Stimulus-Induced Activation of the Glycoprotein Hormone α-Subunit Promoter in Human Placental Choriocarcinoma Cells: Major Role of a tandem cAMP Response Element.

The glycoprotein hormones LH, FSH, TSH and chorionic gonadotropin consist of a common α-subunit and a hormone-specific ß-subunit. The α-subunit is expressed in the pituitary and the placental cells, and its expression is regulated by extracellular signal molecules. Much is known about the regulation of the α-subunit gene in the pituitary, but few studies have addressed the regulation of this gene in trophoblasts. The aim of this study was to characterize the molecular mechanism of stimulus-induced α-subunit gene transcription in JEG-3 cells, a cellular model for human trophoblasts, using chromatin-embedded reporter genes under the control of the α-subunit promoter. The results show that increasing the concentration of the second messengers cAMP or Ca2+, or expressing the catalytic subunit of cAMP-dependent protein kinase in the nucleus activated the α-subunit promoter. Similarly, the stimulation of p38 protein kinase activated the α-subunit promoter, linking α-subunit expression to stress response. The stimulation of a Gαq-coupled designer receptor activated the α-subunit promoter, involving the transcription factor CREB, linking α-subunit expression to hormonal stimulation and an increase in intracellular Ca2+. Deletion mutagenesis underscores the importance of a tandem cAMP response element within the glycoprotein hormone α-subunit promoter, which acts as a point of convergence for a multiple signaling pathway.

Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the ßγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

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

Pancreatic ß-cells synthesize and secrete insulin. A key feature of diabetes mellitus is the loss of these cells. A decrease in the number of ß-cells results in decreased biosynthesis of insulin. Increasing the number of ß-cells should restore adequate insulin biosynthesis leading to adequate insulin secretion. Therefore, identifying proteins that regulate the number of ß-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 ß-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.

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+/calmodulin-dependent 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.

Expression of the C-Terminal Domain of Phospholipase Cß3 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 PLCß has an impact on TRP channel signaling. A genetic approach was used to avoid off-target effects observed when using a pharmacological PLCß inhibitor. In this study, we show that expression of PLCß1ct and PLCß3ct, truncated forms of PLCß1 or PLCß3 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 PLCß3 did not affect Gαq-coupled receptor signaling. PLCß3ct expression impaired signaling of the TRP channels TRPM3 and TRPM8, stimulated with either prognenolone sulfate or icilin. Thus, the C-terminal domain of PLCß3 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. PLCß thus regulates TRPM3 and TRPM8 channels by masking phosphatidylinositol 4,5-bisphosphate with its C-terminal domain.

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 Elk-1, 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.

Regulation of stimulus-induced interleukin-8 gene transcription in human adrenocortical carcinoma cells - Role of AP-1 and NF-κB.

Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1ß (IL-1ß) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1ß-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1ß specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.

Ternary complex factor regulates pancreatic islet size and blood glucose homeostasis in transgenic mice.

A hallmark of diabetes mellitus is the inability of pancreatic ß-cells to secrete sufficient amounts of insulin for maintaining normoglycemia. The formation of smaller islets may underlie the development of a diabetic phenotype, as a decreased ß-cell mass will produce an insufficient amount of insulin. For a pharmacological intervention it is crucial to identify the proteins determining ß-cell mass. Here, we identified the ternary complex factor (TCF) Elk-1 as a regulator of the size of pancreatic islets. Elk-1 mediates, together with a dimer of the serum-response factor (SRF), serum response element-regulated gene transcription. Elk-1 is activated in glucose-treated pancreatic ß-cells but the biological functions of this protein in ß-cells are so far unknown. Elk-1 and homologous TCF proteins are expressed in islets and insulinoma cells. Gene targeting experiments revealed that the TCF proteins show redundant activities. To solve the problem of functional redundancy of these homologous proteins, we generated conditional transgenic mice expressing a dominant-negative mutant of Elk-1 in pancreatic ß-cells. The mutant competes with the wild-type TCFs for DNA and SRF-binding. Expression of the Elk-1 mutant in pancreatic ß-cells resulted in the generation of significantly smaller islets and increased caspase-3 activity, indicating that apoptosis was responsible for the reduction of the pancreatic islet size. Glucose tolerance tests revealed that transgenic mice expressing the dominant-negative mutant of Elk-1 in pancreatic ß-cells displayed impaired glucose tolerance. Thus, we show here for the first time that TCF controls important functions of pancreatic ß-cells in vivo. Elk-1 may be considered as a new therapeutic target for the treatment of diabetes.

Calcineurin controls gene transcription following stimulation of a Gαq-coupled designer receptor.

Stimulation of Gaq-coupled receptors triggers the activation of gene transcription via a rise of intracellular Ca2+. To investigate the role of the Ca2+/calmodulin-dependent phosphatase calcineurin in regulating transcription following Gαq-coupled receptor stimulation, we used a gain-of-function approach and expressed ΔCnA, a constitutively active mutant of calcineurin A. Furthermore, we expressed hM3Dq, a designer receptor that is specifically coupled to Gαq and can be activated by the pharmacological compound clozapine-N-oxide. Stimulation of hM3Dq or expression of ΔCnA induced transcription of a reporter gene controlled by the calcineurin substrate nuclear factor of activated T cells (NFAT), suggesting that calcineurin increased NFAT-regulated gene transcription. In contrast, expression of ΔCnA attenuated hM3Dq-induced biosynthesis of the transcription factors c-Fos and Egr-1 and reduced both c-Fos and Egr-1 promoter activities. A dissection of the c-Fos and Egr-1 promoters revealed that calcineurin inhibited serum response element-mediated transcription. In particular, the expression of ΔCnA reduced the transcriptional activity of the ternary complex factor Elk-1 following stimulation of hM3Dq receptors. Furthermore, ΔCnA reduced the transcriptional activity of the transcription factor CREB and thus attenuated transcription mediated by the cAMP response element. In summary, we show that calcineurin functions as a positive and negative modulator of gene transcription.

Nerve/glial antigen (NG) 2 is a crucial regulator of intercellular adhesion molecule (ICAM)-1 expression.

The proteoglycan nerve/glial antigen (NG) 2 is expressed on multiple cell types and mediates cell proliferation and migration. However, little is known about its function in gene regulation. In this study, we demonstrate that in pericytes and glioblastoma cells intercellular adhesion molecule (ICAM)-1, an essential protein for leukocyte adhesion and transmigration, underlies a NG2-dependent expression. As shown by flow cytometry, Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), silencing of NG2 in human placenta-derived pericytes increased the expression of ICAM-1. Pathway analyses revealed that this is mediated by extracellular-regulated-kinases (ERK) 1/2 signaling. Moreover, leukocyte adhesion to NG2 siRNA-treated pericytes was significantly enhanced when compared to scrambled (scr) siRNA-treated control cells. In vivo, we detected increased ICAM-1 protein levels in the retina of mice lacking NG2 expression. To exclude that this novel mechanism is pericyte-specific, we additionally analyzed the expression of ICAM-1 in dependency of NG2 in two glioblastoma cell lines. We found that A1207 and M059K cells exhibit an inverse expression pattern of NG2 and ICAM-1. Finally, downregulation of NG2 in A1207 cells significantly increased ICAM-1 expression. Taken together, these findings indicate that NG2 may represent a promising target for the modulation of ICAM-1-mediated immune responses.

Resveratrol stimulation induces interleukin-8 gene transcription via NF-κB.

The polyphenol resveratrol activates stimulus-regulated transcription factors, including activator protein-1 (AP-1). As part of a search for resveratrol-regulated target genes we analyzed the gene encoding the chemokine interleukin-8 (IL-8) which is regulated by AP-1. Here, we show that treatment of HEK293 cells with resveratrol induced the expression of IL-8 and activated transcription of a chromatin-embedded IL-8 promoter-controlled reporter gene. Mutational analysis of the IL-8 promoter revealed that it was not the AP-1 binding site, but rather the NF-κB site that was essential to connect resveratrol stimulation with the transcriptional activation of the IL-8 gene. Thus, the NF-κB site of the IL-8 gene functions as resveratrol-responsive element. The analysis of an NF-κB-responsive reporter gene, controlled by the HIV-1 long terminal repeat (LTR), showed that resveratrol stimulation increased the transcriptional activity of NF-κB. These data were corroborated by an experiment showing that incubation of the cells with the NF-κB inhibitor JSH-23 attenuated resveratrol-induced activation of the IL-8 promoter and reduced the cellular NF-κB activity following stimulation of the cells with resveratrol. The protein kinase extracellular signal-regulated protein kinase ERK1/2 was identified to function as signal transducer connecting resveratrol stimulation with the activation of NF-κB and IL-8 promoter-controlled transcription. We conclude that resveratrol, proposed to exhibit anti-inflammatory activity, stimulates expression of the pro-inflammatory chemokine IL-8 via NF-κB, which is known as an important mediator of inflammatory processes.

Specificity of Stress-Responsive Transcription Factors Nrf2, ATF4, and AP-1.

Cellular stress leads to an upregulation of gene transcription. We asked if there is a specificity in the activation of the stress-responsive transcription factors Nrf2, ATF4, and AP-1/c-Jun, or if activation of these proteins is a redundant cellular answer toward extracellular stressors. Here, we show that oxidative stress, induced by stimulation of the cells with the oxidant arsenite, strongly activated gene transcription via the stress-responsive element (StRE), while phorbol ester or tunicamycin, activators of AP-1/c-Jun or ATF4, respectively, activated AP-1 or nutrient-sensing response element-mediated transcription. Preincubation of the cells with N-acetyl-cysteine or overexpression of thioredoxin selectively attenuated arsenite-induced upregulation of StRE-regulated transcription. Expression of either dominant-negative or constitutively active mutants of Nrf2, ATF4, or c-Jun confirmed that distinct transcription units are regulated by these transcription factors. Physiological stimuli involving the activation of either Gαq-coupled designer receptors or the protein kinases c-Jun N-terminal protein kinase or p38 strongly stimulated transcription via AP-1/c-Jun, with minimal effects on Nrf2 or ATF4-responsive promoters. Thus, activation of transcription by extracellular signaling molecules shows specificity at the level of the chemical nature of the signaling molecule, at the level of the intracellular transduction process, and at the level of signal-responsive transcription factors. J. Cell. Biochem. 118: 127-140, 2017. © 2016 Wiley Periodicals, Inc.

Extracellular Signal-Regulated Protein Kinase, c-Jun N-Terminal Protein Kinase, and Calcineurin Regulate Transient Receptor Potential M3 (TRPM3) Induced Activation of AP-1.

Stimulation of transient receptor potential M3 (TRPM3) cation channels with pregnenolone sulfate induces an influx of Ca2+ ions into the cells and a rise in the intracellular Ca2+ concentration, leading to the activation of the activator protein-1 (AP-1) transcription factor. Here, we show that expression of a constitutively active mutant of the Ca2+ /calmodulin-dependent protein phosphatase calcineurin attenuated pregnenolone sulfate-induced AP-1 activation in TRPM3-expressing cells. Likewise, expression of the regulatory B subunit of calcineurin reduced AP-1 activity in the cells following stimulation of TRPM3 channels. MAP kinase phosphatase-1 has been shown to attenuate TRPM3-mediated AP-1 activation. Here, we show that pregnenolone sulfate-induced stimulation of TRPM3 triggers the phosphorylation and activation of the MAP kinase extracellular signal-regulated protein kinase (ERK1/2). Pharmacological and genetic experiments revealed that stimulation of ERK1/2 is essential for the activation of AP-1 in cells expressing stimulated TRPM3 channels. ERK1/2 is required for the activation of the transcription factor c-Jun, a key component of the AP-1 transcription factor, and regulates c-Fos promoter activity. In addition, we identified c-Jun N-terminal protein kinase (JNK1/2) as a second signal transducer of activated TRPM3 channels. Together, the data show that calcineurin and the protein kinases ERK1/2 and JNK1/2 are important regulators within the signaling cascade connecting TRPM3 channel stimulation with increased AP-1-regulated transcription. J. Cell. Biochem. 118: 2409-2419, 2017. © 2017 Wiley Periodicals, Inc.

Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors.

Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin of grapes and other fruits and plants, is a common constituent of our diet and of dietary supplements. Many health-promoting benefits have been connected with resveratrol in the treatment of cardiovascular diseases, cancer, diabetes, inflammation, neurodegeneration, and diseases connected with aging. To explain the pleiotropic effects of resveratrol, the molecular targets of this compound have to be identified on the cellular level. Resveratrol induces intracellular signal transduction pathways which ultimately lead to changes in the gene expression pattern of the cells. Here, we review the effect of resveratrol on the activation of the stimulus-responsive transcription factors CREB, AP-1, Egr-1, Elk-1, and Nrf2. Following activation, these transcription factors induce transcription of delayed response genes. The gene products of these delayed response genes are ultimately responsible for the changes in the biochemistry and physiology of resveratrol-treated cells. The activation of stimulus-responsive transcription factors may explain many of the intracellular activities of resveratrol. However, results obtained in vitro may not easily be transferred to in vivo systems.

Transient receptor potential TRPM3 channels: Pharmacology, signaling, and biological functions.

The transient receptor potential melastatin-3 (TRPM3) channel belongs to the family of transient receptor potential (TRP) cation channels that are expressed in a variety of tissues and cell types, including dorsal root ganglia, cardiomyocytes and pancreatic beta-cells. Although its natural ligands are currently unknown, TRPM3 channels can be activated by the neurosteroid pregnenolone sulfate, the synthetic ligand CIM0216, and by noxious heat. TRPM3 channels are regulated by phosphoinositides, and perhaps by calmodulin. Stimulation of TRPM3 induces an intracellular signaling cascade involving a rise in intracellular Ca2+, activation of the protein kinases Raf, ERK and JNK, and the activation of the stimulus-responsive transcription factors AP-1, CREB, Egr-1, and Elk-1. Functionally, stimulation of TRPM3 channels is connected with heat sensation by somatosensory neurons, insulin secretion by pancreatic beta-cells, regulation of neurotransmitter release, iris constriction, and tumor promotion. With the development of highly specific activators and inhibitors of TRPM3 channels, we expect that additional tissue-specific functions of TRPM3 channels will be discovered, establishing TRPM3 channels as a new therapeutic target.

Inhibition of protein kinase CK2 suppresses tumor necrosis factor (TNF)-α-induced leukocyte-endothelial cell interaction.

Inflammatory endothelial processes are regulated by the nuclear factor-κB (NF-κB) pathway, which involves phosphorylation of p65. Because p65 is a substrate of CK2, we herein investigated, whether this pleiotropic protein kinase may be a beneficial anti-inflammatory target. For this purpose, we analyzed in human dermal microvascular endothelial cells (HDMEC) the effect of CK2 inhibition by quinalizarin and CX-4945 on cell viability, adhesion molecule expression and NF-κB pathway activation. Leukocyte binding to HDMEC was assessed in an in vitro adhesion assay. Dorsal skinfold chambers in BALB/c mice were used to study leukocyte-endothelial cell interaction and leukocyte transmigration by means of repetitive intravital fluorescence microscopy and immunohistochemistry. We found that quinalizarin and CX-4945 effectively suppressed the activity of CK2 in HDMEC without affecting their viability. This was associated with a significant down-regulation of tumor necrosis factor (TNF)-α-induced E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression due to a reduction of shuttling, phosphorylation and transcriptional activity of the NF-κB complex. In consequence, leukocyte binding to quinalizarin- and CX-4945-treated HDMEC was diminished. Finally, CX-4945 treatment significantly decreased the numbers of adherent and transmigrated leukocytes in dorsal skinfold chambers exposed to TNF-α in vivo. These findings indicate that CK2 is a key regulator of leukocyte-endothelial cell interaction in inflammation by regulating the expression of E-selectin, ICAM-1 and VCAM-1 via affecting the transcriptional activity of the NF-κB complex. Accordingly, CK2 represents a promising target for the development of novel anti-inflammatory drugs.

Combining fibroblast isolation with lentiviral gene transfer to validate transgene expression in mice following pronucleus injection.

The binary tetracycline-based expression system in transgenic mice relies on the expression of the tetracycline transactivator (tTA or rtTA) in a particular cell type together with a transcription unit encoding the gene of interest under a tetracycline or doxycycline-responsive promoter. Transgenic mice containing this transcription unit are produced via pronucleus injection. As the chromosomal integration site of the injected DNA influences transgene expression, several founder lines have to be crossed with (r)tTA-expressing mice to find a line showing low background and high transgene expression following doxycycline stimulation. Here, we describe a method to analyze primary fibroblasts derived from the founder lines to quickly test transgene expression and inducibility. Fibroblasts isolated from a small piece of mouse ear were infected with a recombinant lentivirus expressing rtTA. Transgene expression was verified by both RT-PCR and western blot, following stimulation with doxycycline. Transgene expression could easily be detected on the RNA and protein levels in primary fibroblasts derived from transgenic founder lines. An enzymatic function of the transgene was not required for the identification of transgene expression. Thus, the method allows a quick and easy discrimination of transgenic founder lines according to transgene expression and inducibility.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors.

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1.

RE-1 silencing transcription factor (REST): a regulator of neuronal development and neuronal/endocrine function.

RE-1 silencing transcription factor (REST) is a transcriptional repressor that has been proposed to function as a master negative regulator of neurogenesis, as REST target genes encode neuronal receptors, ion channels, neuropeptides and synaptic proteins. During neuronal differentiation, REST expression levels are reduced, allowing expression of selected REST target genes. The analysis of neural stem/progenitor cells that are either devoid of REST or overexpress REST revealed that REST is not the master regulator that is solely responsible for the acquisition of the neuronal fate. Rather, REST provides a regulatory hub that coordinately regulates multiple tiers of neuronal development in vitro. In addition, REST may play an important role for maintaining the integrity of adult neurons. REST confers oxidative stress resistance and is essential for maintaining neuronal viability. Furthermore, the concentration of REST has been reported to influence the pathogenic outcome by neuronal diseases, including stroke, epilepsy and Alzheimer's disease. Experiments performed with PC12 pheochromocytoma cells indicate that REST may function as a key regulator of the neurosecretory phenotype. Moreover, transgenic mice overexpressing REST in pancreatic ß-cells showed impaired insulin secretion leading to significantly reduced plasma insulin levels. Based on the fact that REST plays a prominent role in controlling stimulus-induced secretion in endocrine cells, we propose that REST may also be important for neurotransmitter release via regulation of genes that encode important proteins of the exocytotic machinery.

Expression of the transcription factor Egr-1 in pancreatic acinar cells following stimulation of cholecystokinin or Gαq-coupled designer receptors.

UNLABELLED: BACKGOUND/AIMS: The injection of cerulein, an analogue of the pancreatic secretagogue cholecystokinin (CCK), induces acute pancreatitis in mice that is accompanied by the synthesis of the transcription factor Egr-1. The signaling cascade that connects cerulein stimulation with enhanced Egr-1 biosynthesis was analyzed. METHODS: AR42J rat pancreatic acinar cells were used as a model system to measure cerulein-induced Egr-1 biosynthesis. For comparison, the signaling cascade induced by activation of Gαq-coupled designer receptors with the designer drug clozapine-N-oxide (CNO) was investigated. RESULTS: Stimulation of AR42J cells with cerulein induced a robust and transient biosynthesis of Egr-1. The signaling cascade connecting cerulein stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca(2+) and the activation of the protein kinases PKC, Raf and ERK, while expression of MKP-1 prevented Egr-1 biosynthesis in cerulein-stimulated AR42J cells. In addition, ternary complex factors are required to connect cerulein stimulation with enhanced transcription of the Egr-1 gene. Egr-1 biosynthesis induced in CNO-stimulated AR42J pancreatic acinar cells expressing Gαq-coupled designer receptors required identical signaling molecules, although subtle differences were observed in comparison to cerulein/CCK receptor signaling. CONCLUSION: We propose that overstimulation of the canonical Gαq-induced signaling pathway may be crucial for inducing acute pancreatitis.