Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation.

Adhesion G-protein-coupled receptors (aGPCRs) bear notable similarity to Notch proteins1, a class of surface receptors poised for mechano-proteolytic activation2-4, including an evolutionarily conserved mechanism of cleavage5-8. However, so far there is no unifying explanation for why aGPCRs are autoproteolytically processed. Here we introduce a genetically encoded sensor system to detect the dissociation events of aGPCR heterodimers into their constituent N-terminal and C-terminal fragments (NTFs and CTFs, respectively). An NTF release sensor (NRS) of the neural latrophilin-type aGPCR Cirl (ADGRL)9-11, from Drosophila melanogaster, is stimulated by mechanical force. Cirl-NRS activation indicates that receptor dissociation occurs in neurons and cortex glial cells. The release of NTFs from cortex glial cells requires trans-interaction between Cirl and its ligand, the Toll-like receptor Tollo (Toll-8)12, on neural progenitor cells, whereas expressing Cirl and Tollo in cis suppresses dissociation of the aGPCR. This interaction is necessary to control the size of the neuroblast pool in the central nervous system. We conclude that receptor autoproteolysis enables non-cell-autonomous activities of aGPCRs, and that the dissociation of aGPCRs is controlled by their ligand expression profile and by mechanical force. The NRS system will be helpful in elucidating the physiological roles and signal modulators of aGPCRs, which constitute a large untapped reservoir of drug targets for cardiovascular, immune, neuropsychiatric and neoplastic diseases13.

Functional diversity of PFKFB3 splice variants in glioblastomas.

Tumor cells tend to metabolize glucose through aerobic glycolysis instead of oxidative phosphorylation in mitochondria. One of the rate limiting enzymes of glycolysis is 6-phosphofructo-1-kinase, which is allosterically activated by fructose 2,6-bisphosphate which in turn is produced by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2 or PFKFB). Mounting evidence suggests that cancerous tissues overexpress the PFKFB isoenzyme, PFKFB3, being causing enhanced proliferation of cancer cells. Initially, six PFKFB3 splice variants with different C-termini have been documented in humans. More recently, additional splice variants with varying N-termini were discovered the functions of which are to be uncovered. Glioblastoma is one of the deadliest forms of brain tumors. Up to now, the role of PFKFB3 splice variants in the progression and prognosis of glioblastomas is only partially understood. In this study, we first re-categorized the PFKFB3 splice variant repertoire to simplify the denomination. We investigated the impact of increased and decreased levels of PFKFB3-4 (former UBI2K4) and PFKFB3-5 (former variant 5) on the viability and proliferation rate of glioblastoma U87 and HEK-293 cells. The simultaneous knock-down of PFKFB3-4 and PFKFB3-5 led to a decrease in viability and proliferation of U87 and HEK-293 cells as well as a reduction in HEK-293 cell colony formation. Overexpression of PFKFB3-4 but not PFKFB3-5 resulted in increased cell viability and proliferation. This finding contrasts with the common notion that overexpression of PFKFB3 enhances tumor growth, but instead suggests splice variant-specific effects of PFKFB3, apparently with opposing effects on cell behaviour. Strikingly, in line with this result, we found that in human IDH-wildtype glioblastomas, the PFKFB3-4 to PFKFB3-5 ratio was significantly shifted towards PFKFB3-4 when compared to control brain samples. Our findings indicate that the expression level of distinct PFKFB3 splice variants impinges on tumorigenic properties of glioblastomas and that splice pattern may be of important diagnostic value for glioblastoma.

Prognostic Value of PFKFB3 to PFKFB4 mRNA Ratio in Patients With Primary Glioblastoma (IDH-Wildtype).

A hallmark of glioblastoma is the high level of aerobic glycolysis. PFKFB3 and PFKFB4 are regulatory glycolytic enzymes, which are overexpressed in glioblastomas. Selective inhibition of these enzymes has emerged as a new approach in tumor therapy. We investigated the ratios of PFKFB3 to PFKFB4 mRNA expression in 66 astrocytic tumors of different malignancy grades. PFKFB3 mRNA levels were considerably higher than those of PFKFB4 in all analyzed tumors. IDH-wildtype glioblastomas showed lower PFKFB3 to PFKFB4 mRNA ratios (7.7:1) than IDH-mutant low-grade astrocytomas (36.5:1), indicating a dependency of the ratio on malignancy grade. In IDH-wildtype glioblastomas exhibiting loss of heterozygosity (LOH) of the PFKFB3 gene locus, the decrease of PFKFB3 mRNA levels was accompanied by lower PFKFB4 mRNA levels, but the PFKFB3 to PFKFB4 mRNA ratio did not differ between tumors with or without PFKFB3 LOH. IDH-wildtype primary glioblastoma patients with high PFKFB3 to PFKFB4 mRNA ratios above the average of 7.7:1 had a significantly longer overall survival time (14 months) than patients with lower ratios (9 months). Our results indicate that low PFKFB3 to PFKFB4 expression ratio is a poor prognostic factor in patients with IDH-wildtype primary glioblastoma and that PFKFB3 and PFKFB4 might represent promising targets for astrocytoma and glioblastoma treatment.

Modulation of GLO1 Expression Affects Malignant Properties of Cells.

The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect) characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO). Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1) that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.

Ethyl Pyruvate Combats Human Leukemia Cells but Spares Normal Blood Cells.

Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3ß. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors.

Expression of vascular endothelial growth factor (VEGF) mRNA, VEGF receptor 2 (Flk-1) mRNA, and of VEGF co-receptor neuropilin (Nrp)-1 mRNA in brain tissue of aging Tg2576 mice by in situ hybridization.

Vascular endothelial growth factor (VEGF) has been characterized as a heparin binding angiogenic growth factor displaying high specificity for endothelial cells. It is profoundly accumulated and co-localized with amyloid beta (Aß) plaques in the brain of Alzheimer's disease patients. In order to examine the effect of Aß plaques on the expression level of VEGF mRNA and its receptors, brain tissue of both transgenic Tg2576 and wild type mice at ages ranging from 13 to 22 months was subjected to in situ hybridization followed by densitometric assessment using computer-assisted image analysis. Strong expression of VEGF mRNA, fetal liver kinase (Flk)-1 mRNA, and neuropilin (Nrp)-1 mRNA in the piriform, entorhinal, somatosensory, frontal cortex and hippocampal formation of both transgenic and non-transgenic mice brain was detected. Developmentally, only expression of VEGF mRNA was increased with age in the entorhinal, and somatosensory cortex of wild type mice. In 20-month-old transgenic Tg2576 mice, up-regulation of VEGF mRNA, Flk-1 mRNA, and Nrp-1 mRNA transcripts was observed in the entorhinal cortex compared to age-matched wild type mice. Our data suggest up-regulation of VEGF mRNA, Flk-1 mRNA and Nrp-1 mRNA, at least in the entorhinal cortex at ages when Aß deposition in Tg2576 is typically increasing.

Exploring glyoxalase 1 expression in prostate cancer tissues: targeting the enzyme by ethyl pyruvate defangs some malignancy-associated properties.

BACKGROUND: The glyoxalase (GLO)1 is part of a ubiquitous detoxification system in the glycolytic pathway of normal and tumor cells. It protects against cellular damage caused by cytotoxic metabolites. METHODS: Aiming at exploring the role of GLO1 in prostate cancer, we evaluated and targeted the expression of GLO1 in prostate cancer tissues and cell lines and analyzed its correlation with grading systems and tumor growth indices. RESULTS: Immunohistochemical studies on 37 prostate cancer specimens revealed a positive correlation between Helpap-grading and the cytoplasmic (P = 0.002)/nuclear (P = 0.006) GLO1 level. A positive correlation between Ki-67 proliferation marker and the cytoplasmic GLO1 (P = 0.006) was evident. Furthermore, the highest GLO1 level was detected in the androgen-sensitive LNCaP compared to the androgen-independent Du-145 and PC-3 prostate cell lines and the breast cancer cell MCF-7, both at protein and mRNA level. Treating cancer cells with ethyl pyruvate was found to defang some malignancy-associated properties of cancer cells including proliferation, invasion and anchorage-independent growth. In vitro results revealed that the potency of ethyl pyruvate is increased when cells are metabolically activated by growth stimulators, for example, by fetal calf serum, dihydrotestosterone, tumor growth factor-ß1 and leptin. CONCLUSIONS: The positive correlation of GLO1 expression level in prostate cancer tissues with the pathological grade and proliferation rate may assign GLO1 as a risk factor for prostate cancer development and progression. Furthermore, our data indicate that inhibitors of GLO1 might be useful to decelerate the cancer cell growth by a novel therapeutic approach that we may call "induced metabolic catastrophe."

Glycerophosphoglycerol, Beta-alanine, and pantothenic Acid as metabolic companions of glycolytic activity and cell migration in breast cancer cell lines.

In cancer research, cell lines are used to explore the molecular basis of the disease as a substitute to tissue biopsies. Breast cancer in particular is a very heterogeneous type of cancer, and different subgroups of cell lines have been established according to their genomic profiles and tumor characteristics. We applied GCMS metabolite profiling to five selected breast cancer cell lines and found this heterogeneity reflected on the metabolite level as well. Metabolite profiles of MCF-7 cells belonging to the luminal gene cluster proved to be more different from those of the basal A cell line JIMT-1 and the basal B cell lines MDA-MB-231, MDA-MB-435, and MDA-MB-436 with only slight differences in the intracellular metabolite pattern. Lactate release into the cultivation medium as an indicator of glycolytic activity was correlated to the metabolite profiles and physiological characteristics of each cell line. In conclusion, pantothenic acid, beta-alanine and glycerophosphoglycerol appeared to be related to the glycolytic activity designated through high lactate release. Other physiological parameters coinciding with glycolytic activity were high glyoxalase 1 (Glo1) and lactate dehydrogenase (LDH) enzyme activity as well as cell migration as an additional important characteristic contributing to the aggressiveness of tumor cells. Metabolite profiles of the cell lines are comparatively discussed with respect to known biomarkers of cancer progression.

Effect of VEGF and its receptor antagonist SU-5416, an inhibitor of angiogenesis, on processing of the ß-amyloid precursor protein in primary neuronal cells derived from brain tissue of Tg2576 mice.

A large number of Alzheimer patients demonstrate cerebrovascular pathology, which has been assumed to be related to ß-amyloid (Aß) deposition. Aß peptides have been described to inhibit angiogenesis both in vitro and in vivo, and deregulation of angiogenic factors may contribute to various neurological disorders including neurodegeneration. One of the key angiogenic factor is the vascular endothelial growth factor (VEGF). Increased levels of VEGF have been observed in brains of Alzheimer patients, while the functional significance of VEGF up-regulation in the pathogenesis and progression of AD is still a matter of debate. To test whether VEGF may affect neuronal APP processing, primary neuronal cells derived from brain tissue of E16 embryos of Tg2576 mice were exposed with 1 ng/ml VEGF for 6, 12, and 24h, followed by monitoring formation and secretion of soluble Aß peptides, release of the human APP cleavage products, sAPPßswe and sAPPα, into the culture medium as well as the activities of α- and ß-secretases in neuronal cell extracts. Exposure of primary neuronal cells by VEGF for 24h led to slightly reduced sAPPß release, accompanied by decreased ß-secretase activity 12h after VEGF exposure. Incubation of neurons by the VEGF receptor antagonist and angiogenesis inhibitor SU-5416 for 24h resulted in increased release of sAPPßswe, and strikingly enhanced secretion of Aß peptides into the culture medium, which was accompanied by a significant increase in ß-secretase activity, as compared to control incubations. The SU-5416-induced effects on APP processing could not be suppressed by the additional presence of VEGF, suggesting that SU-5416 affects pathways that are apparently independent of VEGF receptor signaling. The data obtained indicate that VEGF-driven mechanisms may affect APP processing, suggesting a link of angiogenesis and pathogenesis of Alzheimer's disease.

Alpha2-macroglobulin inhibits the malignant properties of astrocytoma cells by impeding beta-catenin signaling.

Targets that could improve the treatment of brain tumors remain important to define. This study of a transformation-associated isoform of alpha2-macroglobulin (A2M*) and its interaction with the low-density lipoprotein receptor-related protein-1 (LRP1) suggests a new mechanism for abrogating the malignant potential of astrocytoma cells. LRP1 bound A2M* found to be associated with an inhibition of tumor cell proliferation, migration, invasion, spheroid formation, and anchorage-independent growth. Transcriptional studies implicated effects on the Wnt/beta-catenin signaling pathway. Notably, LRP1 antibodies could phenocopy the effects of A2M*. Our findings suggest a pathway of tumor suppression in astrocytoma that might be tractable to therapeutic exploitation.

Aberrant methylation of human L- and M-fructose 1,6-bisphosphatase genes in cancer.

A possible epigenetic regulation of the two isoenzymes of fructose 1,6-bisphosphatase (FBPase) was studied in liver, muscle, mamma, breast cancer and in different cancer cell lines. Results obtained after bisulfite sequencing revealed a different CpG methylation of both promoters in liver, muscle and breast tissue which is putatively involved in the cell-type specific gene expression of the two enzymes. In tumor cell lines, demethylation with 5-aza-deoxycytidine activated the expression of both isoenzymes. Additional inhibition of histone deacetylase with trichostatin A further increased FBPase mRNA concentrations. Since cancers typically have an abnormal energy metabolism and exhibit a low gluconeogenic phenotype, it was studied whether promoter methylation contributes to the decreased expression of FBPase in breast cancer. When non-malignant and malignant tissue samples from the same patient were compared a correlation between an increase of FBPase promoter methylation and a decrease of FBPase mRNA levels was observed.

Curcumin inhibits glyoxalase 1: a possible link to its anti-inflammatory and anti-tumor activity.

BACKGROUND: Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor. METHODOLOGY/PRINCIPAL FINDINGS: Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (K(i) = 5.1+/-1.4 microM). Applying a whole blood assay, IC(50) values of pro-inflammatory cytokine release (TNF-alpha, IL-6, IL-8, IL-1beta) were found to be positively correlated with the K(i)-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. CONCLUSIONS/SIGNIFICANCE: The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

Ethyl pyruvate and ethyl lactate down-regulate the production of pro-inflammatory cytokines and modulate expression of immune receptors.

Esters of alpha-oxo-carbonic acids such as ethyl pyruvate (EP) have been demonstrated to exert inhibitory effects on the production of anti-inflammatory cytokines. So far, there is no information about effects, if any, of ethyl lactate (EL), an obviously inactive analogue of EP, on inflammatory immune responses. In the present study, we provide evidence that the anti-inflammatory action of alpha-oxo-carbonic acid esters is mediated by inhibition of glyoxalases (Glo), cytosolic enzymes that catalyse the conversion of alpha-oxo-aldehydes such as methylglyoxal (MGO) into the corresponding alpha-hydroxy acids using glutathione as a cofactor. In vitro enzyme activity measurements revealed the inhibition of human Glo1 by alpha-oxo-carbonic acid esters, whilst alpha-hydroxy-carbonic acid esters such as EL were not inhibitory. In contrast, both EP and EL were shown to suppress the Lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and IL-8 from human immunocompetent cells, and modulated the expression of the immune receptors HLA-DR, CD14 and CD91 on human monocytes. Here, we show a crossing link between glyoxalases and the immune system. The results described herein introduce glyoxalases as a possible target for therapeutic approaches of immune suppression.

Oligomeric beta-amyloid(1-42) induces the expression of Alzheimer disease-relevant proteins in cholinergic SN56.B5.G4 cells as revealed by proteomic analysis.

Alzheimer's disease (AD) is characterized by cholinergic dysfunction and progressive basal forebrain cell loss which has been hypothesized to be associated with extensive accumulation of beta-amyloid (Abeta). To reveal whether oligomeric Abeta displays a particular toxicity for cholinergic neurons, the cholinergic cell line SN56.B5.G4 (SN56) was used as a model. Recently performed microarray analyses demonstrated that genes affected by exposure of SN56 cells with 50 microM oligomeric Abeta(1-42) for 24 h were involved in protein modification and degradation [Heinitz, K., Beck, M., Schliebs, R., Perez-Polo, J.R., 2006. Toxicity mediated by soluble oligomers of beta-amyloid(1-42) on cholinergic SN56.B5.G4 cells. J. Neurochem. 98, 1930-1945]. Using a proteomic approach, we compared the levels of proteins and specially of phosphorylated proteins in cytosolic fractions of cell lysates from cholinergic SN56 cells exposed to 50 microM Abeta(1-42) for 24h to those in control incubations. We show here that the levels of calreticulin, and mitogen-activated protein kinase (MAPK) kinase 6c were up-regulated in cholinergic SN56 cells exposed to Abeta(1-42), while gamma-actin appeared down-regulated. Abeta(1-42) exposure of cholinergic SN56 cells led to decreased phosphorylation of phosphoproteins, such as the Rho GDP dissociation inhibitor, the ubiquitin carboxyl terminal hydrolase-1, and the tubulin alpha-chain isotype Malpha6, as compared to untreated control lysates. The proteins identified have also been reported to be affected in brains of AD patients, suggesting a potential role of Abeta in influencing the integrity and functioning of the proteome in AD.

Developmental impairments of select neurotransmitter systems in brains of Cln3(Deltaex7/8) knock-in mice, an animal model of juvenile neuronal ceroid lipofuscinosis.

The neuronal ceroidlipofuscinoses (NCL) are a group of neurodegenerative disorders and are the most common lysosomal storage diseases of infancy and childhood. Juvenile NCL is caused by CLN3 mutation, producing retinal degeneration, uncontrollable seizures, cognitive and motor decline, and early death before the age of 30 years. To study the pathogenetic mechanisms of the disease, Cln3 knock-in mice (Cln3(Deltaex7/8)) have been generated, which reproduce the 1.02-kb deletion in the CLN3 gene observed in more than 85% of juvenile NCL patients. To characterize the impact of the common Cln3 mutation on development of autofluorescent storage material, gliosis, glucose metabolism, oxidative stress, and transmitter receptors during postnatal brain maturation, brain tissue of Cln3(Deltaex7/8) mice at the ages of 3, 4, 5, 6, 9, and 19 months was subjected to immunocytochemistry to label gliotic markers and nitric oxide synthases; photometric assays to assess enzyme activities of glycolysis and antioxidative defense systems; and level of reactive nitrogen species as well as quantitative receptor autoradiography to detect select cholinergic, glutamatergic, and GABAergic receptor subtypes. The developmental increase in cerebral cortical autofluorescent lipofuscin-like deposition is accompanied by a significant astro- and microgliosis, increased activities of lactate dehydrogenase and phosphofructokinase, decreased level of glutathione peroxidase, enhanced amount of reactive nitrogen species, and lowered binding levels of N-methyl-D-aspartate- and M1-muscarinic acetylcholine receptors in select brain regions but hardly in GABA(A) receptor sites compared with wild-type mice. Detailed elucidation of the sequence of pathological events during postnatal development highlights new potential strategies for symptomatic treatment of the disease.

Expression of purinergic receptors in the hypothalamus of the rat is modified by reduced food availability.

ATP-sensitive P2 receptors are suggested to play an important role in the cerebral signal transduction. We examined the expression of the P2Y1 receptor and the possibly downstream-related neuronal nitric oxide synthase (nNOS) in the hypothalamus of rats food-restricted for 3 or 10 days and rats refed after a restriction of 10 days. The restriction caused a reduction of the body weight and plasma triacylglyceride, an increase of non-esterified fatty acid levels correlating with a decrease of leptin levels and an enhancement of plasma corticosterone. All changes returned to basal levels after refeeding. The restriction induced an enhanced intake within 30 min after food presentation and a reduction in the latency. Interestingly, the latter was not abolished by refeeding. The daily food intake induced by refeeding was enhanced at the first day only. The expression of hypothalamic P2Y1 receptor/nNOS mRNA and protein and of leptin receptor mRNA were enhanced after restricted feeding. These changes were abolished after 3 days of refeeding. Immunofluorescence studies indicated that P2Y1 receptor and nNOS immunoreactivities are present in the dorsomedial, ventromedial and lateral hypothalamus and in the nucleus arcuatus. P2Y1 receptor-positive cells were partially also nNOS-positive. The P2Y1 receptor labeling was restricted to cell bodies of obviously non-glial cells, whereas nNOS labeling could be detected also at cellular processes of these cells. In the nucleus arcuatus, astrocytes were identified, expressing P2Y1 receptors at cell bodies and cellular processes. The data suggest that restricted feeding may enhance the sensitivity of the hypothalamus to extracellular ADP/ATP by regulation of the expression of P2Y1 receptors and possibly of their signal transduction pathway via nitric oxide production.

Differential protein expression in brain capillary endothelial cells induced by hypoxia and posthypoxic reoxygenation.

Cerebral ischemia causes functional alteration of the blood-brain barrier, formed by brain capillary endothelial cells (BCEC). Changes in protein expression and activity of selected differentially expressed enzymes were investigated in BCEC subjected to hypoxia (24 h) alone or followed by a 24-h reoxygenation. BCEC proteins were isolated, separated by 2-DE, and identified by MALDI-MS. Computer-based 2-D gel analysis identified 21 up-regulated proteins and 4 down-regulated proteins after hypoxia alone and 9 proteins that were further up-regulated after posthypoxic reoxygenation. The expression of the majority of hypoxia-induced proteins was reduced toward control levels during reoxygenation. The most prominent changes were identified for glycolytic enzymes (e.g., phosphoglycerate kinase), proteins of the ER (e.g., calreticulin), and cytoskeletal (e.g., vimentin) proteins. The results indicate that BCEC respond to hypoxia/reoxygenation by adaptive up-regulation of proteins involved in the glycolysis, protein synthesis, and stress response.

Characterization of the human P-type 6-phosphofructo-1-kinase gene promoter in neural cell lines.

In humans three isoforms of 6-phosphofructo-1-kinase (PFK) exist. Among them platelet-type PFK (PFKP) is highly abundant in the brain. With its distinct allosteric properties PFKP is regarded to be the key enzyme for the regulation of glycolysis in this organ. We cloned 1.7 kb of the 5' upstream promoter of the human PFKP gene and analyzed the promoter activity by deletion and mutation analysis using a luciferase reporter. The transcription start point was determined at 48 bp upstream of the start codon. In deletion studies the region -65 to +48 turned out to be sufficient for promoter activity while fragment -153 to +48 showed the highest promoter activity. Sequence analysis of the region from -153 to +48 revealed a stretch of eight adjacent putative transcription factor binding sites, seven of which are Sp-family specific sites. Sp1 and Sp3 were shown to bind to most if not all of them. Additionally, an NF-Y binding site was identified. Results of deletion and mutation analysis suggest that all of these transcription factors contribute positively to promoter activity. The methylation status of the promoter region was analyzed in different neural tumor cell lines and compared with that in human leukocytes and muscle.

Aging-related increase in oxidative stress correlates with developmental pattern of beta-secretase activity and beta-amyloid plaque formation in transgenic Tg2576 mice with Alzheimer-like pathology.

The molecular mechanisms of beta-amyloidogenesis in sporadic Alzheimer's disease are still poorly understood. To reveal whether aging-associated increases in brain oxidative stress and inflammation may trigger onset or progression of beta-amyloid deposition, a transgenic mouse (Tg2576) that express the Swedish double mutation of human amyloid precursor protein (APP) was used as animal model to study the developmental pattern of markers of oxidative stress and APP processing. In Tg2576 mouse brain, cortical levels of soluble beta-amyloid (1-40) and (1-42) steadily increased with age, but significant deposition of fibrillary beta-amyloid in cortical areas did not occur before postnatal age of 10 months. The slope of increase in cerebral cortical beta-secretase (BACE1) activities in Tg2576 mice between ages of 9 and 13 months was significantly higher as compared to that of the alpha-secretase, while the expression level of BACE1 protein and mRNA did not change with age. The activities of superoxide dismutase and glutathione peroxidase in cortical tissue from Tg2576 mice steadily increased from postnatal age 9-12 months. The levels of cortical nitric oxide, and reactive nitrogen species demonstrated peak values around 9 months of age, while the level of interleukin-1beta steadily increased from postnatal month 13 onwards. The developmental temporal coincidence of increased levels of reactive nitrogen species and antioxidative enzymes with the onset of beta-amyloid plaque deposition provides further evidence that developmentally and aging-induced alterations in brain oxidative status exhibit a major factor in triggering enhanced production and deposition of beta-amyloid, and potentially predispose to Alzheimer's disease.

Glial cell expression of hepatocyte growth factor in vitreoretinal proliferative disease.

The hepatocyte growth factor (HGF) has been crucially implicated in the development of proliferative retinal diseases; however, it is unclear whether retinal glial cells express or respond to HGF. Therefore, we examined the expression of HGF and of the receptor for HGF, c-Met, by immunohistochemical costaining with glial fibrillary acidic protein (GFAP) in epiretinal membranes of patients with proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), respectively. Furthermore, it was determined whether cells of the human retinal glial cell line, MIO-M1, secrete HGF protein, and whether HGF stimulates proliferation and chemotaxis, and secretion of the vascular endothelial growth factor (VEGF). Neuroretinas of patients with PVR express elevated mRNA level for HGF in comparison to control retinas. In epiretinal membranes of patients with PVR or PDR, immunoreactivity for HGF and for c-Met, respectively, partially colocalized with immunoreactivity for GFAP. Fetal bovine serum and basic fibroblast growth factor, but not heparin-binding epidermal or platelet-derived growth factors, evoked HGF secretion by cultured retinal glial cells. HGF displayed only a marginal effect on cell proliferation while it stimulated chemotaxis. HGF promoted the secretion of VEGF, via activation of the phosphatidylinositol-3 kinase. It is concluded that glial cells in epiretinal membranes express both HGF protein and c-Met receptors. The results suggest an autocrine/paracrine role of HGF in glial cell responses during proliferative vitreoretinal disorders as well as in retinal neovascularization, by stimulating of VEGF release.