Platelet adhesion to podoplanin under flow is mediated by the receptor CLEC-2 and stabilised by Src/Syk-dependent platelet signalling

Platelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in blood-filled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following αIIbβ3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγRIIA receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice.

Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signalling

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

Filamin A (FlnA) cross-links actin filaments and connects the Von Willebrand factor receptor GPIb-IX-V to the underlying cytoskeleton in platelets. Because FlnA deficiency is embryonic lethal, mice lacking FlnA in platelets were generated by breeding FlnA(loxP/loxP) females with GATA1-Cre males. FlnA(loxP/y) GATA1-Cre males have a macrothrombocytopenia and increased tail bleeding times. FlnA-null platelets have decreased expression and altered surface distribution of GPIbalpha because they lack the normal cytoskeletal linkage of GPIbalpha to underlying actin filaments. This results in approximately 70% less platelet coverage on collagen-coated surfaces at shear rates of 1,500/s, compared with wild-type platelets. Unexpectedly, however, immunoreceptor tyrosine-based activation motif (ITAM)- and ITAM-like-mediated signals are severely compromised in FlnA-null platelets. FlnA-null platelets fail to spread and have decreased alpha-granule secretion, integrin alphaIIbbeta3 activation, and protein tyrosine phosphorylation, particularly that of the protein tyrosine kinase Syk and phospholipase C-gamma2, in response to stimulation through the collagen receptor GPVI and the C-type lectin-like receptor 2. This signaling defect was traced to the loss of a novel FlnA-Syk interaction, as Syk binds to FlnA at immunoglobulin-like repeat 5. Our findings reveal that the interaction between FlnA and Syk regulates ITAM- and ITAM-like-containing receptor signaling and platelet function.

Renal cells activate the platelet receptor CLEC-2 through podoplanin.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts.

The translocation of protein kinase C (PKC) isoforms PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from soluble to particulate fractions was studied in ventricular cardiomyocytes cultured from neonatal rats. Endothelin-1 (ET-1) caused a rapid ETA receptor-mediated translocation of PKC-delta and PKC-epsilon (complete in 0.5-1 min). By 3-5 min, both isoforms were returning to the soluble fraction, but a greater proportion of PKC-epsilon remained associated with the particulate fraction. The EC50 of translocation for PKC-delta was 11-15 nM ET-1 whereas that for PKC-epsilon was 1.4-1.7 nM. Phenylephrine caused a rapid translocation of PKC-epsilon (EC50 = 0.9 microM) but the proportion lost from the soluble fraction was less than with ET-1. Translocation of PKC-delta was barely detectable with phenylephrine. Neither agonist caused any consistent translocation of PKC-alpha or PKC-zeta. Activation of p42 and p44 mitogen-activated protein kinase (MAPK) by ET-1 or phenylephrine followed more slowly (complete in 3-5 min). Phosphorylation of p42-MAPK occurred simultaneously with its activation. The proportion of the total p42-MAPK pool phosphorylated in response to ET-1 (50%) was greater than with phenylephrine (20%). In addition to activation of MAPK, an unidentified p85 protein kinase was activated by ET-1 in the soluble fraction whereas an unidentified p58 protein kinase was activated in the particulate fraction.

Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy.

Phenylephrine and noradrenaline (alpha-adrenergic agonism) or isoprenaline (beta-adrenergic agonism) stimulated protein synthesis rates, increased the activity of the atrial natriuretic factor gene promoter and activated mitogen-activated protein kinase (MAPK). The EC50 for MAPK activation by noradrenaline was 2-4 microM and that for isoprenaline was 0.2-0.3 microM. Maximal activation of MAPK by isoprenaline was inhibited by the beta-adrenergic antagonist, propranolol, whereas the activation by noradrenaline was inhibited by the alpha1-adrenergic antagonist, prazosin. FPLC on a Mono-Q column separated two peaks of MAPK (p42MAPK and p44MAPK) and two peaks of MAPK-activating activity (MEK) activated by isoprenaline or noradrenaline. Prolonged phorbol ester exposure partially down-regulated the activation of MAPK by noradrenaline but not by isoprenaline. This implies a role for protein kinase C in MAPK activation by noradrenaline but not isoprenaline. A role for cyclic AMP in activation of the MAPK pathway was eliminated when other agonists that elevate cyclic AMP in the cardiac myocyte did not activate MAPK. In contrast, MAPK was activated by exposure to ionomycin, Bay K8644 or thapsigargin that elevate intracellular Ca2+. Furthermore, depletion of extracellular Ca2+ concentrations with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA) or blocking of the L-type Ca2+ channel with nifepidine or verapamil inhibited the response to isoprenaline without inhibiting the responses to noradrenaline. We conclude that alpha- and beta-adrenergic agonists can activate the MEK/MAPK pathway in the heart by different signalling pathways. Elevation of intracellular Ca2+ rather than cyclic AMP appears important in the activation of MAPK by isoprenaline in the cardiac myocyte.

Stimulation of the p38 mitogen-activated protein kinase pathway in neonatal rat ventricular myocytes by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine: a role in cardiac myocyte hypertrophy?

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by approximately 12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 microM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response.

GPR30 activation decreases anxiety in the open field test but not in the elevated plus maze test in female mice

The GPR30 is a novel estrogen receptor (ER) that is a candidate membrane ER based on its binding to 17beta estradiol and its rapid signaling properties such as activation of the extracellular-regulated kinase (ERK) pathway. Its distribution in the mouse limbic system predicts a role for this receptor in the estrogenic modulation of anxiety behaviors in the mouse. A previous study showed that chronic administration of a selective agonist to the GPR30 receptor, G-1, in the female rat can improve spatial memory, suggesting that GPR30 plays a role in hippocampal-dependent cognition. In this study, we investigated the effect of a similar chronic administration of G-1 on behaviors that denote anxiety in adult ovariectomized female mice, using the elevated plus maze (EPM) and the open field test as well as the activation of the ERK pathway in the hippocampus. Although estradiol benzoate had no effect on behaviors in the EPM or the open field, G-1 had an anxiolytic effect solely in the open field that was independent of ERK signaling in either the ventral or dorsal hippocampus. Such an anxiolytic effect may underlie the ability of G-1 to increase spatial memory, by acting on the hippocampus.

Adenosine receptor type 2a is differently modulated by nicotine in dorsal brainstem cells of Wistar Kyoto and spontaneously hypertensive rats

Hypertension can result from neuronal network imbalance in areas of central nervous system that control blood pressure, such as the nucleus tractus solitarius (NTS). There are several neurotransmitters and neuromodulatory substances within the NTS, such as adenosine, which acts on purinoreceptors A(2a) (A(2a)R). The A(2a)R modulates neurotransmission in the NTS where its activation may induce decrease in blood pressure by different mechanisms. Nicotine is a molecule that crosses the hematoencephalic barrier and acts in several areas of central nervous system including the NTS, where it may interact with some neurotransmitter systems and contributes to the development of hypertension in subjects with genetic predisposition to this disease. In this study we first determined A(2a)R binding, protein, and mRNA expression in dorsomedial medulla oblongata of neonate normotensive (WKY) and spontaneously hypertensive rats (SHR). Subsequently, we analyzed the modulatory effects of nicotine on A(2a)R in cell culture in order to evaluate its possible involvement in the development of hypertension. Data showed a decreased A(2a)R binding and increased protein and mRNA expression in tissue sample and culture of dorsal brainstem from SHR compared with those from WKY rats at basal conditions. Moreover, nicotine modulated A(2a)R binding, protein, and mRNA expression in cells from both strains. Interestingly, nicotine decreased A(2a)R binding and increased protein levels, as well as, induced a differential modulation in A(2a)R mRNA expression. Results give us a clue about the mechanisms involved in the modulatory effects of nicotine on A(2a)R as well as hypothesize its possible contribution to the development of hypertension. In conclusion, we demonstrated that A(2a)R of SHR cells which differ from WKY and nicotine differentially modulates A(2a)R in dorsal brainstem cells of SHR and WKY.

Melatonin and the time window for the expression of the alpha 8 nicotinic acetylcholine receptor in the membrane of chick retinal cells in culture

We have previously shown that melatonin influences the development of alpha 8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing alpha 8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48 h suppresses the development of functional alpha 8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of alpha 8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[(125)I] iodomelatonin (23 pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60 min, or 12 h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of alpha 8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [(125)I]-alpha-bungarotoxin (10 nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing alpha 8 nAChR subtype in full functional form. (C) 2010 ISDN. Published by Elsevier Ltd. All rights reserved.

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Aims Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. Methods and results NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO(2)) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 mu M). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Conclusion Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Prolonged consumption of soy or fish-oil-enriched diets differentially affects the pattern of hypothalamic neuronal activation induced by refeeding in rats

We used c-Fos immunoreactivity to estimate neuronal activation in hypothalamic feeding-regulatory areas of 3-month-old rats fed control or oil-enriched diets (soy or fish) since weaning. While no diet effect was observed in c-Fos immunoreactivity of 24-h fasted animals, the acute response to refeeding was modified by both hyperlipidic diets but with different patterns. Upon refeeding, control-diet rats had significantly increased c-Fos immunoreactivity only in the paraventricular hypothalamic nucleus (PVH, 142%). In soy-diet rats, refeeding with the soy diet increased c-Fos immunoreactivity in dorsomedial hypothalamic nucleus (DMH, 271%) and lateral hypothalamic area (LH, 303%). Refeeding fish-diet rats with the fish diet increased c-Fos immunoreactivity in PVH (161%), DMH (177%), VMH (81%), and ARC (127%). Compared to the fish-diet, c-Fos immunoreactivity was increased in LH by the soy-diet while it was decreased in ventromedial hypothalamic nucleus (VMH) and arcuate hypothalamic nucleus (ARC). Based on the known roles of the activated nuclei, it is suggested that, unlike the fish-diet, the soy-diet induced a potentially obesogenic profile, with high LH and low VMH/PVH activation after refeeding.

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Differential response of AMPA and NMDA glutamate receptors of Purkinje cells to aging of the chicken cerebellum

Aging can lead to cognitive, affective, learning, memory and motor deficits. Since the cerebellum and glutamatergic neurotransmission are involved in several of those functions, the present work aimed at studying the expression of AMPA and NMDA glutamate receptor subunits in the chick cerebellum during aging. Young (30 days old) and aged (ca. 4 years old) chickens (Gallus gallus) were used in order to evaluate the expression of GluR1, GluR2/3 and NR1 subunits. The cerebella of young and aged chickens were subjected to immunohistochemical and immunoblotting techniques. Numbers of GluR1, GluR2/3 and NR1-positive cells and optical density of the immunoblotting data were analyzed and submitted to statistical analysis using ANOVA and the Bonferroni post hoc test. Mean density of Purkinje cells stained for Giemsa, GluR1, GluR2/3 and NR1 in the cerebellum all showed a statistically significant decrease in aged animals when compared to the young animals (Giemsa, P < 0.01; GluRs and NR1, P < 0.03). However, the ratio of GluR1 and GluR2/3-positive Purkinje cells in relation the total number of Purkinje cells found in each time point decreased with aging (ca. 10%), whereas the ratio of NR1-positive cells increased (ca. 9%). The immunoblotting data showed a significant decrease of GluR1 (ca. 66%) and GluR2/3 (ca. 55%) protein expression with aging, but did not reveal changes for NR1. Our data suggest that aging can lead to differential changes in the pattern of expression of glutamate receptor subunits, which can underlie at least part of the cognitive and motor disorders found in aged animals. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

The role of proliferator-activated receptor gamma coactivator-1 alpha in the fatty-acid -dependent transcriptional control of interleukin-10 in hepatic cells of rodents

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1 alpha could be mediated by reducing the transcription of the IL-10 gene Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1 alpha in the control of IL-10 expression in hepatic cells First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1 alpha are increased Inhibiting PGC-1 alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1 alpha with c-Maf and p50-nuclear factor (NF) kappa B, 2 transcription factors known to modulate IL-10 expression In addition, after fatty acid treatment. PGC-1 alpha, c-Maf, and p50-NF kappa B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region Inhibiting NF kappa B activation with salicylate reduces IL-10 expression and the association of PGC-1 alpha with p50-NF kappa B Thus, PGC-1 alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver (C) 2010 Elsevier Inc All rights reserved