Inna M. Yasinska

S-nitrosation of Cys-800 of HIF-1α protein activates its interaction with p300 and stimulates its transcriptional activity

Hypoxia inducible factor 1 (HIF‐1) is a heterodimeric transcriptional complex that plays pivotal role in the regulation of cellular utilization of oxygen as well as glucose and is an essential regulator of angiogenesis in solid tumor and ischemic disorders. Recently HIF‐1α, a subunit of HIF‐1 complex, was characterized as a potential target for S‐nitrosation, providing no information about the impact of this posttranslational modification on the protein transactivation. Cys‐800 of HIF‐1α protein has reactive SH‐group, which is critical for the recruitment of p300 co‐activator that is necessary for transcriptional activity of HIF‐1 complex. Here we report that S‐nitrosation of Cys‐800 activates HIF‐1α–p300 interaction and stimulates protein transactivation. We have found that S‐nitrosation of the HIF‐1α C‐terminal domain by nitric oxide derived from donors and nitric oxide synthase increases protein transcriptional activity. The increase of HIF‐1 transcriptional activity was not observed in the case of Cys‐800 substitution to Ala, though other protein thiol groups were nitrosated. Experiments with GST pull‐down assay suggest that S‐nitrosation of Cys‐800 stimulates the recruitment of p300 co‐activator protein to the HIF‐1α C‐terminal domain.

Role of MAP kinase-dependent apoptotic pathway in innate immune responses and viral infection.

Mitogen‐activated protein (MAP) kinase cascades are multifunctional signalling networks that influence cell growth, differentiation, apoptosis and cellular responses to stress. Apoptosis signal‐regulating kinase 1 (ASK1) is a MAP kinase kinase kinase that triggers apoptogenic kinase cascade leading to the phosphorylation/activation of c‐Jun N‐terminal kinases (JNK) and p38‐MAP kinase, which are responsible to induce apoptotic cell death. This pathway plays a pivotal role in the transduction of signals from different apoptotic stimuli. Recently, it has become evident that ASK1 and its downstream pathway are employed in the transduction of signals from Toll‐like receptors (TLR) – multistep processes that interfere with different intracellular signalling pathways. TLR are the key proteins that allow mammals to detect pathogens and mediate innate immune responses. In addition, ASK1 and its downstream pathway play a target role in the regulation of apoptosis in some cases of viral infection – AIDS, influenza, hepatitis C and others. In the present review, we summarize current knowledge about the role of ASK1 and its downstream pathway in innate immune responses and viral infection.

Gold Nanoparticles Downregulate Interleukin‐1β‐Induced Pro‐Inflammatory Responses

Interleukin 1 beta (IL-1β)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1β-dependent diseases and, though their mechanisms of action are still unclear, there is evidence that gold interferes with the transmission of inflammatory signalling. Here we show for the first time that citrate-stabilized gold nanoparticles, in a size dependent manner, specifically downregulate cellular responses induced by IL-1β both in vitro and in vivo. Our results indicate that the anti-inflammatory activity of gold nanoparticles is associated with an extracellular interaction with IL-1β, thus opening potentially novel options for further therapeutic applications.

Involvement of Hypoxia-Inducible Factor-1 in the Inflammatory Responses of Human LAD2 Mast Cells and Basophils

We recently showed that hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the pro-allergic functions of human basophils by transcriptional control of energy metabolism via glycolysis as well as directly triggering expression of the angiogenic cytokine vascular endothelium growth factor (VEGF). Here, we investigated HIF-1 involvement in controlling the synthesis of angiogenic and inflammatory cytokines from various human effector cells stimulated by IgE-dependent or innate immune triggers. Purified primary human basophils, LAD2 human mast cells and THP-1 human myeloid cells were used for investigations of FcεRI and Toll-like receptor (TLR) ligand-induced responses. In contrast to basophils, LAD2 mast cells expressed background levels of HIF-1α, which was largely independent of the effects of stem cell factor (SCF). Both mast cells and basophils expressed TLR2 and 4, albeit weakly compared to THP-1 cells. Cytokine production in mast cells following TLR ligand stimulation was markedly reduced by HIF-1α knockdown in LAD2 mast cells. In contrast, although HIF-1 is involved in IgE-mediated IL-4 secretion from basophils, it is not clearly induced by peptidoglycan (PGN). HIF-1α accumulation is critical for sustaining human allergic effector cell survival and function. This transcription complex facilitates generation of both pro-angiogenic and inflammatory cytokines in mast cells but has a differential role in basophil stimulation comparing IgE-dependent triggering with innate immune stimuli.

Involvement of xanthine oxidase and hypoxia-inducible factor 1 in Toll-like receptor 7/8-mediated activation of caspase 1 and interleukin-1β

Inflammatory reactions to ssRNA viruses are induced by the endosomal Toll-like receptors (TLRs) 7 and 8. TLR7/8-mediated inflammatory reaction results in activation of the Nalp3 inflammasome via an unknown mechanism. Here we report for the first time that TLR7/8 mediate activation of xanthine oxidase (XOD) in an HIF-1α-dependent manner. XOD produces uric acid and reactive oxygen species, which could activate Nalp3 and therefore induce activation of caspase 1, known to convert inactive pro-IL-1β into active IL-1β. Specific inhibition of the XOD activity attenuates TLR7/8-mediated activation of caspase 1 and IL-1β release. These results were obtained using human THP-1 myeloid macrophages. The findings were verified by conducting in vivo experiments on mice.

Mechanisms of hypoxic signal transduction regulated by reactive nitrogen species

Recent reports devoted to the field of oxygen sensing outline that signalling molecules such as nitric oxide/nitric oxide derived species as well as cytokines and other inflammatory mediators participate in hypoxic signal transduction. In the present review, we summarize the current knowledge about the role of nitric oxide and reactive nitrogen species (RNS) derived from it in hypoxic signal transduction and particularly in accumulation/de‐accumulation of hypoxia inducible factor 1 alpha (HIF‐1α) protein, which is critical not only for cellular adaptation to low oxygen availability but also for generation of inflammatory and innate immune responses. After brief description of nitric oxide and other RNS as multifunctional messengers we analyse and discuss the RNS‐dependent accumulation of HIF‐1α protein under normoxia followed by discussion of the mechanisms of nitric oxide (NO)‐dependent enzyme‐regulated degradation of HIF‐1α protein under low oxygen availability.

Effects of Stem Cell Factor on Hypoxia-Inducible Factor 1 Alpha Accumulation in Human Acute Myeloid Leukaemia and LAD2 Mast Cells

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.

The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a severe and often fatal systemic malignancy. Malignant cells are capable of escaping host immune surveillance by inactivating cytotoxic lymphoid cells. In this work we discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9. This occurs in a protein kinase C and mTOR (mammalian target of rapamycin)-dependent manner. Tim-3 participates in galectin-9 secretion and is also released in a free soluble form. Galectin-9 impairs the anti-cancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. Soluble Tim-3 prevents secretion of interleukin-2 (IL-2) required for the activation of cytotoxic lymphoid cells. These results were validated in ex vivo experiments using primary samples from AML patients. This pathway provides reliable targets for both highly specific diagnosis and immune therapy of AML.

Dysfunctional mitochondria contain endogenous high-affinity human Toll-like receptor 4 (TLR4) ligands and induce TLR4-mediated inflammatory reactions.

Mitochondria, known to share many common features with prokaryotic cells, accumulate several endogenous ligands of the pattern-recognition Toll-like receptor 4 (TLR4), such as the heat shock proteins (Hsp) 70 and 60. TLR4 specifically recognises and responds to LPS of Gram-negative bacteria and participates in both autoimmune reactions and tissue regeneration due to its ability to recognise endogenous ligands. In the present study we show that mitochondria extracts obtained from hydrogen peroxide-dysfunctionalised cells induce a pro-inflammatory response in human THP-1 myeloid leukaemia cells. This inflammatory response was similar to that caused by LPS and much stronger than that induced by the extracts of normal mitochondria. Such reactions include activation of stress-adaptation hypoxia-inducible factor 1 alpha (HIF-1α) and expression/release of the pro-inflammatory cytokines IL-6 and TNF-α. Pre-treatment of THP-1 myeloid macrophages with TLR4-neutralising antibody before exposure to mitochondria extracts or LPS attenuated the inflammatory responses. Signalling pathways recruited by TLR4 in response to LPS and mitochondria-derived ligands were found to be the same. An in vitro ELISA-based TLR4-ligand binding assay, in which the ligand-binding domain of human TLR4 was immobilised, showed that mitochondria extracts contain endogenous TLR4 ligands. These results were verified in surface plasmon resonance experiments in which the affinity of the ligands derived from dysfunctional mitochondria was comparable with that of LPS and was much higher than that observed for normal mitochondria.

Peroxynitrite as an alternative donor of oxygen in HIF-1α proline hydroxylation under low oxygen availability

In the last years, nitric oxide (NO) mediated signaling became an integral component in understanding physiological and pathophysiological processes of cell proliferation, death or cellular adaptation. Among other activities, NO affects multiple targets that allow regulation of gene expression. Recently, NO was found to attenuate accumulation of hypoxia inducible factor-1alpha (HIF-1α) under hypoxic conditions because of several mechanisms: redistribution of oxygen toward non-respiratory oxygen-dependent targets (like HIF-1α proline hydroxylases—PHDs, which perform hydroxylation of Pro402/564 of HIF-1α leading to its proteasomal degradation); in addition, peroxynitrite formed during interactions between NO and mitochondria derived superoxide leads to an increase in cytosolic iron/2-oxoglutarate (2-OG), which required for PHD activation. Here, we propose a hypothesis that peroxynitrite, formed in the cells upon exposure to NO under low oxygen availability, serves as an alternative donor of oxygen for activated PHDs so they can perform HIF-1α proline hydroxylation to de-accumulate the protein.