The execution of a specific pattern of gene expression requires communication between extracellular signals and the nucleus. The objective of this study was to analyze the signaling cascade leading to the expression of the transcription factor Egr-1 in 39M1-81 cells following stimulation with thrombin. 39M1-81 cells are derived from CCL39 cells that have been frequently used as a model to study thrombin induced signal transduction. The biosynthesis of the zinc finger transcription factor Egr-1 is induced by various growth factors, hormones, and neurotransmitters [15
], indicating that the Egr-1 gene is a convergence point for many intracellular signaling cascades. In keratinocytes, thrombin stimulation leads to an upregulation of Egr-1 expression [4
]. Here, we show that Egr-1 expression is strikingly induced in 39M1-81 cells that were stimulated with thrombin. We also demonstrate that the newly synthesized Egr-1 protein was biologically active, using a lentivirus-based technique to implant an Egr-1 responsive reporter gene into the chromatin of 39M1-81 cells.
Recently, we showed that stimulation of 293 cells expressing P2X7
receptors with the ligand BzATP induced a transient expression of Egr-1 [24
receptors are ionotropic receptors that are permeable for small cations following stimulation. In particular, stimulation of P2X7
receptors leads to an influx of extracellular Ca2+
ions into the cytosol [24
]. Experiments using the acetoxymethylester of the cytosolic Ca2+
chelator BAPTA revealed that the rise of cytosolic Ca2+
concentration is essential for induction of Egr-1 biosynthesis following P2X7
receptor stimulation. Likewise, an increase of [Ca2+
is required in carbachol-stimulated neuroblastoma cells and gonadotrophs that express M3
muscarinic acetylcholine receptors to induce the biosynthesis of Egr-1 [22
]. In 39M1-81 cells, thrombin stimulation increased the free cytosolic Ca2+
] and we have shown here that an increase of the intracellular Ca2+
-concentration is essential for the upregulation of Egr-1 gene transcription in thrombin stimulated 39M1-81 cells. Egr-1 is thus a Ca2+
-ion regulated transcription factor – similar to CREB, NFAT, NF-κB and others.
An elevation of the intracellular Ca2+
concentration may trigger an activation of ERK, either via activation of PKC or via the transactivation of the EGF receptor. The thrombin-activated receptors PAR-1, PAR-2, and PAR-3 belong to the G-protein coupled receptor superfamily [6
]. Stimulation of G-protein coupled receptors can induce a transactivation of the EGF receptor [31
]. In human glioma cells, for example, we have shown that stimulation of the G-protein coupled neurokinin receptor-1 by substance P induces the biosynthesis of Egr-1 via the transactivation of the EGF receptor [33
]. Likewise, we have shown that P2X7
receptor stimulation induces an activation of the EGF receptor tyrosine kinase and impairment of this kinase activity interrupts the connection between receptor stimulation and enhanced Egr-1 biosynthesis [24
]. The results presented here show that transactivation of the EGF receptor is necessary in 39M1-81 fibroblasts to connect M1
muscarinic acetylcholine receptor activation with transcription of the Egr-1 gene. In contrast, thrombin-induced signaling in 39M1-81 fibroblasts and 1321N1 astrocytoma cells is independent of EGF receptor transactivation and rather requires the activation of PKC to connect the elevation of the intracellular Ca2+
concentration with an activation of the ERK signaling pathway. These results are in contrast to previously published observation showing that in HaCaT keratinocytes, transactivation of the EGF receptor is an essential event in the thrombin induced signaling cascade [4
]. These results are in line with previous observations showing that EGF receptor transactivation is not necessary for thrombin-induced phosphorylation of ERK in astrocytes [34
]. Thus, cell type-specific variations in the thrombin-elicited signaling cascade are obvious. We further showed that the activation of ERK is of major importance for the signaling cascade that connects thrombin and carbachol stimulation with enhanced Egr-1 gene transcription. These data were corroborated with a cell line where Egr-1 expression was induced following activation of ΔRaf-1:ER, a Raf-1/estrogen receptor fusion protein that specifically activated the ERK signaling pathway.
A major nuclear substrate for ERK is Elk-1, a member of the Ets family of transcription factors. Elk-1 is an essential component of the serum response ternary complex that binds to DNA and to the serum response factor SRF. The transcriptional activity of Elk-1 depends on its phosphorylation-status. Elk-1 is phosphorylated by several protein kinases including ERK, leading to enhanced DNA binding, ternary complex formation and SRE-mediated transcription [35
]. Phosphorylation of Elk-1 connects the ERK signaling cascade with SRE-mediated gene transcription. The human Egr-1 promoter contains five SREs encompassing the consensus sequence CC [A/T]6
GG, known as the CArG box. The SREs occur in two clusters in the Egr-1 promoter, a distal 5' cluster of 3 SREs and a proximal 3' cluster of 2 SREs. In addition, multiple binding sites for Elk-1 and other ternary complex factors are adjacent to the CArG boxes having the Ets consensus core sequence GGAA/T. Transcriptional activation of Egr-1 is often preceded by an activation of Elk-1, indicating that the SREs within the Egr-1 promoter mediate signal-induced activation of Egr-1 gene transcription. For example, in stimulated glutamatergic corticostriatal neurons a strict spatiotemporal connection between Elk-1 activation and Egr-1 mRNA synthesis has been demonstrated [36
]. The analysis of Egr-1 promoter/luciferase reporter genes containing two or five SREs revealed that both SRE clusters connect thrombin stimulation with enhanced Egr-1 gene transcription. These data are in line with an earlier observation that both the distal as well as the proximal SRE clusters couple enhanced ERK activity with transcriptional upregulation [37
]. Similar results were obtained in 4OHT-treated 39M1-81-ΔRaf-1:ER cells that has been infected with lentiviruses expressing the indicated reporter genes. In contrast, in primary human endothelial cells, only the proximal serum response elements are required to increase Egr-1 promoter/luciferase reporter gene transcription after thrombin stimulation [14
]. Thus, our data are clearly different from those reported by these authors. The differences may rely on the analysis of different cell types (endothelial cells or fibroblasts). Alternatively, instead of using transient transfection of reporter plasmids, we used a lentivirus-based technique to implant reporter genes into the chromatin of 39M1-81 cells. The transient transfection of promoter/reporter gene containing plasmids has the disadvantage that the structure of these plasmids may be incompletely organized in comparison to cellular chromatin, and may thus resemble a prokaryotic gene organisation including a nonrestrictive transcriptional ground state. In contrast, the chromatin structure in eukaryotes causes a restrictive ground state, occluding proteins such as RNA polymerases and transcriptional regulators from binding to DNA. Hence, promoter/reporter genes should be integrated into the chromatin to investigate transcriptional regulatory mechanisms. This strategy enabled us to analyse gene transcription of reporter genes that are packed into an ordered chromatin structure.
We have shown that stimulation of 39M1-81 cells with thrombin induces the phosphorylation of Elk-1 at serine residue 383. We then went a step further and performed a loss-of-function experiment to unequivocally prove the key role of ternary complex factor activation for thrombin-induced upregulation of Egr-1 expression. Genetic inactivation of Elk-1 or other ternary complex factors in transgenic mice revealed minimal changes of the phenotype [38
], suggesting that functional redundancy may exist. Therefore, we have assessed the necessity of ternary complex factor activation for thrombin or carbachol-induced Egr-1 biosynthesis by using a dominant negative version of Elk-1 in loss-of-function experiments. Due to its binding to DNA and SRF, the Elk-1 mutant REST/Elk-1ΔC most likely also inhibits the activity of two other ternary complex factors, SAP-1 and SAP-2. These experiments revealed that expression of REST/Elk-1ΔC almost completely blocked the stimulus-induced biosynthesis of Egr-1. Thus, ternary complex factor activation is a key step in connecting thrombin or carbachol stimulation with enhanced Egr-1 biosynthesis.
Using chromatin-integrated reporter genes, controlled by the proximal SREs from the Egr-1 promoter, we directly showed that expression of the dominant-negative mutant of Elk-1 impaired reporter gene transcription after stimulation of the cells with either thrombin or carbachol. As a control, we have analyzed the regulation of the 9E3/cCAF gene. Two Elk-1 binding sites were identified between -534 and -483 of the 9E3/cCAF promotor that functions as thrombin response elements [28
]. Our data show that stimulation of 39M1-81 fibroblasts with either thrombin or carbachol induced transcription of a chromatin-embedded 9E3/cCAF promotor/luciferase reporter gene. This stimulus-induced upregulation of reporter gene expression was impaired in cells that expressed the dominant-negative Elk-1 mutant REST/Elk-1ΔC. These data confirm previous studies showing that Elk-1 is essential for the thrombin-induced expression of the 9E3/cCAF gene [28
]. While the Egr-1 promoter contains five SREs and multiple TCF binding sites, the 9E3/cCAF promoter does not contain a SRE. Thus, the ability of Elk-1 to stimulate transcription does not require the presence of SREs. The experiments presented here show that the dominant-negative mutant REST/Elk-1ΔC blocks Elk-1-regulated gene transcription regardless of the presence or absence of SREs in the regulatory region of Elk-1-controlled genes. Finally, we showed that an impairment of Elk-1 phosphorylation by overexpression of MKP-1 also prevented Egr-1 expression in thrombin or carbachol-stimulated 39M1-81 cells. Together, these data indicate that phosphorylation and activation of Elk-1 is required to connect the ERK signaling cascade with transcription of the Egr-1 gene in thrombin or carbachol-stimulated 39M1-81 cells.
39M1-81 cells express both protease-activated receptors and M1 muscarinic acetylcholine receptors. The data presented here demonstrate that the signaling cascades induced by either thrombin or carbachol stimulation of 39M1-81 cells are indistinguishable, except that activation of M1 muscarinic acetylcholine receptors induces the transactivation of the EGF receptor. Analyzing two different cell lines, 39M1-81 fibroblasts and 1321N1 glioma cells, we show that the thrombin induced signaling cascade is independent of EGF receptor transactivation and rather relies on the activation of PKC to connect the increase of the intracellular Ca2+-concentration with the ERK signaling cascade.