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.

Stimulation of TRPM3 channels increases the transcriptional activation potential of Elk-1 involving cytosolic Ca2+, extracellular signal-regulated protein kinase, and calcineurin.

Stimulation of transient receptor potential M3 (TRPM3) channels with the steroid pregnenolone sulfate increases the transcriptional activation potential of Elk-1, a transcription factor that regulates serum response element-mediated transcription. Here, we show that an influx of Ca2+ ions into the cells is essential for the activation of Elk-1 following stimulation of TRPM3. Using genetically encoded Ca2+ buffers, we show that a rise in cytoplasmic Ca2+ is required for the upregulation of the transcriptional activation potential of Elk-1, while buffering of Ca2+ in the nucleus had no inhibitory effect on the transcriptional activity of Elk-1. Pharmacological and genetic experiments showed that extracellular signal-regulated protein kinase (ERK1/2) functions as signal transducer connecting TRPM3 channels with the Elk-1 transcription factor. Accordingly, dephosphorylation of ERK1/2 in the nucleus by MAP kinase phosphatase attenuated TRPM3-mediated Elk-1 activation. Moreover, we show that the Ca2+/calmodulin-dependent protein phosphatase calcineurin is part of a shut-off-device for the signaling cascade connecting TRPM3 channels with the activation of Elk-1. The fact that TRPM3 channel stimulation activates Elk-1 connects TRPM3 with the biological functions of Elk-1, including the regulation of proliferation, differentiation, survival, transcription, and cell migration.

Pharmacological characterization of the orexin/hypocretin receptor agonist Nag 26.

One promising series of small-molecule orexin receptor agonists has been described, but the molecular pharmacological properties, i.e. ability and potency to activate the different orexin receptor-regulated signal pathways have not been reported for any of these ligands. We have thus here assessed these properties for the most potent ligand of the series, 4'-methoxy-N,N-dimethyl-3'-[N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl sulfamoyl]-(1,1'-biphenyl)-3-carboxamide (Nag 26). Chinese hamster ovary-K1 cells expressing human orexin receptor subtypes OX1 and OX2 were used. Ca2+ elevation and cell viability and death were assessed by fluorescent methods, the extracellular signal-regulated kinase pathway by a luminescent Elk-1 reporter assay, and phospholipase C and adenylyl cyclase activities by radioactive methods. The data suggest that for the Gq-dependent responses, Ca2+, phospholipase C and Elk-1, Nag 26 is a full agonist for both receptors, though of much lower potency. However, saturation was not always reached for OX1, partially due to Nag 26's low solubility and partially because the response decreased at high concentrations. The latter occurs in the same range as some reduction of cell viability, which is independent of orexin receptors. Based on the EC50, Nag 26 was OX2-selective by 20-200 fold in different assays, with some indication of biased agonism (as compared to orexin-A). Nag 26 is a potent orexin receptor agonist with a largely similar pharmacological profile as orexin-A. However, its weaker potency (low-mid micromolar) and low water solubility as well as the non-specific effect in the mid-micromolar range may limit its usefulness under physiological conditions.

Thyroid transcription factor FOXE1 interacts with ETS factor ELK1 to co-regulate TERT.

BACKGROUND: Although FOXE1 was initially recognized for its role in thyroid organogenesis, more recently a strong association has been identified between the FOXE1 locus and thyroid cancer. The role of FOXE1 in adult thyroid, and in particular regarding cancer risk, has not been well established. We hypothesised that discovering key FOXE1 transcriptional partners would in turn identify regulatory pathways relevant to its role in oncogenesis. RESULTS: In a transcription factor-binding array, ELK1 was identified to bind FOXE1. We confirmed this physical association in heterologously transfected cells by IP and mammalian two-hybrid assays. In thyroid tissue, endogenous FOXE1 was shown to bind ELK1, and using ChIP assays these factors bound thyroid-relevant gene promoters TPO and TERT in close proximity to each other. Using a combination of electromobility shift assays, TERT promoter assays and siRNA-silencing, we found that FOXE1 positively regulated TERT expression in a manner dependent upon its association with ELK1. Treating heterologously transfected thyroid cells with MEK inhibitor U0126 inhibited FOXE1-ELK1 interaction, and reduced TERT and TPO promoter activity. METHODOLOGY: We investigated FOXE1 interactions within in vitro thyroid cell models and human thyroid tissue using a combination of immunoprecipitation (IP), chromatin IP (ChIP) and gene reporter assays. CONCLUSIONS: FOXE1 interacts with ELK1 on thyroid relevant gene promoters, establishing a new regulatory pathway for its role in adult thyroid function. Co-regulation of TERT suggests a mechanism by which allelic variants in/near FOXE1 are associated with thyroid cancer risk.

Paradoxical activation of MEK/ERK signaling induced by B-Raf inhibition enhances DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells.

B-Raf inhibitors have been used for the treatment of some B-Raf-mutated cancers. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but paradoxically activates MEK/ERK in Ras-mutated cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. This study focused on determining the effects of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-exposure of Ras-mutated cancer cells to PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively, our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells and hence may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition.

Serum Levels of IL-1 ß , IL-6, TGF- ß , and MMP-9 in Patients Undergoing Carotid Artery Stenting and Regulation of MMP-9 in a New In Vitro Model of THP-1 Cells Activated by Stenting.

Inflammation plays an important role in the pathophysiological process after carotid artery stenting (CAS). Monocyte is a significant source of inflammatory cytokines in vascular remodeling. Telmisartan could reduce inflammation. In our study, we first found that, after CAS, the serum IL-1ß, IL-6, TGF-ß, and MMP-9 levels were significantly increased, but only MMP-9 level was elevated no less than 3 months. Second, we established a new in vitro model, where THP-1 monocytes were treated with the supernatants of human umbilical vein endothelial cells (HUVECs) that were scratched by pipette tips, which mimics monocytes activated by mechanical injury of stenting. The treatment enhanced THP-1 cell adhesion, migration and invasion ability, and the phosphorylation of ERK1/2 and Elk-1 and MMP-9 expression were significantly increased. THP-1 cells pretreated with PD98095 (ERK1/2 inhibitor) attenuated the phosphorylation of ERK1/2 and Elk-1 and upregulation of MMP-9, while pretreatment with telmisartan merely decreased the phosphorylation of Elk-1 and MMP-9 expression. These results suggested that IL-1ß, IL-6, TGF-ß, and MMP-9 participate in the pathophysiological process after CAS. Our new in vitro model mimics monocytes activated by stenting. MMP-9 expression could be regulated through ERK1/2/Elk-1 pathway, and the protective effects of telmisartan after stenting are partly attributed to its MMP-9 inhibition effects via suppression of Elk-1.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors.

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1.

Neurokinin B induces c-fos transcription via protein kinase C and activation of serum response factor and Elk-1 in immortalized GnRH neurons.

Mutations in neurokinin B (NKB) and its receptor, NK3R, were identified in human patients with hypogonadotropic hypogonadism, a disorder characterized by lack of puberty and infertility. Further studies have suggested that NKB acts at the level of the hypothalamus to control GnRH neuron activity, either directly or indirectly. We recently reported that treatment with senktide, a NK3R agonist, induced GnRH secretion and expression of c-fos mRNA in GT1-7 cells. Here, we map the responsive region in the murine c-fos promoter to between -400 and -200 bp, identify the signal transducer and activator of transcription (STAT) (-345) and serum response element (-310) sites as required for induction, a modulatory role for the Ets site (-318), and show that induction is protein kinase C dependent. Using gel shift and Gal4 assays, we further show that phosphorylation of Elk-1 leads to binding to DNA in complex with serum response factor at serum response element and Ets sites within the c-fos promoter. Thus, we determine molecular mechanisms involved in NKB regulation of c-fos induction, which may play a role in modulation of GnRH neuron activation.

Endothelial indoleamine 2,3-dioxygenase protects against development of pulmonary hypertension.

RATIONALE: A proliferative and apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMCs) is key to pathologic vascular remodeling in pulmonary hypertension (PH). Expression of indoleamine-2,3-dioxygenase (IDO) by vascular endothelium is a newly identified vasomotor-regulatory mechanism also involved in molecular signaling cascades governing vascular smooth muscle cell (vSMC) plasticity. OBJECTIVES: To investigate the therapeutic potential of enhanced endothelial IDO in development of PH and its associated vascular remodeling. METHODS: We used loss and gain of function in vivo studies to establish the role and determine the therapeutic effect of endothelial IDO in hypoxia-induced PH in mice and monocrotaline-induced PH in rats. We also studied PASMC phenotype in an IDO-high in vivo and in vitro tissue microenvironment. MEASUREMENTS AND MAIN RESULTS: The endothelium was the primary site for endogenous IDO production within mouse lung, and the mice lacking this gene had exaggerated hypoxia-induced PH. Conversely, augmented pulmonary endothelial IDO expression, through a human IDO-encoding Sleeping Beauty (SB)-based nonviral gene-integrating approach, halted and attenuated the development of PH, right ventricular hypertrophy, and vascular remodeling in both preclinical models of PH. IDO derived from endothelial cells promoted apoptosis in PH-PASMCs through depolarization of mitochondrial transmembrane potential and down-regulated PH-PASMC proliferative/synthetic capacity through enhanced binding of myocardin to CArG box DNA sequences present within the promoters of vSMC differentiation-specific genes. CONCLUSIONS: Enhanced endothelial IDO ameliorates PH and its associated vascular structural remodeling through paracrine phenotypic modulation of PH-PASMCs toward a proapoptotic and less proliferative/synthetic state.

hypoxia-inducible factors activate CD133 promoter through ETS family transcription factors.

CD133 is a cellular surface protein that has been reported to be a cancer stem cell marker, and thus it is considered to be a potential target for cancer treatment. However, the mechanism regulating CD133 expression is not yet understood. In this study, we analyzed the activity of five putative promoters (P1-P5) of CD133 in human embryonic kidney (HEK) 293 cells and colon cancer cell line WiDr, and found that the activity of promoters, particularly of P5, is elevated by overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α). Deletion and mutation analysis identified one of the two E-twenty six (ETS) binding sites (EBSs) in the P5 region as being essential for its promoter activity induced by HIF-1α and HIF-2α. In addition, a chromatin imunoprecipitation assay demonstrated that HIF-1α and HIF-2α bind to the proximal P5 promoter at the EBSs. The immunoprecipitation assay showed that HIF-1α physically interacts with Elk1; however, HIF-2α did not bind to Elk1 or ETS1. Furthermore, knockdown of both HIF-1α and HIF-2α resulted in a reduction of CD133 expression in WiDr. Taken together, our results revealed that HIF-1α and HIF-2α activate CD133 promoter through ETS proteins.

Upregulation of TRPM7 channels by angiotensin II triggers phenotypic switching of vascular smooth muscle cells of ascending aorta.

RATIONALE: Angiotensin II (Ang II) has pleiotropic effects on vascular smooth muscle cells (VSMCs). It has been demonstrated to promote the proliferative phenotype of VSMCs in mouse ascending aorta, but the underlying mechanisms remain incompletely understood. OBJECTIVE: The present study was designed to explore whether the Ca(2+)-permeable transient receptor potential melastatin 7 (TRPM7) channel is involved in Ang II-induced phenotype switching of ascending aortic VSMCs and to dissect the molecular mechanisms by which TRPM7 modulates VSMC phenotype. METHODS AND RESULTS: As revealed by current recording, Ang II infusion increased TRPM7 whole-cell currents in ascending aortic VSMCs. The increase in TRPM7 currents was found to result from enhanced expression of TRPM7 protein rather than elevated single-channel activity (open probability and slope conductance) and/or reduced Mg(2+)-mediated channel block. Mechanistically, Ang II elevated TRPM7 expression via Ang II type 1 receptor-mediated ERK1/2 signaling. As indicated by the expression levels of VSMC differentiation marker genes, phenotypic switching of ascending aorta occurred during Ang II infusion. Meanwhile, ERK1/2-Elk-1 signaling pathway known to suppress VSMC differentiation was activated in Ang II-infused ascending aorta. Knockdown of TRPM7 with small interfering RNA established a causative role of TRPM7 in Ang II-induced phenotypic change and promotion of cell proliferation. Moreover, TRPM7 was shown to be required for Pyk2-ERK1/2-Elk-1 pathway activation by Ang II, which potentiated TRPM7 channel function and thus activated the Ca(2+)-sensitive kinase Pyk2. Finally, TRPM7 knockdown attenuated Ang II-induced displacement of myocardin from SM22 promoter, but the effects could be reversed by expression of constitutively active c-Src. CONCLUSIONS: Our data establish that upregulation of TRPM7 channels by Ang II contributes to the development of the proliferative phenotype of ascending aortic VSMCs, and TRPM7 channel suppresses VSMC gene expression via Ca(2+) influx-mediated activation of Pyk2-ERK1/2-Elk-1 pathway.

Regulation of cancer cell proliferation by caveolin-2 down-regulation and re-expression.

We investigated whether altering caveolin-2 (cav-2) expression affects the proliferation of cancer cells. Cav-2 was not detected in HepG2, SH-SY5Y and LN-CaP cells, and the loss of cav-2 expression was not restored by 5-aza-2'-deoxycytidine treatment. In contrast, C6, HeLa, A549, MCF7 and PC3M cells expressed cav-2. Effects of re-expression of exogenous cav-2 in HepG2, SH-SY5Y and LN-CaP cells, and siRNA-mediated down-regulation of endogenous cav-2 in C6, HeLa, A549, MCF7 and PC3M cells on cancer proliferation were examined by MTT assay, colony formation assay and flow cytometric analysis. Cav-2 transfection in HepG2 hepatocellular carcinoma cells and knockdown in C6 glioma cells caused reduction in cell proliferation and growth with retarded entry into the S phase. Cav-2 re-expression in SH-SY5Y neuroblastoma cells and depletion in HeLa epithelial cervical cancer and A549 lung adenocarcinoma cells promoted cancer cell proliferation. Luciferase reporter assay showed that transcriptional activation of Elk-1 and STAT3 was significantly decreased in cav-2-transfected HepG2 hepatocellular carcinoma and down-regulated C6 glioma cells. Our data suggest that cav-2 acts as a modulator of cancer progression.

ETS-domain transcription factor Elk-1 mediates neuronal survival: SMN as a potential target.

Elk-1 belongs to the ternary complex factors (TCFs) subfamily of the ETS domain proteins, and plays a critical role in the expression of immediate-early genes (IEGs) upon mitogen stimulation and activation of the mitogen-activated protein kinase (MAPK) cascade. The association of TCFs with serum response elements (SREs) on IEG promoters has been widely studied and a role for Elk-1 in promoting cell cycle entry has been determined. However, the presence of the ETS domain transcription factor Elk-1 in axons and dendrites of post-mitotic adult brain neurons has implications for an alternative function for Elk-1 in neurons other than controlling proliferation. In this study, possible alternative roles for Elk-1 in neurons were investigated, and it was demonstrated that blocking TCF-mediated transactivation in neuronal cells leads to apoptosis through a caspase-dependent mechanism. Indeed RNAi-mediated depletion of endogenous Elk-1 results in increased caspase activity. Conversely, overexpression of either Elk-1 or Elk-VP16 fusion proteins was shown to rescue PC12 cells from chemically-induced apoptosis, and that higher levels of endogenous Elk-1 correlated with longer survival of DRGs in culture. It was shown that Elk-1 regulated the Mcl-1 gene expression required for survival, and that RNAi-mediated degradation of endogenous Elk-1 resulted in elimination of the mcl-1 message. We have further identified the survival-of-motor neuron-1 (SMN1) gene as a novel target of Elk-1, and show that the ets motifs in the SMN1 promoter are involved in this regulation.

Epidermal growth factor regulates PAI-1 expression via activation of the transcription factor Elk-1.

PAI-1 (plasminogen activator inhibitor-1) in breast cancer cells is involved in tumour development and metastasis of breast cancer cells. PAI-1 function and the regulation of its expression have been precisely investigated. Here we report that EGF, which promotes breast cancer tumour growth and survival, rapidly induces PAI-1 expression in the breast adenocarcinoma cell line MCF-7 through the activation of the transcription factor Elk-1. We have found that the PAI-1 promoter fragment (-140 to +173) containing the Ets consensus binding site is activated by Elk-1. Chromatin immunoprecipitation analysis confirms in vivo binding of Elk-1 to the PAI-1 promoter and demonstrates that Elk-1 phosphorylation on the Ets binding site is EGF-dependent.

ERK signaling leads to mitochondrial dysfunction in extracellular zinc-induced neurotoxicity.

A zinc-induced signaling pathway leading to extracellular signal-regulated kinase 1/2 (ERK1/2) activation and subsequent neuronal death has been investigated. We find that an extracellular zinc application stimulates biphasic phosphorylation of ERK1/2 and p38 MAPK in rat cultured neurons. The activation of ERK1/2, but not p38, is responsible for zinc neurotoxicity as only U0126, a MEK inhibitor that blocks ERK1/2 phosphorylation, significantly protects cortical neurons from zinc exposure. Over-expression of a dominant negative Ras mutant blocks zinc-induced Elk1-dependent gene expression in neurons, indicating the involvement of Ras activation in the zinc pathway leading to ERK phosphorylation and Elk1 signaling. We also find that zinc treatment results in neuronal mitochondrial hyperpolarization. Importantly, both U0126 and bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, effectively reduce zinc-triggered mitochondrial changes. As bongkrekic acid also prevents zinc-triggered neuronal death but not ERK1/2 phosphorylation, activation of MAPK signaling precedes and is required for mitochondrial dysfunction and cell death. These results provide new insight on the mechanism of extracellular zinc-induced toxicity in which the regulation of mitochondrial function by the Ras/MEK/ERK pathway is closely associated with neuronal viability.

Downstream of FGF during mesoderm formation in Xenopus: the roles of Elk-1 and Egr-1.

Signalling by members of the FGF family is required for induction and maintenance of the mesoderm during amphibian development. One of the downstream effectors of FGF is the SRF-interacting Ets family member Elk-1, which, after phosphorylation by MAP kinase, activates the expression of immediate-early genes. Here, we show that Xenopus Elk-1 is phosphorylated in response to FGF signalling in a dynamic pattern throughout the embryo. Loss of XElk-1 function causes reduced expression of Xbra at neurula stages, followed by a failure to form notochord and muscle and then the partial loss of trunk structures. One of the genes regulated by XElk-1 is XEgr-1, which encodes a zinc finger transcription factor: we show that phosphorylated XElk-1 forms a complex with XSRF that binds to the XEgr-1 promoter. Superficially, Xenopus tropicalis embryos with reduced levels of XEgr-1 resemble those lacking XElk-1, but to our surprise, levels of Xbra are elevated at late gastrula stages in such embryos, and over-expression of XEgr-1 causes the down-regulation of Xbra both in whole embryos and in animal pole regions treated with activin or FGF. In contrast, the myogenic regulatory factor XMyoD is activated by XEgr-1 in a direct manner. We discuss these counterintuitive results in terms of the genetic regulatory network to which XEgr-1 contributes.

Regulation of ERK1/2 phosphorylation by acute and chronic morphine - implications for the role of cAMP-responsive element binding factor (CREB)-dependent and Ets-like protein-1 (Elk-1)-dependent transcription; small interfering RNA-based strategy.

Extracellular signal-regulated kinases (ERKs) have been shown to be activated by opioids and functionally linked to addiction. Morphine-associated changes in ERK activity seem to be the characteristic features of opioid action. In this study, we observed a rapid and severe increase in ERK1/2 activity after a 5 min morphine treatment of HEK-MOR cells (transfected with the rat mu-opioid receptor MOR1) expressing mu-opioid receptor. Cellular adaptations to chronic (72 h) morphine treatment were manifested by a slight and sustained increase in ERK1/2 activity. Withdrawal caused by an opioid receptor antagonist - naloxone - attenuated phosphorylation of ERK1/2. Little information is available on the precise mechanism of ERK activity regulation. Using RNA interference technology, we generated stably transfected cells with silenced expression of cAMP-responsive element binding factor (CREB) and Ets-like protein-1 (Elk-1) transcription factors, which are known targets for activated ERK1/2. In these cells, ERK1/2 activity regulation was altered. Silencing of CREB or Elk-1 significantly increased ERK activation observed after 5 min of morphine stimulation. The initial level of activated ERKs in these cells was also augmented. Moreover, the cellular response to withdrawal signals and chronic opioid treatment was diminished. These differences suggest that both CREB-dependent and Elk-1-dependent transcription contribute to the expression of proteins regulating morphine-induced ERK activity (particular phosphatases, upstream kinases or their activatory proteins).

Regulation of MDMX expression by mitogenic signaling.

MDMX is an important regulator of p53 transcriptional activity and stress response. MDMX overexpression and gene amplification are implicated in p53 inactivation and tumor development. Unlike MDM2, MDMX is not inducible by p53, and little is known about its regulation at the transcriptional level. We found that MDMX levels in tumor cell lines closely correlate with promoter activity and mRNA level. Activated K-Ras and insulin-like growth factor 1 induce MDMX expression at the transcriptional level through mechanisms that involve the mitogen-activated protein kinase and c-Ets-1 transcription factors. Pharmacological inhibition of MEK results in down-regulation of MDMX in tumor cell lines. MDMX overexpression was detected in approximately 50% of human colon tumors and showed strong correlation with increased extracellular signal-regulated kinase phosphorylation. Therefore, MDMX expression is regulated by mitogenic signaling pathways. This mechanism may protect normal proliferating cells from p53 but also hamper p53 response during tumor development.

Transcriptional regulation of inflammatory and extracellular matrix-regulating genes in cerebral arteries following experimental subarachnoid hemorrhage in rats. Laboratory investigation.

OBJECT: Subarachnoid hemorrhage (SAH) results in the expression of inflammatory and extracellular matrix (ECM)-related genes and various G protein-coupled receptors. In the present study, the authors evaluated the time course and sequence of the transduction pathways, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase-1 and 2 (ERK1/2), and associated transcription factor activation as well as gene regulation and associated protein levels. METHODS: Subarachnoid hemorrhage was induced in rats by injecting 250 microl of blood into the suprachiasmatic cistern, and gene regulation in the cerebral arteries was examined at various points in time following SAH by using quantitative polymerase chain reaction (PCR) and immunohistochemistry. RESULTS: Immunohistochemical findings demonstrated that SAH phosphorylates and activates p38 and ERK1/2 as well as the downstream transcription factors Elk-1 and activating transcription factor-2. The pattern of activation consists of a rapid phase within the first few hours and a late phase that occurs from 24 to 48 hours. Activation is followed by an increase in the transcription of the inflammatory and ECM-related genes (IL6, TNFalpha, IL1beta, CXCL1, CXCL2, CCL20, MMP8, MMP9, MMP13, and iNOS), as demonstrated using real-time PCR. For MMP13 and iNOS, the changes in transcription were translated into functional proteins, as revealed on immunohistochemistry. CONCLUSIONS: Activation of the p38 and ERK1/2 signaling pathways and their downstream transcription factors can explain the increase in the transcription of the genes studied. This increase and the subsequent augmentation in protein levels suggest that the inflammatory response may in part explain the remodeling that occurs in cerebral arteries following SAH.

Amitriptyline induces early growth response-1 gene expression via ERK and JNK mitogen-activated protein kinase pathways in rat C6 glial cells.

Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.