Seiko Oda

LPS Induces Hypoxia-Inducible Factor 1 Activation in Macrophage-Differentiated Cells in a Reactive Oxygen Species–Dependent Manner

A prominent feature of various inflamed and diseased tissue is the presence of low oxygen tension (hypoxia). Effector cells of the innate immune system must maintain their viability and physiologic functions in a hypoxic microenvironment. Monocytes circulating in the bloodstream differentiate into macrophages. During this process, cells acquire the ability to exert effects at hypoxic sites of inflammation. The transcription factor hypoxia-inducible factor 1 (HIF-1) mediates adaptive responses to reduced oxygen availability. In this study, we demonstrated that lipopolysaccharide (LPS) induces HIF-1 activation by enhancing both HIF-1alpha protein expression through a translation-dependent pathway and HIF-1alpha transcriptional activity in THP-1 human myeloid cells that have undergone macrophage differentiation but not in undifferentiated monocytic THP-1 cells. LPS-induced HIF-1 activation was blocked by treatment with antioxidant (N-acetylcysteine or thioredoxin-1), NADPH oxidase inhibitor (diphenyleneiodonium), indicating that reactive oxygen species generated in response to LPS are essential in this process. LPS-mediated activation of HIF-1 was independent of NF-kappaB activity. LPS-induced ROS generation and HIF-1 activation required the expression of Toll-like receptor 4 or myeloid differentiation factor (MyD) 88, thus providing a molecular basis for the selective activation of HIF-1 in differentiated THP-1 cells.

Macrophage migration inhibitory factor activates hypoxia-inducible factor in a p53-dependent manner.

Background Macrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner. Methods/Principal Findings The effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly. Conclusion/Significance MIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIFs potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1α protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF.

Persisting mild hypothermia suppresses hypoxia-inducible factor-1alpha protein synthesis and hypoxia-inducible factor-1-mediated gene expression.

The transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in regulating gene expression in response to hypoxia-ischemia. Ischemia causes the tissue not only to be hypoxic but also to be hypothermic because of the hypoperfusion under certain circumstances. On the other hand, the induced hypothermia is one of the most common therapeutic modalities to extend tolerance to hypoxia. Although hypoxia elicits a variety of cellular and systemic responses at different organizational levels in the body, little is known about how hypoxia-induced responses are affected by low temperature. We examined the influence of mild hypothermic conditions (28-32 degrees C) on HIF-1 in both in vitro and in vivo settings. In vitro experiments adopting cultured cells elucidated that hypoxia-induced HIF-1 activation was resistant to 4-h exposure to the low temperature. In contrast, exposure to the low temperature as long as 24 h suppressed HIF-1 activation and the subsequent upregulation of HIF-1 target genes such as VEGF or GLUT-1. HIF-1alpha protein stability in the cell was not affected by hypothermic treatment. Furthermore, intracellular ATP content was reduced under 1% O(2) conditions but was not largely affected by hypothermic treatment. The evidence indicates that reduction of oxygen consumption is not largely involved in suppression of HIF-1. In addition, we demonstrated that HIF-1 DNA-binding activity and HIF-1-dependent gene expressions induced under 10% O(2) atmosphere in mouse brain were not influenced by treatment under 3-h hypothermic temperature but were inhibited under 5-h treatment. On the other hand, we indicated that warming ischemic legs of mice for 24 h preserved HIF-1 activity. In this report we describe for the first time that persisting low temperature significantly reduced HIF-1alpha neosynthesis under hypoxic conditions, leading to a decrease in gene expression for adaptation to hypoxia in both in vitro and in vivo settings.

The intravenous anesthetic propofol inhibits hypoxia-inducible factor 1 activity in an oxygen tension-dependent manner.

Hypoxia elicits a wide range of responses that occur at different organizational levels in the body. Hypoxia is not only a signal for energy conservation and metabolic change, but triggers expression of a select set of genes. The transcription factor hypoxia‐inducible factor 1 (HIF‐1) is now appreciated to be a master factor of the gene induction. Although knowledge on molecular mechanisms of HIF‐1 activation in response to hypoxia is accumulating, the molecular mechanism of maintenance of HIF‐1 activity under normoxic conditions remains to be elucidated. We demonstrate that the intravenous anesthetic propofol reversibly inhibits HIF‐1 activity and the gene expression mediated by HIF‐1 by blocking the synthesis of the HIF‐1α subunit under 20% or 5% O2 conditions, but not under 1% O2 conditions.

The intravenous anesthetic propofol inhibits lipopolysaccharide-induced hypoxia-inducible factor 1 activation and suppresses the glucose metabolism in macrophages

PurposeHypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of propofol on HIF-1 activity is not well documented. In this study, we investigated the effect of propofol on HIF-1 activation using macrophage-differentiated THP-1 cells.MethodsCells were exposed to lipopolysaccharide (LPS) under 20 or 1% O2 conditions with or without propofol treatment. The cell lysate was subjected to Western blot analysis using anti-HIF-1α and HIF-1β antibodies. HIF-1-dependent gene expression was investigated by quantitative real-time reverse-transcriptase PCR analysis and luciferase assay. The amount of cellular lactate and ATP was assayed.ResultsPropofol suppressed HIF-1α protein accumulation induced by LPS, but not by hypoxia in the THP-1 cells in a dose-dependent manner by inhibiting the neo-synthesis of HIF-1α protein. Induction of the HIF-1 downstream gene expression including glucose transporter 1, enolase 1, lactate dehydrogenase A, pyruvate dehydrogenase kinase-1 and vascular endothelial growth factor was inhibited by propofol. Propofol suppressed LPS-induced lactate accumulation and ATP content in THP-1 cells.ConclusionOur experimental results indicate that propofol inhibits HIF-1 activation and downstream gene expression induced by LPS and suppressed HIF-1-dependent glucose metabolic reprogramming. HIF-1 suppression by propofol in macrophages may explain molecular mechanisms behind the inhibitory effect of propofol on cellular inflammatory responses.

n-Propyl gallate activates hypoxia-inducible factor 1 by modulating intracellular oxygen-sensing systems

HIF-1 (hypoxia-inducible factor 1) is a master regulator of cellular adaptive responses to hypoxia. The expression and transcriptional activity of the HIF-1alpha subunit is stringently controlled by intracellular oxygen tension through the action of prolyl and asparaginyl hydroxylases. In the present study we demonstrate that PG (n-propyl gallate) activates HIF-1 and expression of its downstream target genes under normoxic conditions in cultured cells and in mice. The stability and transcriptional activity of HIF-1alpha are increased by PG. PG treatment inhibits the interaction between HIF-1alpha and VHL (von Hippel-Lindau protein) and promotes the interaction between HIF-1alpha and p300, indicating that PG inhibits the activity of both prolyl and asparaginyl HIF-1alpha hydroxylases. We conclude that PG activates HIF-1 and enhances the resultant gene expression by directly affecting the intracellular oxygen sensing system in vitro and in vivo and that PG represents a lead compound for the development of a non-toxic activator of HIF-1.

The intravenous anesthetics barbiturates inhibit hypoxia-inducible factor 1 activation

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of barbiturates on HIF-1 activity has not been reported. In this study, we investigated the effect of thiopental and thiamylal on HIF-1 activity using the neuronal SH-SY5Y cells, the non-neuronal HEK293 cells, and the macrophage-differentiated THP-1 cells. Cells were exposed to 20% or 1% O(2) conditions with or without thiopental or thiamylal treatment. The cell lysate were subjected to Western blot analysis using anti-HIF-1alpha and -HIF-1beta antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time RT-PCR and luciferase assay. Hydroxylation of HIF-1alpha protein was evaluated by in vitro pulldown assay using recombinant protein. Both thiopental and thiamylal reversibly suppressed hypoxia-induced HIF-1 activation in the neuronal and the non-neuronal cells in a dose-dependent manner. Moreover, the barbiturates inhibited lipopolysaccharide-induced HIF-1alpha expression in THP-1 cells. The HIF-1-downstream gene expression was also inhibited by the barbiturates. HIFalpha-hydroxylases activity and HIF-1alpha stability were not affected but the HIF-1alpha protein neosynthesis was inhibited by the barbiturates. Our experimental results indicate that barbiturates inhibit induced HIF-1 activation and downstream genes expression.

The calcium channel blocker cilnidipine selectively suppresses hypoxia-inducible factor 1 activity in vascular cells

Calcium ion is one of the most important second messengers of cellular signal transduction including hypoxia-elicited signals. In this study, we investigated the effects of the L-type calcium channel blockers such as nifedipine, efonidipine cilnidipine, diltiazem, and verapamil, on the activity of hypoxia-inducible factor-1 (HIF-1), a key transcription factor in control of hypoxia-induced gene expression. Using the lung carcinoma cell line A549 cells, human aortic smooth muscle cells, and human umbilical vein endothelial cells, we demonstrated that cilnidipine exclusively suppressed HIF-1 activity and the expressions of downstream genes in a cell-type specific manner. We also demonstrated that cilnidipine blocked the synthesis of the HIF-1alpha protein not by affecting activity of the intracellular hypoxia-sensing element prolyl hydroxylases but inhibiting activity of Akt and mitogen-activated protein kinase and that the inhibition is not dependent on the effect on calcium homeostasis.

The effects of local anesthetics on cellular hypoxia-induced gene responses mediated by hypoxia-inducible factor 1

PurposeHypoxia (reduced oxygen availability) induces a series of adaptive physiological responses. At the cellular level, the adaptation includes a switch of energy metabolism from oxidative phosphorylation to anaerobic glycolysis, increased glucose uptake, and the expression of stress proteins related to cell survival. One of the most important transcription factors that activate the expression of oxygen-regulated genes is hypoxia-inducible factor 1 (HIF-1). We previously reported that halothane inhibits the hypoxia-induced HIF-1 activation. In this study, we investigated the effect of local anesthetics on HIF-1 activation and its downstream gene expression.MethodsThe established cell line Hep3B and SK-N-MC cells were exposed to 1% O2 with or without treatment by either lidocaine or bupivacaine. Expression of subunits of HIF-1, HIF-1α, and HIF-1β was examined by Western blot using specific antibodies. Expression of mRNA of HIF-1 and the HIF-1-dependent genes was investigated by RT-PCR and reporter assay.ResultsNeither of the local anesthetics tested affected the accumulation of HIF-1α induced by hypoxia, nor did they affect NOC18-induced HIF-1α accumulation. Moreover, they had no effects on HIF-1-mediated hypoxia-induced gene expression.ConclusionThe local anesthetics lidocaine and bupivacaine did not affect the HIF-1-dependent cellular hypoxia-induced gene responses.

Opioid receptor stimulation does not affect cellular hypoxia-induced gene responses mediated by hypoxia-inducible factor 1 in cultured cell lines.

Hypoxia induces a series of adaptive physiological responses including gene inductions. Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor that regulates hypoxia-induced gene expression to maintain homeostasis in the living body. Opioids are potent analgesic agents that are widely used in clinical practice. Therefore, we investigated the effect of opioids on HIF-1 activity. SH-SY5Y human neuronal cells, which express opioid receptors intrinsically, were cultured under 1% or 20% O2 conditions with or without treatment by DAGO, DPDPE, or U-50488, which are the selective agonists of µ-, δ-, and κ-opioid receptors, respectively. Expression of subunits of HIF-1, HIF-1α, and HIF-1β were examined by Western blot using specific antibodies. Expression of the HIF-1-dependent gene were investigated by reporter assay. None of the selective agonists of opioid receptors tested affected HIF-1 activation by hypoxia. Therefore, it is suggested that opioid receptor-mediated signals do not affect HIF-1-dependent cellular hypoxia-induced gene responses.