accumulation as well as S2448 phosphorylation. LAD2 cells were cultured for 24 h in the absence or presence of 100 ng/ml SCF. All Western blot data are from one experiment representative of three that gave similar results. Quantitative data are mean values ± S.D. of at least three individual experiments. *P<0.01 vs control. a – differences are significant when comparing two indicated values (P<0.01).
Mentions: Finally, we investigated the impact of ASK1 (as a pro-apoptotic negative regulator of protein synthesis), PKC-δ and XOD on mTOR accumulation and S2448 phosphorylation (XOD contributes to a crucial step of purine nucleotide catabolism, possibly influencing biochemical events associated with the whole transcription/translation system). In these experiments we pre-treated THP-1 cells with 10 µM rapamycin, 100 µM rottlerin or 250 µg/ml allopurinol for 1 h followed by 4 h of exposure to 100 ng/ml SCF. Some of the THP-1 cells were also transfected with the dominant-negative isoform of ASK1 and then exposed for 4 h to 100 ng/ml SCF. The accumulation of mTOR and HIF-1α protein were then analysed. We found that SCF clearly upregulated mTOR accumulation/S2448 phosphorylation in THP-1 cells. These processes were attenuated by rapamycin, rottlerin and allopurinol showing that both PKC-δ and XOD contribute to SCF-induced mTOR activation (Figure 6). Downregulation of ASK1 induced by its dominant-negative form led to a further non-significant increase in intracellular mTOR and its S2448 phosphorylation levels in THP-1 cells exposed to SCF. The effects of the above agents on the mTOR accumulation/S2448-phosphorylation correlated with those observed for HIF-1α (Figure 6). Interestingly, in LAD2 cells, mTOR was constitutively expressed and phosphorylated when they were cultured in the presence of 100 ng/ml SCF or kept for 24 h in the absence of this cytokine, which is consistent with absence in changes in HIF-1α accumulation (Figure 6).

Bottom Line: However, the mechanisms of this pathophysiological effect remain unclear.We demonstrated that LAD2 cells have a more robust glutathione (GSH)-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner.BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD) activity in THP-1 cells and to near attenuation of it in LAD2 cells.

View Article: PubMed Central - PubMed

Affiliation: Medway School of Pharmacy, University of Kent, Kent, United Kingdom. B.F.Gibbs@kent.ac.uk

ABSTRACT
Stem cell factor (SCF) is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1) in hematopoietic cells--a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH)-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD) activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation--an important stage of the myeloid leukaemia cell life cycle.

Show MeSH
Related in: MedlinePlus