Differential control of hypoxia-inducible factor 1 activity during pro-inflammatory reactions of human haematopoietic cells of myeloid lineage

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Abstract

The biological responses of human haematopoietic cells of myeloid lineage following malignant transformation depends crucially on their adaptation to signalling stress. This adaptation is dependent on the activation of the hypoxia-inducible factor 1 (HIF-1) transcription complex, although the mechanisms underlying its control remain unclear. Here, we report that mammalian target of rapamycin (mTOR) kinase, a central regulator of myeloid cell growth and metabolism, is crucially involved in HIF-1α accumulation/HIF-1 activation in human THP-1 myeloid monocytes, LAD2 mast cells and primary basophils in a variety of different settings. In THP-1 cells, mTOR was a major contributor to HIF-1 activation induced by ligands of both membrane-associated and endosomal toll-like receptors that recognise molecular patterns shared by pathogens. In LAD2 mast cells and primary human basophils mTOR was vital for HIF-1α accumulation/HIF-1 activity during immunoglobulin E (IgE)-dependent pro-allergic responses as well as in maintaining constitutive HIF-1α levels in mast cells. Furthermore, we observed that nitric oxide synthase activity was dependent on mTOR in these myeloid cells and involved in HIF-1α accumulation mediated by endosomal toll-like receptor 7/8 triggering in THP-1 cells as well as IgE-dependent basophil, but not mast cell, responses. In both cases the process is likely to be associated with an impact of reactive nitrogen species on HIF-1α-degrading prolyl hydroxylation. These studies underline the importance of mTOR as a fundamental regulator of HIF-1 signalling in myeloid cells.

Introduction

The biological functions of human haematopoietic cells of myeloid lineage include innate immune responses to pathogens and pro-allergic hypersensitivity reactions (Broudy, 1997). During maturation, however, these cells potentially undergo a malignant transformation that causes the development and progression of acute myeloid leukaemia (Broudy, 1997). All these types of responses are induced through highly specific signalling receptors in a ligand-dependent manner. For example, recognition of pathogen-associated molecular patterns is governed by plasma membrane-associated or endosomal Toll-like receptors (TLRs) expressed in myeloid cells (Akira and Takeda, 2004). Mast cells and basophils express the high-affinity IgE receptor (FcɛRI), which recognises IgE and leads to cell activation upon crosslinking by allergens (reviewed in: Crivellato et al., 2010).

In cases of malignant transformation, myeloid cells continue expressing the Kit receptor (CD117), which recognises stem cell factor (SCF) – a cytokine that plays a crucial role in haematopoiesis and melanogenesis. SCF controls the proliferation of leukaemia cells thus acting as a major contributor to a myeloid leukaemia progression (Broudy, 1997, Lee et al., 2007). Unlike other myeloid cells, mast cells continue to express the Kit receptor even without malignant transformation (they are the only terminally differentiated haematopoietic cells that retain Kit receptor expression).

Recent evidence clearly demonstrated that the above mentioned normal and pathological processes cause intracellular signalling stress associated with increased intracellular oxygen and ATP consumption (Sumbayev and Nicholas, 2010, Zarember and Malech, 2005). However, an increased influx of these cells into the site where a disorder is taking place results in a substantial reduction of oxygen availability. Therefore, hypoxic conditions are considered as a physiological environment for both inflammatory reactions of haematopoietic cells of myeloid lineage and acute myeloid leukaemia progression (Zarember and Malech, 2005). Under these settings, cells become more dependent on glycolysis and less on respiration where they utilise glycolytic ATP to maintain mitochondrial membrane potential and thus prevent apoptotic cell death (Garedew et al., 2010).

To adapt to pro-inflammatory or pro-leukaemic stress, cells employ the hypoxia-inducible factor 1 transcription complex (HIF-1) which consists of a constitutive beta and inducible alpha subunit. Under normal oxygen availability the 402nd and 564th proline residues of the HIF-1α subunit undergo hydroxylation by prolyl hydroxylases (PHDs) followed by ubiquitination and proteasomal degradation of the protein (Semenza, 2002). Under hypoxic conditions, prolyl hydroxylation is substantially reduced. This stabilizes the HIF-1α protein which then interacts with its beta subunit and triggers expression of genes that encode for proteins responsible for angiogenesis, glycolysis and cell adhesion (Walmsley et al., 2005). In the case of inflammatory reactions of haematopoietic cells of myeloid lineage induced by TLR ligands and pro-allergic mediators HIF-1α is stabilised via redox-dependent (redox-dependent downregulation of HIF-1α PHD activity) and mitogen-activated protein (MAP) kinase-dependent phosphorylation mechanisms (Sumbayev, 2008, Nicholas and Sumbayev, 2009, Nicholas and Sumbayev, 2010).

Recently, it was demonstrated that HIF-1α accumulation in activated haematopoietic cells is dependent on biosynthetic mechanisms controlled by the mammalian target of rapamycin (mTOR) (Jung et al., 2003, Gibbs et al., 2011). Upon S2448 phosphorylation mTOR gains kinase activity and phosphorylates S6K1 kinase and 4E-BP1 – a physiological inhibitor of eukaryotic initiation factor 4E leading to its release and activation. mTOR serine/threonine kinase is the catalytic subunit of two multi-protein complexes, known as mTORC1 and mTORC2. Signalling downstream of mTORC1 plays a critical role in leukocyte cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. This is particularly important in myeloid leukaemia cells (Grimaldi et al., 2012) where successful progression of different types of myeloid leukaemia depends on mTOR signalling. Additionally, mTOR signalling is also essentially involved in both the initiation as well as suppression of host innate immune responses of myeloid cells. Generation of crucial pro-inflammatory (IL-12, interferons) and anti-inflammatory (IL-10) cytokines is controlled by the mTOR downstream pathway (Dazert and Hall, 2011). It has also been demonstrated that mTOR contributes to interleukin (IL)-1beta (acting via the IL-1 receptor type I, which is highly homologous to plasma membrane-associated TLRs 2 and 4) and SCF-dependent HIF-1α accumulation (Jung et al., 2003, Gibbs et al., 2011). In a number of inflammatory responses, cells depend on enzymatic generation of nitric oxide, which on one the hand downregulates the catalytic activity of HIF-1α PHDs (Sumbayev and Yasinska, 2007, Nicholas and Sumbayev, 2010). On the other hand, nitric oxide activates guanylate cyclase, which generates cGMP from GTP. cGMP acts as an allosteric modulator for protein kinase G (PKG), which activates Ca2+ plasma membrane associated ion channels and thus controls intracellular calcium levels (Sugiya et al., 1998). It has been shown that the intracellular Ca2+ level could be important for HIF-1α accumulation since it influences PHD activity (Berchner-Pfannschmidt et al., 2004, Liu et al., 2004, Werno et al., 2008). Enzymatically generated NO is known to positively impact on TLR7/8 (recognise single-stranded viral RNA)-dependent HIF-1α accumulation (Nicholas and Sumbayev, 2009). However, it does not contribute to the lipopolysaccharide (LPS)-induced TLR4-mediated process (Sumbayev, 2008). The mTOR pathway was found to influence control on intracellular Ca2+ levels (Kisfalvi et al., 2007, Fregeau et al., 2011) which could possibly take place via NOS-mediated actions on mTOR. Generally, although there are differential roles of mTOR and NOS pathways in HIF-1α accumulation in activated haematopoietic cells of myeloid lineage the details of these have not yet been elucidated.

Here we report that mTOR is involved in ligand-induced TLR2/7/8-mediated HIF-1α accumulation in THP-1 cells. However, its contribution in the case of TLR7/8 is moderate compared to the one observed with TLR2 and SCF-induced responses. Involvement of mTOR in HIF-1α accumulation/HIF-1 activity in LAD2 mast cells and primary human basophils during IgE-dependent responses was also demonstrated. In all cases described above, NOS activity was dependent on mTOR. NOS, however, was clearly involved in HIF-1α accumulation/HIF-1 activation mediated by TLR7/8 and IgE-dependent basophil responses. In both cases the process was associated with an impact of reactive nitrogen species (RNS) on HIF-1α PHD.

Section snippets

Materials

RPMI-1640 medium, foetal calf serum and supplements, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) transfection reagent, peptidoglycan (PGN) and most of the pharmacological inhibitors were purchased from Sigma (Suffolk, UK). R848 (TLR7/8 ligand) was purchased from Axxora (Exeter, UK). Maxisorp™ microtitre plates were obtained from Nunc (Roskilde, Denmark). ELISA-based assay kit for detection of VEGF was bought from R&D Systems (Abingdon, UK). Rapamycin and

Involvement of mTOR and NOS pathways in HIF-1 activation during TLR-mediated responses of THP-1 cells

We first investigated the involvement of mTOR and NOS pathways on HIF-1 activation induced by plasma membrane-associated and endosomal TLRs. Homodimeric plasma membrane-associated TLR2 and endosomal TLR7/8 were used as targets. PGN was used as a TLR2 activator and the synthetic ligand R848 was employed to trigger TLR7/8-mediated responses. THP-1 cells were pre-treated for 30 min with 10 μM rapamycin (mTOR inhibitor) and 100 μM l-NG-monomethyl arginine (NMMA, NOS inhibitor) for 30 min followed by 4 h

Discussion

The HIF-1 transcription complex controls adaptation of myeloid haematopoietic cells to inflammatory stress induced by pathogen-associated or pro-allergic mediators (Sumbayev and Nicholas, 2010). HIF-1 activation depends on the accumulation of its inducible alpha subunit, which is controlled via differential mechanisms depending on the type of response and inducing receptor. In the case of plasma membrane-associated TLRs, HIF-1α accumulation is controlled by a redox-dependent mechanism in

Acknowledgements

We thank Prof. Dean Metcalfe and Dr. Arnold Kirshenbaum (NIH, USA) for generously providing us with LAD2 human mast cells. This work was supported by a grant from Asthma UK (grant number 10/065 – to Drs. B.F. Gibbs and V. Sumbayev). We are grateful to masters’ students Olawunmi Shobande, Kadijatu Bundu, Deborah Ologun, Konstantinos Lagouvardos and Judith Akuchie for assistance in analysing the initial experiments reported in this study.

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    These authors contributed equally to this study.

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