Regulation of glucose transporter expression in cardiac myocytes: p38 MAPK is a strong inducer of GLUT4.

OBJECTIVE: In vivo differentiation of cardiac myocytes is associated with downregulation of the glucose transporter isoform GLUT1 and upregulation of the isoform GLUT4. Adult rat cardiomyocytes in primary culture undergo spontaneous dedifferentiation, followed by spreading and partial redifferentiation, which can be influenced by growth factors. We used this model to study the signaling mechanisms modifying the expression of GLUT4 in cardiac myocytes. RESULTS: Adult rat cardiomyocytes in primary culture exhibited spontaneous upregulation of GLUT1 and downregulation of GLUT4, suggesting resumption of a fetal program of GLUT gene expression. Treatment with IGF-1 and, to a minor extent, FGF-2 resulted in restored expression of GLUT4 protein and mRNA. Activation of p38 MAPK mediated the increased expression of GLUT4 in response to IGF-1. Transient transfection experiments in neonatal cardiac myocytes confirmed that p38 MAPK could activate the glut4 promoter. Electrophoretic mobility shift assay in adult rat cardiomyocytes and transient transfection experiments in neonatal cardiac myocytes indicated that MEF2 was the main transcription factor transducing the effect of p38 MAPK activation on the glut4 promoter. CONCLUSION: Spontaneous dedifferentiation of adult rat cardiomyocytes in vitro is associated with downregulation of GLUT4, which can be reversed by treatment with IGF-1. The effect of IGF-1 is mediated by the p38 MAPK/MEF2 axis, which is a strong inducer of GLUT4 expression.

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain.

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.

Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans.

Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

Islet-brain1/JNK-interacting protein-1 is required for early embryogenesis in mice.

Islet-brain1/JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein that organizes the JNK, MKK7, and MLK1 to allow signaling specificity. Targeted disruption of the gene MAPK8IP1 encoding IB1/JIP-1 in mice led to embryonic death prior to blastocyst implantation. In culture, no IB1/JIP-1(-/-) embryos were identified indicating that accelerated cell death occurred during the first cell cycles. IB1/JIP-1 expression was detected in unfertilized oocytes, in spermatozoa, and in different stages of embryo development. Thus, despite the maternal and paternal transmission of the IB1/JIP-1 protein, early transcription of the MAPK8IP1 gene is required for the survival of the fertilized oocytes.

Regulation of epithelial-mesenchymal IL-1 signaling by PPARbeta/delta is essential for skin homeostasis and wound healing.

Skin morphogenesis, maintenance, and healing after wounding require complex epithelial-mesenchymal interactions. In this study, we show that for skin homeostasis, interleukin-1 (IL-1) produced by keratinocytes activates peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) expression in underlying fibroblasts, which in turn inhibits the mitotic activity of keratinocytes via inhibition of the IL-1 signaling pathway. In fact, PPARbeta/delta stimulates production of the secreted IL-1 receptor antagonist, which leads to an autocrine decrease in IL-1 signaling pathways and consequently decreases production of secreted mitogenic factors by the fibroblasts. This fibroblast PPARbeta/delta regulation of the IL-1 signaling is required for proper wound healing and can regulate tumor as well as normal human keratinocyte cell proliferation. Together, these findings provide evidence for a novel homeostatic control of keratinocyte proliferation and differentiation mediated via PPARbeta/delta regulation in dermal fibroblasts of IL-1 signaling. Given the ubiquitous expression of PPARbeta/delta, other epithelial-mesenchymal interactions may also be regulated in a similar manner.

Expressed isolated integrin beta1 subunit cytodomain induces endothelial cell death secondary to detachment.

Expression of isolated beta integrin cytoplasmic domains in cultured endothelial cells was reported to induce cell detachment and death. To test whether cell death was the cause or the consequence of cell detachment, we expressed isolated integrin beta1 cytoplasmic and transmembrane domains (CH1) in cultured human umbilical vein endothelial cells (HUVEC), and monitored detachment, viability, caspase activation and signaling. CH1 expression induced dose-dependent cell detachment. At 24 h over 90% of CH1-expressing HUVEC were detached but largely viable (>85%). No evidence of pro-caspase-8,-3, and PARP cleavage or suppression of phosphorylation of ERK, PKB and Ikappa-B was observed. The caspase inhibitor z-VAD did not prevent cell detachment. At 48 h, however, CH1-expressing cells were over 50% dead. As a comparison trypsin-mediated detachment resulted in a time-dependent cell death, paralleled by caspase-3 activation and suppression of ERK, PKB and Ikappa-B phosphoyrylation at 24 h or later after detachment. HUVEC stimulation with agents that strengthen integrin-mediated adhesion (i.e. PMA, the Src inhibitor PP2 and COMP-Ang1) did not prevent CH1-induced detachment. Expression of CH1 in rat carotid artery endothelial cells in vivo caused endothelial cell detachment and increased nuclear DNA fragmentation among detached cells. A construct lacking the integrin cytoplasmic domain (CH2) had no effect on adhesion and cell viability in vitro and in vivo. These results demonstrate that isolated beta1 cytoplasmic domain expression induces caspase-independent detachment of viable endothelial cells and that death is secondary to detachment (i.e. anoikis). They also reveal an essential role for integrins in the adhesion and survival of quiescent endothelial cells in vivo.

Dlgh1 and Carma1 MAGUK proteins contribute to signal specificity downstream of TCR activation.

Mitogen-activated protein kinases (MAPKs), including p38 and c-Jun N-terminal kinase (JNK), have a key role in T cell receptor (TCR)-induced gene transcription but their precise mechanism of activation is not well understood. The findings of two recent papers provide new insight into the activation of p38 and JNK by the membrane-associated guanylate kinase (MAGUK) family members Dlgh1 and Carma1, respectively, and show how distinct MAGUK proteins control specific aspects of TCR-mediated MAPK activation.

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

Delta-9-tetrahydrocannabinol (THC) protects partly against demyelination by modulating the inflammatory response: an in vitro study in aggregating brain cell cultures

Delta 9-tetrahydrocannabinol (THC) has been proposed as therapeutic agent in the treatment of multiple sclerosis. In the present study, we examined whether a modulation of brain inflammatory by THC may protect against demyelination. Myelinating aggregating brain cell cultures were subjected to demyelination by a repeated treatment (3x) with the two inflammatory agents interferon-y (IFN-y) and lipopolysaccharide (LPS). The effects of THC on an acute inflammatory reponse were also examined by treating the aggregates with a single application of the two inflammatory agents. THC effects on the demyelinating process and on several mediators of the inflammatory reponse were analyzed. THC treatment partially prevented the decreased immunoreactivity for MBP, and the decrease in MBP content measured by immunoblotting. It prevented IFN-y + LPS -induced microglial reactivity; and decreased the IFN-y + LPS-induced i8ncreased phosphorylation of p44/42 MAP kinase. The other inflammatory markers, I-NOS and TNF-a mRNA expression, and p38 MAP kinase phosphorylation of p44/42 MAP kinase. The other inflammatory markers, I-NOS and TNF-a mRNA expression, and p38 MAP kinase phosphorylation were downregulated by THC treatment following a single application of the inflammatory agents, but not after repeated applications. THC protected partially against the IFN-y + LPS-induced demyelination. The protective effect of THC on IFN-y + LPS-induced demyelination may be due to a decrease of the inflammatory reponse. However, the anti-inflammatory effect of THC on some inflammatory markers is lost when the inflammatory response is more proeminent and of longer duration, suggesting either that the anti-inflammatory effect of a molecule may depend on the properties of the inflammatory response, or that the anti-inflammatory potential of THC decreases in case of repeated exposure.

Human high-density lipoprotein particles prevent activation of the JNK pathway induced by human oxidised low-density lipoprotein particles in pancreatic beta cells.

AIMS/HYPOTHESIS: We explored the potential adverse effects of pro-atherogenic oxidised LDL-cholesterol particles on beta cell function. MATERIALS AND METHODS: Isolated human and rat islets and different insulin-secreting cell lines were incubated with human oxidised LDL with or without HDL particles. The insulin level was monitored by ELISA, real-time PCR and a rat insulin promoter construct linked to luciferase gene reporter. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Prolonged incubation with human oxidised LDL particles led to a reduction in preproinsulin expression levels, whereas the insulin level was preserved in the presence of native LDL-cholesterol. The loss of insulin production occurred at the transcriptional levels and was associated with an increase in activator protein-1 transcriptional activity. The rise in activator protein-1 activity resulted from activation of c-Jun N-terminal kinases (JNK, now known as mitogen-activated protein kinase 8 [MAPK8]) due to a subsequent decrease in islet-brain 1 (IB1; now known as MAPK8 interacting protein 1) levels. Consistent with the pro-apoptotic role of the JNK pathway, oxidised LDL also induced a twofold increase in the rate of beta cell apoptosis. Treatment of the cells with JNK inhibitor peptides or HDL countered the effects mediated by oxidised LDL. CONCLUSIONS/INTERPRETATION: These data provide strong evidence that oxidised LDL particles exert deleterious effects in the progression of beta cell failure in diabetes and that these effects can be countered by HDL particles.

Growth promoting signaling by tenascin-C [corrected].

Tenascin-C is an adhesion-modulating extracellular matrix molecule that is highly expressed in tumor stroma and stimulates tumor cell proliferation. Adhesion of T98G glioblastoma cells to a fibronectin substratum is inhibited by tenascin-C. To address the mechanism of action, we performed a RNA expression analysis of T89G cells grown in the presence or absence of tenascin-C and found that tenascin-C down-regulates tropomyosin-1. Upon overexpression of tropomyosin-1, cell spreading on a fibronectin/tenascin-C substratum was restored, indicating that tenascin-C destabilizes actin stress fibers through down-regulation of tropomyosin-1. Tenascin-C also increased the expression of the endothelin receptor type A and stimulated the corresponding mitogen-activated protein kinase signaling pathway, which triggers extracellular signal-regulated kinase 1/2 phosphorylation and c-Fos expression. Tenascin-C additionally caused down-regulation of the Wnt inhibitor Dickkopf 1. In consequence, Wnt signaling was enhanced through stabilization of beta-catenin and stimulated the expression of the beta-catenin target Id2. Finally, our in vivo data derived from astrocytoma tissue arrays link increased tenascin-C and Id2 expression with high malignancy. Because increased endothelin and Wnt signaling, as well as reduced tropomyosin-1 expression, are closely linked to transformation and tumorigenesis, we suggest that tenascin-C specifically modulates these signaling pathways to enhance proliferation of glioma cells.

Paired activation of two components within muscarinic M3 receptor dimers is required for recruitment of beta-arrestin-1 to the plasma membrane.

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

Characterization of Tollip in the Interleulkin-1 Receptor/Toll Like Receptors signaling pathways

SUMMARY IL-1R and TLRs are key players in innate immunity and inflammation. Tollip was identified as a component of IL-1RI, TLR2 and TLR4 signaling complexes that activate NF-κB and MAP kinase pathways. Tollip was previously shown as a negative regulator of NF-κB and MAP Kinase activation. We have characterized the role of Tollip in IL-R/TLRs induced signaling by the analysis of the Tollip deficient mice. We showed that NF-κB and MAPK (p38, JNK, or ERK1/2) signaling appeared normal in Tollip deficient cells following stimulation with IL-1β, lipopolysaccharide (LPS), and other TLR ligands. Also IL-1β and TLRs ligands induced activation of immune cells was indistinguishable from wild-type cells. Strikingly, in Tollip deficient mice the production of the inflammatory cytokines, IL-6 or TNF-α was significantly reduced relative to control mice after treatment with physiological doses of IL-1β or LPS, whereas no difference was observed at high doses of stimulation with LPS or in LPS induced septic shock. Therefore, Tollip could be critical for regulation of optimal responses to IL-1β and LPS, in addition to its role as negative regulator of the signaling. We also studied the role of Tollip as an endocytic adaptor for IL-1R endocytosis. We could show that Il-1R is ubiquitinated after IL-1β stimulation, and that Tollip's CUE domain binds IL-1RI in an ubiquitin-dependent manner. We followed IL-1R internalization and Tollip localization by confocal microscopy. Consistent with a role for Tollip in sorting of ubiquitinated IL-1RI, a significant amount of Tollip was also localized at the late endosomal compartment. We could show that Tollip is required for efficient lysosomal targeting of ubiquitinated IL-1R1, In the absence of Tollip or in Tollip deficient cells reconstituted with a Tollip mutant (defective in ubiquitin binding) IL-1RI accumulates in enlarged late endosomes. In addition, Tollip was shown to interact with, another endocytic adapter, Toml, and both interact with IL-1RI. In conclusion, we showed that Tollip is required for IL-1β and LPS signaling for cytokine production. In addition we showed and that Tollip has a role as an endocytic adapter, necessary for efficient trafficking and lysosomal degradation of IL-1RI. Resumé Le récepteur à l'interleukine-1 (IL-1R) et les récepteurs "Toll-like" (TLRs) sont des acteurs cruciaux de la réponse immunitaire innée et de l'inflammation. La proteine Tollip a été identifiée comme étant un élément des complexes de signalisation, induits par les récepteurs IL-1RI, TLR-2 et TLR-4, qui mènent à l'activation de la voie des MAP kinases et de NF-κB. Dans de précédentes études, il a été montré que Tollip pouvait inhiber ces deux voies de signalisation. Nous avons voulu caractériser plus précisément le rôle de Tollip dans l'activation des voies de signalisation mitées par IL-1R/TLRs en utilisant une lignée murine déficiente pour la protéine Tollip. Ainsi, en absence de Tollip, les cascades d'activation de NF-κB et MAPK (p38, JNK, or ERK1/2) ne semblent pas affectées après stimulation avec IL-1β, lipopolysaccharide (LPS) ou d' autres ligands des TLR. La réponse des cellules du système immunitaire induite par la stimulation avec IL-1β et les ligands des TLR est également comparable entre les souris sauvages et les souris deficientes pour Tollip. Par contre, dans cette lignée murine, la production de cytokines proinflammatoires IL-6 et TNFα induite par la stimulation à dose physiologique de IL-1β or LPS, est réduite. Cependant, lors de stimulation à plus hautes doses de LPS ou pendant un choc septique induit par de LPS, cette réduction n'est pas observée. Ces résultats montrent que Tollip pourrait avoir un rôle déterminant dans l'activation optimale en réponse à l' IL-1β et au LPS qui s'ajoute à sa fonction inhibitrice des mêmes voies de signalisation. Nous avons aussi étudié le rôle de Tollip comme molécule adaptatatrice du mécanisme endocytique d'internalisation de l' IL-1RI. Ainsi, l' IL-1R est ubiquitiné après stimulation par l' IL-1β , permettant à Tollip de se lier au récepteur. Cette interaction est réalisée entre le domaine CUE de Tollip et l'IL-1R via l'ubiquitine. L'internalisation et la localisation intracellulaire de l'IL-1RI et de Tollip ont été observés par microscopie confocale. En accord avec le rôle de Tollip dans le triage et la recirculation des IL-1R ubiquitiné, une quantité importante de Tollip été détectée dans l' endosome tardif. Nous avons pu démontrer que Tollip était nécessaire pour diriger efficacement ubiquitiné vers les lysosomes. Dans des cellules déficientes pour Tollip, ou reconstituées avec un mutant de Tollip (MF/AA) incapable de lier l'ubiquitine, IL-1RI s'accumule dans des vesicules anormales de l'endosome tardif. Dans ce travail, nous avons pu confirmer et préciser la fonction de la protéine Tollip dans l' activation de la production de cytokines induites par l' IL-1p and le LPS lors de l'inflammation et découvrir son rôle d'adaptateur dans l' internalisation et l'endocytose de l' IL-1RI.