AP-2 complex contributes to hyphal-tip-localization of a chitin synthase in the filamentous fungus Aspergillus nidulans.

Filamentous fungi maintain hyphal growth to continually internalize membrane proteins related to cell wall synthesis, transporting them to the hyphal tips. Endocytosis mediates protein internalization via target recognition by the adaptor protein 2 complex (AP-2 complex). The AP-2 complex specifically promotes the internalization of proteins important for hyphal growth, and loss of AP-2 complex function results in abnormal hyphal growth. In this study, deletion mutants of the genes encoding the subunits of the AP-2 complex (α, ß2, µ2, or σ2) in the filamentous fungus Aspergillus nidulans resulted in the formation of conidiophores with abnormal morphology, fewer conidia, and activated the cell wall integrity pathway. We also investigated the localization of ChsB, which plays pivotal roles in hyphal growth in A. nidulans, in the Δµ2 strain. Quantitative analysis suggested that the AP-2 complex is involved in ChsB internalization at subapical collar regions. The absence of the AP-2 complex reduced ChsB localization at the hyphal tips. Our findings suggest that the AP-2 complex contributes to cell wall integrity by properly localizing ChsB to the hyphal tips.

Type II phosphatidylserine decarboxylase is crucial for the growth and morphogenesis of the filamentous fungus Aspergillus nidulans.

Phosphatidylserine decarboxylases (PSDs) catalyze the production of phosphatidylethanolamine (PE) from phosphatidylserine (PS) and are crucial for the maintenance of PE levels in fungi. The PSDs are classified into two types; the type I PSDs are conserved from bacteria to humans, while the type II PSDs exist only in fungi and plants. In yeasts, the deletion of type I PSD-encoding genes causes severe growth retardation. In contrast, the deletion of type II PSD-encoding genes has little or no effect. In this study, we found four genes encoding type II PSD orthologs in the filamentous fungus Aspergillus nidulans; these included psdB, psdC, psdD, and psdE. Deletion of psdB caused severe growth defects on minimal medium and these defects were partially restored by the addition of ethanolamine, choline, PE, or phosphatidylcholine into the medium. The conidiation efficiency of the psdB deletion mutant was dramatically decreased and its conidiophore structures were aberrant. In the psdB deletion mutant, the PE content decreased while the PS content increased. We further showed that PsdB had a major PSD activity. Our findings suggest that the type II PSDs exert important roles in the phospholipid homeostasis, and in the growth and morphogenesis of filamentous fungi.

Effects of Pyrrole-Imidazole Polyamides Targeting Human TGF-ß1 on the Malignant Phenotypes of Liver Cancer Cells.

Synthetic pyrrole-imidazole (PI) polyamides bind to the minor groove of double-helical DNA with high affinity and specificity, and inhibit the transcription of corresponding genes. In liver cancer, transforming growth factor (TGF)-ß expression is correlated with tumor grade, and high-grade liver cancer tissues express epithelial-mesenchymal transition markers. TGF-ß1 was reported to be involved in cancer development by transforming precancer cells to cancer stem cells (CSCs). This study aimed to evaluate the effects of TGF-ß1-targeting PI polyamide on the growth of liver cancer cells and CSCs and their TGF-ß1 expression. We analyzed TGF-ß1 expression level after the administration of GB1101, a PI polyamide that targets human TGF-ß1 promoter, and examined its effects on cell proliferation, invasiveness, and TGF-ß1 mRNA expression level. GB1101 treatment dose-dependently decreased TGF-ß1 mRNA levels in HepG2 and HLF cells, and inhibited HepG2 colony formation associated with downregulation of TGF-ß1 mRNA. Although GB1101 did not substantially inhibit the proliferation of HepG2 cells compared to untreated control cells, GB1101 significantly suppressed the invasion of HLF cells, which displayed high expression of CD44, a marker for CSCs. Furthermore, GB1101 significantly inhibited HLF cell sphere formation by inhibiting TGF-ß1 expression, in addition to suppressing the proliferation of HLE and HLF cells. Taken together, GB1101 reduced TGF-ß1 expression in liver cancer cells and suppressed cell invasion; therefore, GB1101 is a novel candidate drug for the treatment of liver cancer.

Cell division cycle 34 is highly expressed in hepatitis C virus-positive hepatocellular carcinoma with favorable phenotypes.

Despite tremendous efforts to develop curative agents, there are few effective drugs for the treatment of hepatocellular carcinoma (HCC). This is predominantly due to the variations in individual HCC cases. As numerous HCC cases have no mutations in known tumor-associated genes, identification of novel genes involved in the development and progression of human cancers is considered to be an urgent issue. In the present study, surgical specimens of HCC were analyzed for the expression patterns of ubiquitin-conjugating enzyme, cell division cycle 34 (CDC34), which is hypomethylated in its promoter region and exhibits elevated expression levels in mouse skin tumors. The results of the current study clearly indicated that the elevated CDC34 expression level in cancerous regions was significantly associated with favorable clinicopathological features, such as reduced alanine aminotransferase (ALT) levels and histological grades. Similarly, a higher T/N ratio, which is the ratio of CDC34 expression in HCCs to that in non-tumorous tissues, was significantly associated with favorable features, such as a lower indocyanin green retention rate after 15 min (ICG15R), reduced α-fetoprotein and smaller tumor size. These results indicate that the CDC34 expression level in HCC is a marker for predicting the HCC prognosis and that CDC34 acts as a tumor suppressor.

Production of low-Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR-Cas system.

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below µm), cesium (Cs+ ) can be taken up by crops and transported to their edible parts. This plant capacity to take up Cs+ from low concentrations has notably affected the production of rice (Oryza sativa L.) in Japan after the nuclear accident at Fukushima in 2011. Several strategies have been put into practice to reduce Cs+ content in this crop species such as contaminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management, with limited impact or pernicious side-effects. Conversely, the development of biotechnological approaches aimed at reducing Cs+ accumulation in rice remain challenging. Here, we show that inactivation of the Cs+ -permeable K+ transporter OsHAK1 with the CRISPR-Cas system dramatically reduced Cs+ uptake by rice plants. Cs+ uptake in rice roots and in transformed yeast cells that expressed OsHAK1 displayed very similar kinetics parameters. In rice, Cs+ uptake is dependent on two functional properties of OsHAK1: (i) a poor capacity of this system to discriminate between Cs+ and K+ ; and (ii) a high capacity to transport Cs+ from very low external concentrations that is likely to involve an active transport mechanism. In an experiment with a Fukushima soil highly contaminated with 137 Cs+ , plants lacking OsHAK1 function displayed strikingly reduced levels of 137 Cs+ in roots and shoots. These results open stimulating perspectives to smartly produce safe food in regions contaminated by nuclear accidents.

Search for aflatoxin and trichothecene production inhibitors and analysis of their modes of action.

Mycotoxin contamination of crops is a serious problem throughout the world because of its impact on human and animal health as well as economy. Inhibitors of mycotoxin production are useful not only for developing effective methods to prevent mycotoxin contamination, but also for investigating the molecular mechanisms of secondary metabolite production by fungi. We have been searching for mycotoxin production inhibitors among natural products and investigating their modes of action. In this article, we review aflatoxin and trichothecene production inhibitors, including our works on blasticidin S, methyl syringate, cyclo(L-Ala-L-Pro), respiration inhibitors, and precocene II.

Inhibitory effects of respiration inhibitors on aflatoxin production.

Aflatoxin production inhibitors, which do not inhibit the growth of aflatoxigenic fungi, may be used to control aflatoxin without incurring a rapid spread of resistant strains. A respiration inhibitor that inhibits aflatoxin production was identified during a screening process for natural, aflatoxin-production inhibitors. This prompted us to evaluate respiration inhibitors as potential aflatoxin control agents. The inhibitory activities of four natural inhibitors, seven synthetic miticides, and nine synthetic fungicides were evaluated on aflatoxin production in Aspergillus parasiticus. All of the natural inhibitors (rotenone, siccanin, aptenin A5, and antimycin A) inhibited fungal aflatoxin production with IC50 values around 10 µM. Among the synthetic miticides, pyridaben, fluacrypyrim, and tolfenpyrad exhibited strong inhibitory activities with IC50 values less than 0.2 µM, whereas cyflumetofen did not show significant inhibitory activity. Of the synthetic fungicides, boscalid, pyribencarb, azoxystrobin, pyraclostrobin, and kresoxim-methyl demonstrated strong inhibitory activities, with IC50 values less than 0.5 µM. Fungal growth was not significantly affected by any of the inhibitors tested at concentrations used. There was no correlation observed between the targets of respiration inhibitors (complexes I, II, and III) and their IC50 values for aflatoxin-production inhibitory activity. This study suggests that respiration inhibitors, including commonly used pesticides, are useful for aflatoxin control.

Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis.

A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of (13)C-labeled diC8PC ((methyl-(13)C)3-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-(13)C)3-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.

DNA hypermethylation of zygote arrest 1 (ZAR1) in hepatitis C virus positive related hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common human malignancies in the world, and its prognosis is generally poor. Epigenetic alteration such as DNA methylation has been shown to be important in the development of human cancers including HCC. Here, we analyzed the methylation status of ZAR1, which has been reported to be aberrantly methylated in a few human cancers. METHODS: We investigated the methylation status of ZAR1 in 88 HCV-positive HCC and matched nontumorous liver tissue samples and 4 normal liver tissue samples used as a control using MassARRAY EpiTYPER. Further statistical analysis was performed to determine the relationship between methylation level and patient clinicopathological features and prognosis. RESULTS: CpG islands in ZAR1 exon 1 showed a higher methylation level in all 88 HCC than in nontumorous tissues. The hypermethylation group, whose cancer tissues showed a twofold or higher methylation level compared with nontumorous tissues, showed a significantly higher serum AFP (p = 0.018) and lower serum albumin (p = 0.001) and single rather than multiple tumors (p = 0.031) compared with the hypomethylation group. Multivariate regression analyses were performed to identify which of the following factors were the predictors of the hypermethylation group: serum albumin, AFP, and tumor multiplicity. This study showed that patients who had Zar1 hypermethylation in the HCC tissues had a significantly lower serum albumin level than those in the hypomethylation group (p = 0.007). CONCLUSION: Although it is still unknown how ZAR1 hypermethylation affects HCC development, it could be a potential marker to detect HCV-related HCC.

Involvement of Golgi-associated retrograde protein complex in the recycling of the putative Dnf aminophospholipid flippases in yeast.

It is widely accepted that phosphatidylethanolamine (PE) is enriched in the cytosolic leaflet of the eukaryotic plasma membranes. To identify genes involved in the establishment and regulation of the asymmetric distribution of PE on the plasma membrane, we screened the deletion strain collection of the yeast Saccharomyces cerevisiae for hypersensitive mutants to the lantibiotic peptide Ro09-0198 (Ro) that specifically binds to PE on the cell surface and inhibits cellular growth. Deletion mutants of VPS51, VPS52, VPS53, and VPS54 encoding the components of Golgi-associated retrograde protein (GARP) complex, YPT6 encoding a Rab family small GTPase that functions with GARP complex, RIC1 and RGP1 encoding its guanine nucleotide exchange factor (GEF), and TLG2 encoding t-SNARE exhibited hypersensitivity to Ro. The mutants deleted for VPS51, VPS52, VPS53, and VPS54 were impaired in the uptake of fluorescently labeled PE. In addition, aberrant intracellular localization of the EGFP-tagged Dnf2p, the putative inward-directed phospholipid translocase (flippase) of the plasma membrane, was observed in the mutant defective in the GARP complex, Ypt6p, its GEF proteins, or Tlg2p. Our results suggest that the GARP complex is involved in the recycling of Dnf flippases.

High TSC22D3 and low GBP1 expression in the liver is a risk factor for early recurrence of hepatocellular carcinoma.

Recurrence after liver resection for hepatocellular carcinoma (HCC) is a major clinical problem, and prognostic markers for recurrence are urgently required. For 390 HCC cases, segmented linear regression analysis with two segments was performed, and the interval for the early and late recurrence groups was partitioned at the crosspoint (676 days). We investigated whether gene expression in non-tumorous tissues of remnant liver from 39 hepatitis C virus-positive HCC cases may be associated with early recurrence of this disease. By microarray analysis, 21 genes were identified as candidate recurrence-associated genes. Further gene expression analysis was performed, and the localization and expression of the gene products of these candidate genes were immunohistochemically evaluated. Low expression of the GBP1 gene and high expression of the TSC22D3 gene were significantly (both P=0.04) associated with the risk of early recurrence. Through backward step-wise multivariate logistic regression analysis for the 21 candidate genes, high expression of GBP1 reduced [odds ratio (OR)=0.20; 95% confidence interval (CI) 0.06-0.73, P=0.02] and high expression of TSC22D3 increased the risk of early recurrence (OR=19.6; 95% CI 1.14-337.2; P=0.04). Immunohistochemical analysis revealed that hepatocytes showed strong membranous expression for GBP1 in the late recurrence group, but weak membranous expression for GBP1 in the early recurrence group. TSC22D3 was frequently expressed in lymphocytes and in a few hepatocytes in tissues of the early recurrence group. Our observations suggest that the combination of the high expression of the TSC22D3 gene and low expression of the GBP1 gene in the non-tumorous tissue of the remnant liver is significantly associated with early recurrence after surgical resection of HCC.

Inhibition of MMP-9 transcription and suppression of tumor metastasis by pyrrole-imidazole polyamide.

Matrix metalloproteinase (MMP)-9, the 92-kDa type IV collagenase, contributes to tumor invasion and metastases, and strategies to down-regulate its expression could ultimately be of clinical utility. A pyrrole-imidazole (PI) polyamide that targets the activator protein-1 (AP-1)-binding site of the MMP-9 promoter was designed and synthesized as a gene-silencing agent for tumor metastases. The synthesized product showed selective DNA binding ability. The MMP-9 PI polyamide significantly inhibited MMP-9's mRNA expression, protein level, and enzymatic activity in human breast adenocarcinoma cells (MDA-MB-231). Furthermore, the MMP-9 PI polyamide inhibited migration and invasion by in vitro wound-healing and matrigel-invasion assay. The FITC-labeled PI polyamide was localized in nuclei in 45 min of incubation with an MDA-MB-231 cell and remained in the nuclei for up to 96 h after incubation in vitro. It was also quickly localized in the mouse cellular nuclei of many tissues, including liver, kidney, and spleen, after intravenous injection without using any drug-delivery system. Moreover, the polyamide treatment significantly decreased metastasis in a mouse model of liver metastasis. Our results suggest that this PI polyamide, which targets the MMP-9 gene promoter, can be a novel MMP-9 down-regulating molecule for antimetastasis.

Hepatocyte growth factor suppresses ischemic cerebral edema in rats with microsphere embolism.

The present study was aimed at determining whether human recombinant hepatocyte growth factor (HGF) ameliorates cerebral edema induced by microsphere embolism (ME). Rats were injected with 700 microspheres (48 microm in diameter). Continuous administration of HGF at 13 microg/3 days/animal into the right ventricle was started from 10 min after embolism to the end of the experiment by using an osmotic pump. On day 3 after the ME, the rats were anesthetized, and their brains were perfused with an isotonic mannitol solution to eliminate constituents in the vascular and extracellular spaces. Thereafter, tissue water and cation contents were determined. A significant increase in tissue water content of the right hemisphere by ME was seen. This ME-induced increase in water content was associated with increases in tissue sodium and calcium ion contents and decreases in tissue potassium and magnesium ion contents of the right hemisphere. The treatment of the animal with HGF suppressed the increases in water and sodium and calcium ion contents, but not the decreases in potassium and magnesium ion contents. These results suggest that HGF suppresses the formation of ischemic cerebral edema provoked intracellularly in rats with ME.

Hepatocyte growth factor improves synaptic localization of the NMDA receptor and intracellular signaling after excitotoxic injury in cultured hippocampal neurons.

To examine the effects of HGF on synaptic densities under excitotoxic conditions, we investigated changes in the number of puncta detected by double immunostaining with NMDA receptor subunits and presynaptic markers in cultured hippocampal neurons. Exposure of hippocampal neurons to excitotoxic NMDA (100 muM) decreased the synaptic localization of NMDA receptor subunit NR2B, whereas synaptic NR1 and NR2A clusters were not altered. Colocalization of PSD-95, a scaffolding protein of the receptor, with the presynaptic protein synapsin I was also decreased after excitotoxicity. Treatment with HGF attenuated these decreases in number. The decrease in the levels of surface NR2B subunits following the addition of the excitotoxic NMDA was also attenuated by the HGF treatment. The decrease in CREB phosphorylation in response to depolarization-evoked NMDA receptor activation was prevented by the HGF treatment. These results suggest that HGF not only prevented neuronal cell death but also attenuated the decrease in synaptic localization of NMDA receptor subunits and prevented intracellular signaling through the NMDA receptor.

[Ischemic brain injury and hepatocyte growth factor].

Cerebral ischemia causes an irreversible and neurodegenerative disorder that may lead to progressive dementia and global cognitive deterioration. Since the overall process of ischemic brain injuries is extremely complex, treatment with endogenous multifunctional factors would be better choices for preventing complicated ischemic brain injuries. Hepatocyte growth factor, HGF, is a multifunctional cytokine originally identified and purified as a potent mitogen for hepatocyte. The activation of the c-Met/HGF receptor evokes diverse cellular responses, including mitogenic, morphogenic, angiogenic and anti-apoptotic activities in various types of cell. Previous studies showed that HGF and c-Met were expressed in various brain regions under normal conditions and that HGF enhanced the survival of hippocampal and cortical neurons during the aging of cells in culture. The protective effects of HGF on in vivo ischemic brain injuries and their mechanisms have not fully understood. To elucidate therapeutic potencies of HGF for ischemic brain injuries, we examined effects of HGF on ischemia-induced learning and memory dysfunction, neuronal cell death and endothelial cell damage by using the 4-vessel occlusion model and the microsphere embolism model in rats. Our findings suggested that treatment with HGF was capable of protecting hippocampal neurons against ischemia-induced cell death through the prevention of apoptosis-inducing factor translocation to the nucleus. Furthermore, we demonstrated that HGF had the ability to prevent tissue degeneration and improved learning and memory function after cerebral embolism, possibly through prevention of cerebral vessel injuries. As HGF has a potent cerebroprotective effect, it could be a prospective agent for the therapy against complicated ischemic brain diseases.

Hepatocyte growth factor promotes the number of PSD-95 clusters in young hippocampal neurons.

Hepatocyte growth factor (HGF) and its receptor are expressed in various regions of the brain and have protective effects against excitotoxic injuries. However, their effects on synapse formation remain to be elucidated. To determine whether HGF has the ability to alter synaptic function during development, we investigated changes in the number of synapse detected by double immunostaining for NMDA receptor subunits and a presynaptic marker in cultured young hippocampal neurons. Whereas application of HGF increased the number of cluster of synapsin, a presynaptic protein, the clusters of NMDA receptor subunits NR1 and NR2B were not altered. Interestingly, colocalization of PSD-95, a scaffolding protein of the receptor, with synapsin was increased by HGF treatment without a change in the total amount of it. In addition, we investigated the expression of surface NMDA receptor, neuroligin, and neurexin, which were assessed by use of a cell-surface biotinylation assay. The application of HGF did not change the surface expression of these proteins. Furthermore, we determined the release of glutamate in response to depolarization. Treatment with HGF promoted depolarization-evoked release of glutamate. These results suggest that HGF modulates the expression of the scaffolding protein of the NMDA receptor at the synapse and promotes maturation of excitatory synapses in young hippocampal neurons.

Hepatocyte growth factor attenuates cerebral ischemia-induced increase in permeability of the blood-brain barrier and decreases in expression of tight junctional proteins in cerebral vessels.

Hepatocyte growth factor (HGF) exerts its physiological activities as that of an organotropic factor for regeneration and can prevent ischemia-induced injuries; however, its effect and mechanism of action under in vivo pathophysiological conditions remains to be determined. Recently, we demonstrated that treatment with human recombinant HGF (hrHGF) attenuated the disruption of the blood-brain barrier (BBB) observed after microsphere embolism-induced sustained cerebral ischemia. To see if tight junctional proteins were involved in this attenuation, in the present study, we investigated the effects of HGF on the levels of occludin and zonula occludens (ZO)-1 in cerebrovascular endothelial cells after microsphere embolism. Sustained cerebral ischemia was induced by the injection of 700 microspheres (48 microm diameter) into the right internal carotid artery of rats. hrHGF was injected into the right ventricle of the brain by using an osmotic pump at a dose of 30 microg/7 days per animal. The levels of tight junctional proteins in the endothelial cells were examined by immunohistochemical analysis. Treatment with hrHGF attenuated the decrease in the expression of occludin and ZO-1 proteins in the endothelial cells that occurred after sustained cerebral ischemia. Furthermore, treatment with hrHGF resulted in retention of these tight junctional proteins in fluorescein isothiocyanate (FITC)-albumin-perfused cerebral vessels, which did not leak FITC-albumin in the ipsilateral cortex. These results suggest that HGF-mediated maintenance of the tight junctional proteins in the endothelial cells may be a possible mechanism for the protective effect of HGF against the disruption of the BBB after cerebral ischemia.

The protective effect of hepatocyte growth factor against cell death in the hippocampus after transient forebrain ischemia is related to the improvement of apurinic/apyrimidinic endonuclease/redox factor-1 level and inhibition of NADPH oxidase activity.

Early oxidative DNA damage is regarded to be an initiator of neuronal apoptotic cell death after cerebral ischemia. Although evidence suggests that HGF has the ability to protect cells from oxidative stress, it remains unclear as to how HGF suppresses oxidative DNA damage after cerebral ischemia. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) is a multifunctional protein in the DNA base repair pathway that is responsible for repairing apurinic/apyrimidinic sites in DNA after oxidation. We demonstrated that both the immunoreactivity and the number of APE/Ref-1-positive cells in the hippocampal CA1 region were decreased after transient forebrain ischemia and that treatment with HGF suppressed this reduction. The expression of Cu/ZnSOD and MnSOD in the hippocampal CA1 region did not change after ischemia, regardless of treatment with or not with HGF. The activity of NADPH oxidase was increased mainly in glia-like cells in the hippocampal CA1 region after ischemia, and this increase was attenuated by HGF treatment. These results suggest that the protective effects of HGF against cerebral ischemia-induced cell death in the hippocampal CA1 region are related to the improvement of neuronal APE/Ref-1 expression and the inhibition of NADPH oxidase activity in glia-like cells.

Prevention of apoptosis-inducing factor translocation is a possible mechanism for protective effects of hepatocyte growth factor against neuronal cell death in the hippocampus after transient forebrain ischemia.

Hepatocyte growth factor (HGF) is one of the prospective agents for therapy against a variety of neurologic and neurodegenerative disorders, although the precise mechanisms for the effect of HGF remain to be elucidated. We showed that treatment with HGF protected hippocampal cornu ammonis (CA) subregion 1 neurons from apoptotic cell death after transient forebrain ischemia. Accumulating evidence indicates that ischemia-induced neuronal damage occurs via caspase-independent pathways. In the present study, we focused on the localization of apoptosis-inducing factor (AIF), which is an important protein in the signal-transduction system through caspase-independent pathways, to investigate the possible mechanism for the protective effect of HGF after transient forebrain ischemia. Hepatocyte growth factor attenuated the increase in the expression of AIF protein in the nucleus after transient forebrain ischemia. We further explored the upstream components of AIF translocation. Primary DNA damage induced by Ca(2+) influx and subsequent NO formation are thought to be the initial events for AIF translocation, which results in the subsequent DNA damage by AIF. Hepatocyte growth factor prevented the primary oxidative DNA damage, as was estimated by using anti-8-OHdG (8-hydroxy-2'-deoxyguanosine) antibody. Oxidative DNA damage after ischemia is known to lead to the activation of poly(ADP-ribose) polymerase (PARP) and p53, resulting in AIF translocation. Marked increases in the PAR polymer formation and the expression of p53 protein after ischemia were effectively prevented by HGF treatment. In the present study, we first showed that HGF was capable of preventing neuronal cell death by inhibiting the primary oxidative DNA damage and then preventing the activation of the PARP/p53/AIF pathway.

Effects of hepatocyte growth factor on phosphorylation of extracellular signal-regulated kinase and hippocampal cell death in rats with transient forebrain ischemia.

Hepatocyte growth factor (HGF) has been implicated in protection against several types of cell injuries. We investigated the effects of human recombinant HGF (hrHGF) on the selective neuronal cell death in the hippocampal CA1 region after transient forebrain ischemia in rats and explored the nature of the intracellular signaling pathway for the protection against this neuronal injury. hrHGF was injected continuously into the hippocampal CA1 region directly using an osmotic pump from 10 min to 72 h after the start of reperfusion. The marked increase in the number of TUNEL-positive cells found in the CA1 region after ischemia was almost completely abolished by the hrHGF treatment. Akt phosphorylation as well as IkappaB phosphorylation, which has been implicated in events downstream of the Akt, was not affected by hrHGF treatment. Extracellular signal-regulated kinase (ERK) phosphorylation was decreased in the CA1 region with time after ischemia. hrHGF increased or recovered ERK phosphorylation without changing the total amount of ERK protein. Immunohistochemical analysis demonstrated that phosphorylated ERK was colocalized with a neuronal nucleus marker NeuN in the hippocampal CA1 region of ischemic rats with hrHGF treatment at the early period after reperfusion. These results suggest that the protective effects of hrHGF against neuronal death in the hippocampal CA1 after transient forebrain ischemia could be related to an ERK-dependent pathway.