The role of tissue transglutaminase in 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in differentiated human SH-SY5Y neuroblastoma cells

Tissue transglutaminase (TG2) can induce post-translational modification of proteins, resulting in protein cross-linking or incorporation of polyamines into substrates, and can also function as a signal transducing G protein. The role of TG2 in the formation of insoluble cross-links has led to its implication in some neurodegenerative conditions. Exposure of pre-differentiated SH-SY5Y cells to the Parkinsonian neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) resulted in significant dose-dependent reductions in TG2 protein levels, measured by probing Western blots with a TG2-specific antibody. Transglutaminase (TG) transamidating activity, on the other hand, monitored by incorporation of a polyamine pseudo-substrate into cellular proteins, was increased. Inhibitors of TG (putrescine) and TG2 (R283) exacerbated MPP+ toxicity, suggesting that activation of TG2 may promote a survival response in this toxicity paradigm.

Increased TG2 expression can result in induction of transforming growth factor β1, causing increased synthesis and deposition of matrix proteins, which can be regulated by nitric oxide

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-kappaB activation, resulting in the increased expression of transforming growth factor (TGF) beta(1). In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFbeta(1) in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-kappaB and TGFbeta1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-kappaB and TGFbeta(1) production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.

Evidence that interaction between conserved residues in transmembrane helices 2, 3, and 7 are crucial for human VPAC1 receptor activation

The VPAC(1) receptor belongs to family B of G protein-coupled receptors (GPCR-B) and is activated upon binding of the vasoactive intestinal peptide (VIP). Despite the recent determination of the structure of the N terminus of several members of this receptor family, little is known about the structure of the transmembrane (TM) region and about the molecular mechanisms leading to activation. In the present study, we designed a new structural model of the TM domain and combined it with experimental mutagenesis experiments to investigate the interaction network that governs ligand binding and receptor activation. Our results suggest that this network involves the cluster of residues Arg(188) in TM2, Gln(380) in TM7, and Asn(229) in TM3. This cluster is expected to be altered upon VIP binding, because Arg(188) has been shown previously to interact with Asp(3) of VIP. Several point mutations at positions 188, 229, and 380 were experimentally characterized and were shown to severely affect VIP binding and/or VIP-mediated cAMP production. Double mutants built from reciprocal residue exchanges exhibit strong cooperative or anticooperative effects, thereby indicating the spatial proximity of residues Arg(188), Gln(380), and Asn(229). Because these residues are highly conserved in the GPCR-B family, they can moreover be expected to have a general role in mediating function.

The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis

VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.

Acute and long-term effects of peroxisome proliferator-activated receptor-γ activation on the function and insulin secretory responsiveness of clonal beta-cells

Thiazolidinediones (TZDs) are used as antidiabetic therapy. The purpose of the present study was to examine whether the TZD rosiglitazone has direct actions on pancreatic beta-cells that contribute to its overall effects. Effects of acute and prolonged (48 h) exposure to rosiglitazone, as a model glitazone compound, were assessed in clonal pancreatic BRIN-BD11 beta-cells maintained in standard, glucotoxic and lipotoxic cultures. In acute 20-min incubations, rosiglitazone (0.2-100 M) did not alter basal or glucose-stimulated insulin secretion. However, rosiglitazone (6.25 M) enhanced (p

Pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC1) gene is suppressed by transglutaminase 2 activation

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Does metabolic reprogramming underpin age-associated changes in T cell phenotype and function?

T cells are required for an effective adaptive immune response. The principal function of T cells is to promote efficient removal of foreign material by identifying and mounting a specific response to nonself. A decline in T cell function in aging is thought to contribute to reduced response to infection and vaccination and an increase in autoimmunity. This may in part be due to the age-related decrease in naïve CD4+ T cells and increase in antigen-experienced CD4+ T cells, loss of redox homeostasis, and impaired metabolic switching. Switching between subsets is triggered by the integration of extracellular signals sensed through surface receptors and the activation of discrete intracellular metabolic pathways. This article explores how metabolic programming and loss of redox homeostasis during aging may contribute to age-associated changes in T cell phenotype and function. © 2014 Elsevier Inc.

An Ins(1,4,5)P3 receptor in Paramecium is associated with the osmoregulatory system

In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.

Nutritional influences on cognitive function:mechanisms of susceptibility

The impact of nutritional variation, within populations not overtly malnourished, on cognitive function and arousal is considered. The emphasis is on susceptibility to acute effects of meals and glucose loads, and chronic effects of dieting, on mental performance, and effects of cholesterol and vitamin levels on cognitive impairment. New developments in understanding dietary influences on neurohormonal systems, and their implications for cognition and affect, allow reinterpretation of both earlier and recent findings. Evidence for a detrimental effect of omitting a meal on cognitive performance remains equivocal: from the outset, idiosyncrasy has prevailed. Yet, for young and nutritionally vulnerable children, breakfast is more likely to benefit than hinder performance. For nutrient composition, despite inconsistencies, some cautious predictions can be made. Acutely, carbohydrate-rich–protein-poor meals can be sedating and anxiolytic; by comparison, protein-rich meals may be arousing, improving reaction time but also increasing unfocused vigilance. Fat-rich meals can lead to a decline in alertness, especially where they differ from habitual fat intake. These acute effects may vary with time of day and nutritional status. Chronically, protein-rich diets have been associated with decreased positive and increased negative affect relative to carbohydrate-rich diets. Probable mechanisms include diet-induced changes in monoamine, especially serotoninergic neurotransmitter activity, and functioning of the hypothalamic pituitary adrenal axis. Effects are interpreted in the context of individual traits and susceptibility to challenging, even stressful, tests of performance. Preoccupation with dieting may impair cognition by interfering with working memory capacity, independently of nutritional status. The change in cognitive performance after administration of glucose, and other foods, may depend on the level of sympathetic activation, glucocorticoid secretion, and pancreatic β-cell function, rather than simple fuelling of neural activity. Thus, outcomes can be predicted by vulnerability in coping with stressful challenges, interacting with nutritional history and neuroendocrine status. Functioning of such systems may be susceptible to dietary influences on neural membrane fluidity, and vitamin-dependent cerebrovascular health, with cognitive vulnerability increasing with age.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells

The proteinase-activated receptor 2 (PAR-2) expression is increased in endothelial cells derived from women with preeclampsia, characterized by widespread maternal endothelial damage, which occurs as a consequence of elevated soluble vascular endothelial growth factor receptor-1 (sVEGFR-1; commonly known as sFlt-1) in the maternal circulation. Because PAR-2 is upregulated by proinflammatory cytokines and activated by blood coagulation serine proteinases, we investigated whether activation of PAR-2 contributed to sVEGFR-1 release. PAR-2–activating peptides (SLIGRL-NH2 and 2-furoyl-LIGRLO-NH2) and factor Xa increased the expression and release of sVEGFR-1 from human umbilical vein endothelial cells. Enzyme-specific, dominant-negative mutants and small interfering RNA were used to demonstrate that PAR-2–mediated sVEGFR-1 release depended on protein kinase C-ß1 and protein kinase C-e, which required intracellular transactivation of epidermal growth factor receptor 1, leading to mitogen-activated protein kinase activation. Overexpression of heme oxygenase 1 and its gaseous product, carbon monoxide, decreased PAR-2–stimulated sVEGFR-1 release from human umbilical vein endothelial cells. Simvastatin, which upregulates heme oxygenase 1, also suppressed PAR-2–mediated sVEGFR-1 release. These results show that endothelial PAR-2 activation leading to increased sVEGFR-1 release may contribute to the maternal vascular dysfunction observed in preeclampsia and highlights the PAR-2 pathway as a potential therapeutic target for the treatment of preeclampsia.

Regulation of neovascularization by S-glutathionylation via the Wnt5a/sFlt-1 pathway

S-glutathionylation occurs when reactive oxygen or nitrogen species react with protein-cysteine thiols. Glutaredoxin-1 (Glrx) is a cytosolic enzyme which enzymatically catalyses the reduction in S-glutathionylation, conferring reversible signalling function to proteins with redox-sensitive thiols. Glrx can regulate vascular hypertrophy and inflammation by regulating the activity of nuclear factor κB (NF-κB) and actin polymerization. Vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) migration is inhibited by Glrx overexpression. In mice overexpressing Glrx, blood flow recovery, exercise function and capillary density were significantly attenuated after hindlimb ischaemia (HLI). Wnt5a and soluble Fms-like tyrosine kinase-1 (sFlt-1) were enhanced in the ischaemic-limb muscle and plasma respectively from Glrx transgenic (TG) mice. A Wnt5a/sFlt-1 pathway had been described in myeloid cells controlling retinal blood vessel development. Interestingly, a Wnt5a/sFlt-1 pathway was found also to play a role in EC to inhibit network formation. S-glutathionylation of NF-κB components inhibits its activation. Up-regulated Glrx stimulated the Wnt5a/sFlt-1 pathway through enhancing NF-κB signalling. These studies show a novel role for Glrx in post-ischaemic neovascularization, which could define a potential target for therapy of impaired angiogenesis in pathological conditions including diabetes.

Novel glucagon-like peptide-1 (GLP-1) analog (Val8)GLP-1 results in significant improvements of glucose tolerance and pancreatic β-cell function after 3-week daily administration in obese diabetic (ob/ob) mice

This study evaluates the antidiabetic potential of an enzyme-resistant analog, (Val8)GLP-1. The effects of daily administration of a novel dipeptidyl peptidase IV-resistant glucagon-like peptide-1 (GLP-1) analog, (Val8)GLP-1, on glucose tolerance and pancreatic β-cell function were examined in obese-diabetic (ob/ob) mice. Acute intraperitoneal administration of (Val8)GLP-1 (6.25-25 nmol/kg) with glucose increased the insulin response and reduced the glycemic excursion in a dose-dependent manner. The effects of (Val8)GLP-1 were greater and longer lasting than native GLP-1. Once-daily subcutaneous administration of (Val8)GLP-1 (25 nmol/kg) for 21 days reduced plasma glucose concentrations, increased plasma insulin, and reduced body weight more than native GLP-1 without a significant change in daily food intake. Furthermore, (Val8)GLP-1 improved glucose tolerance, reduced the glycemic excursion after feeding, increased the plasma insulin response to glucose and feeding, and improved insulin sensitivity. These effects were consistently greater with (Val8)GLP-1 than with native GLP-1, and both peptides retained or increased their acute efficacy compared with initial administration. (Val8)GLP-1 treatment increased average islet area 1.2-fold without changing the number of islets, resulting in an increased number of larger islets. These data demonstrate that (Val8)GLP-1 is more effective and longer acting than native GLP-1 in obese-diabetic ob/ob mice.

Differential impact of amino acid substitutions on critical residues of the human glucagon-like peptide-1 receptor involved in peptide activity and small-molecule allosterys

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor systemis highly complex, able to be activated by both endogenous [GLP-1(1-36)NH2, GLP-1(1-37), GLP-1(7-36)NH2, GLP-1(7-37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tertbutylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptidemediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca2+ mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.

On the Approximation of the Generalized Cut Function of Degree p+1 By Smooth Sigmoid Functions

We introduce a modification of the familiar cut function by replacing the linear part in its definition by a polynomial of degree p + 1 obtaining thus a sigmoid function called generalized cut function of degree p + 1 (GCFP). We then study the uniform approximation of the (GCFP) by smooth sigmoid functions such as the logistic and the shifted logistic functions. The limiting case of the interval-valued Heaviside step function is also discussed which imposes the use of Hausdorff metric. Numerical examples are presented using CAS MATHEMATICA.