Oliver G. Rössler

Epidermal-growth-factor-induced proliferation of astrocytes requires Egr transcription factors

Stimulation of astrocytes with epidermal growth factor (EGF) induced proliferation and triggered the biosynthesis of the transcription factor Egr-1, involving the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. No differences in the proliferation rate of astrocytes prepared from wild-type or Egr-1-deficient mice were detected. However, expression of a dominant-negative mutant of Egr-1 that interfered with DNA-binding of all Egr proteins prevented EGF-induced proliferation of astrocytes. Site-directed mutagenesis of two crucial cysteine residues within the zinc finger DNA-binding domain revealed that DNA-binding of the Egr-1 mutant was essential to inhibit proliferation of EGF-stimulated astrocytes. Expression of NAB2 (a negative co-regulator of Egr-1, Egr-2 and Egr-3) or a dominant-negative mutant of Elk-1 (a key regulator of Egr-1 biosynthesis) abolished EGF-induced proliferation of astrocytes. Chromatin immunoprecipitation experiments showed that Egr-1, Egr-2 and Egr-3 bound to the gene expressing basic fibroblast growth factor (bFGF) in EGF-stimulated astrocytes. Egr-2 and Egr-3 also interacted with the bFGF gene in EGF-stimulated astrocytes prepared from Egr-1-deficient mice, indicating that loss of Egr-1 is compensated by other Egr proteins. Together, these data show that Egr transcription factors are essential for conversion of the mitogenic signal of EGF into a proliferative response.

Glutamate-induced cell death of immortalized murine hippocampal neurons: neuroprotective activity of heme oxygenase-1, heat shock protein 70, and sodium selenite.

HT22 immortalized hippocampal neurons serve as a cellular model system to study oxidative stress, an imbalance of cellular redox homeostasis. Glutamate induces HT22 cell death by inhibiting the uptake of cystine into the cells via the cystine/glutamate transport system xc-, thus leading to reduced levels of glutathione. Here, we show that glutamate-induced cell death is attenuated in HT22 cells overexpressing heat shock protein 70 or heme oxygenase-1. Moreover, supplementing the culture medium with sodium selenite completely protected HT22 against oxidative glutamate toxicity. In contrast, neither heat shock protein 70 nor heme oxygenase-1 expression or increased concentrations of sodium selenite protected HT22 cells against serum withdrawal-induced cell death. These data indicate that glutamate-induced cell death differs substantially from that induced by growth factor deprivation.

Neuronal cell death induced by antidepressants: lack of correlation with Egr-1, NF-κB and extracellular signal-regulated protein kinase activation

The tricyclic antidepressants (TCA) amitriptyline and desipramine and the serotonin reuptake inhibitor fluoxetine induce, at microM concentrations, cell death in HT22 immortalized hippocampal neurons and PC12 pheochromocytoma cells. Here, we show that these neurotoxic effects are accompanied by a selective activation of extracellular signal-regulated protein kinase (ERK), the biosynthesis of the transcription factor Egr-1 and an increase in the transcriptional activity of NF-kappa B. However, an impairment of both ERK activation and Egr-1 biosynthesis by the MAP kinase kinase-1 (MEK-1) inhibitor PD98059 did not block cell death. Moreover, stimulation of ERK phosphorylation and Egr-1 biosynthesis by sphingosine-1-phosphate did not induce cell death, indicating that stimulation of the ERK signaling pathway and Egr-1 biosynthesis are not required for neuronal cell death induced by antidepressants. Likewise, attenuation of antidepressant-induced NF-kappa B activity by elevation of the intracellular cAMP concentration or by retroviral driven expression of the non-degradable superrepressor I kappa B alpha S32A/S36A demonstrated that the elevation of NF-kappa B activity by amitriptyline, desipramine and fluoxetine is not an integral part of the apoptotic signaling cascade triggered by these compounds.

Up‐regulation of tyrosine hydroxylase gene transcription by tetradecanoylphorbol acetate is mediated by the transcription factors Ets‐like protein‐1 (Elk‐1) and Egr‐1

Tyrosine hydroxylase is the rate‐limiting enzyme in the biosynthesis of catecholamines. Expression of the tyrosine hydroxylase gene is regulated at the transcriptional level by extracellular signalling molecules, including epidermal growth factor (EGF), nerve growth factor (NGF) and glucocorticoids. We have analysed the stimulation of tyrosine hydroxylase gene transcription by the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in noradrenergic locus coeruleus‐like CATH.a cells and observed a striking enhancement of the transcriptional activation potential of the ternary complex factor Ets‐like protein‐1 (Elk‐1), a key transcriptional regulator of serum response element‐driven gene transcription. Likewise, TPA strongly up‐regulated the biosynthesis of the transcription factor Egr‐1 via distal serum response elements within the Egr‐1 5′‐flanking region. Subsequently, enhancement of the transcriptional activation potential of Egr‐1 was observed. Overexpression of Egr‐1 was sufficient to activate transcription of a tyrosine hydroxylase promoter/reporter gene, corroborating the view that the tyrosine hydroxylase gene is a target gene of Egr‐1. Expression of dominant‐negative mutants of Elk‐1 or Egr‐1 impaired TPA‐induced stimulation of a tyrosine hydroxylase promoter/reporter gene transcription. In contrast, dominant‐negative mutants of the transcription factors activating transcription factor (ATF)‐2, ATF4, cAMP response element‐binding protein, c‐Jun and CCAAT/enhancer binding protein (C/EBP) did not change TPA‐induced tyrosine hydroxylase promoter activity, indicating that these proteins are not part of the TPA‐mediated signalling cascade directed towards the tyrosine hydroxylase gene.

Egr-1—A Ca2+-regulated transcription factor

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

Thapsigargin Induces Expression of Activating Transcription Factor 3 in Human Keratinocytes Involving Ca2+ Ions and c-Jun N-Terminal Protein Kinase

Thapsigargin is a specific inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase of the endoplasmic reticulum. Here, we show that stimulation of human HaCaT keratinocytes with nanomolar concentrations of thapsigargin triggers expression of activating transcription factor (ATF) 3, a basic-region leucin zipper transcription factor. ATF3 expression was also up-regulated in thapsigargin-stimulated glioma cells, hepatoma cells, retinal pigment epithelial cells, and airway epithelial cells. Thapsigargin-induced up-regulation of ATF3 expression in keratinocytes was attenuated by BAPTA-acetoxymethyl ester or by expression of the Ca2+-binding protein parvalbumin in the cytosol of HaCaT cells but not by a panel of pharmacological agents that chelate extracellular Ca2+ (EGTA) or inhibit either ryanodine receptors (dantrolene) or voltage-gated Ca2+ channels (nifedipine). Hence, elevated levels of intracellular Ca2+, released from intracellular stores, are essential for the effect of thapsigargin on the biosynthesis of ATF3. The thapsigargin-induced signaling pathway was blocked by expression of either mitogen-activated protein kinase phosphatase-1 or -5. Experiments involving pharmacological and genetic tools revealed the importance of c-Jun N-terminal protein kinase (JNK) within the signaling cascade, whereas inhibition of extracellular signal-regulated protein kinase or p38 protein kinase did not attenuate thapsigargin-induced expression of ATF3. Functional studies showed that treatment of HaCaT keratinocytes with thapsigargin led to a 2-fold induction of caspase-3/7 activity. The up-regulation of caspase-3/7 activity in thapsigargin-stimulated HaCaT cells was attenuated by inhibition of JNK. Together, these data show that stimulation of HaCaT cells with thapsigargin induces a specific signaling pathway in keratinocytes involving activation of JNK, biosynthesis of ATF3, and up-regulation of caspase-3/7 activity.

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

BackgroundThe serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts.ResultsStimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK). Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M1 muscarinic acetylcholine receptors required the transactivation of the EGF receptor, while thrombin signaling did not.ConclusionThis study shows that stimulus-transcription coupling in thrombin-treated lung fibroblasts relies on the elevation of the intracellular Ca2+-concentration and the activation of PKC and ERK. In the nucleus, ternary complex factors function as key proteins linking the intracellular signaling cascade with enhanced transcription of the Egr-1 gene. This study further shows that the dominant-negative Elk-1 mutant is a valuable tool to study Elk-1-mediated gene transcription.

P2X7 receptor stimulation upregulates Egr‐1 biosynthesis involving a cytosolic Ca2+ rise, transactivation of the EGF receptor and phosphorylation of ERK and Elk‐1

The P2X7 receptor is an ATP‐gated ionotropic receptor that is permeable for small cations including Ca2+ ions. Using 293 cells expressing P2X7 receptors, we show that the P2X7 receptor‐specific ligand 2′,3′‐O‐(4‐benzoyl‐benzoyl)‐ATP (BzATP) induces a signaling cascade leading to the biosynthesis of biologically active Egr‐1, a zinc finger transcription factor. BzATP‐triggered Egr‐1 biosynthesis was attenuated by the mitogen‐activated protein kinase kinase inhibitor PD98059, by BAPTA‐AM, the acetoxymethylester of the cytosolic Ca2+ chelator BAPTA, and by an epidermal growth factor (EGF) receptor‐specific tyrosine kinase inhibitor (AG1478). These results indicate that phosphorylation and activation of extracellular signal‐regulated protein kinase ERK, elevated levels of intracellular Ca2+ and the transactivation of the EGF receptor are essential for BzATP‐induced upregulation of Egr‐1. The requirement of Ca2+ within the signaling cascade was upstream of Raf kinase activation. Lentiviral‐mediated expression of MAP kinase phosphatase‐1 (MKP‐1), a dual‐specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, inhibited Egr‐1 biosynthesis following BzATP stimulation, indicating that MKP‐1 functions as a nuclear shut‐off device. Furthermore, the ternary complex factor Elk‐1 was phosphorylated and the transcriptional activation potential of Elk‐1 was enhanced following P2X7 receptor stimulation. Expression of a dominant‐negative mutant of Elk‐1 impaired BzATP‐induced upregulation of Egr‐1 biosynthesis. Thus, Elk‐1 connects the intracellular signaling cascade elicited by activation of P2X7 receptors with the transcription of the Egr‐1 gene. J. Cell. Physiol. 213: 36–44, 2007.

Immediate-Early Transcriptional Response to Angiotensin II in Human Adrenocortical Cells

Angiotensin II binds to the angiotensin II receptors type 1 (AT1 receptors) in adrenocortical cells and triggers an intracellular signaling cascade leading to changes in the gene expression pattern. Here, we show that stimulation with angiotensin II induces the expression of biologically active early growth response (Egr)-1, a zinc finger transcription factor, in human H295R adrenocortical cells. Expression of a dominant-negative mutant of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression in angiotensin II-stimulated H295R cells, indicating that Ets-like protein-1 (Elk-1) or related ternary complex factors connect the intracellular signaling cascade elicited by activation of AT1 receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that angiotensin II stimulation increased the transcription activation potential of Elk-1. In addition, activator protein-1 transcriptional activity was significantly elevated in angiotensin II-treated H295R cells. Expression of c-Jun and c-Fos was increased as well as the transcription activation potential of c-Fos. Expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression in angiotensin II-stimulated adrenocortical cells, suggesting that the serum response element within the c-Fos promoter functions as an angiotensin II-response element. Expression of a dominant-negative mutant of c-Jun reduced activator protein-1 activity in angiotensin II-stimulated adrenocortical cells and reduced the up-regulation of c-Jun after angiotensin II stimulation. Thus, c-Jun regulates its own expression in adrenocortical cells. Together, the data show that angiotensin II stimulation activates the transcription factors Egr-1, Elk-1, c-Jun, and c-Fos in adrenocortical cells, leading to stimulus-dependent changes in the gene expression pattern.

Regulation of cellular proliferation, differentiation and cell death by activated Raf

The protein kinases Raf-1, A-Raf and B-Raf connect receptor stimulation with intracellular signaling pathways and function as a central intermediate in many signaling pathways. Gain-of-function experiments shed light on the pleiotropic biological activities of these enzymes. Expression experiments involving constitutively active Raf revealed the essential functions of Raf in controlling proliferation, differentiation and cell death in a cell-type specific manner.