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.

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.

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.

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.

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.

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.

Dihydrotestosterone activates AP-1 in LNCaP prostate cancer cells.

A cross-talk between androgen/androgen receptor signaling and the AP-1 transcription factor has been proposed. In this study, we asked whether activation of AP-1 modifies androgen-responsive gene transcription, and whether androgens effect AP-1-regulated gene transcription. We show that activation of AP-1 via expression of a constitutively active mutant of mitogen-activated/extracellular signal responsive kinase kinase (MEK) kinase-1 did not increase the activity of the androgen-responsive probasin promoter. Likewise, expression of a constitutively active mutant of the transcription factor c-Jun, which is a major constitutent of AP-1, did not increase the activity of the probasin promoter. In contrast, 5α-dihydrotestosterone (DHT) activated both the probasin promoter and the AP-1-regulated collagenase promoter in LNCaP prostate cancer cells. The AP-1 binding site within the collagenase promoter was identified as DHT-responsive element. In line with this, DHT increased the activities of the c-Jun promoter and the tumor necrosis factor alpha promoter, which both contain AP-1 binding sites. The signal transduction pathway coupling DHT stimulation with AP-1 activation required c-Jun, MAP kinases and androgen receptors, but was independent of transient receptor potential melastatin-8 (TRPM8) channels, proposed to function as ionotropic testosterone receptors. Expression of the GTPase activating protein RGS2 attenuated DHT-induced activation of AP-1, indicating that the DHT-induced signaling cascade involves G proteins.

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.

Resveratrol stimulates c-Fos gene transcription via activation of ERK1/2 involving multiple genetic elements.

The polyphenol resveratrol is found in many plant and fruits and is a constituent of our diet. Resveratrol has been proposed to have chemopreventive and anti-inflammatory activities. On the cellular level, resveratrol activates stimulus-regulated transcription factors. To identify resveratrol-responsive elements within a natural gene promoter, the molecular pathway leading to c-Fos gene expression by resveratrol was dissected. The c-Fos gene encodes a basic region leucine zipper transcription factor and is a prototype of an immediate-early gene that is regulated by a wide range of signaling molecules. We analyzed chromatin-integrated c-Fos promoter-luciferase reporter genes where transcription factor binding sites were destroyed by point mutations or deletion mutagenesis. The results show that mutation of the binding sites for serum response factor (SRF), activator protein-1 (AP-1) and cAMP response element binding protein (CREB) significantly reduced reporter gene transcription following stimulation of the cells with resveratrol. Inactivation of the binding sites for signal transducer and activator of transcription (STAT) or ternary complex factors did not influence resveratrol-regulated c-Fos promoter activity. Thus, the c-Fos promoter contains three resveratrol-responsive elements, the cAMP response element (CRE), and the binding sites for SRF and AP-1. Moreover, we show that the transcriptional activation potential of the c-Fos protein is increased in resveratrol-stimulated cells, indicating that the biological activity of c-Fos is elevated by resveratrol stimulation. Pharmacological and genetic experiments revealed that the protein kinase ERK1/2 is the signal transducer that connects resveratrol treatment with the c-Fos gene.

Regulation of Gene Transcription Following Stimulation of Transient Receptor Potential (TRP) Channels.

Transient receptor potential (TRP) channels belong to a heterogeneous superfamily of cation channels that are involved in the regulation of numerous biological functions, including regulation of Ca2+ and glucose homeostasis, tumorigenesis, temperature, and pain sensation. To understand the functions of TRP channels, their associated intracellular signaling pathways and molecular targets have to be identified on the cellular level. Stimulation of TRP channels frequently induces an influx of Ca2+ ions into the cells and the subsequent activation of protein kinases. These intracellular signal transduction pathways ultimately induce changes in the gene expression pattern of the cells. Here, we review the effects of TRPC6, TRPM3, and TRPV1 channel stimulation on the activation of the stimulus-responsive transcription factors AP-1, CREB, Egr-1, Elk-1, and NFAT. Following activation, these transcription factors induce the transcription of delayed response genes. We propose that many biological functions of TRP channels can be explained by the activation of stimulus-responsive transcription factors and their delayed response genes. The proteins encoded by those delayed response genes may be responsible for the biochemical and physiological changes following TRP channel activation.

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.

Stimulation of B-Raf increases c-Jun and c-Fos expression and upregulates AP-1-regulated gene transcription in insulinoma cells.

Stimulation of pancreatic ß-cells with glucose activates the protein kinases B-Raf and extracellular signal-regulated protein kinase that participate in glucose sensing. Inhibition of both kinases results in impairment of glucose-regulated gene transcription. To analyze the signaling pathway controlled by B-Raf, we expressed a conditionally active form of B-Raf in INS-1 insulinoma cells. Here, we show that stimulation of B-Raf strongly activated the transcription factor AP-1 which is accompanied by increased c-Jun and c-Fos promoter activities, an upregulation of c-Jun and c-Fos biosynthesis, and elevated transcriptional activation potentials of c-Jun and c-Fos. Mutational analysis identified the AP-1 sites within the c-Jun promoter and the serum response element (SRE) within the c-Fos promoter as the essential genetic elements connecting B-Raf stimulation with AP-1 activation. In line with this, the transcriptional activation potential of the SRE-binding protein Elk-1 was increased following B-Raf activation. The signal pathway from B-Raf to AP-1 required the activation of c-Jun. We identified the cyclin D1 gene as a delayed response gene for AP-1 following stimulation of B-Raf in insulinoma cells. Moreover, MAP kinase phosphatase-1 and the Ca2+/calmodulin-dependent protein phosphatase calcineurin were identified to function as shut-off-devices for the signaling cascade connecting B-Raf stimulation with the activation of AP-1. The fact that stimulation with glucose, activation of L-type voltage-gated Ca2+ channels, and stimulation of B-Raf all trigger an activation of AP-1 indicates that AP-1 is a point of convergence of signaling pathways in ß-cell.

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.

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.