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Seth Malmersjö,
Paola Rebellato,
Erik Smedler,
Henrike Planert,
Shigeaki Kanatani,
Isabel Liste,
Evanthia Nanou,
Hampus Sunner,
Shaimaa Abdelhady,
Songbai Zhang,
Michael Andäng,
Abdeljabbar El Manira,
Gilad Silberberg,
Ernest Arenas, Per Uhlén
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ABSTRACT: Coherent network activity among assemblies of interconnected cells is essential for diverse functions in the adult brain. However, cellular networks before formations of chemical synapses are poorly understood. Here, embryonic stem cell-derived neural progenitors were found to form networks exhibiting synchronous calcium ion (Ca(2+)) activity that stimulated cell proliferation. Immature neural cells established circuits that propagated electrical signals between neighboring cells, thereby activating voltage-gated Ca(2+) channels that triggered Ca(2+) oscillations. These network circuits were dependent on gap junctions, because blocking prevented electrotonic transmission both in vitro and in vivo. Inhibiting connexin 43 gap junctions abolished network activity, suppressed proliferation, and affected embryonic cortical layer formation. Cross-correlation analysis revealed highly correlated Ca(2+) activities in small-world networks that followed a scale-free topology. Graph theory predicts that such network designs are effective for biological systems. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation.
Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor
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ABSTRACT: BACKGROUND: Polycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca2+) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells. METHODS: We investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca2+ signaling. Plasma membrane (PM) Ca2+ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy. RESULTS: We demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca2+ signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca2+ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca2+ permeability. Intracellular Ca2+ buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca2+/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM. CONCLUSIONS: These novel findings demonstrate intracellular Ca2+-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.
BMC Nephrology 02/2013; 14(1):34. · 2.18 Impact Factor
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Konstantin Gaengel,
Colin Niaudet,
Kazuhiro Hagikura,
Bàrbara Laviña Siemsen,
Lars Muhl,
Jennifer J Hofmann,
Lwaki Ebarasi,
Staffan Nyström,
Simin Rymo,
Long Long Chen, [......],
Jimmy Larsson,
Mats Hellström,
Jonas Fuxe, Per Uhlén,
Ralf Adams,
Lars Jakobsson,
Arindam Majumdar,
Dietmar Vestweber,
Anne Uv,
Christer Betsholtz
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ABSTRACT: Angiogenesis, the process by which new blood vessels arise from preexisting ones, is critical for embryonic development and is an integral part of many disease processes. Recent studies have provided detailed information on how angiogenic sprouts initiate, elongate, and branch, but less is known about how these processes cease. Here, we show that S1PR1, a receptor for the blood-borne bioactive lipid sphingosine-1-phosphate (S1P), is critical for inhibition of angiogenesis and acquisition of vascular stability. Loss of S1PR1 leads to increased endothelial cell sprouting and the formation of ectopic vessel branches. Conversely, S1PR1 signaling inhibits angiogenic sprouting and enhances cell-to-cell adhesion. This correlates with inhibition of vascular endothelial growth factor-A (VEGF-A)-induced signaling and stabilization of vascular endothelial (VE)-cadherin localization at endothelial junctions. Our data suggest that S1PR1 signaling acts as a vascular-intrinsic stabilization mechanism, protecting developing blood vessels against aberrant angiogenic responses.
Developmental cell 09/2012; 23(3):587-99. · 13.36 Impact Factor
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ABSTRACT: Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance ex vivo, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.
Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for ex vivo culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.
The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.
BMC Physiology 03/2012; 12:3.
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ABSTRACT: The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca(2+)) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca(2+) from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca(2+) and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015.
PLoS ONE 01/2012; 7(2):e31258. · 4.09 Impact Factor
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ABSTRACT: Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca(2+)) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) are intracellular Ca(2+) release channels that mediate Ca(2+) release from endoplasmic reticulum (ER) Ca(2+) stores. The three IP(3)R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP(3)R by the endogenous modulators IP(3), Ca(2+), protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP(3)R subtype in shaping cytosolic Ca(2+) oscillations.
Neurochemical Research 07/2011; 36(7):1175-85. · 2.24 Impact Factor
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ABSTRACT: Mitochondrial morphology is controlled by two opposing processes: fusion and fission. Drp1 (dynamin-related protein 1) and hFis1 are two key players of mitochondrial fission, but how Drp1 is recruited to mitochondria and how Drp1-mediated mitochondrial fission is regulated in mammals is poorly understood. Here, we identify the vertebrate-specific protein MIEF1 (mitochondrial elongation factor 1; independently identified as MiD51), which is anchored to the outer mitochondrial membrane. Elevated MIEF1 levels induce extensive mitochondrial fusion, whereas depletion of MIEF1 causes mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria in a manner independent of hFis1, Mff (mitochondrial fission factor) and Mfn2 (mitofusin 2), but inhibits Drp1 activity, thus executing a negative effect on mitochondrial fission. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reverse the MIEF1-induced fusion phenotype. In addition to inhibiting Drp1, MIEF1 also actively promotes fusion, but in a manner distinct from mitofusins. In conclusion, our findings uncover a novel mechanism which controls the mitochondrial fusion-fission machinery in vertebrates. As MIEF1 is vertebrate-specific, these data also reveal important differences between yeast and vertebrates in the regulation of mitochondrial dynamics.
The EMBO Journal 06/2011; 30(14):2762-78. · 9.20 Impact Factor
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ABSTRACT: ATP-Binding Cassette (ABC) transporters are efflux pumps frequently associated with multidrug resistance in many biological systems, including malaria. Antimalarial drug-resistance involves an ABC transporter, PfMDR1, a homologue of P-glycoprotein in humans. Twenty years of research have shown that several single nucleotide polymorphisms in pfmdr1 modulate in vivo and/or in vitro drug susceptibility. The underlying physiological mechanism of the effect of these mutations remains unclear. Here we develop structural models for PfMDR1 in different predicted conformations, enabling the study of transporter motion. Such analysis of functional polymorphisms allows determination of their potential role in transport and resistance. The bacterial MsbA ABC pump is a PfMDR1 homologue. MsbA crystals in different conformations were used to create PfMDR1 models with Modeller software. Sequences were aligned with ClustalW and analysed by Ali2D revealing a high level of secondary structure conservation. To validate a potential drug binding pocket we performed antimalarial docking simulations. Using aminoquinoline as probe drugs in PfMDR1 mutated parasites we evaluated the physiology underlying the mechanisms of resistance mediated by PfMDR1 polymorphisms. We focused on the analysis of well known functional polymorphisms in PfMDR1 amino acid residues 86, 184, 1034, 1042 and 1246. Our structural analysis suggested the existence of two different biophysical mechanisms of PfMDR1 drug resistance modulation. Polymorphisms in residues 86/184/1246 act by internal allosteric modulation and residues 1034 and 1042 interact directly in a drug pocket. Parasites containing mutated PfMDR1 variants had a significant altered aminoquinoline susceptibility that appears to be dependent on the aminoquinoline lipophobicity characteristics as well as vacuolar efflux by PfCRT. We previously described the in vivo selection of PfMDR1 polymorphisms under antimalarial drug pressure. Now, together with recent PfMDR1 functional reports, we contribute to the understanding of the specific structural role of these polymorphisms in parasite antimalarial drug response.
PLoS ONE 01/2011; 6(9):e23875. · 4.09 Impact Factor
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ABSTRACT: Early telencephalic development is dependent on the spatially and temporally coordinated regulation by essential signaling factors. For example, members of the Bone Morphogenetic Protein (BMP) family, such as BMP4, are crucial for proper development of dorsal telencephalic structures. Stimulation of multipotent telencephalic neural stem cells (NSCs) with BMP4 induces differentiation primarily into astrocytic and mesenchymal cells. However, BMP4-mediated mesenchymal differentiation is inhibited at certain culture conditions of NSCs, corresponding to in vivo developmental contexts. These inhibitory mechanisms are not fully understood and the terminal fate of non-astrocytic BMP4 treated NSCs under these conditions is unclear. Here we show that secreted factors inhibited BMP4-mediated mesenchymal differentiation of telencephalic NSCs. BMP4 mediated a dramatic and direct up-regulation of endogenous noggin levels, that in turn exerted a concentration-dependent inhibition of BMP4-mediated mesenchymal differentiation of NSCs. Instead, BMP4 exposure of NSCs induced neuronal differentiation in mesenchyme-preventing conditions, whereas treatment with recombinant noggin alone did not. Wnt signaling is known to be essential for the development of neurons derived from the dorsal telencephalon, and co-stimulation of NSCs with BMP4+Wnt3a resulted in a synergistic effect yielding significantly increased number of mature neurons compared to stimulation with each factor alone. Thus whereas only a subset of BMP4-induced neurons derived from telencephalic NSCs, responded to glutamate receptor (GluR) agonists, over 80% of BMP4+Wnt3a-induced neurons responded appropriately to GluR-agonists. Our results increase the understanding of the role for BMP4 in differentiation of telencephalic multipotent progenitors, and reveal novel implications for noggin and Wnt3a in these events.
Molecular and Cellular Neuroscience 01/2011; 47(1):10-8. · 3.66 Impact Factor
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ABSTRACT: Cellular calcium uptake is a controlled physiological process mediated by multiple ion channels. The exposure of cells to either one of the protein kinase C (PKC) inhibitors, staurosporine (STS) or PKC412, can trigger Ca²(+) influx leading to cell death. The precise molecular mechanisms regulating these events remain elusive. In this study, we report that the PKC inhibitors induce a prolonged Ca²(+) import through hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) in lung carcinoma cells and in primary culture of cortical neurons, sufficient to trigger apoptosis-inducing factor (AIF)-mediated apoptosis. Downregulation of HCN2 prevented the drug-induced Ca²(+) increase and subsequent apoptosis. Importantly, the PKC inhibitors did not cause Ca²(+) entry into HEK293 cells, which do not express the HCN channels. However, introduction of HCN2 sensitized them to STS/PKC412-induced apoptosis. Mutagenesis of putative PKC phosphorylation sites within the C-terminal domain of HCN2 revealed that dephosphorylation of Thr⁵⁴⁹ was critical for the prolonged Ca²(+) entry required for AIF-mediated apoptosis. Our findings demonstrate a novel role for the HCN2 channel by providing evidence that it can act as an upstream regulator of cell death triggered by PKC inhibitors.
The EMBO Journal 10/2010; 29(22):3869-78. · 9.20 Impact Factor
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ABSTRACT: Perception of the environment relies on somatosensory neurons. Mechanosensory, proprioceptor and many nociceptor subtypes of these neurons have specific mechanosensitivity profiles to adequately differentiate stimulus patterns. Nevertheless, the cellular basis of differential mechanosensation remains largely elusive. Successful transduction of sensory information relies on the recruitment of sensory neurons and mechanosensation occurring at their peripheral axonal endings in vivo. Conspicuously, existing in vitro models aimed to decipher molecular mechanisms of mechanosensation test single sensory neuron somata at any one time. Here, we introduce a compartmental in vitro chamber design to deliver precisely controlled mechanical stimulation of sensory axons with synchronous real-time imaging of Ca(2+) transients in neuronal somata that reliably reflect action potential firing patterns. We report of three previously not characterized types of mechanosensitive neuron subpopulations with distinct intrinsic axonal properties tuned specifically to static indentation or vibration stimuli, showing that different classes of sensory neurons are tuned to specific types of mechanical stimuli. Primary receptor currents of vibration neurons display rapidly adapting conductance reliably detected for every single stimulus during vibration and are consistently converted into action potentials. This result allows for the characterization of two critical steps of mechanosensation in vivo: primary signal detection and signal conversion into specific action potential firing patterns in axons.
Proceedings of the National Academy of Sciences 09/2010; 107(37):16336-41. · 9.68 Impact Factor
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ABSTRACT: Human embryonic stem (hES) cell differentiation into dopamine neurons is considered a promising strategy for cell replacement therapy in Parkinson's disease, yet the functional properties of hES cell-derived dopamine neurons remain poorly defined. The objective of this study was to characterize intracellular calcium (Ca(2+)) and sub-plasma membrane cyclic AMP-signaling properties in hES cell-derived dopamine neurons. We found that hES cell-derived dopamine neurons and neural progenitors raised Ca(2+) from intra- and extracellular compartments in response to depolarization, glutamate, ATP, and dopamine D(2) receptor activation, while undifferentiated hES cells only mobilized Ca(2+) from intracellular stores in response to ATP and D(2) receptor-induced activation. Interestingly, we also found that hES cell-derived dopamine neurons in addition to primary ventral midbrain dopamine neurons were more prone to release Ca(2+) from intracellular stores than non-dopamine neurons following treatment with the neuropeptide neurotensin. Furthermore, hES cell-derived dopamine neurons showed cAMP elevations in response to forskolin and 3-isobutyl-methylxanthine, similar to primary dopamine neurons. Taken together, these results unravel the temporal sequence by which hES cells acquire Ca(2+) and cAMP signaling competence during dopamine differentiation.
Stem cells and development 09/2010; 19(9):1355-64. · 4.15 Impact Factor
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ABSTRACT: Cytosolic calcium (Ca2+) oscillations are vastly flexible cell signals that convey information regulating numerous cellular processes. The frequency and amplitude of the oscillating signal can be varied infinitely by concerted actions of Ca2+ transporters and Ca2+-binding proteins to encode specific messages that trigger downstream molecular events. High frequency cytosolic Ca2+ oscillations regulate fast responses, such as synaptic transmission and secretion, whereas low frequency oscillations regulate slow processes, such as fertilization and gene transcription. Thus, the cell exploits Ca2+ oscillations as a signalling carrier to transduce vital information that controls its behaviour. Here, we review the underlying biochemical mechanisms responsible for generating and discriminating cytosolic Ca2+ oscillations.
Biochemical and Biophysical Research Communications 05/2010; 396(1):28-32. · 2.48 Impact Factor
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ABSTRACT: We have previously shown that angiomotin (Amot) is essential for endothelial cell migration during mouse embryogenesis. However, approximately 5% of Amot knockout mice survived without any detectable vascular defects. Angiomotin-like protein 1 (AmotL1) potentially compensates for the absence of Amot as it is 62% homologous to Amot and exhibits similar expression pattern in endothelial cells.
Here, we report the identification of a novel isoform of AmotL1 that controls endothelial cell polarization and directional migration.
Small interfering RNA-mediated silencing of AmotL1 in mouse aortic endothelial cells caused a significant reduction in migration. In confluent mouse pancreatic islet endothelial cells (MS-1), AmotL1 colocalized with Amot to tight junctions. Small interfering RNA knockdown of both Amot and AmotL1 in MS-1 cells exhibited an additive effect on increasing paracellular permeability compared to that of knocking down either Amot or AmotL1, indicating both proteins were required for proper tight junction activity. Moreover, as visualized using high-resolution 2-photon microscopy, the morpholino-mediated knockdown of amotl1 during zebrafish embryogenesis resulted in vascular migratory defect of intersegmental vessels with strikingly decreased junction stability between the stalk cells and the aorta. However, the phenotype was quite distinct from that of amot knockdown which affected polarization of the tip cells of intersegmental vessels. Double knockdown resulted in an additive phenotype of depolarized tip cells with no or decreased connection of the stalk cells to the dorsal aorta.
These results cumulatively validate that Amot and AmotL1 have similar effects on endothelial migration and tight junction formation in vitro. However, in vivo Amot appears to control the polarity of vascular tip cells whereas AmotL1 mainly affects the stability of cell-cell junctions of the stalk cells.
Circulation Research 08/2009; 105(3):260-70. · 9.49 Impact Factor
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Luc Desfrere,
Marie Karlsson,
Hiromi Hiyoshi,
Seth Malmersjö,
Evanthia Nanou,
Manuel Estrada,
Ayako Miyakawa,
Hugo Lagercrantz,
Abdeljabbar El Manira,
Mark Lal, Per Uhlén
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ABSTRACT: Dendritic growth is pivotal in the neurogenesis of cortical neurons. The sodium pump, or Na,K-ATPase, is an evolutionarily conserved protein that, in addition to its central role in establishing the electrochemical gradient, has recently been reported to function as a receptor and signaling mediator. Although a large body of evidence points toward a dual function for the Na,K-ATPase, few biological implications of this signaling pathway have been described. Here we report that Na,K-ATPase signal transduction triggers dendritic growth as well as a transcriptional program dependent on cAMP response element binding protein (CREB) and cAMP response element (CRE)-mediated gene expression, primarily regulated via Ca(2+)/calmodulin-dependent protein (CaM) kinases. The signaling cascade mediating dendritic arbor growth also involves intracellular Ca(2+) oscillations and sustained phosphorylation of mitogen-activated protein (MAP) kinases. Thus, our results suggest a novel role for the Na,K-ATPase as a modulator of dendritic growth in developing neurons.
Proceedings of the National Academy of Sciences 02/2009; 106(7):2212-7. · 9.68 Impact Factor
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ABSTRACT: Increasing evidence suggests that alpha-chemokines serve several important functions in the nervous system, including regulation of neuroimmune responses, neurotransmission, neuronal survival, and central nervous system development. In this study, we first examined the function of two alpha-chemokines, chemokine ligand (CXCL) 6 and CXCL8, and their receptors, CXCR1 and CXCR2, in the developing rat ventral midbrain (VM). We found that CXCR2 and CXCL6 are regulated during VM development and that CXCL6 promotes the differentiation of nurr77-related receptor (Nurr1)+ precursors into dopaminergic (DA) neurons in vitro. Intriguingly, CXCL8, a ligand expressed only in Homo sapiens, enhanced progenitor cell division, neurogenesis, and tyrosine hydroxylase-positive (TH+) cell number in rodent precursor and neurosphere cultures. CXCL1, the murine ortholog of CXCL8, was developmentally regulated in the VM and exhibited activities similar but not identical to those of CXCL8. TH+ cells derived from chemokine-treated VM neurospheres coexpressed Nurr1 and VMAT and were functionally active, as shown by calcium (Ca(2+)) fluxes in response to AMPA. In conclusion, our data demonstrate that CXCL1, CXCL6, and CXCL8 increase the number of DA neurons in VM precursor and neurosphere cultures by diverse mechanisms. Thus, alpha-chemokines may find an application in the preparation of cells for drug development or Parkinson's disease cell replacement therapy.
Stem Cells 05/2008; 26(7):1891-900. · 7.78 Impact Factor
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ABSTRACT: Calcium (Ca2+) oscillations play fundamental roles in various cell signaling processes and have been the subject of numerous modeling studies. Here we have implemented a general mathematical model to simulate the impact of store-operated Ca2+ entry on intracellular Ca2+ oscillations. In addition, we have compared two different models of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and their influences on intracellular Ca2+ oscillations. Store-operated Ca2+ entry following Ca2+ depletion of endoplasmic reticulum (ER) is an important component of Ca2+ signaling. We have developed a phenomenological model of store-operated Ca2+ entry via store-operated Ca2+ (SOC) channels, which are activated upon ER Ca2+ depletion. The depletion evokes a bi-phasic Ca2+ signal, which is also produced in our mathematical model. The IP3R is an important regulator of intracellular Ca2+ signals. This IP3 sensitive Ca2+ channel is also regulated by Ca2+. We apply two IP3R models, the Mak-McBride-Foskett model and the De Young and Keizer model, with significantly different channel characteristics. Our results show that the two separate IP3R models evoke intracellular Ca2+ oscillations with different frequencies and amplitudes. Store-operated Ca2+ entry affects the oscillatory behavior of these intracellular Ca2+ oscillations. The IP3 threshold is altered when store-operated Ca2+ entry is excluded from the model. Frequencies and amplitudes of intracellular Ca2+ oscillations are also altered without store-operated Ca2+ entry. Under certain conditions, when intracellular Ca2+ oscillations are absent, excluding store-operated Ca2+ entry induces an oscillatory response. These findings increase knowledge concerning store-operated Ca2+ entry and its impact on intracellular Ca2+ oscillations.
Mathematical Biosciences 01/2007; 204(2):232-49. · 1.54 Impact Factor
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ABSTRACT: Mounting evidence suggests that the ion pump, Na,K-ATPase, can, in the presence of ouabain, act as a signal transducer. A prominent binding motif linking the Na,K-ATPase to intracellular signaling effectors has, however, not yet been identified. Here we report that the N-terminal tail of the Na,K-ATPase catalytic alpha-subunit (alphaNT-t) binds directly to the N terminus of the inositol 1,4,5-trisphosphate receptor. Three amino acid residues, LKK, conserved in most species and most alpha-isoforms, are essential for the binding to occur. In wild-type cells, low concentrations of ouabain trigger low frequency calcium oscillations that activate NF-kappaB and protect from apoptosis. All of these effects are suppressed in cells overexpressing a peptide corresponding to alphaNT-t but not in cells overexpressing a peptide corresponding to alphaNT-t deltaLKK. Thus we have identified a well conserved Na,K-ATPase motif that binds to the inositol 1,4,5-trisphosphate receptor and can trigger an anti-apoptotic calcium signal.
Journal of Biological Chemistry 09/2006; 281(31):21954-62. · 4.77 Impact Factor
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ABSTRACT: Gain-of-function mutations in SHP-2/PTPN11 cause Noonan syndrome, a human developmental disorder. Noonan syndrome is characterized by proportionate short stature, facial dysmorphia, increased risk of leukemia, and congenital heart defects in approximately 50% of cases. Congenital heart abnormalities are common in Noonan syndrome, but the signaling pathway(s) linking gain-of-function SHP-2 mutants to heart disease is unclear. Diverse cell types coordinate cardiac morphogenesis, which is regulated by calcium (Ca2+) and the nuclear factor of activated T-cells (NFAT). It has been shown that the frequency of Ca2+ oscillations regulates NFAT activity. Here, we show that in fibroblasts, Ca2+ oscillations in response to FGF-2 require the phosphatase activity of SHP-2. Conversely, gain-of-function mutants of SHP-2 enhanced FGF-2-mediated Ca2+ oscillations in fibroblasts and spontaneous Ca2+ oscillations in cardiomyocytes. The enhanced frequency of cardiomyocyte Ca2+ oscillations induced by a gain-of-function SHP-2 mutant correlated with reduced nuclear translocation and transcriptional activity of NFAT. These data imply that gain-of-function SHP-2 mutants disrupt the Ca2+ oscillatory control of NFAT, suggesting a potential mechanism for congenital heart defects in Noonan syndrome.
Proceedings of the National Academy of Sciences 03/2006; 103(7):2160-5. · 9.68 Impact Factor
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ABSTRACT: Epithelial cells are vital to sense the presence of bacteria, thereby initiating a proper innate immune response. This occurs via different mechanisms, e.g. recognition by pattern recognition receptors (TLR), or alteration of the cellular Ca2+ homeostasis. The Escherichia coli toxin cytolysin A (ClyA) is naturally delivered to target cells as active pore assemblies within outer membrane vesicles (OMVs), and we here investigate a possible role of ClyA-containing OMVs (ClyA+ (OMV)) for induction of proinflammatory responses via the above-mentioned mechanisms. We report that low, sublytic concentrations of ClyA+ (OMV) affect the Ca2+ homeostasis in epithelial cells by induction of slow, intracellular Ca2+ oscillations, while increased concentrations act cytolytically. Thus, ClyA belongs to the novel group of pore-forming toxins shown to elicit such biphasic responses. Ca2+ waves in the minute range have been shown to regulate gene transcription of, e.g. interleukin (IL)-6 and -8. While the periodicity of ClyA+ (OMV)-induced Ca2+ waves (22.9 +/- 0.9 min) fail to induce an IL-8 response, our data fit to the general concept of frequency-specific gene expression. Molecular investigations of the signal transduction pathway reveals that ClyA+ (OMV) utilize a different one as compared with those previously reported for other toxins causing Ca2+ waves. The ClyA protein per se and ClyA pore assemblies are non-immunogenic, while lipopolysaccharide present on the OMVs induces a TLR4-dependent proinflammatory response as expected. Additional membrane components of the OMV, e.g. OmpW, was also found to elicit proinflammatory responses that was independent of TLR4 and Ca2+ signalling.
Cellular Microbiology 07/2005; 7(6):779-88. · 5.46 Impact Factor
