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)

Constitutive activation of the MAPK/ERK kinase (MEK)1-ERK2 signaling module in Madin-Darby canine kidney (MDCK)-C7 cells disrupts their ability to form cyst-like structures in collagen gels and induces an invasive, myofibroblast-like phenotype. However, the reversibility of these cellular events, as well as the relative role of both MEK isoforms (MEK1 and MEK2) and both ERK isoforms (ERK1 and ERK2) during these processes, has not yet been investigated. We now report that loss of constitutively active MEK1 (caMEK1) and, thus, loss of active ERK1/2 in C7caMEK1 cells is associated with increased MEK2 protein expression, reexpression of ERK1 protein, and epithelial redifferentiation of these cells. The morphological changes toward an epithelial phenotype in these revertant cell lines (C7rev4, C7rev5, C7rev7) are reflected by the upregulation of epithelial marker proteins, such as E-cadherin, beta-catenin, and cytokeratin, by the loss of alpha-smooth muscle actin expression, and by the ability of these epithelial revertants to form well-organized spherical cysts when grown in three-dimensional collagen gels. Further evidence for a role of the MEK1-ERK1/2 module in epithelial-mesenchymal transition was obtained from the analysis of two novel, spontaneously transdifferentiated MDCK-C7 cell clones (C7e1 and C7e2 cells). In these clones, increased MEK1/2-ERK1/2 phosphorylation, reduced MEK2 protein expression, and loss of ERK1 protein expression is associated with phenotypic alterations similar to those observed in transdifferentiated C7caMEK1 cells. C7e1 cells at least partially regained some of their epithelial characteristics at higher passages. In contrast, C7e2 cells maintained a transdifferentiated phenotype at high passage, were unable to generate cyst-like epithelial structures, and retained invasive properties when grown on a three-dimensional collagen matrix. We conclude that in renal epithelial MDCK-C7 cells, stable epithelial-to-mesenchymal transition (EMT) is associated with loss of ERK1 protein expression, reduced MEK2 protein expression, and increased basal ERK2 phosphorylation. In contrast, loss of active MEK1-ERK1/2 results in increased MEK2 protein expression and reexpression of ERK1 protein, concomitant with the restoration of epithelial phenotype and the ability to form cystic structures.
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PMID:Loss of active MEK1-ERK1/2 restores epithelial phenotype and morphogenesis in transdifferentiated MDCK cells. 1290 Mar 89

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2-5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I-III, laminin beta2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I-III production and significantly decreased laminin beta2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.
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PMID:Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids. 1290 81

We previously reported that human interleukin (IL)-2 dependent T cell lines derived from very late antigen (VLA)-1(+) CD45RO(+) peripheral blood (PB) T-cells adhere constitutively to collagen type IV, whereas lines from VLA-1(-) PB lymphocytes (L) adhere weakly. Here we report that the latter are induced to adhere by phorbol 12-myristate 13-acetate (PMA). Both PMA dependent and constitutive adhesion, including that of a Herpes Virus Saimiri (HVS) infected CD4(+)VLA-1(+) clone (HVST) were inhibited by anti-VLA-1 monoclonal antibodies (mAb), by inhibitors of phospholipase C (PLC)gamma and by lovastatin but not by a MEK1 inhibitor, whereas only PMA induced adhesion was blocked by inhibition of protein-kinase (PK) C. Furthermore, lovastatin enhanced PLCgamma and anti VLA-1 mAb blockade, and its effect was not reversed by mevalonic acid (MVA). Lovastatin also inhibited interferon (IFN)gamma secretion by T cells triggered with anti-CD3 and in cells detaching from collagen IV. These results suggest new ways for functional modulation of activated T-cells interacting with collagen.
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PMID:Lovastatin and phospholipase Cgamma regulate constitutive and protein kinase C dependent integrin mediated interactions of human T-cells with collagen. 1291 56

Background/AIMS: Hepatic stellate cells (HSCs) play a key role in the production and degradation of extracellular matrix (ECM) in the liver. In the present study, we investigated the interaction between ECM and HSCs in vitro with emphasis on the modulation of matrix metalloproteinases (MMPs) by ECM. METHODS: Freshly isolated rat HSCs were cultured in several conditions on type I collagen- or matrigel-coated dishes, on thick matrigel or in three-dimensional type I collagen (3D-gel), and MMPs expression in HSCs was examined. In addition, activation and signaling pathway of MMP-9 expression modulated by 3D-gel in HSCs were examined. RESULTS: Increased expression of MMP-3, -9, -13 and -14 was markedly detected only in the 3D-gel-treated HSCs. Zymography demonstrated that only 3D-gel-treated cells showed active gelatinase activity of MMP-9 at 82 kDa. MMP-9 expression was inhibited by neutralizing antibody against integrin alpha2beta1, tyrosine kinase inhibitors, or MEK1,2 inhibitor PD 98059, but not by p38 inhibitor SB 203580. Western blotting also showed phosphorylated p38, ERK1,2, and JUN/SAPK was quickly induced in HSCs by 3D-gel. CONCLUSIONS: MMP-9 expression and activation is induced in HSCs by 3D-gel and this observed collagen-dependent induction of MMP-9 requires ERK1,2 activity.
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PMID:Modulation of matrix metalloproteinase-9 in hepatic stellate cells by three-dimensional type I collagen: its activation and signaling pathway. 1296 32

MMP-9 (92 kDa) is the major gelatinase able to degrade collagen IV, secreted by keratinocytes that are actively involved in wound-healing or tumorigenesis. Since the invasive phenotype of cancers is dependent on MMP-9 expression, it appeared of interest to precisely characterize which signal transduction pathways activated by TNF-alpha are involved in MMP-9 up-regulation induced by TNF-alpha. In HaCaT cells, activation of MMP-9 occurs at the transcriptional level. Inhibition of the MAPK pathway using specific inhibitors of the Ras, Raf, MEK1/2, and Erk1/2 cascade was correlated with a marked inhibition of MMP-9 activity, as determined by gene and protein expression. MAPK pathway activation via TNF-alpha was confirmed by marked AP-1 activation detected in EMSA. Under our experimental conditions, p38 MAPK and SAPK/JNK pathways were not activated. Gene and protein expression of other MMPs that regulate MMP-9, such as MMP-1 and MMP-13, were also up-regulated by TNF-alpha and inhibited by UO126, providing evidence that the MAPK pathway plays a fundamental role in the regulation of MMP-9 secretion by keratinocytes. As TNF-alpha is known to be a main activator of NF-kappaB pathway, the effects of campthothecin and caffeic acid were investigated, such as, TNF-alpha campthothecin up-regulated MMP-9 activity but caffeic acid only weakly inhibited MMP-9 activation induced by TNF-alpha. However, NF-kappaB is activated as shown from immunostaining data, a nuclear staining and higher Western blotting expression of p50 and p65 NF-kappaB subunits were detected after TNF-alpha treatment. A higher specific signal was also detected in EMSA for TNF-alpha-treated cells.
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PMID:The inhibition of MAPK pathway is correlated with down-regulation of MMP-9 secretion induced by TNF-alpha in human keratinocytes. 1451 92

Multiple lines of evidence establish that angiotensin II (Ang II) induces not only hypertension but also directly contributes to cardiac diseases. Apoptosis signal-regulating kinase 1 (ASK1), one of mitogen-activated protein kinase kinase kinases, plays a key role in stress-induced cellular responses. However, nothing is known about the role of ASK1 in cardiac hypertrophy and remodeling in vivo. In this study, by using mice deficient in ASK1 (ASK1-/- mice), we investigated the role of ASK1 in cardiac hypertrophy and remodeling induced by Ang II. Left ventricular (LV) ASK1 was activated by Ang II infusion in wild-type mice, which was mediated by angiotensin II type 1 receptor and superoxide. Although Ang II-induced hypertensive effect was comparable to wild-type and ASK1-/- mice, LV ASK1 activation by Ang II was not detectable in ASK1-/- mice, and p38 and c-Jun N-terminal kinase (JNK) activation was lesser in ASK-/- mice than in wild-type mice. Elevation of blood pressure by continuous Ang II infusion was comparable between ASK1-/- and wild-type mice. However, Ang II-induced cardiac hypertrophy and remodeling, including cardiomyocyte hypertrophy, cardiac hypertrophy-related mRNA upregulation, cardiomyocyte apoptosis, interstitial fibrosis, coronary arterial remodeling, and collagen gene upregulation, was significantly attenuated in ASK1-/- mice compared with wild-type mice. These results provided the first in vivo evidence that ASK1 is the critical signaling molecule for Ang II-induced cardiac hypertrophy and remodeling. Thus, ASK1 is proposed to be a potential therapeutic target for cardiac diseases.
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PMID:Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensin II-induced cardiac hypertrophy and remodeling. 2694 26

Glomerular epithelial cell (GEC) injury and apoptosis may contribute to sclerosis in glomerulonephritis. The present study addresses signals that regulate survival of GEC in culture and in the acute puromycin aminonucleoside nephrosis (PAN) model of GEC injury in vivo. Compared with GEC on plastic substratum, adhesion to collagen increased activation of focal adhesion kinase (FAK), c-Src, and ERK and facilitated survival (prevented apoptosis). GEC on plastic exhibited increased caspase-8 and -9 activities, increased expression of the proapoptotic protein, Bax, and decreased the antiapoptotic protein, Bcl-XL, compared with collagen. Stable expression of constitutively active mutants of FAK (CD2-FAK) or MEK (R4F-MEK) activated the ERK pathway and supplanted the requirement of collagen for survival. In contrast, expression of a Ras mutant that activates phosphatidylinositol 3-kinase but blocks ERK activation or pharmacological inhibition of the ERK pathway decreased survival on collagen. Glomeruli isolated from rats with PAN revealed increased beta1-integrin expression, along with increased activation of FAK, c-Src, and ERK, compared with controls. EGF receptor activation was undetectable in PAN. Therefore, adhesion to collagen, resulting in activation of FAK and the Ras-ERK pathway, supports GEC survival. Analogous signals for GEC survival are activated in PAN.
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PMID:Extracellular matrix regulates glomerular epithelial cell survival and proliferation. 1455 18

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.
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PMID:Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation. 1460 60

Activated receptor tyrosine kinases bind the Shc adaptor protein through its N-terminal phosphotyrosine-binding (PTB) and C-terminal Src homology 2 (SH2) domains. After binding, Shc is phosphorylated within the central collagen-homology (CH) linker region on Tyr-317, a residue remote to both the PTB and SH2 domains. Shc phosphorylation plays a pivotal role in the initiation of mitogenic signaling through the Ras/Raf/MEK/ERK pathway, but it is unclear if Tyr-317 phosphorylation affects Shc-receptor interactions through the PTB and SH2 domains. To investigate the structural impact of Shc phosphorylation, molecular dynamics simulations were carried out using special-purpose Molecular Dynamics Machine-Grape computers. After a 1-nanosecond equilibration, atomic motions in the structures of unphosphorylated Shc and Shc phosphorylated on Tyr-317 were calculated during a 2-nanosecond period. The results reveal larger phosphotyrosine-binding domain fluctuations and more structural flexibility of unphosphorylated Shc compared with phosphorylated Shc. Collective motions between the PTB-SH2, PTB-CH, and CH-SH2 domains were highly correlated only in unphosphorylated Shc. Dramatic changes in domain coupling and structural rigidity, induced by Tyr-317 phosphorylation, may alter Shc function, bringing about marked differences in the association of unphosphorylated and phosphorylated Shc with its numerous partners, including activated membrane receptors.
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PMID:Tyr-317 phosphorylation increases Shc structural rigidity and reduces coupling of domain motions remote from the phosphorylation site as revealed by molecular dynamics simulations. 1461 32

Cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, has been shown to be elevated in the serum of patients with ischemic heart disease and valvular heart disease, and induces cardiomyocyte hypertrophy in vitro. We investigated expression of CT-1 in post-MI rat heart and the effect of CT-1 on cultured primary adult rat cardiac fibroblasts. Elevated CT-1 expression was observed in the infarct zone at 24 h and continued through 2, 4 and 8 weeks post-MI, compared to sham-operated animals. CT-1 induced rapid phosphorylation of Jak, Jak2, STAT1, STAT3, p42/44 MAPK and Akt in cultured adult cardiac fibroblasts. CT-1 induced cardiac fibroblast protein synthesis and proliferation. Protein and DNA synthesis were dependent on activation of Jak/STAT, MEK1/2, PI3K and Src pathways as evidenced by decreased 3H-leucine and 3H-thymidine incorporation after pretreatment with AG490, PD98059, LY294002 and genistein respectively. Furthermore, CT-1 treatment increased procollagen-1-carboxypropeptide (PICP) synthesis, a marker of mature collagen synthesis. CT-1 induced cell migration of rat cardiac fibroblasts. Our results suggest that CT-1, as expressed in post-MI heart, may play an important role in infarct scar formation and ongoing remodeling of the scar. CT-1 was able to initiate each of the processes considered important in the formation of infarct scar including cardiac fibroblast migration as well as fibroblast proliferation and collagen synthesis. Further work is required to determine factors that induce CT-1 expression and interplay with other mediators of cardiac infarct wound healing in the setting of acute cardiac ischemia and chronic post-MI heart failure.
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PMID:Cardiotrophin-1: expression in experimental myocardial infarction and potential role in post-MI wound healing. 1467 4


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