Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

<p>Abstract</p> <p>Background</p> <p>The serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts.</p> <p>Results</p> <p>Stimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca²⁺, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK). Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M₁muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M₁muscarinic acetylcholine receptors required the transactivation of the EGF receptor, while thrombin signaling did not.</p> <p>Conclusion</p> <p>This study shows that stimulus-transcription coupling in thrombin-treated lung fibroblasts relies on the elevation of the intracellular Ca²⁺-concentration and the activation of PKC and ERK. In the nucleus, ternary complex factors function as key proteins linking the intracellular signaling cascade with enhanced transcription of the Egr-1 gene. This study further shows that the dominant-negative Elk-1 mutant is a valuable tool to study Elk-1-mediated gene transcription.</p

TRPM3-Induced Gene Transcription Is under Epigenetic Control

Transient receptor potential M3 (TRPM3) cation channels regulate numerous biological functions, including gene transcription. Stimulation of TRPM3 channels with pregnenolone sulfate activates stimulus-responsive transcription factors, which bind to short cognate sequences in the promoters of their target genes. In addition, coregulator proteins are involved that convert the chromatin into a configuration that is permissive for gene transcription. In this study, we determined whether TRPM3-induced gene transcription requires coactivators that change the acetylation pattern of histones. We used compound A485, a specific inhibitor of the histone acetyltransferases CBP and p300. In addition, the role of bromodomain proteins that bind to acetylated lysine residues of histones was analyzed. We used JQ1, an inhibitor of bromodomain and extra terminal domain (BET) family proteins. The results show that both compounds attenuated the activation of AP-1 and CREB-regulated gene transcription following stimulation of TRPM3 channels. Inhibition of CBP/p300 and BET proteins additionally reduced the transcriptional activation potential of the transcription factors c-Fos and Elk-1. Transcriptional upregulation of the interleukin-8 gene was attenuated by A485 and JQ1, indicating that proinflammatory cytokine expression is controlled by CBP/p300 and bromodomain proteins. We conclude that TRPM3-induced signaling involves transcriptional coactivators and acetyl-lysine-bound bromodomain proteins for activating gene transcription

Glucose Homeostasis and Pancreatic Islet Size Are Regulated by the Transcription Factors Elk-1 and Egr-1 and the Protein Phosphatase Calcineurin

Pancreatic β-cells synthesize and secrete insulin. A key feature of diabetes mellitus is the loss of these cells. A decrease in the number of β-cells results in decreased biosynthesis of insulin. Increasing the number of β-cells should restore adequate insulin biosynthesis leading to adequate insulin secretion. Therefore, identifying proteins that regulate the number of β-cells is a high priority in diabetes research. In this review article, we summerize the results of three sophisticated transgenic mouse models showing that the transcription factors Elk-1 and Egr-1 and the Ca2+/calmodulin-regulated protein phosphatase calcineurin control the formation of sufficiently large pancreatic islets. Impairment of the biological activity of Egr-1 and Elk-1 in pancreatic β-cells leads to glucose intolerance and dysregulation of glucose homeostasis, the process that maintains glucose concentration in the blood within a narrow range. Transgenic mice expressing an activated calcineurin mutant also had smaller islets and showed hyperglycemia. Calcineurin induces dephosphorylation of Elk-1 which subsequently impairs Egr-1 biosynthesis and the biological functions of Elk-1 and Egr-1 to regulate islet size and glucose homeostasis

Expression of the C-Terminal Domain of Phospholipase Cβ3 Inhibits Signaling via Gαq-Coupled Receptors and Transient Receptor Potential Channels

Transient receptor potential (TRP) channels are cation channels that play a regulatory role in pain and thermosensation, insulin secretion, and neurotransmission. It has been proposed that activation of TRP channels requires phosphatidylinositol 4,5-bisphosphate, the major substrate for phospholipase C (PLC). We investigated whether inhibition of PLCβ has an impact on TRP channel signaling. A genetic approach was used to avoid off-target effects observed when using a pharmacological PLCβ inhibitor. In this study, we show that expression of PLCβ1ct and PLCβ3ct, truncated forms of PLCβ1 or PLCβ3 that contain the C-terminal membrane binding domains, almost completely blocked the signal transduction of a Gαq-coupled designer receptor, including the phosphorylation of ERK1/2. In contrast, expression of the helix-turn-helix motif (Hα1—Hα2) of the proximal C-terminal domain of PLCβ3 did not affect Gαq-coupled receptor signaling. PLCβ3ct expression impaired signaling of the TRP channels TRPM3 and TRPM8, stimulated with either prognenolone sulfate or icilin. Thus, the C-terminal domain of PLCβ3 interacts with plasma membrane targets, most likely phosphatidylinositol 4,5-bisphosphate, and in this way blocks the biological activation of TRPM3 and TRPM8, which require interaction with this phospholipid. PLCβ thus regulates TRPM3 and TRPM8 channels by masking phosphatidylinositol 4,5-bisphosphate with its C-terminal domain

Theoretical study of interacting hole gas in p-doped bulk III-V semiconductors

We study the homogeneous interacting hole gas in $p$-doped bulk III-V semiconductors. The structure of the valence band is modelled by Luttinger's Hamiltonian in the spherical approximation, giving rise to heavy and light hole dispersion branches, and the Coulomb repulsion is taken into account via a self-consistent Hartree-Fock treatment. As a nontrivial feature of the model, the self-consistent solutions of the Hartree-Fock equations can be found in an almost purely analytical fashion, which is not the case for other types of effective spin-orbit coupling terms. In particular, the Coulomb interaction renormalizes the Fermi wave numbers for heavy and light holes. As a consequence, the ground state energy found in the self-consistent Hartree-Fock approach and the result from lowest-order perturbation theory do not agree. We discuss the consequences of our observations for ferromagnetic semiconductors, and for the possible observation of the spin-Hall effect in bulk $p$-doped semiconductors. Finally, we also investigate elementary properties of the dielectric function in such systems.Comment: 9 pages, 5 figures, title slightly changed in the course of editorial process, a few references added, version to appear in Phys. Rev.

Ca2+ Microdomains, Calcineurin and the Regulation of Gene Transcription

Ca2+ ions function as second messengers regulating many intracellular events, including neurotransmitter release, exocytosis, muscle contraction, metabolism and gene transcription. Cells of a multicellular organism express a variety of cell-surface receptors and channels that trigger an increase of the intracellular Ca2+ concentration upon stimulation. The elevated Ca2+ concentration is not uniformly distributed within the cytoplasm but is organized in subcellular microdomains with high and low concentrations of Ca2+ at different locations in the cell. Ca2+ ions are stored and released by intracellular organelles that change the concentration and distribution of Ca2+ ions. A major function of the rise in intracellular Ca2+ is the change of the genetic expression pattern of the cell via the activation of Ca2+-responsive transcription factors. It has been proposed that Ca2+-responsive transcription factors are differently affected by a rise in cytoplasmic versus nuclear Ca2+. Moreover, it has been suggested that the mode of entry determines whether an influx of Ca2+ leads to the stimulation of gene transcription. A rise in cytoplasmic Ca2+ induces an intracellular signaling cascade, involving the activation of the Ca2+/calmodulin-dependent protein phosphatase calcineurin and various protein kinases (protein kinase C, extracellular signal-regulated protein kinase, Ca2+/calmodulin-dependent protein kinases). In this review article, we discuss the concept of gene regulation via elevated Ca2+ concentration in the cytoplasm and the nucleus, the role of Ca2+ entry and the role of enzymes as signal transducers. We give particular emphasis to the regulation of gene transcription by calcineurin, linking protein dephosphorylation with Ca2+ signaling and gene expression

Insulin-Responsive Transcription Factors

The hormone insulin executes its function via binding and activating of the insulin receptor, a receptor tyrosine kinase that is mainly expressed in skeletal muscle, adipocytes, liver, pancreatic β-cells, and in some areas of the central nervous system. Stimulation of the insulin receptor activates intracellular signaling cascades involving the enzymes extracellular signal-regulated protein kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase, protein kinase B/Akt, and phospholipase Cγ as signal transducers. Insulin receptor stimulation is correlated with multiple physiological and biochemical functions, including glucose transport, glucose homeostasis, food intake, proliferation, glycolysis, and lipogenesis. This review article focuses on the activation of gene transcription as a result of insulin receptor stimulation. Signal transducers such as protein kinases or the GLUT4-induced influx of glucose connect insulin receptor stimulation with transcription. We discuss insulin-responsive transcription factors that respond to insulin receptor activation and generate a transcriptional network executing the metabolic functions of insulin. Importantly, insulin receptor stimulation induces transcription of genes encoding essential enzymes of glycolysis and lipogenesis and inhibits genes encoding essential enzymes of gluconeogenesis. Overall, the activation or inhibition of insulin responsive transcription factors is an essential aspect of orchestrating a wide range of insulin-induced changes in the biochemistry and physiology of insulin-responsive tissues

Network synchronization of groups

In this paper we study synchronized motions in complex networks in which there are distinct groups of nodes where the dynamical systems on each node within a group are the same but are different for nodes in different groups. Both continuous time and discrete time systems are considered. We initially focus on the case where two groups are present and the network has bipartite topology (i.e., links exist between nodes in different groups but not between nodes in the same group). We also show that group synchronous motions are compatible with more general network topologies, where there are also connections within the groups

Adaptive Detection of Instabilities: An Experimental Feasibility Study

We present an example of the practical implementation of a protocol for experimental bifurcation detection based on on-line identification and feedback control ideas. The idea is to couple the experiment with an on-line computer-assisted identification/feedback protocol so that the closed-loop system will converge to the open-loop bifurcation points. We demonstrate the applicability of this instability detection method by real-time, computer-assisted detection of period doubling bifurcations of an electronic circuit; the circuit implements an analog realization of the Roessler system. The method succeeds in locating the bifurcation points even in the presence of modest experimental uncertainties, noise and limited resolution. The results presented here include bifurcation detection experiments that rely on measurements of a single state variable and delay-based phase space reconstruction, as well as an example of tracing entire segments of a codimension-1 bifurcation boundary in two parameter space.Comment: 29 pages, Latex 2.09, 10 figures in encapsulated postscript format (eps), need psfig macro to include them. Submitted to Physica

Spatial patterns of desynchronization bursts in networks

We adapt a previous model and analysis method (the {\it master stability function}), extensively used for studying the stability of the synchronous state of networks of identical chaotic oscillators, to the case of oscillators that are similar but not exactly identical. We find that bubbling induced desynchronization bursts occur for some parameter values. These bursts have spatial patterns, which can be predicted from the network connectivity matrix and the unstable periodic orbits embedded in the attractor. We test the analysis of bursts by comparison with numerical experiments. In the case that no bursting occurs, we discuss the deviations from the exactly synchronous state caused by the mismatch between oscillators