Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HL-60 cells treated by PMA develop the monocyte adherent phenotype and synthesize plasminogen activator inhibitor type-1 (PAI-1). We focused our study on the identification of the PMA-activated protein kinase C (PKC) isoform and its downstream transduction pathway activating PAI-1 synthesis. Acquisition of the monocytic phenotype was evidenced by cell adherence (90-95%) and a sharp increase of CD 36 and receptor for urokinase plasminogen activator (uPAR) surface expression. Ro 31-8220, a specific inhibitor of PKC, prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion. To identify the PKC isoform, we took advantage of the HL-525 cell line, an HL-60 cell variant deficient in PKCbeta gene expression. This defect prevents PMA to induce the differentiation process. HL-525 stimulated by PMA did not synthesize PAI-1 nor become adherent. However, in HL-525 cells either pretreated by retinoic acid that reinduces PKCbeta gene expression or transfected with PKCbeta cDNA, PMA significantly activated PAI-1 synthesis and adhesion of cells. Immunoblotting of active Mitogen Activated Protein Kinase (MAPK) p42/p44 in HL-60 cells showed a preferential and sustained activation of the p42 isoform by PMA over the p44 isoform. Ro 31-8220 significantly attenuated this activation. PD 098059 and U0126, both highly specific MEK inhibitors, efficiently prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion whereas SB203580, a specific inhibitor of stress-activated MAPK p38, did not. Results obtained from HL-60 and HL-525 cells indicate that the PMA-activated transduction pathway of uPAR expression involves a PKC isoform other than PKCbeta. In conclusion, we propose that the pathway PKCbeta-MEK-MAPK p42 is a potential linear route for PAI-1 synthesis leading to morphological changes and adherence linked to PMA-induced differentiation in HL-60 cells.
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PMID:Activation of plasminogen activator inhibitor-1 synthesis by phorbol esters in human promyelocyte HL-60--roles of PCKbeta and MAPK p42. 1010 71

Plasminogen activator inhibitor 1 (PAI-1) is a major inhibitor of urokinase-type plasminogen activator (uPA). In this study, we explored the role of PAI-1 in cell signaling. In MCF-7 cells, PAI-1 did not directly activate the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2, but instead altered the response to uPA so that ERK phosphorylation was sustained. This effect required the cooperative function of uPAR and the very low density lipoprotein receptor (VLDLr). When MCF-7 cells were treated with uPA-PAI-1 complex in the presence of the VLDLr antagonist, receptor-associated protein, or with uPA-PAI-1(R76E) complex, which binds to the VLDLr with greatly decreased affinity, transient ERK phosphorylation (<5 min) was observed, mimicking the uPA response. ERK phosphorylation was not induced by tissue-type plasminogen activator-PAI-1 complex or by uPA-PAI-1 complex in the presence of antibodies that block uPA binding to uPAR. uPA-PAI-1 complex induced tyrosine phosphorylation of focal adhesion kinase and Shc and sustained association of Sos with Shc, whereas uPA caused transient association of Sos with Shc. By sustaining ERK phosphorylation, PAI-1 converted uPA into an MCF-7 cell mitogen. This activity was blocked by receptor-associated protein and not observed with uPA-PAI-1(R76E) complex, demonstrating the importance of the VLDLr. uPA promoted the growth of other cells in which ERK phosphorylation was sustained, including beta3 integrin overexpressing MCF-7 cells and HT 1080 cells. The MEK inhibitor, PD098059, blocked the growth-promoting activity of uPA and uPA-PAI-1 complex in these cells. Our results demonstrate that PAI-1 may regulate uPA-initiated cell signaling by a mechanism that requires VLDLr recruitment. The kinetics of ERK phosphorylation in response to uPAR ligation determine the function of uPA and uPA-PAI-1 complex as growth promoters.
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PMID:Plasminogen activator inhibitor 1 functions as a urokinase response modifier at the level of cell signaling and thereby promotes MCF-7 cell growth. 1126 65

Impairment of the fibrinolytic system, mostly due to elevated plasma levels of plasminogen activator inhibitor 1 (PAI-1), is often associated with metabolic disorders such as diabetes mellitus and insulin-resistance syndrome. Moreover, insulin, as we have previously shown, directly stimulates PAI-1 production with a mechanism underlying a complex signaling network which ultimately leads to ERK activation. In this study we have analyzed the effects of agonists of the peroxisome proliferator-activated receptor (PPAR) alpha and gamma on PAI-1 biosynthesis in HepG2 cells in the presence or absence of insulin. The high affinity PPARalpha agonist, Wy-14,643, increased basal and insulin-stimulated PAI-1 antigen release with a mechanism involving gene transcription. We then investigated whether the MAP kinase pathway also plays a role in the stimulatory properties of Wy-L4,643. Wy-L4,643 increases phosphorylation of ERK and p38 in a time-dependent manner without affecting that of SAPK/JNK or ERK5. Moreover, the MEK (ERK kinase) inhibitors, PD98059 and UO126, completely prevented PAI-1 induction by Wy-14,643 without inhibiting the activation of a reporter gene carrying the PPRE element. Interestingly, the addition of p38 inhibitor followed by insulin and Wy-14,643 resulted in a greater than additive stimulation of PAI-1 secretion acting through ERK1/2 phosphorylation. In contrast, the synthetic PPARgamma agonist, rosiglitazone, did not change PAI-1 level, although this compound induced transcription from the PPRE-driven luciferase reporter construct. In conclusion, Wy-14,643 induces PAI-1 gene expression, in the presence or absence of insulin, with a mechanism which is independent on PPARalpha activation and requires signaling through the ERK1/2 signaling pathway.
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PMID:Induction of plasminogen activator inhibitor I by the PPARalpha ligand, Wy-14,643, is dependent on ERK1/2 signaling pathway. 1451 81

The type-I plasminogen activator inhibitor (PAI-1), the primary inhibitor of both tissue-type and urokinase-type plasminogen activators (t-PA, u-PA), is the primary regulator of plasminogen activation and possibly of extracellular proteolysis. In anchorage-dependent cells, the PAI-1 gene is regulated by cell adhesion. PAI-1 gene expression is induced more evidently in cells that adhered to the culture plate than in those that did not adhere. In this study, we further demonstrate that the PAI-1 gene expression associated with cell adhesion is elicited through the activation of MEK and p42/p44 mitogen-activated protein (MAP) kinase (MAPK; ERK) signal pathways. We found that the MEK inhibitors, PD98059 and U0126, inhibited the induction of PAI-1 gene and protein expression during cell adhesion, PD98059 also inhibited the adhesion of cells to the culture plate, and cell adhesion elicited the kinase activities of MEK and ERK. In addition, we illustrate that two transcription response elements, the serum response element (SRE) and the hypoxia response element (HRE), which exist in the PAI-1 promoter, might be correlated with PAI-1 gene expression during cell adhesion. We discovered that the binding ability of nucleoproteins to both SRE and HRE was enhanced by cell adhesion and was dependent on MEK. Based on these results, we suggest that both MEK and ERK are involved in the induction of PAI-1 gene expression during cell adhesion. Furthermore, the subsequent downstream molecules, Elk-1 and HIF-1, may also participate.
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PMID:The plasminogen activator inhibitor-1 gene is induced by cell adhesion through the MEK/ERK pathway. 1463 Nov 13

Several proteases and their specific inhibitors modulate the interdependent processes of cell migration and matrix proteolysis as part of the global program of trauma repair. Expression of plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor (SERPIN) important in the control of barrier proteolysis and cell-to-matrix adhesion, for example, is spatially-temporally regulated following epithelial denudation injury in vitro as well as in vivo. PAI-1 mRNA/protein synthesis was induced early after epidermal monolayer scraping and restricted to keratinocytes comprising the motile cohort closely recapitulating, thereby, similar events during cutaneous healing. The time course of PAI-1 promoter-driven PAI-1-GFP fusion "reporter" expression in wound-juxtaposed cells approximated that of the endogenous PAI-1 gene confirming the location-specificity of gene regulation in this model. ERK activation was evident within 5 min after injury and particularly prominent in cells residing at the scrape-edge (suggesting a possible role in PAI-1 induction and/or the motile response) as was myosin light chain (MLC) phosphorylation. Indeed, MEK blockade with PD98059 or U0126 attenuated keratinocyte migration (by > or =60%), as did transient transfection of a dominant-negative ERK1 construct (40% decrease in monolayer repair), and completely inhibited PAI-1 transcript expression. Anti-sense down-regulation of PAI-1 synthesis (by 80-85%), or addition of PAI-1 neutralizing antibodies also inhibited injury site closure over a 24 h period establishing that PAI-1 was required for efficient long-term planar motility in this system. PAI-1 anti-sense transfection or actinomycin D transcriptional blockade, in contrast, did not affect the initial migratory response suggesting that residual PAI-1 protein levels (at least in transfectant cells and actinomycin D-treated cultures) may be sufficient to support early cell movement. Pharmacologic inhibition of keratinocyte MEK signaling effectively ablated scrape-induced PAI-1 mRNA expression but failed to attenuate wound-associated increases in cellular PAI-1 protein levels soon after monolayer injury. Collectively, these data suggest that basal PAI-1 transcripts may be mobilized for initial PAI-1 synthesis and, perhaps, the early motile response while maintenance of the normal rate of migration requires the prolonged PAI-1 expression that typically accompanies the repair response. To assess this possibility, scrape site closure studies were designed using keratinocytes isolated from PAI-1-/- mice. PAI-1-/- keratinocytes, in fact, had a significant wound healing defect evident even within the first 6 h following monolayer denudation injury. Addition of active PAI-1 protein to PAI-/- keratinocytes rescued the migratory phenotype that that approximating wild-type cells. These findings validate use of the present keratinocyte model to investigate injury-related controls on PAI-1 gene regulation and, collectively, implicate participation of PAI-1 in two distinct phases of epidermal wound repair.
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PMID:PAI-1 expression is required for epithelial cell migration in two distinct phases of in vitro wound repair. 1517

GnRH analog (GnRHa) and TGF-beta act directly on leiomyoma/myometrial smooth muscle cells (LSMCs and MSMCs) regulating diverse activities resulting in leiomyoma growth and regression. Because GnRH and TGF-beta receptor signaling is in part mediated through the MAPK pathway, we determined whether the contribution of MAPK/ERK and transcriptional activation of c-fos and c-jun, result in differential regulation of type I collagen, fibronectin, and plasminogen activator inhibitor 1 (PAI-1) gene expression, whose products are known to influence extracellular matrix turnover, which is critical in leiomyoma growth and GnRHa-induced regression. We found that GnRHa and TGF-beta in a dose- and time-dependent manner increased the level of phosphorylated ERK1/2 (pERK1/2) in LSMCs and MSMCs. GnRHa and TGF-beta increased ERK1/2 nuclear accumulation resulting in differential regulation of c-fos and c-jun mRNA expression via downstream signaling from MAPK kinase (MEK)1/2, because pretreatment with U0126, a synthetic inhibitor of MEK1/2, abolished basal and GnRHa- and TGF-beta-induced pERK1/2 and the expression of c-fos and c-jun. LSMCs and MSMCs also express fibronectin, type I collagen, and PAI-1 mRNA, and GnRHa and TGF-beta altered their expression in a cell-specific manner through MEK1/2. We concluded that GnRHa and TGF-beta acting through a MAPK/ERK pathway and transcriptional activation of c-fos/c-jun results in differential regulation of specific genes whose products may in part influence the outcome of leiomyoma growth and regression.
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PMID:Gonadotropin releasing hormone and transforming growth factor beta activate mitogen-activated protein kinase/extracellularly regulated kinase and differentially regulate fibronectin, type I collagen, and plasminogen activator inhibitor-1 expression in leiomyoma and myometrial smooth muscle cells. 1553 10

Transforming growth factor-beta1 (TGF-beta1) stimulates expression of plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor (SERPIN) important in the control of stromal barrier proteolysis and cell-to-matrix adhesion. Pharmacologic agents that target MEK (PD98059, U0126) or src family (PP1) kinases attenuated TGF-beta1-dependent PAI-1 transcription in R22 aortic smooth muscle cells. Pretreatment with PP1 at concentrations that inhibited TGF-beta1-dependent PAI-1 expression also blocked ERK1/2 activation/nuclear accumulation suggesting that the required src kinase activity is upstream of ERK1/2 in the TGF-beta1-initiated signaling cascade. The IC(50) of the PP1-sensitive kinase, furthermore, specifically implied involvement of pp60(c-src) in PAI-1 induction. Indeed, addition of TGF-beta1 to quiescent R22 cells resulted in a 3-fold increase in pp60(c-src) autophosphorylation and kinase activity. Transfection of a dominant-negative pp60(c-src) construct, moreover, reduced TGF-beta1-induced PAI-1 expression levels to that of unstimulated controls or PP1-pretreated cells. A >/=170 kDa protein that co-immunoprecipitated with TGF-beta1-activated pp60(c-src) was also phosphorylated transiently in response to TGF-beta1. TGF-beta1 is known to transactivate the 170 kDa EGF receptor (EGFR) by autocrine HB-EGF or TGF-alpha mechanisms suggesting involvement of EGFR activation in certain TGF-beta1-initiated responses. Incubation of quiescent R22 cells with the EGFR-specific inhibitor AG1478 prior to growth factor (EGF or TGF-beta1) addition effectively blocked EGFR activation as determined by direct visualization of receptor internalization. AG1478 suppressed (in a dose-dependent fashion) EGF-induced PAI-1 protein levels and, at a final concentration of 2.5 muM, virtually eliminated EGF-dependent PAI-1 synthesis. More importantly, AG1478 similarly repressed inducible PAI-1 levels in TGF-beta1-stimulated R22 cultures. PP1, PD98059, and U0126 also inhibited TGF-beta1-dependent cell motility at concentrations that significantly attenuated PAI-1 expression. Consistent with the AG1478-associated reductions in EGF- and TGF-beta1-stimulated PAI-1 expression, pretreatment of R22 cell cultures with AG1478 effectively suppressed growth factor-stimulated cell motility. These data indicate that two major phenotypic characteristics of TGF-beta1-exposure (i.e., transcription of specific target genes [e.g., PAI-1], increased cell motility) are linked in the R22 vascular smooth muscle cell system, require pp60(c-src) kinase activity and MEK signaling and involve activation of an AG1478-sensitive (likely EGFR-dependent) pathway.
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PMID:Plasminogen activator inhibitor type-1 gene expression and induced migration in TGF-beta1-stimulated smooth muscle cells is pp60(c-src)/MEK-dependent. 1562 20

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation and survival/or apoptosis of many cells. Knock-out experiments in mice for the three isoforms of TGF-beta have demonstrated their importance in regulating inflammation and tissue repair. TGF-beta is implicated in the pathogenesis of human diseases, including tissue fibrosis and carcinogenesis. TGF-beta receptors act through multiple intracellular pathways. Upon binding of TGF-beta with its receptor, receptor-regulated Smad2/3 proteins become phosphorylated and associate with Smad4. Such complex translocates to the nucleus, binds to DNA and regulates transcription of specific genes. Negative regulation of TGF-beta/Smad signalling may occur through the inhibitory Smad6/7. Furthermore, TGF-beta-activated kinase-1 (TAK1) is a component of TGF-beta signalling and activates stress-activated kinases: p38 through MKK6 or MKK3 and c-Jun N-terminal kinases (JNKs) via MKK4. In the brain TGF-beta, normally expressed at the very low level, increases dramatically after injury. Increased mRNA levels of the three TGF-beta isoforms correlate with the degree of malignancy of human gliomas. TGF-betas are secreted as latent precursors requiring activation into the mature form. TGF-beta may contribute to tumour pathogenesis by direct support of tumour growth and influence on local microenvironment, resulting in immunosuppression, induction of angiogenesis, and modification of the extracellular matrix. TGF-beta1,2 may stimulate production of vascular endothelial growth factor (VEGF) as well as plasminogen activator inhibitor (PAI-I), that are involved in vascular remodelling occurring during angiogenesis. Blocking of TGF-beta action inhibits tumour viability, migration, metastases in mammary cancer, melanoma and prostate cancer model. Reduction of TGF-beta production and activity may be a promising target of therapeutic strategies to control tumour growth.
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PMID:TGF beta signalling and its role in tumour pathogenesis. 1599 Sep 18

Upregulation of plasminogen activator inhibitor type 1 (PAI-1) expression is a critical mechanism through which transforming growth factor-beta1 (TGF-beta1) accelerates intimal growth. The aim of this study was to identify signaling pathways through which TGF-beta1 upregulates PAI-1 expression in endothelial cells (EC) and test interventions for blocking these pathways. We transduced cultured bovine EC with an adenoviral vector containing the PAI-1 promoter fused to a beta-galactosidase reporter gene. We used these cells, along with vectors expressing potential modifiers of TGF-beta1 signaling and pharmacologic antagonists of mitogen-activated protein kinase (MAPK) pathways to identify key mediators of basal and TGF-beta1-regulated PAI-1 expression. Basal activity of the PAI-1 promoter was directly correlated with Ras activation and was blocked by a dominant negative (DN) type I TGF-beta receptor. TGF-beta1-stimulated activity of the PAI-1 promoter did not require Ras activation, and was lessened or eliminated by expression of either DN type I or type II TGF-beta receptors and by inhibition of either of two MAPKs: MEK and p38. Our results suggest unanticipated pathways of TGF-beta1 signaling in EC and point to new strategies to limit TGF-beta1-induced vascular disease.
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PMID:Identification of intracellular pathways through which TGF-beta1 upregulates PAI-1 expression in endothelial cells. 1613 37

13-Deoxytedanolide is a structurally unique macrolide with strong antitumor activity isolated from a marine sponge. Recently, we showed that 13-deoxytedanolide bound to the large subunit of the yeast ribosome and inhibited polypeptide elongation in vitro, but the mechanism by which it exerts antitumor activity is still unknown. Here we show that 13-deoxytedanolide strongly induces plasminogen activator inhibitor 1 (PAI-1) promoter-derived gene expression. 13-Deoxytedanolide, unlike TGF-beta, did not cause apparent nuclear translocation of Smad2/3, but it relocalized the temperature-sensitive mutant of mouse p53 (p53Val153) from the cytoplasm to the nucleus at a nonpermissive temperature, suggesting that 13-deoxytedanolide inhibits protein synthesis. Indeed, the drug inhibited in vivo protein synthesis at low nanomolar concentrations and strongly activated stress-activated protein kinases such as p38 mitogen-activated protein kinase and Jun NH2-terminal protein kinase (JNK). Anisomycin, a well-known inducer of ribotoxic stress that activates both p38 and JNK, also activated PAI-1 gene expression, while other protein synthesis inhibitors that do not activate the kinases failed to do so. PAI-1 gene expression by 13-deoxytedanolide and anisomycin was blocked by SB202190, a specific inhibitor of p38, and SP600125, an inhibitor of both p38 and JNK. 13-Deoxytedanolide and anisomycin caused activation of apoptosis signal-regulating kinase 1, MKK3/MKK6, and SEK1/MKK4, the regulatory kinases upstream of p38 and JNK. These results suggest that 13-deoxytedanolide, like anisomycin, triggers a ribotoxic stress response that activates stress-activated protein kinase cascades, thereby inducing PAI-1 gene expression and apoptosis.
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PMID:Induction of a ribotoxic stress response that stimulates stress-activated protein kinases by 13-deoxytedanolide, an antitumor marine macrolide. 1642 34


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