ERK Mitogen-activated Protein Research Articles

MAPKs-NF-kappaB Pathway Plays a Crucial Role in the Antiinflammatory Effects of Amentoflavone in Lipopolysaccharide-treated BV2 Microglia

Amentoflavone also known as didemethyl-ginkgetin, 3’,8”-biapigenin, is a plant bioflavonoid found in serval plants, with a number of pharmacological effects including antiinflammatory, antioxidant, antiviral, neuroprotective, and anticancer. The present study revealed that secretion of prostaglandin E2 and nitric oxide were inhibited by amentoflavone in a concentration-dependent manner in the lipopolysaccharide/interferon gamma-stimulated BV2 microglial cells. Meanwhile, protein expression of inducible nitric oxide synthase and cyclooxygenase-2 induced by LPS/INF-gamma were inhibited by amentoflavone in the same concentration range. Moreover, amentoflavone not only reduced the phosphorylation of nuclear factor-kappaB but also inhibited the phosphorylation of mitogen-activated protein kinases, including ERK, JNK, and p38 mitogen-activated protein kinases induced by lipopolysaccharide. In addition, a parallel concentration-dependent manner was observed in the inhibition of secretion of prostaglandin E2 and NO, expression of inducible nitric oxide synthase and cyclooxygenase-2, and phosphorylation of mitogen-activated protein kinases and nuclear factor-kappaB pathway. These results suggested that amentoflavone possessed the potential to act against lipopolysaccharide/interferon gamma-induced secretion of prostaglandin E2 and NO via downregulation of inducible nitric oxide synthase and cyclooxygenase-2 expressions by blocking the activation of nuclear factor-kappaB pathway via phosphorylation of mitogen-activated protein kinases.

Response of Mammalian Macrophages to Challenge with the Chlorovirus Acanthocystisturfacea Chlorella Virus 1

It was recently reported that 44% of the oropharyngeal samples from the healthy humans in a study cohort had DNA sequences similar to that of the chlorovirus ATCV-1 (Acanthocystis turfacea chlorella virus 1, family Phycodnaviridae) and that these study subjects had decreases in visual processing and visual motor speed compared with individuals in whom no virus was detected. Moreover, mice inoculated orally with ATCV-1 developed immune responses to ATCV-1 proteins and had decreases in certain cognitive domains. Because heightened interleukin-6 (IL-6), nitric oxide (NO), and ERK mitogen-activated protein (MAP) kinase activation from macrophages are linked to cognitive impairments, we evaluated cellular responses and viral PFU counts in murine RAW264.7 cells and primary macrophages after exposure to ATCV-1 in vitro for up to 72 h after a virus challenge. Approximately 8% of the ATCV-1 inoculum was associated with macrophages after 1 h, and the percentage increased 2- to 3-fold over 72 h. Immunoblot assays with rabbit anti-ATCV-1 antibody detected a 55-kDa protein consistent with the viral capsid protein from 1 to 72 h and increasing de novo synthesis of a previously unidentified 17-kDa protein beginning at 24 h. Emergence of the 17-kDa protein did not occur and persistence of the 55-kDa protein declined over time when cells were exposed to heat-inactivated ATCV-1. Moreover, starting at 24 h, RAW264.7 cells exhibited cytopathic effects, annexin V staining, and cleaved caspase 3. Activation of ERK MAP kinases occurred in these cells by 30 min postchallenge, which preceded the expression of IL-6 and NO. Therefore, ATCV-1 persistence in and induction of inflammatory factors by these macrophages may contribute to declines in the cognitive abilities of mice and humans. Virus infections that persist in and stimulate inflammatory factors in macrophages contribute to pathologies in humans. A previous study showed that DNA sequences homologous to the chlorovirus ATCV-1 were found in a significant fraction of oropharyngeal samples from a healthy human cohort. We show here that ATCV-1, whose only known host is a eukaryotic green alga (Chlorella heliozoae) that is an endosymbiont of the heliozoon Acanthocystis turfacea, can unexpectedly persist within murine macrophages and trigger inflammatory responses including factors that contribute to immunopathologies. The inflammatory factors that are produced in response to ATCV-1 include IL-6 and NO, whose induction is preceded by the activation of ERK MAP kinases. Other responses of ATCV-1-challenged macrophages include an apoptotic cytopathic effect, an innate antiviral response, and a metabolic shift toward aerobic glycolysis. Therefore, mammalian encounters with chloroviruses may contribute to chronic inflammatory responses from macrophages.

PG201 downregulates the production of nitrite by upregulating heme oxygenase-1 expression through the control of phosphatidylinositol 3-kinase and NF-E2-related factor 2

PG201 is an ethanol extract prepared from a specially designed botanical formulation and has previously been shown to contain strong anti-arthritic activities by controlling inflammation and cartilage destruction in two animal models [1,2]. In the present study, we evaluated the effects of PG201 on the expression of heme oxygenase-1 (HO-1). The treatment of Raw264.7 cells (a murine macrophage cell line) and bone marrow-derived macrophages (BMDMs) with PG201 increased the protein and RNA levels of HO-1. The results from a reporter plasmid assay indicated that PG201 induced HO-1 promoter activity through the stress response element present in the two enhancers of the HO-1 promoter. The treatment of cells with PG201 increased the total amount and the nuclear level of NF-E2-related factor 2 (Nrf2). Protein analysis using BMDMs from Nrf2 knockout mice showed that Nrf2 was necessary for the PG201-mediated induction of HO-1 expression. The PG201-mediated induction of these anti-oxidative stress factors was inhibited by a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), but not by inhibitors of p38, ERK and JNK mitogen-activated protein kinases. Furthermore, the results from an experiment involving a specific siRNA and chemical inhibitors for HO-1 showed that the PG201-mediated increase of the HO-1 protein contributed to the suppression of inducible nitric oxide synthase (iNOS) and nitrite production stimulated by lipopolysaccharide. Taken together, these results suggest that PG201 activates Nrf2 through the PI3K signal transduction pathway, increases the expression of HO-1, and subsequently decreases the production of iNOS and nitrite, eventually exerting anti-inflammatory activities.

Rit-mediated Stress Resistance Involves a p38-Mitogen- and Stress-activated Protein Kinase 1 (MSK1)-dependent cAMP Response Element-binding Protein (CREB) Activation Cascade

The cAMP response element (CRE)-binding protein (CREB) is a key regulatory factor of gene transcription, and plays an essential role in development of the central nervous system and for neuroprotection. Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both ERK and p38 mitogen-activated protein (MAP) kinases cascades. Recent studies have identified the Ras-related small G-protein, Rit, as a central regulator of a p38-MK2-HSP27 signaling cascade that functions as a critical survival mechanism for cells adapting to stress. Here, we examine the contribution of Rit-p38 signaling to the control of stress-dependent gene transcription. Using a pheochromocytoma cell model, we find that a novel Rit-p38-MSK1/2 pathway plays a critical role in stress-mediated CREB activation. RNAi-mediated Rit silencing, or inhibition of p38 or MSK1/2 kinases, was found to disrupt stress-mediated CREB-dependent transcription, resulting in increased cell death. Furthermore, ectopic expression of active Rit stimulates CREB-Ser133 phosphorylation, induces expression of the anti-apoptotic Bcl-2 and Bcl(XL) proteins, and promotes cell survival. These data indicate that the Rit-p38-MSK1/2 signaling pathway may have an important role in the stress-dependent regulation of CREB-dependent gene expression.

Abstract 3248: New inhibitors of the Shp2 phosphatase

Abstract The Shp2 protein tyrosine phosphatase (PTP) mediates signal transduction of growth factor receptors and regulates cellular activities critical to tumor growth and metastasis. In the basal state, the interactions between the N-SH2 and PTP domains keep the phosphatase in an auto inhibited closed conformation. Upon growth factor or cytokine stimulation, the SH2 domains of Shp2 binds to tyrosine phosphorylated docking proteins such as Gab1 and Gab2, which activates Src, Ras, and the Erk1/2 (Erk) mitogen-activated protein (MAP) kinase pathway. Shp2 gain-of-function mutations are found in leukemias and solid tumors and are linked to Noonan syndrome. Although Shp2 is involved in pathogenesis of human cancers, it is not clear how Shp2 mediates tumorigenesis. Shp2 is therefore believed to be an important enzyme for targeted cancer therapy. Since no potent and selective Shp2 PTP inhibitor is currently available for chemical biology studies and experimental therapy, we and others have been actively engaged in the search for such agents. In our continuing efforts to develop potent and selective Shp2 inhibitors to serve as both chemical probes to further our understanding of the role and signaling mechanisms of Shp2 in human diseases and as potential drugs for cancer therapy. We will report the screening of a focused library of a set of commercially available salicylic acid derivatives using the in vitro DIFMUP phosphatase assay for their ability to inhibit the Shp2 phosphatase domain. Activity of hits was confirmed using the Biomol Green assay to determine inhibition of dephosphorylation of a Shp2 peptide substrate derived from EphR. One such hit was a thiazole containing salicylic acid that inhibits Shp2 with an IC50 (DIFMUP) of 100 µM. We will report the design and synthesis of salicylic acid libraries along with their benzoic acid counterparts. The most potent compound to date is HM2-08-4 which has an IC50 (DIFMUP) 1.1 µM. Emerging structure-activity relationships as well as modeling and X-ray studies will be discussed. The preliminary use of selected compounds as tools to probe the pharmacological inhibition of Shp2 and elucidate its role in cancer will be described. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3248. doi:10.1158/1538-7445.AM2011-3248

The Third Conformation of p38α MAP Kinase Observed in Phosphorylated p38α and in Solution

MAPKs engage substrates, MAP2Ks, and phosphatases via a docking groove in the C-terminal domain of the kinase. Prior crystallographic studies on the unphosphorylated MAPKs p38α and ERK2 defined the docking groove and revealed long-range conformational changes affecting the activation loop and active site of the kinase induced by peptide. Solution NMR data presented here for unphosphorylated p38α with a MEK3b-derived peptide (p38α/pepMEK3b) validate these findings. Crystallograhic data from doubly phosphorylated active p38α (p38α/T∗GY∗/pepMEK3b) reveal a structure similar to unphosphorylated p38α/MEK3b, and distinct from phosphorylated p38γ (p38γ/T∗GY∗) and ERK2 (ERK2/T∗EY∗). The structure supports the idea that MAP kinases adopt three distinct conformations: unphosphorylated, phosphorylated, and a docking peptide-induced form.

Interleukin-1 Drives Cerebrovascular Inflammation via MAP Kinase-Independent Pathways

Cerebrovascular inflammation is triggered by diverse central nervous system (CNS) insults and contributes to disease pathogenesis. The pro-inflammatory cytokine interleukin (IL)-1 is central to this cerebrovascular inflammatory response and understanding the underlying signalling mechanisms of IL-1 actions in brain endothelium may provide therapeutic targets for disease intervention. For the first time, we compare the contributions of p38, JNK and ERK mitogen-activated protein (MAP) kinase and NF-kB pathways to IL-1-induced brain endothelial activation. In cultures of primary mouse brain endothelium and the rat brain endothelial GPNT cell line, interleukin-1β (IL-1β induced a rapid (within 5 minutes) and transient activation of p38 and JNK (but not ERK) MAP kinases. IL-1β also induced nuclear recruitment of nuclear factor (NF)-kB p65. IL-1β-induced brain endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was insensitive to MAP kinase inhibitors. IL-1β-induced brain endothelial expression of ICAM-1 and VCAM-1 was inhibited (80-88 %) by the proteasome inhibitor MG132 or the antioxidant caffeic acid phenethyl ester (CAPE), effects suggested to be NF-kB-dependent. IL-1β-induced brain endothelial CXCL1 expression was partially inhibited by JNK MAP kinase or MG132 (62 or 56 %, respectively). However, CXCL1 secretion from brain endothelium was reduced (65 %) only by MG132, and not MAP kinase inhibitors. Similarly, IL-1β-induced neutrophil transendothelial migration was reduced (77-89 %) by MG132, but not MAP kinase inhibitors. In summary, we show that several key components of IL-1β-induced brain endothelial activation (CAM, CXCL1 expression or release and neutrophil transmigration) are largely independent of MAP kinase activity but are reduced by proteasome inhibition, possibly reflecting a requirement for NF-kB activity. Similar mechanisms may contribute to cerebrovascular inflammation in response to CNS injury.

Abstract 734: Development of small molecule inhibitors of the phosphatase Shp2 by fragment screening

Abstract Protein tyrosine phosphorylation, which is controlled by tyrosine kinase (PTK) and tyrosine phosphatase (PTP), regulates numerous cellular processes, including proliferation, survival, differentiation, migration and apoptosis. Accumulating evidence has shown that defective function of PTPs leads to aberrant protein phosphorylation, which is associated with many humane diseases, including cancer. Src homology 2 phosphatase-2 (Shp2) is a cytoplasmic, nonreceptor protein tyrosine phophatase. It is the first proto-oncogene identified in the PTP superfamily. The structure of Shp2 consists of two SH2 (N-SH2 and C-SH2) domains, a single PTP region and a C-terminal hydrophilic tail. In the basal state, Shp2 displays a low catalytic activity due to close interactions between the N-SH2 and PTP domains that keep the phosphatase in an autoinhibited closed conformation. Upon growth factor or cytokine stimulation, the SH2 domains of Shp2 binds to tyrosine phosphorylated docking proteins such as Gab1 and Gab2, which activates Src, Ras, and the Erk1/2 (Erk) mitogen-activated protein (MAP) kinase pathway. Shp2 gain-of-function mutations are found in leukemias and solid tumors and are linked to Noonan syndrome. Although Shp2 is involved in pathogenesis of human cancers, it is not clear how Shp2 mediates tumorigenesis. To aid chemical biology studies and ultimately experimental therapy, we have been actively engaged in the search potent and selective Shp2 inhibitors. We will describe the results of a fragment-based screen as a method for identifying Shp2 inhibitors. The fragment libraries were both purchased and constructed in-house with a bias towards compounds that would potentially function as phosphotyrosine mimics. This has revealed compounds with moderate to low activity (IC50 200-500 µM) that have served as the starting pints for fragment-to-lead development. The emerging structure-activity relationships will be discussed as well as synthesis, modeling and X-ray studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 734.

TNF-ALPHA-STIMULATED REGULATION OF NF-κB-DEPENDENT GENE TRANSCRIPTION IS MEDIATED BY P38 AND ERK MAP KINASES IN ISOLATED ACINAR CELLS

A transcription factor that is necessary for the transcription of many proinflammatory cytokine genes is nuclear factor kappaB (NFkB). We have previously shown that NFkB-dependent gene transcription is mediated by both p38 and ERK mitogen-activated protein (MAP) kinases in an exocrine pancreatic cancer cell line (AR42J cells). Here we evaluate the role of MAP kinases in regulating tumor necrosis factor-alpha (TNFa)-stimulated NFkB-dependent transcription in acinar cells isolated from pancreata of healthy mice. We hypothesize that both the p38 and ERK MAP kinases are necessary for NFkB-dependent transcription in acinar cells stimulated with TNFa. Using a promoter construct driven only by NFkB, we infected isolated acinar cells with a replication-deficient adenoviral vector containing either an empty vector, a dominant negative (DN) p38, or a DN ERK expression vector. Stimulation of native TNFa receptors promoted a significant increase in NFkB-dependent gene expression, as measured by luciferase activity, in cells expressing the empty vector. In both unstimulated and TNFa-stimulated cells, over expression of either DN p38 or DN ERK expression vectors significantly abrogated NFkB-dependent luciferase activity. These findings indicate that TNFa-stimulated NFkB-dependent transcription in mouse acinar cells is attenuated by p38 and/or ERK MAP kinase inhibition. These findings support our hypothesis that both the p38 and ERK MAP kinases are involved in the activation of proinflammatory nuclear transcription factors, such as NFkB, in pancreatic exocrine cells. In addition, isolated acinar cells provide an ideal model to study NFkB-dependent gene expression mechanistically prior to performing studies in vivo in an experimental model of pancreatitis.

DNA Vector Augments Inflammation in Epithelial Cells via EGFR-Dependent Regulation of TLR4 and TLR2

Gene delivery applications to treat lung diseases are, in some instances, suboptimal due to deleterious host inflammatory reactions. Current DNA plasmids (pDNA) exert toxicity in part via unmethylated CpG motifs that stimulate Toll-like receptor (TLR)9-expressing leukocytes; however, the airway epithelial response has not been well defined. Bronchial epithelial cells (BEAS-2B) were exposed to pDNA complexes and inflammatory mediators were measured. As patients with inflammatory lung disease are susceptible to infectious exacerbations, we also evaluated the reciprocal inflammatory response to pDNA and bacterial components lipopolysaccharide (LPS) and lipoteichoic acid (LTA), recognized by TLR4 and TLR2, respectively. Cells primed with pDNA synergistically expressed IL-8 mRNA and protein in response to LPS and LTA (3- to 5-fold). A similar induction was also observed for IL-1beta, IL-6, colony-stimulating factor (CSF)-1, and granulocyte macrophage-CSF. Their synergistic elevation was associated with an increase in TLR4 and TLR2 levels. Methylation of pDNA only partially reduced (25-30%) IL-8 release; hence, signaling occurs via CpG/TLR9-dependent and -independent modules. As epidermal growth factor receptor (EGFR) signaling has been implicated in bronchial IL-8 expression, we assessed whether pDNA priming events were coordinated via EGFR. AG1478 (EGFR inhibitor) restored normal TLR4/2 levels and also suppressed synergistic release of IL-8. The extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase inhibitor also blocked IL-8 release, implicating Erk as a key mediator of EGFR signaling. Our findings identify a novel EGFR-dependent mechanism for regulating TLR, and show that targeted disruption of EGFR signaling ameliorates the airway epithelial inflammatory response to pDNA. Targeting the EGFR system may improve the efficiency, tolerability, and safety of gene therapy strategies.

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.

IL-1 and TNF Induction of Matrix Metalloproteinase-3 by c-Jun N-Terminal Kinase in Trabecular Meshwork

The cytokines TNF and IL-1 mediate the MMP-3 increase that occurs in response to trabecular meshwork (TM) treatment by laser trabeculoplasty. This MMP-3 increase appears to play a key role in the efficacy of this treatment for open-angle glaucoma. Protein kinase Cmu and the Erk mitogen-activated protein (MAP) kinases are essential signaling components in transducing MMP-3 increases produced by treatment of TM cells with these cytokines. Here, the involvement of the JNK-MAP kinase pathway in this process was evaluated. Porcine TM cells were treated with TNFalpha, IL-1alpha, or IL-1beta. Changes in MMP-3 and MMP-9 protein levels in the media were then determined by Western immunoblot. The effect of JNK inhibitor 2 was evaluated. Changes in the level of phosphorylation of JNK, c-Jun, ATF-2, MKK4, and MKK7 were also determined at various times after TNFalpha or IL-1alpha treatment. A 2.3-kb MMP-3 promoter fragment was cloned into a secreted alkaline phosphatase reporter vector. This reporter construct was cotransfected into TM cells with a mammalian expression vector containing a dominant-negative mutant of JNK. The involvement of JNK activity in the TNFalpha and IL-1alpha induction of MMP-3 expression was then evaluated. TNFalpha, IL-1alpha, and IL-1beta increase media MMP-3 and MMP-9 protein levels, and JNK inhibitor 2 blocks these increases. JNK1/2, MKK4, c-Jun, and ATF-2 phosphorylation levels increase in response to TNFalpha and IL-1alpha treatment. JNK inhibitor 2 pretreatment blocks these c-Jun and ATF-2 phosphorylation increases. Dominant-negative JNK dramatically reduces the MMP-3 promoter-driven reporter activity induced by these cytokines. JNK activity is necessary for the induction of MMP-3 and MMP-9 by TNFalpha, IL-1alpha, or IL-1beta in TM cells. Phosphorylation of components of the JNK signaling pathway and of the transcription factors c-Jun and ATF-2 support a role for this pathway in the induction of MMP-3 and MMP-9 in the TM in response to these cytokines. Thus, at least three separate signal transduction pathways are necessary in this signaling event in TM cells.

Phosphorylation of Phosphoprotein Enriched in Astrocytes (PEA-15) Regulates Extracellular Signal-regulated Kinase-dependent Transcription and Cell Proliferation

Cell cycle progression is dependent on the nuclear localization and transcriptional effects of activated extracellular signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinases (ERK1/2). Phosphoprotein enriched in astrocytes (PEA-15) binds ERK1/2 and inhibits their nuclear localization, thus blocking cell proliferation. Here, we report that phosphorylation of PEA-15 blocks its interaction with ERK1/2 in vitro and in vivo and that phosphorylation of both Ser104 and Ser116 is required for this effect. Using phosphomimetic and nonphosphorylatable mutants of PEA-15, we found that PEA-15 phosphorylation abrogates its capacity to block the nuclear localization and transcriptional activities of ERK1/2; this phosphorylation therefore enables the proliferation of cells that express high levels of PEA-15. Additionally, we report that PEA-15 phosphorylation can modulate nontranscriptional activities of ERK1/2, such as the modulation of the affinity of integrin adhesion receptors. Finally, we used a novel anti-phospho-specific PEA-15 antibody to establish that PEA-15 is phosphorylated in situ in normal mammary epithelium. These results define a novel posttranslational mechanism for controlling the subcellular localization of ERK1/2 and for specifying the output of MAP kinase signaling.

Specific inactivation and nuclear anchoring of extracellular signal-regulated kinase 2 by the inducible dual-specificity protein phosphatase DUSP5.

The mechanisms which determine the nuclear accumulation and inactivation of the extracellular signal-regulated kinase 1 (ERK1) or ERK2 mitogen-activated protein (MAP) kinases are poorly understood. Here we demonstrate that DUSP5, an inducible nuclear phosphatase, interacts specifically with ERK2 via a kinase interaction motif (KIM) within its amino-terminal noncatalytic domain. This binding determines the substrate specificity of DUSP5 in vivo, as it inactivates ERK2 but not Jun N-terminal protein kinase or p38 MAP kinase. Using green fluorescent protein fusions, we identify within this same domain of DUSP5 a functional nuclear localization signal (NLS) which functions independently of the KIM. Moreover, we demonstrate that the expression of DUSP5 causes both nuclear translocation and sequestration of inactive ERK2. Nuclear anchoring is ERK2 specific and requires both interactions between the DUSP5 KIM and the common docking site of ERK2 and a functional NLS within DUSP5. Finally, the expression of a catalytically inactive mutant of DUSP5 also tethers ERK2 within the nucleus. Furthermore, this nuclear ERK2 is phosphorylated by MAP kinase kinase in response to growth factors and also activates transcription factor Elk-1. We conclude that DUSP5 is an inducible nuclear ERK-specific MAP kinase phosphatase that functions as both an inactivator of and a nuclear anchor for ERK2 in mammalian cells. In addition, our data indicate that the cytoplasm may not be an exclusive site of MAP kinase activation.

Leukemia inhibitory factor-transfected embryonic fibroblasts and vascular endothelial growth factor successfully improve the skin substitute wound healing by increasing angiogenesis and matrix production

Background and objective : The combined application of cytokines on embryonic fibroblasts and dermal substitute were studied for optimal skin defect coverage. The mechanism of combined treatment of leukemia inhibitory factor (LIF)-transfected embryonic fibroblasts and vascular endothelial growth factor (VEGF) were elucidated and subsequently the in vivo applications of both were tested in an artificial dermal substitute. Methods : Mouse embryonic fibroblast cells, BALB-3T3, were stably transfected with mouse full-length LIF cDNA and added to various doses of VEGF for detection of signaling interaction. LIF-transfected cells and VEGF treatment were tested with pig-tendon derived collagen dermal substitute in the backs of BALB/c male mice up to for 14 days. Results : LIF-transfected cells as well as vector-transfected fibroblasts significantly proliferated by 1, 10, or 100 ng VEGF on days 3 and 5. Erk mitogen-activated protein (MAP) kinase phosphorylation was observed from 1 to 30 min in LIF-transfected and 10 ng of VEFG, and 1 to 60 min in LIF-transfected and 100 ng VEFG treatments. The cellular fibronectin levels also increased in LIF-transfected cells with 10 and 100 ng VEGF additions. In in vivo analyses, LIF-transfected embryonic fibroblasts with 50 μg of VEGF markedly enhanced collagen I expression and CD34 angiogenic marker on days 7 and 14. Conclusion : LIF transfection and VEGF treatment enhanced phosphorylated-Erk-MAP kinase in vitro. In vivo study revealed that the combined application of LIF transfection of embryonic fibroblasts with an angiogenic factor such as VEGF in the template of a dermal substitute induced greater skin collagen production and angiogenesis in the dermal substitute.

Upregulation of the Catalytic Telomerase Subunit by the Transcription Factor ER81 and Oncogenic HER2/Neu, Ras, or Raf.

One hallmark of tumor formation is the transcriptional upregulation of human telomerase reverse transcriptase, hTERT, and the resultant induction of telomerase activity. However, little is presently understood about how hTERT is differentially activated in tumor cells versus normal somatic cells. Specifically, it is unclear if oncoproteins can directly elicit hTERT expression. To this end, we now show that three oncoproteins, HER2/Neu, Ras, and Raf, stimulate hTERT promoter activity via the ETS transcription factor ER81 and ERK mitogen-activated protein (MAP) kinases. Mutating ER81 binding sites in the hTERT promoter or suppression of ERK MAP kinase-dependent phosphorylation of ER81 rendered the hTERT promoter unresponsive to HER2/Neu. Further, expression of dominant-negative ER81 or inhibition of HER2/Neu significantly attenuated telomerase activity in HER2/Neu-overexpressing SKBR3 breast cancer cells. Moreover, HER2/Neu, Ras, and Raf collaborated with ER81 to enhance endogenous hTERT gene transcription and telomerase activity in hTERT-negative, nonimmortalized BJ foreskin fibroblasts. Accordingly, hTERT expression was increased in HER2/Neu-positive breast tumors and breast tumor cell lines relative to their HER2/Neu-negative counterparts. Collectively, our data elucidated a mechanism whereby three prominent oncoproteins, HER2/Neu, Ras, and Raf, may facilitate tumor formation by inducing hTERT expression in nonimmortalized cells via the transcription factor ER81.

RSK2 Activity Is Regulated by Its Interaction with PEA-15

The ERK MAP (mitogen-activated protein) kinase cascade modulates many cellular processes including transcription, adhesion, growth, survival, and proliferation. One target substrate of ERK involved in regulating transcription is the p90 ribosomal S6 kinase (RSK) isozyme, RSK2. Here we demonstrate that a small death effector domain-containing protein called PEA-15 binds RSK2. RSK2 and PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) co-precipitated from cells and were colocalized in the cytoplasm. Furthermore, purified PEA-15 bound in vitro translated RSK2, suggesting that these proteins interact directly. PEA-15 does not bind to RSK1 and therefore exhibits some binding specificity. RSK2 binds the COOH terminus of PEA-15 and does not interact with its NH2-terminal death effector domain. We show that this interaction has functional consequences including the inhibition of RSK2-dependent CREB transcription. PEA-15 expression also blocks histone H3 phosphorylation, an RSK2-dependent event that may contribute to effects on gene expression. These results can be attributed to two effects of PEA-15 on RSK2. First, PEA-15 blocks nuclear accumulation of RSK2 after epidermal growth factor stimulation. Second, PEA-15 inhibits RSK2 kinase activity by 50%. A mutant of PEA-15 that binds RSK2 but is localized to the nucleus had no effect on RSK2-dependent transcription. Interestingly, this mutant also did not affect RSK2 kinase activity. This may indicate that cytoplasmic retention of RSK2 is also required for PEA-15 to impair kinase activity. PEA-15 does not alter ERK phosphorylation of RSK2 and is not itself a substrate of RSK2. Hence the effects of PEA-15 on RSK2 represent a novel mechanism for the regulation of RSK2-mediated signaling.

Derivatives of Vitamins D2 and D3 Activate Three MAPK Pathways and Upregulate pRb Expression in Differentiating HL60 Cells

Analogs of vitamin D have been synthesized which have reduced calcemic activities yet increased anti-proliferative and differentiation-inducing properties, raising expectations that they will be useful for treatment of human neoplastic diseases. In the present study we compared the abilities of three such analogs, 24a, 24b-dihomo-1,25-dihydroxyvitamin D3 (PRI-1890), 24-ene-1,25-dihydroxyvitamin D2(PRI-1906) and (24R)-1,24-dihydroxy-vitamin D3 (PRI-2191) to induce markers (CD14, CD11b and MSE) of differentiation, G1 phase block, and associated molecular events in human promyeloblastic leukemia cells HL60. We found that the potencies of the analogs to induce differentiation paralleled their activation of Erk, JNK and p38 mitogen-activated protein kinase (MAPK) pathways, and the anti-proliferative activity closely correlated with the extent of hypophosphorylation of retinoblastoma protein (pRb). Interestingly, low concentrations of derivatives of vitamin D, which were insufficient to induce any detectable changes in the cell cycle traverse, markedly increased the levels of total pRb, which was highly phosphorylated. These results suggest that pRb may have an unsuspected role in monocytic differentiation, perhaps to increase the sensitivity of the G1 checkpoint, by increasing the amount of substrate for cyclin-dependent kinases.

Non-steroidal Anti-inflammatory Drugs Stimulate Secretion of Non-amyloidogenic Precursor Protein

Chronic inflammatory processes are associated with the pathophysiology of Alzheimer's disease (AD), and it has been proposed that treatment with non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk for AD. Here we report that various NSAIDs, such as the cyclooxygenase inhibitors, nimesulide, ibuprofen and indomethacin, as well as thalidomide (Thal) and its non-teratogenic analogue, supidimide, significantly stimulated the secretion of the non-amyloidogenic alpha-secretase form of the soluble amyloid precursor protein (sAPP alpha) into the conditioned media of SH-SY5Y neuroblastoma and PC12 cells. These NSAIDs markedly reduced the levels of the cellular APP holoprotein, further accelerating non-amyloidogenic processes. sAPP alpha release, induced by nimesulide and Thal, was modulated by inhibitors of protein kinase C and Erk mitogen-activated protein (MAP) kinase. Furthermore, in results complementary to the inhibitor studies, we show for the first time that NSAIDs can activate the Erk MAP kinase signaling cascade, thus identifying a novel pharmacology mechanism of NSAIDs. Our findings suggest that NSAIDs and Thal might prove useful to favor non-amyloidogenic APP processing by enhancing alpha-secretase activity, thereby reducing the formation of amyloidogenic derivatives, and therefore are of potential therapeutic value in AD.

Monocyte adhesion and transmigration induce tissue factor expression: role of the mitogen-activated protein kinases.

The expression of tissue factor (TF) by monocytes that have transmigrated across the endothelium to sites of extravascular inflammation acts both to focus and amplify the inflammatory response. Because clustering of the integrins responsible for endothelial adhesion and transmigration induces tyrosine phosphorylation and activation of the mitogen-activated protein (MAP) kinases, we postulated that transmigration might lead to monocyte activation and TF production. Monocytes were migrated across TNFalpha-primed ECV304 cells grown on fibronectin-coated Transwell chambers in response to FMLP (10(-8) M). After transmigration, monocytes showed a time-dependent increase in surface TF expression and biological procoagulant activity. TF expression was dependent on monocyte adhesion to ECV304 cells. Specifically, TF was not induced by FMLP treatment of suspended monocytes, by migration across fibronectin alone, or by soluble factors induced during migration, whereas monocyte-ECV304 adhesion was sufficient to stimulate TF. Antibodies against CD29 (beta1 integrin), but not against CD18 (beta2 integrin) or CD31 (PECAM-1), inhibited TF expression. Monocyte adhesion to ECV304 cells induced tyrosine phosphorylation of cellular proteins and specifically of the ERK and p38 MAP kinases. Tyrosine kinase inhibition with genistein (10 microg/mL) blocked transmigration, whereas selective ERK inhibition with PD98059 (50 microM) or p38 inhibition with SB203580 (20 microM) did not. However, both ERK and p38 inhibition dose dependently abolished TF expression. These studies suggest that an extravascular focus of infection or inflammation can promote both intravascular thrombosis and extravascular fibrin deposition during the process of adhesion and transmigration across the endothelial barrier. The selective inhibition of the mitogen-activated protein kinases may offer a novel therapeutic means of modulating this inflammatory sequence.