Gerald Thiel

Elk‐1, CREB, and MKP‐1 regulate Egr‐1 expression in gonadotropin‐releasing hormone stimulated gonadotrophs

Stimulation of gonadotropin‐releasing hormone (GnRH) receptors with the GnRH analogue buserelin enhances expression of the zinc finger transcription factor Egr‐1 in a pituitary gonadotroph cell line. The signaling cascade is blocked by overexpression of MAP kinase phosphatase‐1 that dephosphorylates extracellular signal‐regulated protein kinase in the nucleus. Chromatin immunoprecipitation experiments revealed that the phosphorylated form of Elk‐1, a key regulator of gene transcription driven by serum response element (SRE), binds to the 5′‐upstream region of the Egr‐1 gene in buserelin‐stimulated gonadotrophs. Expression of a dominant‐negative mutant of Elk‐1 completely blocked Egr‐1 expression, indicating that Elk‐1 connects the intracellular signaling cascade elicited by activation of GnRH receptors with transcription of the Egr‐1 gene. GnRH receptor activation additionally induced the phosphorylation of CREB, which in its phosphorylated form bound to the Egr‐1 gene. Expression of a dominant‐negative mutant of CREB reduced GnRH receptor‐induced upregulation of Egr‐1 expression, indicating that CREB plays a role in the signaling pathway that regulates Egr‐1 expression in gonadotrophs. We further identified the genes encoding basic fibroblast growth factor, tumor necrosis factor α, and transforming growth factor β as bona fide target genes of Egr‐1 in gonadotrophs. The analysis of gonadotroph cells that express—in addition to GnRH receptors—muscarinic M3 acetylcholine receptors revealed that the nuclear events connecting GnRH receptors and muscarinic M3 acetylcholine receptors with the Egr‐1 gene are indistinguishable. J. Cell. Biochem. 105: 1267–1278, 2008.

CREB-1α Is Recruited to and Mediates Upregulation of the Cytochrome c Promoter during Enhanced Mitochondrial Biogenesis Accompanying Skeletal Muscle Differentiation

ABSTRACT To further understand pathways coordinating the expression of nuclear genes encoding mitochondrial proteins, we studied mitochondrial biogenesis during differentiation of myoblasts to myotubes. This energy-demanding process was accompanied by a fivefold increase of ATP turnover, covered by an eightfold increase of mitochondrial activity. While no change in mitochondrial DNA copy number was observed, mRNAs as well as proteins for nucleus-encoded cytochrome c, cytochrome c oxidase subunit IV, and mitochondrial transcription factor A (TFAM) increased, together with total cellular RNA and protein levels. Detailed analysis of the cytochrome c promoter by luciferase reporter, binding affinity, and electrophoretic mobility shift assays as well as mutagenesis studies revealed a critical role for cyclic AMP responsive element binding protein 1 (CREB-1) for promoter activation. Expression of two CREB-1 isoforms was observed by using specific antibodies and quantitative reverse transcription-PCR, and a shift from phosphorylated CREB-1Δ in myoblasts to phosphorylated CREB-1α protein in myotubes was shown, while mRNA ratios remained unchanged. Chromatin immunoprecipitation assays confirmed preferential binding of CREB-1α in situ to the cytochrome c promoter in myotubes. Overexpression of constitutively active and dominant-negative forms supported the key role of CREB-1 in regulating the expression of genes encoding mitochondrial proteins during myogenesis and probably also in other situations of enhanced mitochondrial biogenesis.

Regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper transcription factors

Tetrahydrobiopterin is an essential cofactor for the phenylalanine, tyrosine and tryptophan hydroxylases, and the family of nitric oxide synthases. The initial and rate‐limiting enzyme in the biosynthesis of tetrahydrobiopterin is GTP cyclohydrolase I. The proximal promoter of the human GTP cyclohydrolase I gene contains the sequence motif 5′‐TGACGCGA‐3′, resembling a cAMP response element (CRE). The objective of this study was to analyze the regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper (bZIP) transcription factors. A constitutively active mutant of the cAMP response element binding (CREB) protein strongly stimulated GTP cyclohydrolase I promoter activity, indicating that the CRE in the context of the GTP cyclohydrolase I gene is functional. Likewise, GTP cyclohydrolase I promoter/luciferase gene transcription was stimulated following nuclear expression of the catalytic subunit of cAMP‐dependent protein kinase. Constitutively active mutants of activating transcription factor 2 (ATF2) and c‐Jun additionally stimulated GTP cyclohydrolase I promoter activity, but to a lesser extent than the constitutively active CREB mutant. The fact that stress‐activated protein kinases target the GTP cyclohydrolase I gene was corroborated by expression experiments involving p38 and MEKK1 protein kinases. We conclude that signaling pathways involving either the cAMP‐dependent protein kinase or stress‐activated protein kinases converge to the GTP cyclohydrolase I gene. Hence, enzymatic reactions that require tetrahydrobiopterin as cofactor are therefore indirectly controlled by signaling cascades involving the signal‐responsive transcription factors CREB, c‐Jun, and ATF2. J. Cell. Biochem.

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

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

cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

BackgroundThe enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE).ResultsThe cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP) family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2), known to compete with CREB for binding to the canonical CRE sequence 5-TGACGTCA-3, did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene.ConclusionsUsing a constitutively active CREB2/CREB fusion protein and a mutant of the PKA catalytic subunit that is targeted to the nucleus, we have shown that the glucose-6-phosphatase gene has two distinct genetic elements that function as bona fide CRE. This study further shows that the expression vectors encoding C2/CREB and catalytic subunit of PKA are valuable tools for the study of CREB-mediated gene transcription and the biological functions of CREB.

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

Graphical abstract Figure. No Caption available. Abstract 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.

CREB, AP-1, ternary complex factors and MAP kinases connect transient receptor potential melastatin-3 (TRPM3) channel stimulation with increased c-Fos expression.

The rise in intracellular Ca2+ stimulates the expression of the transcription factor c‐Fos. Depending on the mode of entry of Ca2+ into the cytosol, distinct signal transducers and transcription factors are required. Here, we have analysed the signalling pathway connecting a Ca2+ influx via activation of transient receptor potential melastatin‐3 (TRPM3) channels with enhanced c‐Fos expression.

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.

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.

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.