Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently identified that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in mice. The present study was designed to examine whether this is also the case in humans. Bradykinin elicited endothelium-dependent relaxations and hyperpolarizations in the presence of indomethacin and N(omega)-nitro-l-arginine, which thus were attributed to EDHF, in human mesenteric arteries. The EDHF-mediated relaxations were significantly inhibited by catalase, an enzyme that specifically decomposes H(2)O(2), whereas catalase did not affect endothelium-independent hyperpolarizations to levcromakalim. Exogenous H(2)O(2) elicited relaxations and hyperpolarizations in endothelium-stripped arteries. Gap junction inhibitor 18alpha-glycyrrhetinic acid partially inhibited, whereas inhibitors of cytochrome P450 did not affect the EDHF-mediated relaxations. These results indicate that H(2)O(2) is also a primary EDHF in human mesenteric arteries with some contribution of gap junctions.     

Leukemia inhibitory factor: Recent advances and implications in biotechnology

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with several functions in health and disease ranging from inflammation to cancer. LIF is also a potential target and/or therapeutic agent for diseases such as multiple sclerosis, stroke and even psychological disorders, where the function of LIF as a neurotrophic factor has only recently been explored. In recent years, a limited number of LIF clinical trials have been completed, which partially explains the shortage of effective applications as a therapeutic agent. With the increasing interest from biotechnology companies producing recombinant LIF, this status quo will certainly change, and the potential impact of LIF in terms of disease diagnosis, treatment and management will be realized.     

Leukemia inhibitory factor and interleukin-11: Critical regulators in the establishment of pregnancy

Blastocyst implantation into a receptive endometrium is critical to the establishment of pregnancy and is tightly regulated by factors within the blastocyst–endometrial micro-environment. Leukemia inhibitory factor (LIF) and interleukin-11 (IL11) have key roles during implantation. Female mice with a null mutation in the LIF or IL11RA gene are infertile due to a complete failure of implantation or a defective differentiation/decidualization response to the implanting blastocyst, respectively. LIF and IL11 deficiency during pregnancy is associated with infertility and miscarriage in women. Numerous cell populations at the maternal–fetal interface are regulated by LIF/IL11 including the endometrial epithelium, decidualizing stroma, placental trophoblasts and leukocytes. This review focuses on the roles of LIF/IL11 during early pregnancy and highlights their potential as contraceptive targets and therapeutic agents for infertility.     

Homology modelling and H NMR studies of human leukaemia inhibitory factor

Human leukaemia inhibitory factor (LIF) is a glycoprotein with a diverse range of activities on many cell types. A molecular model of LIF has been constructed based mainly on the structure of the related cytokine granulocyte colony-stimulating factor, and refined using simulated annealing and molecular dynamics in water. The model was stable during molecular dynamics refinement and is consistent with known stereochemical data on proteins. It has been assessed by comparison with ¹H NMR data on the ionization behaviour of the six histidine residues in LIF, the imidazolium pK_a values of which range from 3.6 to 7.4. These pK_a values were assigned to individual histidine residues from NMR studies on a series of His → Ala mutants. The environments of the histidine residues in the model account very well for their observed ionization behaviour. Furthermore, the model is consistent with mutagenesis studies which have defined a group of amino acid residues involved in receptor binding.     

Characterization of the leukemia inhibitory factor receptor in the goldfish (Carassius auratus)

The cytokine leukemia inhibitory factor (LIF) and its receptor LIFR have been extensively characterized in mammals. LIF has been shown to mediate the proliferation, differentiation and activation of a number of cell types in various tissues. This paper reports on the identification of a novel LIFR isolated from goldfish (Carassius auratus) macrophages. Goldfish LIFR shares a 26% amino acid sequence identity with mammalian LIFR sequences; however it retains all of the conserved amino acid motifs that identify a functional LIFR such as the cytokine binding domains and the box-1 and box-2 motifs. The goldfish LIFR phylogenetically groups with the other identified LIFRs from human, mouse, rat and chicken, and it appears to be ancestral to the divergence of the oncostatin M receptor (OSMR). The tissue expression of goldfish LIFR is observed in the gill, kidney and brain as well as sorted goldfish macrophages which exhibit higher expression than monocytes and early progenitor cells.     

Leukemia inhibitory factor influences the timing of programmed synapse withdrawal from neonatal muscles

We show that leukemia inhibitory factor (LIF) plays a physiological role in the programmed withdrawal of synapses form neonatal muscles. First, LIF mRNA is present in embryonic skeletal muscle and is developmentally regulated. We detect high levels of LIF mRNA at embryonic day 17 (E17) in mouse hind leg muscles. The content of LIF mRNA falls 10-fold between E17 and birth and then remains low in the neonate and adult. The decrease in LIF mRNA in skeletal muscle coincides with the end of secondary myogenesis and the completion of the adult number of myofibers. Second, treatment of the mouse tensor fascia latae (TFL), a superficial muscle of the hind leg, with LIF from birth (100 ng/day), transiently delays the withdrawal of excess inputs from polyneuronally innervated myofibers by approximately 3 days. The midpoint of the process is shifted from 7.5 ± 0.5 to 10.2 ± 0.6 days of age. LIF treatment delays synapse withdrawal by altering its timing without an appreciable effect on its rate. Third, in mice homozygous for a distruption of the LIF gene, the midpoint in the reduction of multiply innervated TFL myofibers occurs 1 day earlier, at 6.5 ± 0.5 days of age. Muscle fiber number is unchanged in LIF null mice. Treatment with LIF does not alter the rate of neonatal growth, the number of muscle fibers in the TFL, or the reappearance of inputs that have been eliminated. Instead, LIF appears to delay maturation of the motor unit by transiently delaying the onset of synapse withdrawal. We hypothesize that this is a necessary component of a selective process that will operate simultaneously and equally on multiple, competing motor units. © 1995 John Wiley & Sons, Inc.     

Adaptive mechanism of Echinochloa crus-galli Beauv. var. formosensis Ohwi under salt stress: Effect of salicylic acid on salt sensitivity

Our previous study revealed that salicylic acid (SA) accumulates in salt-stressed rice (Oryza sativa L. cv. Nipponbare) seedlings, and we hypothesized that the accumulation of SA might potentiate oxidative injury in rice seedlings since the inhibition of SA synthesis alleviated the growth inhibition under high salinity. To further clarify the action of SA under salt stress, we investigated the changes in the SA content, the activities of the antioxidative enzymes, and the effects of exogenous SA on barnyardgrass (Echinochloa crus-galli Beauv. var. formosensis Ohwi), a gramineous weed which shows lower SA content and is more salt tolerant than rice. In E. crus-galli seedlings exposed to high salinity, neither free nor conjugated SA content showed any increase, while the fresh weight of the shoot and chlorophyll fluorescence (Φ_PSII) slightly decreased. When E. crus-galli seedlings were treated with salt after foliar application of SA, the absorbed SA resulted in the enhancement of the salt-induced growth inhibition and a striking reduction of the Φ_PSII value. Catalase (CAT) and superoxide dismutase (SOD) activities of E. crus-galli seedlings were induced by the salt treatment. However, SA pre-treatment suppressed such an induction of CAT activity and further promoted SOD activity, both of which led to the elevation of the leaf hydrogen peroxide (H₂O₂) level. The present results suggested that enlargement of the cellular SA pool facilitates the generation of H₂O₂ through the suppression of CAT activity and through a remarkable promotion of SOD activity, and thereby enhances the oxidative injury caused by salt stress.     

Decreased catalase activity is the underlying mechanism of oxidant susceptibility in glucose-6-phosphate dehydrogenase-deficient erythrocytes

Historically, it has been theorized that the oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes arises as a direct consequence of an inability to maintain cellular gluthione (GSH) levels. This study alternatively hypothesizes that decreased NADPH concentration leads to impaired to catalase activity which, in turn, underlies the observed oxidant susceptibility. To investigate this hypothesis, normal and G6PD-deficient erythrocytes and hemolysates were challenged with a H₂O₂-generating agent. The results of this study demonstrated that catalase activity was severely impaired upon H₂O₂ challenge in the G6PD-deficient cell whiel only decrease was observed in normal cells. Supplmentation of either normal or G6PD-deficient hemolysates with purified NADPH was found to significantly (P < 0.001) inhibit catalase inactivation upon oxidant challenge while addition of NADP⁺ had no effect. Analysis of these results demonstrated direct correlation between NADPH concentration and catalase activity (r = 0.881) and an inverse correlation between catalase activity and erythrocyte oxidant sensitivity (r = 0.906). In contrast, no correlation was found to exist between glutathione concentration (r = 0.170) and oxidant sensitivity. Analysis of NADPH/NADPt ration in acatalasemic mouse erythrocytes demonstrated that NADPH maintenance alone was not sufficient to explain oxidant resistance, and that catalase activity was required. This study supports the hypothesis that impaired catalase activity underlies the enhanced oxidant sensitivity of G6PD-deficient erythrocytes and elucidates the importance of NADPH in the maintenance of normal catalase activity.     

Hepatocyte growth factor prevents multiple organ injuries in endotoxemic mice through a heme oxygenase-1-dependent mechanism

Acute renal failure (ARF) and acute respiratory distress syndrome (ARDS) are still lethal diseases during sepsis, whereas heme oxygenase-1 (HO-1) elicits a host defense response to sepsis. Herein, we provide evidence that hepatocyte growth factor (HGF) prevents ARF and ARDS via enhanced induction of HO-1. Lipopolysaccharide (LPS)-treated mice manifested renal and pulmonary injuries similar to those observed in septic patients, while HGF enhanced the HO-1 induction in renal tubular cells and in lung macrophages. As a result, onsets of ARF and ARDS were blocked by HGF in septic mice. Notably, an HO-1 inhibitor (SnPP) diminished the protective effects of HGF on LPS-induced organ injuries. Furthermore, the inhibitory effect of HGF on up-regulation of interleukin-1β and interleukin-18 was largely restored by SnPP. This is the first report showing that “growth factor therapy” successfully inhibits both ARDS and ARF during endotoxemia, partially via HO-1-dependent suppression of hyper-cytokinemia.     

Leukemia inhibitory factor coordinates the down-regulation of the visual cycle in the retina and retinal-pigmented epithelium

Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-cis-retinal chromophore to photoreceptors in vivo. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-cis-retinal.     

Leukemia inhibitory factor contributes to hepatocyte-like differentiation of human bone marrow mesenchymal stem cells

The current majority of protocols for hepatocyte differentiation of mesenchymal stem cells (MSCs) are conducted using oncostatin M (OSM) as an inducer of hepatocyte-like maturation. As leukemia inhibitory factor (LIF) and OSM share similar signaling pathways, we examined whether LIF could play a role in the hepatocyte differentiation process. A differentiation protocol was designed using LIF as a maturation cytokine and this was compared with standard and control protocols applied to human MSCs of bone marrow origin. We observed that mesenchymal-derived hepatocyte-like cells (MDHLCs) acquired similar morphological changes when exposed to LIF or to OSM. Using protein and gene expression assays, we noticed a comparable hepatic marker expression in both differentiation conditions. Furthermore, LIF and OSM allowed the acquisition of equivalent levels of hepatocyte-like functionality as attested by evaluation of urea secretion and glycogen deposition. However, no increase in the expression of hepatocyte-like features could be observed in MDHLCs after a combined exposition to LIF and OSM. In conclusion, we demonstrated that LIF can play a similar role as OSM in the hepatocyte differentiation process of human MSCs.     

Association of macrophage migration inhibitory factor (MIF) polymorphisms with risk of meningitis from Streptococcus pneumoniae

Macrophage migration inhibitory factor (MIF) is an upstream proinflammatory cytokine encoded by a functionally polymorphic locus. This study of 119 patients explored the potential relationship between MIF genotype and invasive Streptococcus pneumoniae infections. We observed an association between a high-expression MIF allele and occurrence of pneumococcal meningitis.     

Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution

The chemical shift assignments and secondary structure of a murine–human chimera,MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa,have been determined from multidimensional heteronuclear NMR spectra acquired on auniformly 13C,15N-labelled sample. Secondary structure elements were defined on the basisof chemical shifts, NH-CH coupling constants, medium-range NOEs and the location ofslowly exchanging amide protons. The protein contains four -helices, the relativeorientations of which were determined on the basis of long-range, interhelical NOEs. The fourhelices are arranged in an up-up-down-down orientation, as found in other four-helical bundlecytokines. The overall topology of MH35-LIF is similar to that of the X-ray crystallographicstructure for murine LIF [Robinson et al. (1994) Cell, 77, 1101–1116]. Differencesbetween the X-ray structure and the solution structure are evident in the N-terminal tail, wherethe solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystalstructure and the small antiparallel -sheet encompassing residues in the N-terminus andCD loop in the crystal structure is less stable.     

The oxidative mechanism of action of ortho-quinone inhibitors of protein-tyrosine phosphatase alpha is mediated by hydrogen peroxide

Here, we report the identification and characterization of five ortho-quinone inhibitors of PTPalpha. We observed that the potency of these compounds in biochemical assays was markedly enhanced by the presence of DTT. A kinetic analysis suggested that they were functioning as irreversible inhibitors and that the inhibition was targeted to the catalytic site of PTPalpha. The inhibition observed by these compounds was sensitive to superoxide dismutase and catalase, suggesting that reactive oxygen species may be mediators of their inhibition. We observed that in the presence of DTT, these compounds would produce up to 2.5mM hydrogen peroxide (H(2)O(2)). The levels of H(2)O(2) produced were sufficient to completely inactivate PTPalpha. In contrast, without a reducing agent the compounds did not generate H(2)O(2) and showed little activity towards PTPalpha. In addition, these compounds inhibited PTPalpha-dependent cell spreading in NIH 3T3 cells at concentrations that were similar to their activity in biochemical assays. The biological implications of these results are discussed as they support growing evidence that H(2)O(2) is a key regulator of PTPs.     

Leukemia inhibitory factor inhibits the proliferation of gastric cancer by inducing G1-phase arrest

Leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family, plays a complex role in cancer. LIF inhibits the proliferation and survival of several myeloid leukemia cells but promotes tumor progression and metastasis in many solid tumors. However, the relationship between LIF and gastric cancer has not been well understood. LIF was downregulated in gastric cancer as detected by western blot analysis and immunohistochemistry (IHC). Notably, LIF was downregulated in approximately 70% (56/80) of primary gastric cancers, in which it was significantly associated with advanced clinical stage, lymph node metastasis, and poor overall survival (median 5-year survival = 26 vs. 43 months for patients with high LIF expression and low LIF expression gastric cancer, respectively). To study the potential function of LIF in the downregulation of gastric cancer, we monitored the behavior using proliferation, cell cycle, and flow cytometry analysis. Overexpression of LIF inhibited the gastric cancer cell cycle in the G1 phase. In our experiment, overexpression of LIF by lentivirus upregulated P21 and downregulated cyclin D1. Recombinant human LIF also downregulated P21 and cyclin D1 at various times. A further in vivo tumor formation study in nude mice indicated that overexpression of LIF in gastric cancer significantly delayed the progress of tumor formation. These findings indicate that LIF may serve as a negative regulator of gastric cancer.     

ortho-Quinone tanshinones directly inhibit telomerase through an oxidative mechanism mediated by hydrogen peroxide

The tanshinone natural products possess a variety of pharmacological properties including anti-bacterial, anti-inflammatory, anti-oxidant, and anti-neoplastic activity. The molecular basis of these effects, however, remains largely unknown. In the present study, we explored the direct effect of tanshinones on the enzyme telomerase. Telomerase is up-regulated in the majority of cancer cells and is essential for their survival, making it a potential anti-cancer drug target. We found that the ortho-quinone tanshinone II-A inhibits telomerase in a time- and DTT-dependent fashion, and the hydrogen peroxide scavenger catalase protected telomerase from inactivation. These findings demonstrate that ortho-quinone containing tanshinones can inhibit telomerase owing to their ability to generate reactive oxygen species. The results also provide evidence that telomerase is directly and negatively regulated by reactive oxygen species.     

Apoptosis-inducing factor plays a critical role in caspase-independent, pyknotic cell death in hydrogen peroxide-exposed cells

It has been proposed that continuously generated hydrogen peroxide (H₂O₂) inhibits typical apoptosis and instead initiates an alternate, apoptosis-inducing factor (AIF)-dependent process. Aside from the role of AIF, however, the detailed morphological characterization of H₂O₂-induced cell death is not complete. This study examined the cellular mechanism(s) by which the continuous presence of H₂O₂ induces cell death. We also further analyzed the precise role of AIF by inhibiting its expression with siRNA. Exposure of cells to H₂O₂ generated by glucose oxidase caused mitochondrion-mediated, caspase-independent cell death. In addition, H₂O₂ exposure resulted in cell shrinkage and chromatin condensation without nuclear fragmentation, indicating that H₂O₂ stimulates a pyknotic cell death. Further analysis of AIF-transfected cells clearly demonstrated that nuclear translocation of AIF is the most important event required for nuclear condensation, phosphatidyl serine translocation, and ultimately cell death in H₂O₂-exposed cells. Furthermore, ATP was rapidly and severely depleted in cells exposed to H₂O₂ generated by glucose oxidase but not by H₂O₂ added as a bolus. Suppression of the H₂O₂-mediated ATP depletion by 3-aminobenzamide led to a significant increase of nuclear fragmentation in glucose oxidase-exposed cells. Collectively, these findings suggest that an acute energy reduction by H₂O₂ causes caspase-independent and AIF-dependent cell death.