Isabelle Müller

Egr-1—A Ca2+-regulated transcription factor

The biosynthesis of the zinc finger transcription factor Egr-1 is stimulated by many extracellular signaling molecules including hormones, neurotransmitters, growth and differentiation factors. The Egr-1 gene represents a convergence point for many intracellular signaling cascades. An increase of the intracellular Ca(2+) concentration, by activating ionotropic or Galpha(q/11)-coupled receptors or voltage-gated L-type Ca(2+) channels, is often the prerequisite for enhanced Egr-1 gene transcription. This increase has been observed following stimulation with extracellular signaling molecules including ATP, glutamate, thrombin, carbachol, gonadotropin-releasing hormone, or glucose. Egr-1 is thus a Ca(2+) regulated transcription factor - similar to CREB, NFAT, NF-kappaB and others. This review also discusses the importance of the cytoplasmic and nuclear Ca(2+) concentration in transcriptional regulation of the Egr-1 gene.

Signal Transduction of Pregnenolone Sulfate in Insulinoma Cells ACTIVATION OF EGR-1 EXPRESSION INVOLVING TRPM3, VOLTAGE-GATED CALCIUM CHANNELS, ERK, AND TERNARY COMPLEX FACTORS

The neurosteroid pregnenolone sulfate acts on the nervous system by modifying neurotransmission and receptor functions, thus influencing synaptic strength, neuronal survival, and neurogenesis. Here we show that pregnenolone sulfate induces a signaling cascade in insulinoma cells leading to enhanced expression of the zinc finger transcription factor Egr-1 and Egr-1-responsive target genes. Pharmacological and genetic experiments revealed that influx of Ca2+ ions via transient receptor potential M3 and voltage-gated Ca2+ channels, elevation of the cytosolic Ca2+ level, and activation of ERK are essential for connecting pregnenolone sulfate stimulation with enhanced Egr-1 biosynthesis. Expression of a dominant-negative mutant of Elk-1, a key regulator of gene transcription driven by a serum response element, attenuated Egr-1 expression following stimulation, indicating that Elk-1 or related ternary complex factors connect the transcription of the Egr-1 gene with the pregnenolone sulfate-induced intracellular signaling cascade elicited by the initial influx of Ca2+. The newly synthesized Egr-1 was biologically active and bound under physiological conditions to the regulatory regions of the Pdx-1, Synapsin I, and Chromogranin B genes. Pdx-1 is a major regulator of insulin gene transcription. Accordingly, elevated insulin promoter activity and increased mRNA levels of insulin could be detected in pregnenolone sulfate-stimulated insulinoma cells. Likewise, the biosynthesis of synapsin I, a synaptic vesicle protein that is found at secretory granules in insulinoma cells, was stimulated in pregnenolone sulfate-treated INS-1 cells. Together, these data show that pregnenolone sulfate induces a signaling cascade in insulinoma cells that is very similar to the signaling cascade induced by glucose in β-cells.

Regulation of AP‐1 Activity in Glucose‐Stimulated Insulinoma Cells

The transcriptional activity of AP‐1 has been analyzed in glucose‐stimulated INS‐1 insulinoma cells using a chromosomally embedded AP‐1‐responsive reporter gene. We show that AP‐1 activity was significantly elevated in glucose‐treated INS‐1 cells. Preincubation of the cells with nifedipine or expression of the Ca2+ binding protein parvalbumin in the cytoplasm of INS‐1 cells reduced AP‐1 activity. Thus, activation of L‐type Ca2+ channels and an elevated cytoplasmic Ca2+ concentration are crucial to connecting glucose stimulation with enhanced AP‐1 activity. Expression of dominant negative forms of A‐Raf, MKK4 or MKK6 and pharmacological inhibition of MEK and p38 revealed that extracellular signal‐regulated protein kinase, p38 and c‐Jun NH2‐terminal protein kinase participate in the upregulation of AP‐1 activity. Expression of dominant‐negative mutants of c‐Jun and Elk‐1 reduced AP‐1 transcriptional activity in INS‐1 cells indicating that c‐Jun and ternary complex factors are involved in the regulation of AP‐1 activity in glucose‐stimulated insulinoma cells. J. Cell. Biochem. 110: 1481–1494, 2010.

CK2 phosphorylation of Pdx-1 regulates its transcription factor activity.

The duodenal homeobox-1 protein Pdx-1 is one of the regulators for the transcription of the insulin gene. Pdx-1 is a phosphoprotein, and there is increasing evidence for the regulation of some of its functions by phosphorylation. Here, we asked whether protein kinase CK2 might phosphorylate Pdx-1 and how this phosphorylation could be implicated in the functional regulation of Pdx-1. We used fragments of Pdx-1 as well as phosphorylation mutants for experiments with protein kinase CK2. Transactivation was measured by reporter assays using the insulin promoter. Our data showed that Pdx-1 is phosphorylated by protein kinase CK2 at amino acids thr231 and ser232, and this phosphorylation was implicated in the regulation of the transcription factor activity of Pdx-1. Furthermore, inhibition of protein kinase CK2 by specific inhibitors led to an elevated release of insulin from pancreatic β-cells. Thus, these findings identify CK2 as a novel mediator of the insulin metabolism.

Efficient genetic manipulation of 1321N1 astrocytoma cells using lentiviral gene transfer

1321N1 astrocytoma cells are frequently used to analyze stimulus-induced intracellular signaling. These experiments require genetic manipulation of the cells and several chemical and physical methods have been employed in the past. Recently, microporation has been suggested as the best method to transfect 1321N1 astrocytoma cells. Here, we demonstrate that lentiviral gene transfer into 1321N1 cells is highly efficient, cheap and non-toxic. In addition, lentiviral gene transfer efficiently facilitates stable expression of small hairpin RNAs. Finally, lentiviral gene transfer can be used to implant promoter/luciferase reporter genes into the chromatin of the cells, allowing promoter studies using templates that are embedded into the nucleosomal structure of the chromatin.

Pregnenolone Sulfate Activates Basic Region Leucine Zipper Transcription Factors in Insulinoma Cells: Role of Voltage-Gated Ca2+ Channels and Transient Receptor Potential Melastatin 3 Channels

The neurosteroid pregnenolone sulfate activates a signaling cascade in insulinoma cells involving activation of extracellular signal-regulated protein kinase and enhanced expression of the transcription factor Egr-1. Here, we show that pregnenolone sulfate stimulation leads to a significant elevation of activator protein-1 (AP-1) activity in insulinoma cells. Expression of the basic region leucine zipper (bZIP) transcription factors c-Jun and c-Fos is up-regulated in insulinoma cells and pancreatic β-cells in primary culture after pregnenolone sulfate stimulation. Up-regulation of a chromatin-embedded c-Jun promoter/luciferase reporter gene transcription in pregnenolone sulfate-stimulated insulinoma cells was impaired when the AP-1 binding sites were mutated, indicating that these motifs function as pregnenolone sulfate response elements. In addition, phosphorylation of cAMP response element (CRE)-binding protein is induced and transcription of a CRE-controlled reporter gene is stimulated after pregnenolone sulfate treatment, indicating that the CRE functions as a pregnenolone sulfate response element as well. Pharmacological and genetic experiments revealed that both L-type Ca2+ channels and transient receptor potential melastatin 3 (TRPM3) channels are essential for connecting pregnenolone sulfate stimulation with enhanced AP-1 activity and bZIP-mediated transcription in insulinoma cells. In contrast, pregnenolone sulfate stimulation did not enhance AP-1 activity or c-Jun and c-Fos expression in pituitary corticotrophs that express functional L-type Ca2+ channels but only trace amounts of TRPM3. We conclude that expression of L-type Ca2+ channels is not sufficient to activate bZIP-mediated gene transcription by pregnenolone sulfate. Rather, additional expression of TRPM3 or depolarization of the cells is required to connect pregnenolone sulfate stimulation with enhanced gene transcription.

Critical role of Egr transcription factors in regulating insulin biosynthesis, blood glucose homeostasis, and islet size.

Expression of early growth response protein (Egr)-1, a protein of the Egr family of zinc finger transcription factors, is stimulated in glucose-treated pancreatic β-cells and insulinoma cells. The purpose of this study was to elucidate the role of Egr transcription factors in pancreatic β-cells in vivo. To overcome the problem associated with redundancy of functions between Egr proteins, conditional transgenic mice were generated expressing a dominant-negative mutant of Egr-1 in pancreatic β-cells. The Egr-1 mutant interferes with DNA binding of all Egr proteins and thus impairs the biological functions of the entire Egr family. Expression of the Egr-1 mutant reduced expression of TGFβ and basic fibroblast growth factor, known target genes of Egr-1, whereas the expression of Egr-1, Egr-3, Ets-like gene-1 (Elk-1), and specificity protein-3 was not changed in the presence of the Egr-1 mutant. Expression of the homeobox protein pancreas duodenum homeobox-1, a major regulator of insulin biosynthesis, was reduced in islets expressing the Egr-1 mutant. Accordingly, insulin mRNA and protein levels were reduced by 75 or 25%, respectively, whereas expression of glucagon and somatostatin was not altered after expression of the Egr-1 mutant in β-cells. Glucose tolerance tests revealed that transgenic mice expressing the Egr-1 mutant in pancreatic β-cells displayed impaired glucose tolerance. In addition, increased caspase-3/7 activity was detected as a result of transgene expression, leading to a 20% decrease of the size of the islets. These results show that Egr proteins play an important role in controlling insulin biosynthesis, glucose homeostasis, and islet size of pancreatic β-cells in vivo.

Signal transduction via TRPM3 channels in pancreatic β-cells

Transient receptor potential melastatin 3 (TRPM3) channels are non-selective cation channels that are expressed in insulinoma cells and pancreatic β-cells. Stimulation of TRPM3 with the neurosteroid pregnenolone sulfate induces an intracellular signaling cascade, involving a rise in intracellular Ca(2)(+) concentration, activation of the protein kinases Raf and ERK, and a change in the gene expression pattern of the cells. In particular, biosynthesis of insulin is altered following activation of TRPM3 by pregnenolone sulfate. Moreover, a direct effect of TRPM3 stimulation on insulin secretion has been reported. The fact that stimulation of TRPM3 induces a signaling cascade that is very similar to the signaling cascade induced by glucose in β-cells suggests that TRPM3 may influence main functions of pancreatic β-cells. The view that TRPM3 represents an ionotropic steroid receptor of pancreatic β-cells linking insulin release with steroid hormone signaling is discussed.

Ca2+ signaling and gene transcription in glucose-stimulated insulinoma cells.

Glucose stimulation induces expression of the transcription factors Egr-1 and c-Jun as well as phosphorylation of CREB and c-Jun in insulinoma cells. The activator protein-1 (AP-1) binding sites within the c-Jun promoter, the serum response elements (SREs) within the Egr-1 promoter, and cyclic AMP response elements (CREs) function as glucose responsive element. Glucose-induced transcriptional regulation was attenuated by inhibition of L-type Ca(2+) channels, or chelating cytoplasmic Ca(2+). It has been proposed that a rise in nuclear Ca(2+) is required for CREB-mediated transcription of CRE-regulated genes, while elevated cytoplasmic Ca(2+) levels trigger an upregulation of SRE-containing genes. Here, we show that a rise in cytoplasmic Ca(2+) is required for AP-1, CRE, and SRE mediated transcription in glucose-stimulated insulinoma cells. Buffering Ca(2+) in the nucleus or the endoplasmic reticulum had no inhibitory effect upon transcription. However, overexpression of the mitochondrial protein Fis-1 or inhibition of the GTPase Drp-1 impaired glucose-stimulated gene transcription in insulinoma cells, suggesting that the mitochondria play an important role in regulating Ca(2+) mediated gene transcription. The extracellular signal-regulated protein kinase functions as an essential link connecting glucose stimulation, the rise in cytoplasmic Ca(2+), and enhanced transcription in insulinoma cells.

Role of protein kinase CK2 in the dynamic interaction of platelets, leukocytes and endothelial cells during thrombus formation

INTRODUCTION Thrombus formation is a complex process, which is characterized by the dynamic interaction of platelets, leukocytes and endothelial cells. The activation of these cells is strictly mediated by different phospho-regulated signaling pathways. Recently, it has been reported that inhibition of protein kinase CK2 affects platelet function by suppressing phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K) signaling. Based on this finding, we herein analyzed whether CK2 acts as a crucial regulator of thrombus formation. MATERIALS AND METHODS We examined the effect of CK2 inhibition on platelet activation and aggregation, the formation of platelet-leukocyte aggregates (PLA), the endothelial expression of von Willebrand factor (vWF), intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, and the subcellular localization of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and phospho-p65 in human dermal microvascular endothelial cells (HDMEC). Dorsal skinfold chambers were prepared in BALB/c mice to analyze in vivo the effect of CK2 inhibition on photochemically induced thrombus formation using intravital fluorescence microscopy. RESULTS CK2 inhibition by CX-4945 suppressed adenosin diphosphate (ADP)- and proteinase-activated receptor-1-peptide (PAR-1-AP)-stimulated platelet aggregation, which was associated with down-regulation of P-selectin, GPIIb/IIIa and a reduced formation of PLA. Expression and secretion of vWF was diminished in CX-4945-treated HDMEC. Moreover, CK2 inhibition attenuated the endothelial expression of VCAM-1, whereas the expression of ICAM-1 was not affected. Finally, CX-4945-treated mice exhibited a significantly delayed photochemically induced thrombus formation when compared to vehicle-treated controls. CONCLUSION These results indicate that CK2 is a pleiotropic regulator of thrombus formation, affecting multiple interactions of platelets, leukocytes and endothelial cells.