The role of the glycome in symbiotic host-microbe interactions.

Glycosylation plays a crucial role in many aspects of cell biology, including cellular and organismal integrity, structure-and-function of many glycosylated molecules in the cell, signal transduction, development, cancer, and in a number of diseases. Besides, at the inter-organismal level of interaction, a variety of glycosylated molecules are involved in the host-microbiota recognition and initiation of downstream signalling cascades depending on the outcomes of the glycome-mediated ascertainment. The role of glycosylation in host-microbe interactions is better elaborated within the context of virulence and pathogenicity in bacterial infection processes but the symbiotic host-microbe relationships also involve substantive glycome-mediated interactions. The works in the latter field have been reviewed to a much lesser extent, and the main aim of this mini-review is to compensate for this deficiency and summarise the role of glycomics in host-microbe symbiotic interactions.

Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and ß-catenin signaling.

The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric G(q), G(i), and G(12/13) proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of ß-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and ß-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G(12/13) signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/ß-catenin and Notch1 signaling, and in particular the role of G(q) and G(12/13) in mediating PMT effects on Rho/ROCK and Notch1 signaling.

Selective inhibition of hypoxia-inducible factor (HIF) prolyl-hydroxylase 1 mediates neuroprotection against normoxic oxidative death via HIF- and CREB-independent pathways.

Oxidative stress contributes to tissue injury in conditions ranging from cardiovascular disease to stroke, spinal cord injury, neurodegeneration, and perhaps even aging. Yet the efficacy of antioxidants in human disease has been mixed at best. We need a better understanding of the mechanisms by which established antioxidants combat oxidative stress. Iron chelators are well established inhibitors of oxidative death in both neural and non-neural tissues, but their precise mechanism of action remains elusive. The prevailing but not completely substantiated view is that iron chelators prevent oxidative injury by suppressing Fenton chemistry and the formation of highly reactive hydroxyl radicals. Here, we show that iron chelation protects, rather unexpectedly, by inhibiting the hypoxia-inducible factor prolyl 4-hydroxylase isoform 1 (PHD1), an iron and 2-oxoglutarate-dependent dioxygenase. PHD1 and its isoforms 2 and 3 are best known for stabilizing transcriptional regulators involved in hypoxic adaptation, such as HIF-1alpha and cAMP response element-binding protein (CREB). Yet we find that global hypoxia-inducible factor (HIF)-PHD inhibition protects neurons even when HIF-1alpha and CREB are directly suppressed. Moreover, two global HIF-PHD inhibitors continued to be neuroprotective even in the presence of diminished HIF-2alpha levels, which itself increases neuronal susceptibility to oxidative stress. Finally, RNA interference to PHD1 but not isoforms PHD2 or PHD3 prevents oxidative death, independent of HIF activation. Together, these studies suggest that iron chelators can prevent normoxic oxidative neuronal death through selective inhibition of PHD1 but independent of HIF-1alpha and CREB; and that HIF-2alpha, not HIF-1alpha, regulates susceptibility to normoxic oxidative neuronal death.

Small molecule activation of adaptive gene expression: tilorone or its analogs are novel potent activators of hypoxia inducible factor-1 that provide prophylaxis against stroke and spinal cord injury.

A major challenge for neurological therapeutics is the development of small molecule drugs that can activate a panoply of downstream pathways without toxicity. Over the past decade our group has shown that a family of enzymes that regulate posttranscriptional and transcriptional adaptive responses to hypoxia are viable targets for neuronal protection and repair. The family is a group of iron, oxygen, and 2-oxoglutarate-dependent dioxygenases, known as the HIF prolyl 4-hydroxylases (HIF PHDs). We have previously shown that pluripotent protection offered by iron chelators is mediated, in part, via the ability of these agents to inhibit the HIF PHDs. Our group and others have implicated the transcriptional activator HIF-1 in some of the salutary effects of iron chelation-induced PHD inhibition. While some iron chelators are currently employed in humans for conditions such as hemochromatosis, the diverse utilization of iron in physiological processes in the brain makes the development of HIF activators that do not bind iron a high priority. Here we report the development of a high throughput screen to develop novel HIF activators and/or PHD inhibitors for therapeutic use in the central nervous system (CNS). We show that tilorone, a low-molecular weight, antiviral, immunomodulatory agent is the most effective activator of the HIF pathway in a neuronal line. We also show that tilorone enhances HIF protein levels and increases the expression of downstream target genes independent of iron chelation and HIF PHD inhibition in vitro. We further demonstrate that tilorone can activate an HIF-regulated reporter gene in the CNS. These studies confirm that tilorone can penetrate the blood-brain barrier to activate HIF in the CNS. As expected from these findings, we show that tilorone provides effective prophylaxis against permanent ischemic stroke and traumatic spinal cord injury in male rodents. Altogether these findings identify tilorone as a novel and potent modulator of HIF-mediated gene expression in neurons with neuroprotective properties.

Antioxidants, HIF prolyl hydroxylase inhibitors or short interfering RNAs to BNIP3 or PUMA, can prevent prodeath effects of the transcriptional activator, HIF-1alpha, in a mouse hippocampal neuronal line.

Hypoxia-inducible factor (HIF) is a transcriptional activator that promotes death or survival in neurons. The regulators and targets of HIF-1alpha-mediated death remain unclear. We found that prodeath effects of HIF-1 are not attributable to an imbalance in HIF-1alpha and HIF-1beta expression. Rather, the synergistic death caused by oxidative stress and by overexpression of an oxygen-resistant HIF-VP16 in neuroblasts was attributable to transcriptional upregulation of BH3-only prodeath proteins, PUMA or BNIP3. By contrast, overexpression of BNIP3 was not sufficient to potentiate oxidative death. As acidosis is known to activate BNIP3-mediated death, we examined other secondary stresses, such as oxidants or prolyl hydroxylase activity are necessary for exposing the prodeath functions of HIF in neurons. Antioxidants or prolyl hydroxylase inhibition prevented potentiation of death by HIF-1alpha. Together, these studies suggest that antioxidants and PHD inhibitors abrogate the ability of HIF-mediated transactivation of BH3-only proteins to potentiate oxidative death in normoxia. The findings offer strategies for minimizing the prodeath effects of HIF-1 in neurologic conditions associated with hypoxia and oxidative stress, such as stroke and spinal cord injury.

The C3 domain of Pasteurella multocida toxin is the minimal domain responsible for activation of Gq-dependent calcium and mitogenic signaling.

The large 1285-amino-acid protein toxin from Pasteurella multocida (PMT) is a multifunctional single-chain polypeptide that binds to and enters eukaryotic cells and acts intracellularly to promote G(q) and G(12/13) protein-dependent calcium and mitogenic signal transduction. Previous studies indicated that the intracellular activity domain responsible for PMT action was located within the C-terminal 600-700 amino acids. In this study, we have exogenously expressed a series of N- and C-terminal PMT fragments directly in mammalian cells and have used the dual luciferase reporter system to assay for toxin-mediated activation of calcium-calcineurin-NFAT signaling (NFAT-luciferase) and mitogenic serum response signaling (SRE-luciferase). Using this approach, we have defined the last 180 amino acids, which encompass the C3 domain in the crystal structure, as the minimum domain sufficient to activate both NFAT and SRE signaling pathways.

Prolyl 4-hydroxylase activity-responsive transcription factors: from hydroxylation to gene expression and neuroprotection.

Most homeostatic processes including gene transcription occur as a result of deviations in physiological tone that threatens the survival of the organism. A prototypical homeostatic stress response includes changes in gene expression following alterations in oxygen, iron or 2-oxoglutarate levels. Each of these cofactors plays an important role in cellular metabolism. Accordingly, a family of enzymes known as the Prolyl 4-hydroxylase (PHD) enzymes are a group of dioxygenases that have evolved to sense changes in 2-oxoglutarate, oxygen and iron via changes in enzyme activity. Indeed, PHDs are a part of an established oxygen sensor system that regulates transcriptional regulation of hypoxia/stress-regulated genes and thus are an important component of events leading to cellular rescue from oxygen, iron or 2-oxoglutarate deprivations. The ability of PHD activity to regulate homeostatic responses to oxygen, iron or 2-oxoglutarate metabolism has led to the development of small molecule inhibitors of the PHDs as a strategy for activating or augmenting cellular stress responses. These small molecules are proving effective in preclinical models of stroke and Parkinson's disease. However the precise protective pathways engaged by PHD inhibition are only beginning to be defined. In the current review, we summarize the role of iron, 2-oxoglutarate and oxygen in the PHD catalyzed hydroxylation reaction and provide a brief discussion of some of the transcription factors that play an effective role in neuroprotection against oxidative stress as a result of changes in PHD activity.

Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin: suppression of Notch1, stabilization of beta-catenin and pre-adipocyte factor 1.

Pasteurella multocida toxin (PMT) is a potent mitogen and a specific activator of Gq-dependent signalling pathways. PMT impairs osteoblast differentiation and causes bone loss and fat reduction in vivo. We examined the effect of PMT on cell signalling pathways involved in 3T3-L1 adipocyte differentiation. We demonstrate that PMT treatment before or together with differentiation induction factors inhibits adipogenesis and prevents upregulation of important adipocyte markers - peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha). Moreover, PMT completely downregulates PPARgamma and C/EBPalpha expression in mature adipocytes. Differentiation of pre-adipocytes into adipocytes requires the suppression of pre-adipocyte factor 1 (Pref1) and Wnt signalling, along with the degradation of beta-catenin. PMT prevents downregulation of Pref1 and beta-catenin under differentiation-inducing conditions. In addition, PMT treatment downregulates expression of Notch1, a protein responsible for cell fate decision and implicated in regulation of adipogenesis in 3T3-L1 cells. PMT action on adipogenesis was not reversed by cyclosporin A, an inhibitor of Galphaq-PLC-calcium-dependent calcineurin activation. Our results reveal new pathways involved in PMT action on cellular physiology and differentiation. Our study further demonstrates that the effect of PMT on Pref1/PPARgamma/C/EBPalpha expression and adipogenesis does not occur just through activation of the Galphaq-calcium-calcineurin pathway, but involves Wnt/beta-catenin and Notch1 signalling pathways, two signalling pathways strongly linked to cancer predisposition, neurological and immunological dysfunctions, and fat and bone development.

Hypoxia inducible factor prolyl 4-hydroxylase enzymes: center stage in the battle against hypoxia, metabolic compromise and oxidative stress.

Studies of adaptive mechanisms to hypoxia led to the discovery of the transcription factor called hypoxia inducible factor (HIF). HIF is a ubiquitously expressed, heterodimeric transcription factor that regulates a cassette of genes that can provide compensation for hypoxia, metabolic compromise, and oxidative stress including erythropoietin, vascular endothelial growth factor, or glycolytic enzymes. Diseases associated with oxygen deprivation and consequent metabolic compromise such as stroke or Alzheimer's disease may result from inadequate engagement of adaptive signaling pathways that culminate in HIF activation. The discovery that HIF stability and activation are governed by a family of dioxygenases called HIF prolyl 4 hydroxylases (PHDs) identified a new target to augment the transcriptional activity of HIF and thus the adaptive machinery that governs neuroprotection. PHDs lose activity when cells are deprived of oxygen, iron or 2-oxoglutarate. Inhibition of PHD activity triggers the cellular homeostatic response to oxygen and glucose deprivation by stabilizing HIF and other proteins. Herein, we discuss the possible role of PHDs in regulation of both HIF-dependent and -independent cell survival pathways in the nervous system with particular attention to the co-substrate requirements for these enzymes. The emergence of neuroprotective therapies that modulate genes capable of combating metabolic compromise is an affirmation of elegant studies done by John Blass and colleagues over the past five decades implicating altered metabolism in neurodegeneration.

Hypoxia-inducible factor prolyl 4-hydroxylase inhibition. A target for neuroprotection in the central nervous system.

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron- and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1alpha and HIF-2alpha. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1alpha and up-regulate HIF-dependent target genes (e.g. enolase, p21(waf1/cip1), vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress.

Prosurvival and prodeath effects of hypoxia-inducible factor-1alpha stabilization in a murine hippocampal cell line.

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in adaptation to hypoxic stress. Previous studies from our laboratory demonstrated that pharmacological activators of HIF-1 (e.g. deferoxamine, cobalt chloride) could also protect cultured primary neurons or an immortalized hippocampal neuroblast line (HT22) from oxidative stress-induced death. However, whether HIF-1 activation is sufficient to abrogate neuronal death resulting from oxidative stress or other hypoxia-independent death inducers remains unclear. To address this question we utilized a HIF-1alpha fusion protein that partially lacks the domain required for oxygen-dependent degradation of HIF-1alpha and that has a VP16 transcriptional activation domain from herpes simplex virus. HT22 cells were infected with a retrovirus encoding either the HIF-1alpha-VP16 fusion protein or the activation domain of the VP16 protein alone as a control. Expression of HIF-1alpha-VP16, but not VP16 alone, increased luciferase activity driven by a canonical hypoxia response element, increased mRNA of established HIF-1 target genes, and increased activity of one of these HIF-1 target genes. Unexpectedly, enhanced HIF-1 activity in HT22 cells enhanced sensitivity to oxidative death induced by glutathione depletion. Accordingly, suppression of HIF-1alpha expression using RNA interference prevented oxidative death. By contrast, HIF-1alpha-VP16-expressing HT22 cells were more resistant to DNA damage (induced by camptothecin) or endoplasmic reticulum stress (induced by thapsigargin and tunicamycin) than were VP16-expressing cells, and suppression of HIF-1alpha expression using RNA interference rendered HT22 cells more sensitive to death induced by DNA damage or endoplasmic reticulum stress. Together, these data demonstrate that HIF-1 can mediate prodeath or prosurvival responses in the same cell type depending on the injury stimulus.

Translation of ischemic preconditioning to the patient: prolyl hydroxylase inhibition and hypoxia inducible factor-1 as novel targets for stroke therapy.

Effective therapies for stroke must interdict multiple parallel and sequential pathophysiological events. A paradigm which offers insight into multivalent but thoughtfully coordinated protective programs is ischemic preconditioning. A central hypothesis of our group and others is that pharmacological agents that activate programs of gene expression normally induced by ischemic preconditioning will be effective agents for the prevention and treatment of stroke. Inhibitors of a class of enzymes, the hypoxia inducible factor-1 (HIF-1) prolyl hydroxylases stabilize the transcriptional activator HIF-1 and activate target genes involved in compensation for ischemia, including erythropoeitin (Epo) and vascular endothelial growth factor (VEGF). Here, we review evidence suggesting that the HIF-1 prolyl hyroxylases are inhibited during ischemic preconditioning and that pharmacological inhibitors of these enzymes are viable targets for stroke therapy.

Fusarenon-X induced apoptosis in HL-60 cells depends on caspase activation and cytochrome c release.

Fusarenon-X (FX), a trichothecene mycotoxin, is well known to be cytotoxic to mammalian cells. Our previous study revealed that FX induced apoptosis in mouse thymocytes both in vivo and in vitro. We investigated the mode of apoptosis induced by FX using HL-60 cell culture. When FX at a final concentration of 0.5 microg/ml was added, cell degradation was observed 5 h after exposure, and most of the cells had fallen into apoptosis 24 h after exposure. DNA fragmentation into 180-bp multimers was observed 5 h after exposure, and its dose-dependency was clear in the cells treated with 0.1 microg/ml and higher doses. The percentage of apoptotic cells (sub-G(0) population) increased dose- and time-dependently after exposure, when analyzed using flow cytometry. The activities of caspase-3, -8, and -9 were elevated within 2 h by exposure to FX. DNA fragmentation and an increase in the apoptotic population were abrogated by pre-treating the cells with broad-spectrum caspase inhibitors Z-VAD-fmk or Z-Asp-CH(2)-DCB. Cytochrome c release from mitochondria to cytoplasm was observed clearly, and this release occurred caspase-independently. These findings suggest that FX induces apoptosis in HL-60 cells by stimulating cytochrome c release followed by its downstream events including the activation of multiple caspases.