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Pharmacological Inhibition (pharmacological + inhibition)
Selected AbstractsThe mammalian KIR2.x inward rectifier ion channel family: expression pattern and pathophysiologyACTA PHYSIOLOGICA, Issue 3 2010T. P. De Boer Abstract Inward rectifier currents based on KIR2.x subunits are regarded as essential components for establishing a stable and negative resting membrane potential in many excitable cell types. Pharmacological inhibition, null mutation in mice and dominant positive and negative mutations in patients reveal some of the important functions of these channels in their native tissues. Here we review the complex mammalian expression pattern of KIR2.x subunits and relate these to the outcomes of functional inhibition of the resultant channels. Correlations between expression and function in muscle and bone tissue are observed, while we recognize a discrepancy between neuronal expression and function. [source] Plectin deposition at podosome rings requires myosin contractilityCYTOSKELETON, Issue 8 2008Annica Gad Abstract Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Myosin-II negatively regulates minor process extension and the temporal development of neuronal polarityDEVELOPMENTAL NEUROBIOLOGY, Issue 5 2009K.M. Kollins Abstract The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated "minor processes" (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live-cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA-kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell-permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] Signalling pathways involved in retinal endothelial cell proliferation induced by advanced glycation end products: inhibitory effect of gliclazideDIABETES OBESITY & METABOLISM, Issue 2 2004J.-C. Mamputu Aim:, We have previously demonstrated that advanced glycation end products (AGEs) stimulate bovine retinal endothelial cell (BREC) proliferation through induction of vascular endothelial growth factor (VEGF) production by these cells. We have also shown that gliclazide, a sulfonylurea which decreases oxidative stress, inhibits this effect. The aim of the present study was to characterize the signalling pathways involved in AGE-induced BREC proliferation and VEGF production and mediating the inhibitory effect of gliclazide on these biological events. Methods:, BRECs were treated or not treated with AGEs in the presence or absence of gliclazide, antioxidants, protein kinase C (PKC), mitogen-activated protein kinase (MAPK) or nuclear factor-,B (NF-,B) inhibitors. BREC proliferation was assessed by measuring [3H]-thymidine incorporation into DNA. Activation of PKC, MAPK and NF-,B signal transduction pathways and determination of VEGF expression were assessed by Western blot analysis using specific antibodies. MAPK activity was also determined by an in vitro kinase assay. Results:, Treatment of BRECs with AGEs significantly increased cell proliferation and VEGF expression. AGEs induced PKC-, translocation, extracellular signal-regulated protein kinase 1/2 and NF-,B activation in these cells. Pharmacological inhibition of these signalling pathways abolished AGE effects on cell proliferation and VEGF expression. Exposure of BRECs to gliclazide or antioxidants such as vitamin E or N -acetyl- l -cysteine resulted in a significant decrease in AGE-induced activation of PKC-, MAPK- and NF-,B-signalling pathways. Conclusions:, Our results demonstrate the involvement of PKC, MAPK and NF-,B in AGE-induced BREC proliferation and VEGF expression. Gliclazide inhibits BREC proliferation by interfering with these intracellular signal transduction pathways. [source] Microsomal triglyceride transfer protein regulates endogenous and exogenous antigen presentation by group,1 CD1 moleculesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2008Arthur Kaser Abstract Lipid antigens are presented to T cells by the non-polymorphic MHC class,I-related CD1 molecules. Microsomal triglyceride transfer protein (MTP) is an endoplasmic reticulum (ER)-resident chaperone that has been shown to lipidate the group,2 CD1 molecule CD1d and thus to regulate its function. We now report that MTP also regulates the function of group,1 CD1 molecules CD1a, CD1b, and CD1c. Pharmacological inhibition of MTP in monocyte-derived dendritic cells and lymphoblastoid B cell lines transfected with group,1 CD1 resulted in a substantial decrease in endogenous self lipid antigen presentation to several CD1-restricted T cell lines. Silencing MTP expression in CD1c-transfected HeLa cells similarly resulted in decreased self reactivity. Unexpectedly, inhibition of ER-resident MTP, which was confirmed by confocal microscopy, also markedly decreased presentation of exogenous, endosomally loaded, mycobacterial lipid antigens by CD1a and CD1c to T cells. Thus, these studies indicate that MTP, despite its ER localization, regulates endogenous as well as exogenous lipid antigen presentation, and suggest a broad role for MTP in the regulation of CD1 antigen presentation. [source] Mirk/Dyrk1B in cancerJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2007Eileen Friedman Abstract Mirk/Dyrk1B is a member of a conserved family of serine/threonine kinases which are activated by intramolecular tyrosine phosphorylation, and which mediate differentiation in different tissues,Mirk in skeletal muscle, Dyrk1A in the brain, etc. One role of Mirk in skeletal muscle differentiation is to block cycling myoblasts in the G0 quiescent state by modification of cell cycle regulators, while another role of Mirk is to limit apoptosis in fusing myoblasts. Amplification of the Mirk gene, upregulation of Mirk expression and/or constitutive activation of this kinase have been observed in several different types of cancer. If coupled with a stress condition such as serum starvation which induces a quiescent state, depletion of Mirk by RNA interference using either synthetic duplex RNAi's or pSilencer-encoded RNAi's have decreased colony formation of different cancer cell lines and enhanced apoptosis induced by chemotherapeutic drugs. Mirk is activated by phosphorylation by the stress-activated SAPK kinases MKK3 and MKK6. Our working hypothesis is that Mirk is activated by this pathway in response to various stresses, and then acts as a checkpoint kinase to arrest damaged tumor cells in a quiescent state and allow cellular repair. Pharmacological inhibition of Mirk may enhance the anti-tumor effect of chemotherapeutic drugs. J. Cell. Biochem. 102: 274,279, 2007. © 2007 Wiley-Liss, Inc. [source] Regulation of axotomy-induced dopaminergic neuron death and c-Jun phosphorylation by targeted inhibition of cdc42 or mixed lineage kinaseJOURNAL OF NEUROCHEMISTRY, Issue 2 2006Stephen J. Crocker Abstract Mechanical transection of the nigrostriatal dopamine pathway at the medial forebrain bundle (MFB) results in the delayed degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). We have previously demonstrated that c-Jun activation is an obligate component of neuronal death in this model. Here we identified the small GTPase, cdc42, and mixed lineage kinases (MLKs) as upstream factors regulating neuronal loss and activation of c-Jun following MFB axotomy. Adenovirus-mediated expression of a dominant-negative form of cdc42 in nigral neurons blocked MFB axotomy-induced activation (phosphorylation) of MAP kinase kinase 4 (MKK4) and c-Jun, resulting in attenuation of SNpc neuronal death. Pharmacological inhibition of MLKs, MKK4-activating kinases, significantly reduced the phosphorylation of c-Jun and abrogated dopaminergic neuronal degeneration following MFB axotomy. Taken together, these findings suggest that death of nigral dopaminergic neurons following axotomy can be attenuated by targeting cell signaling events upstream of c-Jun N-terminal mitogen-activated protein kinase/c-Jun. [source] Neuroprotection by stem cell factor in rat cortical neurons involves AKT and NF,BJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Krishnan M. Dhandapani Abstract Stem cell factor (SCF) is a highly expressed cytokine in the central nervous system. In the present study, we demonstrate a neuroprotective role for SCF and its tyrosine kinase receptor, c-kit, against camptothecin-induced apoptosis and glutamate excitotoxicity in rat cortical neurons. This protection was blocked by pharmacological or molecular inhibition of either the MEK/ERK or PI3K/Akt signaling pathways. The importance of these pathways was further confirmed by the activation of both ERK, in a MEK-dependent manner, and Akt, via PI3K. Activation of Akt increased the binding of the p50 and p65 subunits of NF,B, which was also important for neuroprotection. Akt inhibition prevented NF,B binding, suggesting a role for Akt in SCF-induced NF,B. Pharmacological inhibition of NF,B or dominant negative I,B also prevented neuroprotection by SCF. SCF up-regulated the anti-apoptotic genes, bcl-2 and bcl-xL in an NF,B-dependent manner. Together, these findings demonstrate a neuroprotective role for SCF in cortical neurons, an effect that was mediated by Akt and ERK, as well as NF,B-mediated gene transcription. SCF represents a novel therapeutic target in the treatment of neurodegenerative disease. [source] Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathwaysJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2007M. HASMIM Summary.,Background: Recent evidence indicates that zoledronate, a nitrogen-containing bisphosphonate used to treat conditions of increased bone resorption, may have anti-angiogenic activity. The endothelial cells signaling events modulated by zoledronate remain largely elusive. Objectives: The aim of this work was to identify signaling events suppressed by zoledronate in endothelial cells and responsible for some of its biological effects. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to zoledronate, isoprenoid analogs (i.e. farnesol and geranylgeraniol) and various inhibitors of signaling, and the effect on adhesion, survival, migration, actin cytoskeleton and signaling events characterized. Results: Zoledronate reduced Ras prenylation, Ras and RhoA translocation to the membrane, and sustained ERK1/2 phosphorylation and tumor necrosis factor (TNF) induced JNK phosphorylation. Isoprenoid analogs attenuated zoledronate effects on HUVEC adhesion, actin stress fibers and focal adhesions, migration and survival. Isoprenoid analogs also restored Ras prenylation, RhoA translocation to the membrane, sustained FAK and ERK1/2 phosphorylation and prevented suppression of protein kinase B (PKB) and JNK phosphorylation in HUVEC exposed to TNF in the presence of zoledronate. Pharmacological inhibition of Rock, a RhoA target mediating actin fiber formation, phosphatidylinositol 3-kinase, an activator of PKB, MEK1/2, an activator of ERK1/2, and JNK, recapitulated individual zoledronate effects, consistent with the involvement of these molecules and pathways and their inhibition in the zoledronate effects. Conclusions: This work has demonstrated that zoledronate inhibits HUVEC adhesion, survival, migration and actin stress fiber formation by interfering with protein prenylation and has identified ERK1/2, JNK, Rock, FAK and PKB as kinases affected by zoledronate in a prenylation-dependent manner. [source] Orexins/hypocretins control bistability of hippocampal long-term synaptic plasticity through co-activation of multiple kinasesACTA PHYSIOLOGICA, Issue 3 2010O. Selbach Abstract Aim:, Orexins/hypocretins (OX/Hcrt) are hypothalamic neuropeptides linking sleep,wakefulness, appetite and neuroendocrine control. Their role and mechanisms of action on higher brain functions, such as learning and memory, are not clear. Methods:, We used field recordings of excitatory post-synaptic potentials (fEPSP) in acute mouse brain slice preparations to study the effects of orexins and pharmacological inhibitors of multiple kinases on long-term synaptic plasticity in the hippocampus. Results:, Orexin-A (OX-A) but not orexin-B (OX-B) induces a state-dependent long-term potentiation of synaptic transmission (LTPOX) at Schaffer collateral-CA1 synapses in hippocampal slices from adult (8- to 12-week-old) mice. In contrast, OX-A applied to slices from juvenile (3- to 4-week-old) animals causes a long-term depression (LTDOX) in the same pathway. LTPOX is blocked by pharmacological inhibition of orexin receptor-1 (OX1R) and plasticity-related kinases, including serine/threonine- (CaMKII, PKC, PKA, MAPK), lipid- (PI3K), and receptor tyrosine kinases (Trk). Inhibition of OX1R, CaMKII, PKC, PKA and Trk unmasks LTDOX in adult animals. Conclusion:, Orexins control not only the bistability of arousal states and threshold for appetitive behaviours but, in an age- and kinase-dependent manner, also bidirectional long-term synaptic plasticity in the hippocampus, providing a possible link between behavioural state and memory functions. [source] The energetic cost of activation in mouse fast-twitch muscle is the same whether measured using reduced filament overlap or N -benzyl- p -toluenesulphonamideACTA PHYSIOLOGICA, Issue 4 2008C. J. Barclay Abstract Aim:, Force generation and transmembrane ion pumping account for the majority of energy expended by contracting skeletal muscles. Energy turnover for ion pumping, activation energy turnover (EA), can be determined by measuring the energy turnover when force generation has been inhibited. Most measurements show that activation accounts for 25,40% of isometric energy turnover. It was recently reported that when force generation in mouse fast-twitch muscle was inhibited using N -benzyl- p -toluenesulphonamide (BTS), activation accounted for as much as 80% of total energy turnover during submaximal contractions. The purpose of this study was to compare EA measured by inhibiting force generation by: (1) the conventional method of reducing contractile filament overlap; and (2) pharmacological inhibition using BTS. Methods:, Experiments were performed in vitro using bundles of fibres from mouse fast-twitch extensor digitorum longus (EDL) muscle. Energy turnover was quantified by measuring the heat produced during 1-s maximal and submaximal tetanic contractions at 20 and 30 °C. Results:,EA measured using reduced filament overlap was 0.36 ± 0.04 (n = 8) at 20 °C and 0.31 ± 0.05 (n = 6) at 30 °C. The corresponding values measured using BTS in maximal contractions were 0.46 ± 0.06 and 0.38 ± 0.06 (n = 6 in both cases). There were no significant differences among these values. EA was also no different when measured using BTS in submaximal contractions. Conclusion:, Activation energy turnover is the same whether measured using BTS or reduced filament overlap and accounts for slightly more than one-third of isometric energy turnover in mouse EDL muscle. [source] Chemokines integrate JAK/STAT and G-protein pathways during chemotaxis and calcium flux responsesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2003Silvia Abstract The JAK/STAT (Janus kinase / signaling transducer and activator of transcription) signaling pathway is implicated in converting stationary epithelial cells to migratory cells. In mammals, migratory responses are activated by chemoattractant proteins, including chemokines. We found that by binding to seven-transmembrane G-protein-coupled receptors, chemokines activate the JAK/STAT pathwayto trigger chemotactic responses. We show that chemokine-mediated JAK/STAT activation is critical for G-protein induction and for phospholipase C-, dependent Ca2+ flux; in addition, pharmacological inhibition of JAK or mutation of the JAK kinase domain causes defects in both responses. Furthermore, G,i association with the receptor is dependent on JAK activation, andthe chemokine-mediated Ca2+ flux that requires phospholipase C-, activity takes place downstream of JAK kinases. The chemokines thus employ a mechanism that links heterologous signaling pathways , G proteins and tyrosine kinases , in a network that may be essential for mediating their pleiotropic responses. [source] Fibrinogen-CD11b/CD18 interaction activates the NF-,B pathway and delays apoptosis in human neutrophilsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2003Carolina Rubel Abstract The regulation of neutrophil half-life by members of the coagulation cascade is critical for the resolution of the inflammatory response. We have demonstrated that soluble fibrinogen (sFbg) delays human neutrophil (PMN) apoptosis through a mechanism that involves CD11b interactions, and phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase,1/2 (ERK1/2). Since NF-,B is a key element in the regulation of apoptotic mechanisms in several immune cells, we investigated whether NF-,B is involved in the control of PMN survival by sFbg. We showthat sFbg triggers inhibitor protein ,B (I,B-,) degradation and NF-,B activation. Furthermore, pharmacological inhibition of NF-,B abrogates sFbg effects on apoptosis. In addition, specific inhibition of MAPK ERK1/2 significantly reduces NF-,B translocation by sFbg, suggesting a relationship between ERK1/2 and NF-,B activation. Similar results are obtained when granulocytic-differentiated HL-60 cells are treated with sFbg, making this model highly attractive for integrin-induced gene expression studies. It can be concluded that NF-,B participates in the prevention of apoptosis induced by sFbg with the participation of MAPK ERK1/2. These results shed light on the molecular mechanisms that control human granulocyte apoptosis, and suggest that NF-,B regulation may be of benefit for the resolution of the inflammatory response. [source] A putative role for cell cycle-related proteins in microtubule-based neuroplasticityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2009Stefanie Schmetsdorf Abstract Cyclins and cyclin-dependent kinases (Cdks) are the main components that control the orderly progression through cell cycle. In the mature nervous system, terminally differentiated neurons are permanently withdrawn from cell cycle, as mitotic quiescence is essential for the functional stability of the complexly wired neuronal system. Recently, we characterized the expression and colocalization of cyclins and Cdks in terminally differentiated pyramidal neurons. The functional impact of the expression of cell cycle-related proteins in differentiated neurons, however, has not been elucidated yet. In the present study, we show by immunoelectron microscopy and immunobiochemical methods an association of cyclins and Cdks with the microtubule network. Cyclins D, E, A and B as well as Cdks 1, 2 and 4 were also found to be associated with the microtubule-associated protein tau. Cyclin/Cdk complexes, in addition, exhibit kinase activity towards tau. In vitro, downregulation of cyclins and Cdks by a siRNA approach and by pharmacological inhibition promotes neurite extension. Taken together, these results indicate that the expression of cell cycle-related proteins in terminal differentiated neurons is associated with physiological functions beyond cell cycle control that might be involved in microtubule-based mechanisms of neuroplasticity. [source] Sodium/bicarbonate cotransporter NBCn1/slc4a7 increases cytotoxicity in magnesium depletion in primary cultures of hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2009Deborah S. Cooper Abstract Growing evidence suggests that pharmacological inhibition of Na/H exchange and Na/HCO3 transport provides protection against damage or injury in cardiac ischemia. In this study, we examined the contribution of the sodium/bicarbonate cotransporter NBCn1 (slc4a7) to cytotoxicity in cultured hippocampal neurons of rats. In neurons exposed to extracellular pH (pHo) ranging from 6.2 to 8.3, NBCn1 protein expression increased by fivefold at pH < 6.5 compared to the expression at pHo 7.4. At pHo 6.5, the intracellular pH of neurons was ,1 unit lower than that at pH 7.4. Immunochemistry showed a marked increase in NBCn1 immunofluorescence in plasma membranes and cytosol of the soma as well as in dendrites, at pHo 6.5. NBCn1 expression also increased by 40% in a prolonged Mg2+ -free incubation at normal pHo. Knockdown of NBCn1 in neurons had negligible effect on cell viability. The effect of NBCn1 knockdown on cytotoxicity was then determined by exposing neurons to 0.5 mm glutamate for 10 min and measuring lactate dehydrogenase (LDH) release from neurons. Compared to normal incubation (pHo 7.2 for 6 h) after glutamate exposure, acidic incubation (pHo 6.3 for 6 h) reduced cytotoxicity by 75% for control neurons and 78% for NBCn1-knockdown neurons. Thus, both controls and knockdown neurons showed acidic protection from cytotoxicity. However, in Mg2+ -free incubation after glutamate exposure, NBCn1 knockdown progressively attenuated cytotoxicity. This attenuation was unaffected by acidic preincubation before glutamate exposure. We conclude that NBCn1 has a dynamic upregulation in low pHo and Mg2+ depletion. NBCn1 is not required for acidic protection, but increases cytotoxicity in Mg2+ -free conditions. [source] Dominant-negative Rac increases both inherent and ionizing radiation-induced cell migration in C6 rat glioma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 8 2006So-Young Hwang Abstract Rho-like GTPases, including Cdc42, Rac1 and RhoA, regulate distinct actin cytoskeleton changes required for cell adhesion, migration and invasion. In the present study, we examined the role of Rac signaling in inherent migration, as well as radiation-induced migration, of rat glioma cells. Stable overexpression of dominant-negative Rac1N17 in a C6 rat glioma cell line (C6-RacN17) promoted cell migration, and ionizing radiation further increased this migration. Migration was accompanied by decreased expression of the focal adhesion molecules FAK and paxillin. Focal contacts and actin stress fibers were also reduced in C6-RacN17 cells. Downstream effectors of Rac include JNK and p38 MAP kinases. Irradiation transiently activated p38, JNK and ERK1/2 MAP kinases in C6-RacN17 cells, while p38 and JNK were constitutively activated in C6 control cells. Blocking JNK activity with JNK inhibitor SP600125 inhibited migration, suggesting that the JNK pathway may regulate radiation-induced, as well as inherent, migration of C6-RacN17 cells. Additionally, the radiation-induced migration increase was also inhibited by SB203580, a specific inhibitor of p38 MAP kinase. However, PD98059, a MEK kinase 1 inhibitor, failed to influence migration. This is the first evidence that suppression of Rac signaling may be involved in invasion or metastasis of glioma cells before and/or after radiotherapy. These data further suggest that radiotherapy for malignant glioma needs to be used with caution because of the potential for therapy-induced cell migration or invasion and that pharmacological inhibition of cell migration and invasion through targeting the Rac signaling pathway may represent a new approach for improving the therapeutic efficacy of radiotherapy for malignant glioma. © 2005 Wiley-Liss, Inc. [source] Expression of Acid-Sensing Ion Channel 3 (ASIC3) in Nucleus Pulposus Cells of the Intervertebral Disc Is Regulated by p75NTR and ERK Signaling,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2007Yoshiyasu Uchiyama Abstract Although a recent study has shown that skeletal tissues express ASICs, their function is unknown. We show that intervertebral disc cells express ASIC3; moreover, expression is uniquely regulated and needed for survival in a low pH and hypoeromsotic medium. These findings suggest that ASIC3 may adapt disc cells to their hydrodynamically stressed microenvironment. Introduction: The nucleus pulposus is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. Because the tissue is hyperosmotic and avascular, the pH of the nucleus pulposus is low. To determine the mechanisms by which the disc cells accommodate to the low pH and hypertonicity, the expression and regulation of the acid sensing ion channel (ASIC)3 was examined. Materials and Methods: Expression of ASICs in cells of the intervertebral disc was analyzed. To study its regulation, we cloned the 2.8-kb rat ASIC3 promoter and performed luciferase reporter assays. The effect of pharmacological inhibition of ASICs on disc cell survival was studied by measuring MTT and caspase-3 activities. Results: ASIC3 was expressed in discal tissues and cultured disc cells in vitro. Because studies of neuronal cells have shown that ASIC3 expression and promoter activity is induced by nerve growth factor (NGF), we examined the effect of NGF on nucleus pulposus cells. Surprisingly, ASIC3 promoter activity did not increase after NGF treatment. The absence of induction was linked to nonexpression of tropomyosin-related kinase A (TrkA), a high-affinity NGF receptor, although a modest expression of p75NTR was seen. When treated with p75NTR antibody or transfected with dominant negative-p75NTR plasmid, there was significant suppression of ASIC3 basal promoter activity. To further explore the downstream mechanism of control of ASIC3 basal promoter activity, we blocked p75NTR and measured phospho extracellular matrix regulated kinase (pERK) levels. We found that DN-p75NTR suppressed NGF mediated transient ERK activation. Moreover, inhibition of ERK activity by dominant negative-mitogen activated protein kinase kinase (DN-MEK) resulted in a dose-dependent suppression of ASIC3 basal promoter activity, whereas overexpression of constitutively active MEK1 caused an increase in ASIC3 promoter activity. Finally, to gain insight in the functional importance of ASIC3, we suppressed ASIC activity in nucleus pulposus cells. Noteworthy, under both hyperosmotic and acidic conditions, ASIC3 served to promote cell survival and lower the activity of the pro-apoptosis protein, caspase-3. Conclusions: Results of this study indicate that NGF serves to maintain the basal expression of ASIC3 through p75NTR and ERK signaling in discal cells. We suggest that ASIC3 is needed for adaptation of the nucleus pulposus and annulus fibrosus cells to the acidic and hyperosmotic microenvironment of the intervertebral disc. [source] Novel Inhibitors of Alkaline Phosphatase Suppress Vascular Smooth Muscle Cell Calcification,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2007Sonoko Narisawa Abstract We report three novel inhibitors of the physiological pyrophosphatase activity of alkaline phosphatase and show that these compounds are capable of reducing calcification in two models of vascular calcification (i.e., they suppress in vitro calcification by cultured Enpp1,/, VSMCs and they inhibit the increased pyrophosphatase activity in a rat aortic model). Introduction: Genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) leads to accumulation of the calcification inhibitor inorganic pyrophosphate (PPi). TNALP deficiency ameliorates the hypermineralization phenotype in Enpp1,/, and ank/ank mice, two models of osteoarthritis and soft tissue calcification. We surmised that the pharmacological inhibition of TNALP pyrophosphatase activity could be used to prevent/suppress vascular calcification. Materials and Methods: Comprehensive chemical libraries were screened to identify novel drug-like compounds that could inhibit TNALP pyrophosphatase function at physiological pH. We used these novel compounds to block calcification by cultured vascular smooth muscle cells (VSMCs) and to inhibit the upregulated pyrophosphatase activity in a rat aortic calcification model. Results: Using VSMC cultures, we determined that Enpp1,/, and ank/ank VSMCs express higher TNALP levels and enhanced in vitro calcification compared with wildtype cells. By high-throughput screening, three novel compounds, 5361418, 5923412, and 5804079, were identified that inhibit TNALP pyrophosphatase function through an uncompetitive mechanism, with high affinity and specificity when measured at both pH 9.8 and 7.5. These compounds were shown to reduce the calcification by Enpp1,/, VSMCs. Furthermore, using an ex vivo rat whole aorta PPi hydrolysis assay, we showed that pyrophosphatase activity was inhibited by all three lead compounds, with compound 5804079 being the most potent at pH 7.5. Conclusions: We conclude that TNALP is a druggable target for the treatment and/or prevention of ectopic calcification. The lead compounds identified in this study will serve as scaffolds for medicinal chemistry efforts to develop drugs for the treatment of soft tissue calcification. [source] Cyclo(His-Pro) promotes cytoprotection by activating Nrf2-mediated up-regulation of antioxidant defenceJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6 2009Alba Minelli Abstract Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and ,-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence. [source] Src and FAK mediate cell,matrix adhesion-dependent activation of met during transformation of breast epithelial cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Angela Y. Hui Abstract Cell,matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand-independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non-neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto-activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with ,1 integrins and is co-localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src-dependent phosphorylation of Met requires cell,matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src-induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF-dependent and Src/integrin-dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells. J. Cell. Biochem. 107: 1168,1181, 2009. © 2009 Wiley-Liss, Inc. [source] Chronic myelogenous leukaemia , new therapeutic principlesJOURNAL OF INTERNAL MEDICINE, Issue 1 2001Michael E. O'Dwyer O'Dwyer ME, Druker BJ (Oregon Health Sciences University, Portland, USA). Chronic myelogenous leukaemia , new therapeutic principles. J. Intern Med 2001; 250: 3,9 The deregulated tyrosine kinase activity of the BCR-ABL fusion protein is the cause of malignant transformation in almost all cases of chronic myelogenous leukaemia (CML), making BCR-ABL an ideal target for pharmacological inhibition. Signal transduction inhibitor (STI571) (formerly CGP57 148B), is an ABL specific, tyrosine kinase inhibitor. In preclinical studies, it has been shown to selectively kill BCR-ABL expressing cells, both in-vitro and in vivo. The results of clinical studies to date are highly encourageing and STI571 promises to be an important addition to the therapy of CML. [source] A role for glutamate in growth and invasion of primary brain tumorsJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Harald Sontheimer Abstract The vast majority of primary brain tumors derive from glial cells and are collectively called gliomas. While, they share some genetic mutations with other cancers, they do present with a unique biology and have developed adaptations to meet specific biological needs. Notably, glioma growth is physically restricted by the skull, and, unless normal brain cells are destroyed, tumors cannot expand. To overcome this challenge, glioma cells release glutamate which causes excitotoxic death to surrounding neurons, thereby vacating room for tumor expansion. The released glutamate also explains peritumoral seizures which are a common symptom early in the disease. Glutamate release occurs via system Xc, a cystine,glutamate exchanger that releases glutamate in exchange for cystine being imported for the synthesis of the cellular antioxidant GSH. It protects tumor cells from endogenously produced reactive oxygen and nitrogen species but also endows tumors with an enhanced resistance to radiation- and chemotherapy. Pre-clinical data demonstrates that pharmacological inhibition of system Xc causes GSH depletion which slows tumor growth and curtails tumor invasion in vivo. An Food and Drug Administration approved drug candidate is currently being introduced into clinical trials for the treatment of malignant glioma. [source] Ras/ERK signalling in cannabinoid tolerance: from behaviour to cellular aspectsJOURNAL OF NEUROCHEMISTRY, Issue 4 2005Tiziana Rubino Abstract We investigated the role of the Ras/extracellular-regulated kinase (ERK) pathway in the development of tolerance to ,9 -tetrahydrocannabinol (THC)-induced reduction in spontaneous locomotor activity by a genetic (Ras-specific guanine nucleotide exchange factor (Ras-GRF1) knock-out mice) and pharmacological approach. Pre-treatment of wild-type mice with SL327 (50 mg/kg i.p.), a specific inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase of ERK, fully prevented the development of tolerance to THC-induced hypolocomotion. We investigated the impact of the inhibition of ERK activation on the biological processes involved in cannabinoid tolerance (receptor down-regulation and desensitization), by autoradiographic cannabinoid CB1 receptor and cannabinoid-stimulated [35S]GTP,S binding studies in subchronically treated mice (THC, 10 mg/kg s.c., twice a day for 5 days). In the caudate putamen and cerebellum of Ras-GRF1 knock-out mice and SL327 pre-treated wild-type mice, CB1 receptor down-regulation and desensitization did not occur, suggesting that ERK activation might account for CB1 receptor plasticity involved in the development of tolerance to THC hypolocomotor effect. In contrast, the hippocampus and prefrontal cortex showed CB1 receptor adaptations regardless of the genetic or pharmacological inhibition of the ERK pathway, suggesting regional variability in the cellular events underlying the altered CB1 receptor function. These findings suggest that at least in the caudate putamen and cerebellum, the Ras/ERK pathway is essential for triggering the alteration in CB1 receptor function responsible for tolerance to THC-induced hypomotility. [source] Levodopa treatment reverses endocannabinoid system abnormalities in experimental parkinsonismJOURNAL OF NEUROCHEMISTRY, Issue 4 2003Mauro Maccarrone Abstract Cannabinoid receptors and their endogenous ligands are potent inhibitors of neurotransmitter release in the brain. Here, we show that in a rat model of Parkinson's disease induced by unilateral nigral lesion with 6-hydroxydopamine (6-OHDA), the striatal levels of the endocannabinoid anandamide (AEA) were increased, while the activity of its membrane transporter and hydrolase (fatty-acid amide hydrolase, FAAH) were decreased. These changes were not observed in the cerebellum of the same animals. Moreover, the frequency and amplitude of glutamate-mediated spontaneous excitatory post-synaptic currents were augmented in striatal spiny neurones recorded from parkinsonian rats. Remarkably, the anomalies in the endocannabinoid system, as well as those in glutamatergic activity, were completely reversed by chronic treatment of parkinsonian rats with levodopa, and the pharmacological inhibition of FAAH restored a normal glutamatergic activity in 6-OHDA-lesioned animals. Thus, the increased striatal levels of AEA may reflect a compensatory mechanism trying to counteract the abnormal corticostriatal glutamatergic drive in parkinsonian rats. However, this mechanism seems to be unsuccessful, since spontaneous excitatory activity is still higher in these animals. Taken together, these data show that anomalies in the endocannabinoid system induced by experimental parkinsonism are restricted to the striatum and can be reversed by chronic levodopa treatment, and suggest that inhibition of FAAH might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinson's disease. [source] Increased CNS uptake and enhanced antinociception of morphine-6-glucuronide in rats after inhibition of P-glycoproteinJOURNAL OF NEUROCHEMISTRY, Issue 2 2002Jörn Lötsch Abstract Morphine-6-glucuronide (M6G) is a substrate of P-glycoprotein (P-gp), which forms an outward transporter at the blood,brain barrier. Inhibition of P-gp may therefore be expected to cause increased CNS uptake of M6G. We directly assessed the spinal concentrations of M6G and its antinociceptive effects in rats following pharmacological inhibition of P-gp. Spinal cord tissue concentrations of M6G were assessed by microdialysis with probes transversally implanted through the dorsal horns of the spinal cord at level L4. Ten rats received M6G intravenously (0.018 mg/kg loading dose plus 0.00115 mg/kg/min for an 8-h infusion), five of them together with PSC833 to inhibit P-gp (32-h infusion, starting 24 h before the addition of M6G). Antinociceptive effects were explored by means of formalin tests. After having obtained evidence for enhanced CNS uptake and antinociception of M6G in the presence of PSC833, additional behavioural experiments were performed in another 32 rats to assess the dose dependency of the antinociceptive effects of M6G either with or without PSC833 in comparison with both PSC833 alone and placebo. Inhibition of P-gp increased the M6G concentrations in the spinal cord approximately three-fold whereas the plasma concentrations were increased only by a factor of 1.4, which resulted in a more than doubled spinal cord/plasma concentration ratio (from 0.08 ± 0.03 for M6G alone to 0.17 ± 0.08 for M6G plus PSC833). Antinociceptive effects of M6G were significantly enhanced by inhibition of P-gp. Inhibition of P-gp alters the transport of M6G across the blood,brain barrier, resulting in enhanced spinal cord uptake and enhanced antinociception. [source] Facilitation of Myocardial PI3K/Akt/nNOS Signaling Contributes to Ethanol-Evoked Hypotension in Female RatsALCOHOLISM, Issue 7 2009Mahmoud M. El-Mas Background:, The mechanism by which ethanol reduces cardiac output (CO) and blood pressure (BP) in female rats remains unclear. We tested the hypothesis that enhancement of myocardial phosphatidylinositol 3-kinase (PI3K)/Akt signaling and related neuronal nitric oxide synthase (nNOS) and/or endothelial nitric oxide synthase (eNOS) activity constitutes a cellular mechanism for the hemodynamic effects of ethanol. Methods:, We measured the level of phosphorylated eNOS (p-eNOS) and p-nNOS in the myocardium of ethanol (1 g/kg intragastric, i.g.) treated female rats along with hemodynamic responses [BP, CO, stroke volume, (SV), total peripheral resistance, (TPR)], and myocardial nitrate/nitrite levels (NOx) levels. Further, we investigated the effect of selective pharmacological inhibition of nNOS with N, -propyl- l -arginine (NPLA) or eNOS with N5 -(1-iminoethyl)- l -ornithine (l -NIO) on cellular, hemodynamic, and biochemical effects of ethanol. The effects of PI3K inhibition by wortmannin on the cardiovascular actions of ethanol and myocardial Akt phosphorylation were also investigated. Results:, The hemodynamic effects of ethanol (reductions in BP, CO, and SV) were associated with significant increases in myocardial NOx and myocardial p-nNOS and p-Akt expressions while myocardial p-eNOS remained unchanged. Prior nNOS inhibition by NPLA (2.5 or 12.5 ,g/kg) attenuated hemodynamic effects of ethanol and abrogated associated increases in myocardial NOx and cardiac p-nNOS contents. The hemodynamic effects of ethanol and increases in myocardial p-Akt phosphorylation were reduced by wortmannin (15 ,g/kg). On the other hand, although eNOS inhibition by l -NIO (4 or 20 mg/kg) in a dose-dependent manner attenuated ethanol-evoked hypotension, the concomitant reductions in CO and SV remained unaltered. Also, selective eNOS inhibition uncovered dramatic increases in TPR in response to ethanol, which appeared to have offset the reduction in CO. Neither NPLA nor l -NIO altered plasma ethanol levels. Conclusions:, These findings implicate the myocardial PI3K/Akt/nNOS signaling in the reductions in BP and CO produced by ethanol in female rats. [source] Ultraviolet radiation stimulates expression of Snail family transcription factors in keratinocytesMOLECULAR CARCINOGENESIS, Issue 4 2007Laurie G. Hudson Abstract The related zinc finger transcription factors Slug and Snail modulate epithelial mesenchymal transformation (EMT), the conversion of sessile epithelial cells into migratory fibroblast-like cells. EMT occurs during development, wound healing, and tumor progression. Growth factors, acting through mitogen-activated protein kinase (MAPK) cascades, regulate expression of Slug and Snail. Expression of Snail family transcription factors appears to be elevated in UVR-induced murine squamous cell carcinomas (SCC). We report here that ultraviolet radiation (UVR), which activates MAPK cascades, also stimulates Snail and Slug expression in epidermal keratinocytes. UVR exposure transiently elevated Slug and Snail mRNA expression in human keratinocytes in vitro and mouse epidermis in vivo. This induction was mediated, at least in part, through the ERK and p38 MAPK cascades, as pharmacological inhibition of these cascades partially or completely blocked Slug and Snail induction by UVR. On the other hand, UVR induction of Slug and Snail was enhanced by inhibition of JNK. Slug appears to play a functional role in the acute response of keratinocytes to UVR, as UVR induction of keratin 6 in the epidermis of Slug knockout mice was markedly delayed compared to wild-type mice. Slug and Snail are known to regulate molecules important in the cytoskeleton, intercellular adhesion, cell motility, and apoptosis, thus it seems probable that transiently or persistently elevated expression of these factors fosters the progression of UVR-induced SCC. © 2007 Wiley-Liss, Inc. [source] Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cellsMOLECULAR CARCINOGENESIS, Issue 3 2005Dong Xiao Abstract The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in apoptosis induction by phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived cancer chemopreventive agent, with DU145 and LNCaP human prostate cancer cells as a model. The MAPK family of serine/threonine kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c- jun N-terminal kinase1/2/3 (JNK1/2/3), and p38 MAPK play an important role in cell proliferation and apoptosis in response to different stimuli. Exposure of DU145 and LNCaP cells to growth suppressive concentrations of PEITC resulted in activation of ERK1/2 and JNKs, but not p38 MAPK, in both cell lines. In DU145 cells, the apoptosis induction by PEITC was statistically significantly attenuated by pharmacological inhibition of JNKs with SP600125. Adenovirus-mediated overexpression of Flag-tagged JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), an inhibitor of JNK, also inhibited PEITC-induced apoptosis in DU145 cells. On the other hand, inhibition of ERK1/2 activation with MEK1 inhibitor PD98059 failed to offer protection against PEITC-induced apoptosis in DU145 cells. In LNCaP cells, the PEITC-induced cell death was not affected by either pretreatment with PD98059 or SP600125 or overexpression of JBD of JIP-1. These results indicate that involvement of MAPKs in apoptosis induction by PEITC in human prostate cancer cells is cell line-specific. © 2005 Wiley-Liss, Inc. [source] The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin functionMOLECULAR MICROBIOLOGY, Issue 5 2003Peter R. Kraus Summary Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37°C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a ,-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall. [source] Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovaniPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2003Meike Bente Abstract In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control. [source] | |||||||||||||