61 research outputs found
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 Ca<sup>2+</sup>, 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 M<sub>1 </sub>muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M<sub>1 </sub>muscarinic 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 Ca<sup>2+</sup>-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
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 CÎł 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
Calmodulin Regulates Transient Receptor Potential TRPM3 and TRPM8-Induced Gene Transcription
Calmodulin is a small protein that binds Ca2+ ions via four EF-hand motifs. The Ca2+/
calmodulin complex as well as Ca2+-free calmodulin regulate the activities of numerous enzymes
and ion channels. Here, we used genetic and pharmacological tools to study the functional role of
calmodulin in regulating signal transduction of TRPM3 and TRPM8 channels. Both TRPM3 and
TRPM8 are important regulators of thermosensation. Gene transcription triggered by stimulation of
TRPM3 or TRPM8 channels was significantly impaired in cells expressing a calmodulin mutant with
mutations in all four EF-hand Ca2+ binding motifs. Similarly, incubation of cells with the calmodulin
inhibitor ophiobolin A reduced TRPM3 and TRPM8-induced signaling. The Ca2+/calmodulindependent protein phosphatase calcineurin was shown to negatively regulate TRPM3-induced gene
transcription. Here, we show that TRPM8-induced transcription is also regulated by calcineurin. We
propose that calmodulin plays a dual role in regulating TRPM3 and TRPM8 functions. Calmodulin is
required for the activation of TRPM3 and TRPM8-induced intracellular signaling, most likely through
a direct interaction with the channels. Ca2+ influx through TRPM3 and TRPM8 feeds back to TRPM3
and TRPM8-induced signaling by activation of the calmodulin-regulated enzyme calcineurin, which
acts as a negative feedback loop for both TRPM3 and TRPM8 channel signaling
Critical Protein–Protein Interactions Determine the Biological Activity of Elk-1, a Master Regulator of Stimulus-Induced Gene Transcription
Elk-1 is a transcription factor that binds together with a dimer of the serum response factor
(SRF) to the serum-response element (SRE), a genetic element that connects cellular stimulation
with gene transcription. Elk-1 plays an important role in the regulation of cellular proliferation
and apoptosis, thymocyte development, glucose homeostasis and brain function. The biological
function of Elk-1 relies essentially on the interaction with other proteins. Elk-1 binds to SRF and
generates a functional ternary complex that is required to activate SRE-mediated gene transcription.
Elk-1 is kept in an inactive state under basal conditions via binding of a SUMO-histone deacetylase
complex. Phosphorylation by extracellular signal-regulated protein kinase, c-Jun N-terminal protein
kinase or p38 upregulates the transcriptional activity of Elk-1, mediated by binding to the mediator
of RNA polymerase II transcription (Mediator) and the transcriptional coactivator p300. Strong
and extended phosphorylation of Elk-1 attenuates Mediator and p300 recruitment and allows the
binding of the mSin3A-histone deacetylase corepressor complex. The subsequent dephosphorylation
of Elk-1, catalyzed by the protein phosphatase calcineurin, facilitates the re-SUMOylation of Elk1, transforming Elk-1 back to a transcriptionally inactive state. Thus, numerous protein–protein
interactions control the activation cycle of Elk-1 and are essential for its biological function
Exchange-correlation vector potentials and vorticity-dependent exchange-correlation energy densities in two-dimensional systems
We present a new approach how to calculate the scalar exchange-correlation
potentials and the vector exchange-correlation potentials from current-carrying
ground states of two-dimensional quantum dots. From these exchange-correlation
potentials we derive exchange-correlation energy densities and examine their
vorticity (or current) dependence. Compared with parameterizations of
current-induced effects in literature we find an increased significance of
corrections due to paramagnetic current densities.Comment: 5 figures, submitted to PR
Regulation of cellular proliferation, differentiation and cell death by activated Raf
The protein kinases Raf-1, A-Raf and B-Raf connect receptor stimulation with intracellular signaling pathways and function as a central intermediate in many signaling pathways. Gain-of-function experiments shed light on the pleiotropic biological activities of these enzymes. Expression experiments involving constitutively active Raf revealed the essential functions of Raf in controlling proliferation, differentiation and cell death in a cell-type specific manner
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