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.

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.

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.

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.

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.

Tyrosine hydroxylase gene promoter activity is upregulated in female catecholaminergic neuroblastoma cells following activation of a Gαq-coupled designer receptor.

Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis that catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine. The tyrosine hydroxylase gene is regulated by extracellular signaling molecules such as epidermal growth factor, nerve growth factor and steroids. Here, we investigated whether the activity of the tyrosine hydroxylase gene promoter is upregulated by activation of G protein-coupled receptors, the largest group of plasma membrane receptors. We used catecholaminergic neuroblastoma cells as a cellular model and chromatin-integrated tyrosine hydroxylase promoter-luciferase reporter genes. The results show that stimulation of Rαq, a Gαq-coupled designer receptor, triggered transcription of a reporter gene driven by the tyrosine hydroxylase promoter. Transcription was attenuated by overexpression of regulator of G-protein signaling-2, which activates the GTPase activity of the G protein α-subunit, and by a truncated, dominant-negative mutant of phospholipase Cß3. Extracellular signal-regulated protein kinase was identified as the signal transducer. At the transcriptional level, tyrosine hydroxylase promoter activity was found to be controlled by the transcription factor CREB. Expression experiments with the adenoviral regulator protein E1A, an inhibitor of CBP/p300 histone acetyltransferases, showed that transcription of the reporter gene controlled by the tyrosine hydroxylase is under epigenetic control. We identified the protein phosphatases MAP kinase phosphatase-1 and calcineurin as part of a shutdown device of the signaling cascade linking Rαq designer receptor activation to tyrosine hydroxylase gene transcription. We conclude that tyrosine hydroxylase promoter activity is controlled by Gαq-coupled receptors.

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.

Regulation of c-Fos gene transcription by stimulus-responsive protein kinases.

The basic region leucin zipper (bZIP) protein c-Fos constitutes together with other bZIP proteins the AP-1 transcription factor complex. Expression of the c-Fos gene is regulated by numerous extracellular signaling molecules including mitogens, metabolites, and ligands for receptor tyrosine kinases, G protein-coupled receptors, and cytokine receptors. Here, we analyzed the effects of the stimulus-responsive MAP kinases ERK1/2 (extracellular signal-regulated protein kinase), JNK (c-Jun N-terminal protein kinase) and p38 protein kinase on transcription of the c-Fos gene. We used chromatin-integrated c-Fos promoter-luciferase reporter genes containing inactivating point mutations of DNA binding sites for distinct transcription factors. ERK1/2, JNK, and p38 protein kinases were specifically activated following expression of either a mutant of B-Raf, a truncated version of mitogen-activated/extracellular signal responsive kinase kinase kinase-1 (MEKK1), or a mutant of MAP kinase kinase-6 (MKK6), respectively. The results show that the DNA binding sites for serum response factor (SRF) and for the ternary complex factor (TCF) are of major importance for stimulating c-Fos promoter activity by MAP kinases. ERK1/2 and p38-induced stimulation of the c-Fos promoter additionally required the DNA binding site for the transcription factor AP-1. Mutation of the DNA binding site for STAT had no or only a small effect on c-Fos promoter activity. We conclude that MAP kinases do not activate distinct transcription factors involving distinct genetic elements. Rather, these kinases mainly target SRF and TCF proteins, leading to an activation of transcription of the c-Fos gene via the serum response element.

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 ß-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.

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 and function of AP-1 in insulinoma cells and pancreatic ß-cells.

Cav1.2 L-type voltage-gated Ca2+ channels play a central role in pancreatic ß-cells by integrating extracellular signals with intracellular signaling events leading to insulin secretion and altered gene transcription. Here, we investigated the intracellular signaling pathway following stimulation of Cav1.2 Ca2+ channels and addressed the function of the transcription factor activator protein-1 (AP-1) in pancreatic ß-cells of transgenic mice. Stimulation of Cav1.2 Ca2+ channels activates AP-1 in insulinoma cells. Pharmacological and genetic experiments identified c-Jun N-terminal protein kinase as a signal transducer connecting Cav1.2 Ca2+ channel activation with gene transcription. Moreover, the basic region-leucine zipper proteins ATF2 and c-Jun or c-Jun-related proteins were involved in stimulus-transcription coupling. We addressed the functions of AP-1 in pancreatic ß-cells analyzing a newly generated transgenic mouse model. These transgenic mice expressed A-Fos, a mutant of c-Fos that attenuates DNA binding of c-Fos dimerization partners. In insulinoma cells, A-Fos completely blocked AP-1 activation following stimulation of Cav1.2 Ca2+ channels. The analysis of transgenic A-Fos-expressing mice revealed that the animals displayed impaired glucose tolerance. Thus, we show here for the first time that AP-1 controls an important function of pancreatic ß-cells in vivo, the regulation of glucose homeostasis.

Immediate-early transcriptional response to insulin receptor stimulation.

Insulin binding to the insulin receptor triggers intracellular signaling cascades involving the activation of protein and lipid kinases. As a result, multiple biological functions of the cells are changed. Here, we analyzed the regulation and signaling cascades leading to insulin-induced activation of the stimulus-responsive transcription factors. For the analyses, we used chromatin-embedded reporter genes having a cellular nucleosomal organisation, and fibroblasts expressing human insulin receptors (HIRcB cells). The results show that stimulation of the insulin receptor induced the expression of the transcription factor Egr-1. Attenuation of Egr-1 promoter activation was observed following expression of a dominant-negative mutant of the ternary complex factor Elk-1. These data were corroborated by experiments showing that insulin receptor stimulation increased the transcriptional activation potential of Elk-1. In addition, the transcriptional activity of AP-1 was significantly elevated in insulin-stimulated HIRcB cells. Expression of the dominant-negative mutant of Elk-1 reduced insulin-induced activation of AP-1, indicating that Elk-1 controls both serum response element and AP-1-regulated transcription. Moreover, we show that stimulation of the insulin receptor activates cyclic AMP response element (CRE)-controlled transcription, involving the transcription factor CREB. Insulin-induced transcription of Elk-1 and CREB-controlled reporter genes was attenuated by overexpression of MAP kinase phosphatase-1 or a constitutively active mutant of calcineurin A, indicating that both phosphatases are part of a negative feedback loop for reducing insulin-mediated gene transcription. Finally, we show that expression of the adenoviral protein E1A selectively reduced CRE-mediated transcription following stimulation of the insulin receptor. These data indicate that insulin-regulated transcription of CRE-containing genes is under epigenetic control.

Chromatin-embedded reporter genes: Quantification of stimulus-induced gene transcription.

Receptors and ion channels expressed on the cell surface ensure proper communication between the cells and the environment. In multicellular organism, stimulus-regulated gene transcription is the basis for communication with the environment allowing individual cells to respond to stimuli such as nutrients, chemical stressors and signaling molecules released by other cells of the organism. Hormones, cytokines, and mitogens bind to receptors and ion channels and induce intracellular signaling cascades involving second messengers, kinases, phosphatases, and changes in the concentration of particular ions. Ultimately, the signaling cascades reach the nucleus. Transcription factors are activated that respond to cellular stimulation and induce changes in gene transcription. Investigating stimulus-transcription coupling combines cell biology with genetics. In this review, we discuss the molecular biology of stimulus-induced transcriptional activators and their responsiveness to extracellular and intracellular signaling molecules and to epigenetic regulators. Stimulus-induced gene expression is measured by several methods, including detection of nuclear translocation of transcription factors, phosphorylation or DNA binding. In this article, we emphasize that the most reliable method to directly measure transcriptional activation involves the use of chromatin-embedded reporter genes.

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.

Zn2+ ions inhibit gene transcription following stimulation of the Ca2+ channels Cav1.2 and TRPM3.

Zinc, a trace element, is necessary for the correct structure and function of many proteins. Therefore, Zn2+ has to be taken up by the cells, using specific Zn2+ transporters or Ca2+ channels. In this study, we have focused on two Ca2+ channels, the L-type voltage-gated Cav1.2 channel and the transient receptor potential channel TRPM3. Stimulation of either channel induces an intracellular signaling cascade leading to the activation of the transcription factor AP-1. The influx of Ca2+ ions into the cytoplasm is essential for this activity. We asked whether extracellular Zn2+ ions affect Cav1.2 or TRPM3-induced gene transcription following stimulation of the channels. The results show that extracellular Zn2+ ions reduced the activation of AP-1 by more than 80% following stimulation of either voltage-gated Cav1.2 channels or TRPM3 channels. Experiments performed with cells maintained in Ca2+-free medium revealed that Zn2+ ions cannot replace Ca2+ ions in inducing gene transcription via stimulation of Cav1.2 and TRPM3 channels. Re-addition of Ca2+ ions to the cell culture medium, however, restored the ability of these Ca2+ channels to induce a signaling cascade leading to the activation of AP-1. Secretory cells, including neurons and pancreatic ß-cells, release Zn2+ ions during exocytosis. We propose that the released Zn2+ ions function as a negative feedback loop for stimulus-induced exocytosis by inhibiting Ca2+ channel signaling.

Pharmacological and genetic inhibition of TRPC6-induced gene transcription.

Transient receptor potential canonical-6 (TRPC6) channels are non-selective cation channels that can be activated by hyperforin, a constituent of Hypericum perforatum. TRPC6 activation has been linked to a variety of biological functions and pathologies, including focal segmental glomerulosclerosis and the development of various tumor entities. Thus, TRPC6 is an interesting drug target, and a specific pharmacological inhibitor would be very valuable for both basic research and therapy of TRPC6-mediated human pathologies. Here, we assessed the biological activity of various TRP channel inhibitors on hyperforin-stimulated TRPC6 channel signaling. Hyperforin stimulates the activity of the transcription factor AP-1 via TRPC6. Expression experiments involving a TRPC6-specific small hairpin RNA confirmed that hyperforin-induced gene transcription requires TRPC6. Cellular AP-1 activity was measured to assess which compound interrupted the TRPC6-induced intracellular signaling cascade. The results show that the compounds 2-APB, clotrimazole, BCTC, TC-I 2014, SAR 7334, and larixyl acetate blocked TRPC6-mediated activation of AP-1. In contrast, the TRPM8-specific inhibitor RQ-00203078 did not inhibit TRPC6-mediated signaling. 2-APB, clotrimazole, BCTC, and TC-I 2014 are broad-spectrum Ca2+ channel inhibitors, while SAR 7334 and larixyl acetate have been proposed to function as rather TRPC6-specific inhibitors. In this study it is shown that both compounds, in addition to inhibiting TRPC6-induced signaling, completely abolished pregnenolone sulfate-mediated signaling via TRPM3 channels. Thus, SAR 7334 and larixyl acetate are not TRPC6-specific inhibitors.

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.

The super-cooling compound icilin stimulates c-Fos and Egr-1 expression and activity involving TRPM8 channel activation, Ca2+ ion influx and activation of the ternary complex factor Elk-1.

The TRPM8 cation channel can be activated by the cooling compound icilin. Recently, we showed that stimulation of TRPM8 channels induces a signaling cascade leading to the activation of the transcription factor AP-1. Additionally, expression of the AP-1 constituent c-Fos has been shown to be induced following TRPM8 stimulation. c-Fos is frequently used as a marker for neuronal activity. Here, we have analyzed the mechanism connecting TRPM8 stimulation and c-Fos expression. Furthermore, we analyzed the expression of the neuronal activity-responsive transcription factor Egr-1 following TRPM8 activation. The results show that icilin-induced stimulation of TRPM8 channels increased c-Fos promoter activity and induced c-Fos expression. Moreover, icilin stimulation increased Egr-1 promoter activity and induced the expression of Egr-1. Pharmacological inhibition of TRPM8 blocked the icilin-induced expression of Egr-1 and c-Fos. An influx of Ca2+ ions into the cells via TRPM8 was necessary to stimulate Egr-1 and c-Fos expression following icilin treatment. Genetic experiments revealed that serum response elements within the Egr-1 and c-Fos promoters are crucial to couple TRPM8 stimulation with enhanced transcription of both the Egr-1 and c-Fos genes. These data were corroborated by experiments showing that TRPM8 stimulation increased the transcriptional activation potential of Elk-1, a SRE binding protein. c-Fos is important for neuronal excitability and survival. Egr-1 plays an important role in synaptic plasticity, consolidation and reconsolidation of long-term memory. Elk-1 may preserve neurons against toxic insults but may also induce depressive behaviour. The fact that TRPM8 stimulation activates the transcription factors c-Fos, Egr-1, and Elk-1 connects TRPM8 signaling with maintaining important brain functions.

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.

Pharmacological inhibition of TRPM8-induced gene transcription.

Transient receptor potential melastatin-8 (TRPM8) channels are activated by cold temperature, menthol and icilin, leading to cold sensation. TRPM8 activation is connected with various diseases, indicating that a specific pharmacological antagonist, allowing nongenetic channel suppression, would be a valuable tool for therapy and basic research. Here, we assessed the biological activity and specificity of various TRPM8 inhibitors following stimulation of TRPM8 channels with either icilin or menthol. Recently, we showed that icilin strikingly upregulates the transcriptional activity of AP-1. By measuring AP-1 activity, we assessed which compound interrupted the TRPM8-induced intracellular signaling cascade from the plasma membrane to the nucleus. We tested the specificity of various TRPM8 inhibitors by analyzing AP-1 activation following stimulation of TRPM3 and TRPV1 channels, L-type voltage-gated Ca2+ channels, and Gαq-coupled receptors, either in the presence or absence of a particular TRPM8 inhibitor. The results show that the TRPM8 inhibitors BCTC, RQ-00203078, TC-1 2014, 2-APB, and clotrimazole blocked TRPM8-mediated activation of AP-1. However, only the compound RQ-00203078 showed TRPM8-specificity, while the other compounds function as broad-spectrum Ca2+ channel inhibitors. In addition, we show that progesterone interfered with TRPM8-induced activation of AP-1, as previously shown for TRPM3 and TRPC6 channels. TRPM8-induced transcriptional activation of AP-1 was additionally blocked by the compound PD98059, indicating that extracellular signal-regulated protein kinase-1/2 is essential to couple TRPM8 stimulation with transcriptional activation of AP-1. Moreover, an influx of Ca2+-ions is essential to induce the intracellular signaling cascade leading to the activation of AP-1.