Satoshi Takabuchi

Nitric Oxide Induces Hypoxia-inducible Factor 1 Activation That Is Dependent on MAPK and Phosphatidylinositol 3-Kinase Signaling

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular adaptive responses to hypoxia. Levels of the HIF-1α subunit increase under hypoxic conditions. Exposure of cells to certain nitric oxide (NO) donors also induces HIF-1α expression under nonhypoxic conditions. We demonstrate that exposure of cells to the NO donor NOC18 or S-nitrosoglutathione induces HIF-1α expression and transcriptional activity. In contrast to hypoxia, NOC18 did not inhibit HIF-1α hydroxylation, ubiquitination, and degradation, indicating an effect on HIF-1α protein synthesis that was confirmed by pulse labeling studies. NOC18 stimulation of HIF-1α protein and HIF-1-dependent gene expression was blocked by treating cells with an inhibitor of the phosphatidylinositol 3-kinase or MAPK-signaling pathway. These inhibitors also blocked NOC18-induced phosphorylation of the translational regulatory proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus providing a mechanism for the modulation of HIF-1α protein synthesis. In addition, expression of a dominant-negative form of Ras significantly suppressed HIF-1 activation by NOC18. We conclude that the NO donor NOC18 induces HIF-1α synthesis under conditions of NO formation during normoxia and that hydroxylation of HIF-1α is not regulated by NOC18.

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.

Hydrogen Sulfide Inhibits Hypoxia- But Not Anoxia-Induced Hypoxia-Inducible Factor 1 Activation in a von Hippel-Lindau- and Mitochondria-Dependent Manner

AIMS In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H(2)S) is an endogenously synthesized gaseous molecule that acts as an important signaling molecule in the living body. Transcription factor hypoxia-inducible factor 1 (HIF-1) is known to respond to intracellular reduced oxygen (O(2)) availability, which is regulated by an elaborate balance between O(2) supply and demand. However, the effect of H(2)S on HIF-1 activity under hypoxic conditions is largely unknown in mammalian cells. In this study, we tried to elucidate the effect of H(2)S on hypoxia-induced HIF-1 activation adopting cultured cells and mice. RESULTS The H(2)S donors sodium hydrosulfide and sodium sulfide in pharmacological concentrations reversibly reduced cellular O(2) consumption and inhibited hypoxia- but not anoxia-induced HIF-1α protein accumulation and expression of genes downstream of HIF-1 in established cell lines. H(2)S did not affect HIF-1 activation induced by the HIF-α hydroxylases inhibitors desferrioxamine or CoCl(2). Experimental evidence adopting von Hippel-Lindau (VHL)- or mitochondria-deficient cells indicated that H(2)S did not affect neosynthesis of HIF-1α protein but destabilized HIF-1α in a VHL- and mitochondria-dependent manner. We also demonstrate that exogenously administered H(2)S inhibited HIF-1-dependent gene expression in mice. INNOVATION For the first time, we show that H(2)S modulates intracellular O(2) homeostasis and regulates activation of HIF-1 and the subsequent gene expression induced by hypoxia by using an in vitro system with established cell lines and an in vivo system in mice. CONCLUSIONS We demonstrate that H(2)S inhibits hypoxia-induced HIF-1 activation in a VHL- and mitochondria-dependent manner.

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.

Fentanyl activates hypoxia-inducible factor 1 in neuronal SH-SY5Y cells and mice under non-hypoxic conditions in a μ-opioid receptor-dependent manner

Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor responsible for hypoxia-induced gene expression. Perioperative drugs including anesthetics have been reported to affect HIF-1 activity. However, the effect of fentanyl on HIF-1 activity is not well documented. In this study, we investigated the effect of fentanyl and other opioids on HIF-1 activity in human SH-SY5Y neuroblastoma cells, hepatoma Hep3B cells, lung adenocarcinoma A549 cells and mice. Cells were exposed to fentanyl, and HIF-1 protein expression was examined by Western blot analysis using anti-HIF-1α and β antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time reverse transcriptase (RT)-PCR (qRT-PCR) and luciferase assay. Furthermore, fentanyl was administered intraperitoneally and HIF-1-dependent gene expression was investigated by qRT-PCR in the brains and kidneys of mice. A 10-μM concentration of fentanyl and other opioids, including 1 μM morphine and 4 μM remifentanil, induced HIF-1α protein expression and HIF-1 target gene expression in an opioid receptor-dependent manner in SH-SY5Y cells with activity peaking at 24h. Fentanyl did not augment HIF-1α expression during hypoxia-induced induction. HIF-1α stabilization assays and experiments with cycloheximide revealed that fentanyl increased translation from HIF-1α mRNA but did not stabilize the HIF-1α protein. Furthermore, fentanyl induced HIF-1 target gene expression in the brains of mice but not in their kidneys in a naloxone-sensitive manner. In this report, we describe for the first time that fentanyl, both in vitro and in vivo, induces HIF-1 activation under non-hypoxic conditions, leading to increases in expression of genes associated with adaptation to hypoxia.

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.