The suppression of maternal-fetal leukemia inhibitory factor signal relay pathway by maternal immune activation impairs brain development in mice.

Recent studies in rodents suggest that maternal immune activation (MIA) by viral infection is associated with schizophrenia and autism in offspring. Although maternal IL-6 is though t to be a possible mediator relating MIA induced these neuropsychiatric disorders, the mechanism remains to be elucidated. Previously, we reported that the maternal leukemia inhibitory factor (LIF)-placental ACTH-fetal LIF signaling relay pathway (maternal-fetal LIF signal relay) promotes neurogenesis of fetal cerebrum in rats. Here we report that the maternal-fetal LIF signal relay in mice is suppressed by injection of polyriboinosinic-polyribocytidylic acid into dams, which induces MIA at 12.5 days post-coitum. Maternal IL-6 levels and gene expression of placental suppressor of cytokine signaling 3 (Socs3) increased according to the severity of MIA and gene expression of placental Socs3 correlated with maternal IL-6 levels. Furthermore, we show that MIA causes reduction of LIF level in the fetal cerebrospinal fluid, resulting in the decreased neurogenesis in the cerebrum. These findings suggest that maternal IL-6 interferes the maternal-fetal LIF signal relay by inducing SOCS3 in the placenta and leads to decreased neurogenesis.

Melanocortins contribute to sequential differentiation and enucleation of human erythroblasts via melanocortin receptors 1, 2 and 5.

In this study, we showed that adrenocorticotropic hormone (ACTH) promoted erythroblast differentiation and increased the enucleation ratio of erythroblasts. Because ACTH was contained in hematopoietic medium as contamination, the ratio decreased by the addition of anti-ACTH antibody (Ab). Addition of neutralizing Abs (nAbs) for melanocortin receptors (MCRs) caused erythroblast accumulation at specific stages, i.e., the addition of anti-MC2R nAb led to erythroblast accumulation at the basophilic stage (baso-E), the addition of anti-MC1R nAb caused accumulation at the polychromatic stage (poly-E), and the addition of anti-MC5R nAb caused accumulation at the orthochromatic stage (ortho-E). During erythroblast differentiation, ERK, STAT5, and AKT were consecutively phosphorylated by erythropoietin (EPO). ERK, STAT5, and AKT phosphorylation was inhibited by blocking MC2R, MC1R, and MC5R, respectively. Finally, the phosphorylation of myosin light chain 2, which is essential for the formation of contractile actomyosin rings, was inhibited by anti-MC5R nAb. Taken together, our study suggests that MC2R and MC1R signals are consecutively required for the regulation of EPO signal transduction in erythroblast differentiation, and that MC5R signal transduction is required to induce enucleation. Thus, melanocortin induces proliferation and differentiation at baso-E, and polarization and formation of an actomyosin contractile ring at ortho-E are required for enucleation.

Significance of sugar chain recognition by galectins and its involvement in disease-associated glycosylation.

Galectins are ß-galactoside-binding lectins that participate in a wide range of biological processes. Galectins are distributed both inside and outside cells and are believed to have roles in both intra- and extracellular milieus. One of the well-recognized functions of galectins is stabilization of glycoproteins on the cell surface, thereby promoting stable signal transduction and transport of substances such as glucose. Glycoprotein-associated diseases, including congenital disorder of glycosylation (CDG, previously called carbohydrate-deficient glycoprotein syndrome), comprise a disease family established only in the last decade. Although numerous in vitro glycobiology studies have been performed, including investigation of glycan-galectin interactions and of galectin action in cultured cells, a few in vivo studies have investigated molecular mechanisms of galectin actions in animal models. Both in vitro and in vivo studies are needed in order to better determine the biological significance of sugar chain recognition. Hitherto, some reports have focused on the role of impaired sugar chain recognition and galectin function in the development of diverse diseases, including rheumatoid arthritis, diabetes mellitus, colitis, and cancer. We recently focused on the function of galectins in immunity and embryogenesis, and in this review we summarize the diseases related to disorders of sugar chain-galectin interaction and discuss the role of galectins as potential risk factors for some congenital and acquired diseases. These diseases are disorders of immunity, metabolism, and cell differentiation. This approach to understanding the significance of sugar chain recognition by galectins may open up a new field into the nature of glycoprotein-related diseases, including CDG.

Effects of melanocortins on fetal development.

Melanocortins, adrenocorticotropic hormone (ACTH) and α-, ß-, and γ-melanocyte-stimulating hormone (MSH) are produced in the placenta and secreted into embryos/fetuses. ACTH concentrations are higher in fetal plasma than in maternal plasma and peak at mid-gestation in rats, whereas ACTH production starts in the anterior lobe of the fetal pituitary at later stages. Melanocortin receptors (MC1-5R), receptors for ACTH and α-, ß- and γ-MSH, are expressed in various adult organs. The specific function of these receptors has been well examined in the hypothalamic-pituitary-adrenocortical (HPA) axis and the HPA axis-like network in the skin, and anti-inflammatory effects for white blood cells have also been investigated. MC2R and/or MC5R are also expressed in the testis, lung, kidney, adrenal, liver, pancreas, brain and blood cells at different stages in mouse and rat embryos/fetuses. Melanocortins in embryos and fetuses promote maturation of the HPA axis and also contribute to the development of lung, testis, brain and blood cells. Recently, a unique ACTH function was revealed in fetuses: placental ACTH, which is secreted by the maternal leukemia inhibitory factor (LIF), and induces LIF secretion from fetal nucleated red blood cells. Finally, the maternal LIF-placental ACTH-fetal LIF signal relay regulates the LIF level and promotes neurogenesis in fetuses, which suggests that ACTH acts as a signal transducer or effector for fetal development in the maternal-fetal signal pathway.

Maternal leukemia inhibitory factor (LIF) promotes fetal neurogenesis via a LIF-ACTH-LIF signaling relay pathway.

Leukemia inhibitory factor (LIF) promotes the proliferation of neuronal progenitor cells in the cerebrum. However, it remains unclear how fetal LIF level is regulated. Here we show evidence that maternal LIF signals drive fetal LIF levels via the placenta, thereby promoting neurogenesis in the fetal brain in rats. Chronological changes showed that LIF concentration in fetal sera (FS) and fetal cerebrospinal fluid peaked at gestational day (GD) 15.5, after the peak of maternal LIF at GD14.5. LIF injection into rat dams at GD15.5 increased the level of ACTH in FS and subsequently increased LIF levels in FS and fetal cerebrospinal fluid. The elevation of fetal LIF after LIF injection into dams was inhibited by in utero injection of anti-ACTH antibody into fetuses. Cultured syncytiotrophoblasts, which express the LIF receptor and glycoprotein 130, were induced to secrete ACTH and up-regulate Pomc expression by the addition of LIF. Nucleated red blood cells from fetuses at GD15.5, but not GD13.5 or GD17.5, displayed LIF secretion in response to ACTH. Moreover, injection of LIF into dams at GD13.5 or GD17.5 did not result in elevation of ACTH or LIF in fetuses. The labeling index of 5-bromo-2'-deoxyuridine-positive cells in the ventricular zone of the cerebral neocortex increased 24 h after injection of LIF into dams at GD15.5 but not GD13.5 or GD17.5. These results suggest that in rats maternal LIF induces ACTH from the placenta, which in turn induces fetal nucleated red blood cells to secrete LIF that finally increases neurogenesis in fetuses around GD15.

Mitochondrial voltage-dependent anion channels (VDACs) as novel pharmacological targets for anti-cancer agents.

Recently, it was demonstrated that some anti-cancer agents used mitochondrial voltage-dependent anion channels (VDAC1-3 isoforms) as their pharmacological target. VDACs are expressed more highly in cancer cells than normal cells; thus the VDAC-dependent cytotoxic agents can have cancer-selectivity. Furanonaphthoquinones (FNQs) induced caspase-dependent apoptosis via the production of NADH-dependent reactive oxygen species (ROS) by VDAC1. The ROS production and the anti-cancer activity of FNQs were increased by VDAC1 overexpression. Meanwhile, erastin induced RAS-RAF-MEK-dependent non-apoptotic cell death via VDAC2. On the other hand, VDACs were needed for transporting ATP to hexokinase (HK), which was highly expressed in cancer cells. We hypothesized that the high glycolysis might induce up-regulation of VDAC. In this review, we propose that VDACs are novel candidates for effective pharmacological targets of anti-cancer drugs.

Bioreductive activation of quinone antitumor drugs by mitochondrial voltage-dependent anion channel 1.

The authors recently demonstrated that the mitochondrial voltage-dependent anion channel 1 (VDAC1) is involved in the sensitivity of cancer cells to furanonaphthoquinone (FNQ). The aim of the present study was to investigate whether mitochondrial VDAC1 reduces quinone antitumor drugs. The VDAC1 purified by immunoprecipitation reduced FNQ in the presence of nicotinamide adenine dinucleotide (NADH) and produced H(2)O(2). Blue native polyacrylamide gel electrophoresis demonstrated that the band that reduced FNQ NADH-dependently mainly included VDAC1. Because H(2)O(2) generation in catalyzing FNQ with NADH caused mitochondrial damage, the cytotoxic activity of FNQ was induced by VDAC1. In the quinone antitumor drugs, menadione (VK3), adriamycin and mitomycin C, mitochondrial VDAC1 bioreductively activated VK3. These results demonstrate that mitochondrial VDAC1 is a pharmacologic target for the treatment of tumor.

Furanonaphthoquinones cause apoptosis of cancer cells by inducing the production of reactive oxygen species by the mitochondrial voltage-dependent anion channel.

The mitochondrial production of reactive oxygen species has been implicated in the anticancer activity of furanonaphthoquinone. However, the mechanism of the activation remains elusive. In the current study, we found that treatment of HeLa cells with 2-methyl-5(or -8)-hydroxy-furanonaphthoquinone (FNQ13) induces mitochondrial swelling, followed by apoptosis. This toxic effect of FNQ13 was reduced by the radical scavengers alpha-tocopherol and trolox. Cytochemical experiments in isolated mitochondria showed that a combination of FNQ13 and NADH induces the production of H(2)O(2) at the exterior mitochondrial membrane surface. This production of H(2)O(2) was reduced by an antibody to the voltage-dependent anion channel (VDAC). Overexpression of the VDAC by transfection with vdac1 cDNA increased the production of H(2)O(2) by HeLa cells, whereas transfection with a small interfering RNA to VDAC reduced FNQ13-induced H(2)O(2) production and cell death due to an almost complete knockdown of VDAC expression. We also found significant correlations between the expression of VDAC and the induction of H(2)O(2) production and cell death by FNQ13 in 11 human cancer cell lines. These results indicate that the anticancer activity of furanonaphthoquinones depends on the production of reactive oxygen species by mitochondrial permeability transition pores including the VDAC.

The synthetic furanonaphthoquinone induces growth arrest, apoptosis and differentiation in a variety of leukaemias and multiple myeloma cells.

2-methyl-naphtho[2,3-b]furan-4,9-dione (FNQ3), a synthetic analogue of the quinone kigelinone, has demonstrated a real potential for use in the treatment of a variety of solid tumours. Unlike other quinones, such as mitomycin-C and adriamycin, the cytotoxicity of FNQ3 is often 10- to 14-fold more potent towards the tumour cells than their normal counterparts. We report, for the first time, that the drug had activity against a broad spectrum of leukaemias and multiple myeloma cells. It decreased the growth of acute myeloid leukaemia (AML) and multiple myeloma cell lines in a dose-dependent fashion (50% inhibitory concentration approximately 1.25 microg/ml against most of the leukaemia cell lines). This dose apparently initiated mitochondrial collapse as measured by depolarisation of the mitochondrial membrane. FNQ3 potentiated the differentiation of HL-60 myeloid cells in the presence of either 1alpha, 25(OH)(2) dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or all-trans-retinoic acid (ATRA). FNQ3 inhibited the proliferation of primary AML cells while inducing apoptosis. Eleven of 14 (79%) AML marrow samples had a prominent decrease in their clonogenic growth when cultured in the presence of the drug. In summary, this drug has growth inhibitory, apoptotic and differentiative effects against myeloid leukaemias and multiple myeloma cells. FNQ3 may represent a new therapeutic approach to these malignancies.

Mitochondrial damage prior to apoptosis in furanonaphthoquinone treated lung cancer cells.

The mechanisms of the antitumor reactions of 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) to human lung adenocarcinoma A549 cells were investigated. A549 cells that received 1.25 microg/ml FNQ3 (IC(50) at 0.35 microg/ml) developed intensive mitochondrial H(2)O(2) production at 1 h. Selective structural mitochondrial swelling, alteration of mitochondrial membrane potential, and cytochrome c and caspase-9 release from the mitochondria occurred 18-24 h later. alpha-Tocopherol inhibited the alteration of both mitochondrial permeability and the leakage of procaspase-9. The caspase-9 was then activated in the cytosol. The expression of Bcl-2 oncoprotein was suppressed by FNQ3, and resulted in apoptosis. The higher dose of 5 microg/ml induced necrosis via severe mitochondrial breakage. These results showed that FNQ3 targets the mitochondria of A549 cells to produce a reactive oxygen species resulting in apoptosis and necrosis.

Alpha-tocopherol protects cultured human cells from the acute lethal cytotoxicity of dioxin.

The possible protection of cultured human cells from acute dioxin injury by antioxidants was investigated. The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), caused vacuolization of the smooth endoplasmic reticulum and Golgi apparatus in cultured human conjunctival epithelial cells and cervical cancer cells. Subsequent nuclear damage included a deep irregular indentation resulting in cell death. A dosage of 30-40 ng/mL TCDD induced maximal intracellular production of H2O2 at 30 minutes and led to severe cell death (0-31% survival) at two hours. A dose of 1.7 mM alpha-tocopherol or 1 mM L-dehydroascorbic acid significantly protected human cells against acute TCDD injuries (78-97% survivals), but vitamin C did not provide this protection. These results indicate that accidental exposure to fatal doses of TCDD causes cytoplasmic free radical production within the smooth endoplasmic reticular systems, resulting in severe cytotoxicity, and that vitamin E and dehydroascorbic acid can protect against TCDD-induced cell damage.