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)

Repetitive mechanical deformation may stimulate intestinal epithelial proliferation. Because the extracellular matrix modulates static intestinal epithelial biology, we examined whether matrix proteins influence intestinal epithelial responses to deformation. Human Caco-2BBE cells and nontransformed human enterocytes (HIPEC) were subjected to 10% average cyclic strain at 10 cycles/min on flexible membranes precoated with matrix proteins without or with plasma fibronectin or functional anti-integrin antibodies in the medium. Strain stimulated proliferation, focal adhesion kinase, extracellular signal-regulated protein kinase (ERK), p38, and Jun N-terminal kinase similarly on collagen I or IV, and more weakly on laminin, but had no effect on fibronectin. MEK blockade (PD98059) prevented strain-stimulated proliferation on collagen but did not affect proliferation on fibronectin. Adding tissue fibronectin to a collagen substrate or plasma fibronectin to the media suppressed strain s mitogenic and signal effects, but not those of epidermal growth factor. Functional antibodies to the alpha5 or alpha(v) integrin subunit blocked strain's effects on Caco-2 proliferation and ERK activation, although ligation of the alpha2 or alpha6 subunit did not. Repetitive strain also stimulated, and fibronectin inhibited, human intestinal primary epithelial cell proliferation. Repetitive deformation stimulates transformed and nontransformed human intestinal epithelial proliferation in a matrix-dependent manner. Tissue or plasma fibronectin may regulate the intestinal epithelial response to strain via integrins containing alpha5 or alpha(v).
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PMID:Regulation of the intestinal epithelial response to cyclic strain by extracellular matrix proteins. 1262 37

Recently, we have shown that autocrine transforming growth factor-alpha (TGF-alpha) controls the expression of integrin alpha2, cell adhesion to collagen IV and motility in highly progressed HCT116 colon cancer cells (Sawhney, R. S., Zhou, G-H. K., Humphrey, L. E., Ghosh, P., Kreisberg, J. I., and Brattain, M. G. (2002) J. Biol. Chem. 277, 75-86). We now report that expression of basal integrin alpha2 and its biological effects are controlled by constitutive activation of the extracellular signal-regulated/mitogen-activated protein kinase (ERK/MAPK) pathway. Treatment of cells with selective mitogen-activated protein kinase kinase (MEK) inhibitors PD098059 and U0126 showed that integrin alpha2 expression, cell adhesion, and activation of ERK are inhibited in a parallel concentration-dependent fashion. Moreover, autocrine TGF-alpha-mediated epidermal growth factor receptor activation was shown to control the constitutive activation of the ERK/MAPK pathway, since neutralizing antibody to the epidermal growth factor receptor was able to block basal ERK activity. TGF-alpha antisense-transfected cells also showed attenuated activation of ERK. Using a real time electric cell impedance sensing technique, it was shown that ERK-dependent integrin alpha2-mediated cell micromotion signaling is controlled by autocrine TGF-alpha. Thus, this study implicates ERK/MAPK signaling activated by endogenous TGF-alpha as one of the mechanistic features controlling metastatic spread.
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PMID:Integrin alpha2 and extracellular signal-regulated kinase are functionally linked in highly malignant autocrine transforming growth factor-alpha-driven colon cancer cells. 1265 25

Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. Studies on cells in three-dimensional collagen matrices have shown that fibroblasts switch between proliferative and quiescence phenotypes, depending upon whether matrices are attached or floating during matrix remodeling. Previous work showed that cell signaling through the ERK pathway was decreased in fibroblasts in floating matrices. In the current research, we extend the previous findings to show that serum stimulation of fibroblasts in floating matrices does not result in ERK translocation to the nucleus. In addition, there was decreased serum activation of upstream members of the ERK signaling pathway, MEK and Raf, even though Ras became GTP loaded. The findings suggest that quiescence of fibroblasts in floating collagen matrices may result from a defect in Ras coupling to its downstream effectors.
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PMID:Fibroblast quiescence in floating collagen matrices: decrease in serum activation of MEK and Raf but not Ras. 1266 62

Tumstatin and endostatin are two inhibitors of angiogenesis derived from precursor human collagen molecules known as alpha 3 chain of type IV collagen and alpha1 chain of type XVIII collagen, respectively. Although both these inhibitors are noncollagenous (NC1) domain fragments of collagens, they only share a 14% amino acid homology. In the present study we evaluated the functional receptors, mechanism of action, and intracellular signaling induced by these two collagen-derived inhibitors. Human tumstatin prevents angiogenesis via inhibition of endothelial cell proliferation and promotion of apoptosis with no effect on migration, whereas human endostatin prevents endothelial cell migration with no effect on proliferation. We demonstrate that human tumstatin binds to alpha v beta 3 integrin in a vitronectin/fibronectin/RGD cyclic peptide independent manner, whereas human endostatin competes with fibronectin/RGD cyclic peptide to bind alpha 5 beta 1 integrin. The activity of human tumstatin is mediated by alpha v beta 3 integrin, whereas the activity of human endostatin is mediated by alpha 5 beta 1 integrin. Additionally, although human tumstatin binding to alpha v beta 3 integrin leads to the inhibition of Cap-dependent translation (protein synthesis) mediated by focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathway, human endostatin binding to alpha 5 beta 1 integrin leads to the inhibition of focal adhesion kinase/c-Raf/MEK1/2/p38/ERK1 mitogen-activated protein kinase pathway, with no effect on phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 and Cap-dependent translation. Collectively, such distinct properties of human tumstatin and human endostatin provide the first insight into their diverse antiangiogenic actions and argue for combining them for targeting tumor angiogenesis.
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PMID:Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins. 3174 8

The activation of human platelets by a variety of agonists is accompanied by the phosphorylation of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein (MAP) kinases. However, the role(s) of, and the substrate(s) for, these enzymes in platelet function remain unclear. Studies on ERKs in platelets have relied on pharmacological tools, including an inhibitor of ERK activation, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene]. In the present study, the effects of U0126 and its "inactive" analogue, U0125 [1,4-diamino-2,3-dicyano-1,4-bis(phenylthio)butadiene], on human platelet aggregation and MAP kinase activity were examined. Several agonists with a variety of signaling pathways were studied including thrombin, a thromboxane analogue, arachidonic acid, collagen, calcium ionophores, and the phorbol ester phorbol myristate acetate (PMA). U0126, at concentrations consistent with inhibition of the isolated enzyme, inhibited ERK phosphorylation, and therefore MEK activation, in response to each agonist. Under such conditions, U0126 did not affect the phosphorylation of a second MAP kinase, p38(MAPK); however, platelet aggregation was also unaffected. Higher concentrations of U0126, and of U0125, inhibited platelet aggregation in response to collagen and PMA with no effect on that induced by the other agonists. These results dissociate ERK activation from platelet aggregation, suggesting an alternative role for ERKs in platelet function. In addition, the effects of higher concentrations of U0126 are likely due to an action on protein kinase C, likely unrelated to ERK inhibition, suggesting that the inhibitor concentration is crucial to the interpretation of such studies.
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PMID:Inhibition of the MEK/ERK pathway has no effect on agonist-induced aggregation of human platelets. 1269 65

We used both a gene knockout approach and pharmacologic modulation to study the implication of the JNK pathway in regulating fibroblast motility, capacity to contract mechanically unloaded collagen gels, and type I collagen gene expression in vitro. These parameters, which are important for tissue repair, are positively regulated by transforming growth factor (TGF)-beta, a cytokine viewed as playing a master role during wound healing. We demonstrate that basal JNK activity is critical for fibroblast motility because (a) mouse embryo jnk-/- fibroblasts exhibit significantly lower ability to close mechanically induced cell layer wounds than their wild-type (wt) counterparts, and (b) wound closure by human dermal fibroblasts is dramatically impaired by the specific JNK inhibitor SP600125. junAA fibroblasts, in which amino acids Ser63 and Ser73 of c-Jun are replaced by two Ala residues so that c-Jun cannot be phosphorylated by JNK, also exhibited impaired motility, suggesting that c-Jun phosphorylation by JNK is critical for fibroblast migration. In sharp contrast to their lesser motility on plastic, jnk-/- and junAA fibroblasts contracted free-floating, mechanically unloaded, collagen lattices markedly faster than wt fibroblasts. Furthermore, basal mRNA steady-state levels for types I and III collagen genes were similar in jnk-/- and wt fibroblasts. Likewise, overexpression of a dominant-negative mutant form of MKK4 in dermal fibroblasts did not affect collagen expression. We also demonstrate that basal JNK activity does not affect either TGF-beta-induced collagen gene expression or lattice contraction, whereas on the other hand, the blockage of motility initiated by JNK inhibition cannot be overcome by TGF-beta. Together these results demonstrate discrete, yet significant and highly specific, regulation of fibroblast functions important for wound healing by basal JNK activity.
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PMID:Disruption of basal JNK activity differentially affects key fibroblast functions important for wound healing. 1273 Feb 13

Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.
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PMID:Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly. 1278 34

Adhesion to type 1 collagen can elicit different cellular responses dependent upon whether the collagen is in a fibrillar form (gel) or monomeric form (film). Hepatocytes adherent to collagen film spread extensively, express cyclin D1, and increase DNA synthesis in response to epidermal growth factor, whereas hepatocytes adherent to collagen gel have increased differentiated function, but lower DNA synthesis. The signaling mechanisms by which different forms of type I collagen modulate cell cycle progression are unknown. When ERK MAP kinase activation was analyzed in hepatocytes attached to collagen film, two peaks of ERK activity were demonstrated. Only the second peak, which correlated with an increase of cyclin D1, was required for G1-S progression. Notably, this second peak of ERK activity was absent in cells adherent to collagen gel, but not required in the presence of exogenous cyclin D1. Expression of activated mutants of the Ras/Raf/MEK signaling pathway in cells adherent to collagen gel restored ERK phosphorylation and DNA synthesis, but differentially affected cell shape. Although Ras, Raf, and MEK all increased expression of cyclin D1 on collagen film, only Ras and Raf significantly up-regulated cyclin D1 levels on collagen gel. These results demonstrate that adhesion to polymerized collagen induces growth arrest by inhibiting the Ras/ERK-signaling pathway to cyclin D1 required in late G1.
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PMID:The role of collagen structure in mitogen stimulation of ERK, cyclin D1 expression, and G1-S progression in rat hepatocytes. 1279 85

Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 activated extracellular signal-regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant-negative ERK blockade reduced TGF-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK-dependent activation of Smad2/3 occurs only in certain cell types. TGF-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 on collagen promoter activity were additive. These results indicate that ERK-dependent R-Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.
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PMID:Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells. 1282 91

Bone morphogenetic proteins (BMPs) are shown to potentiate NGF-induced neuronal differentiation in PC12 phaeochromocytoma cells grown on collagen under low-serum conditions. Whereas, cell bodies remained rounded in control medium or with only BMPs present, addition of BMP4 or BMP6 robustly increased the neuritogenic effect of NGF within 2 days. NGF-increased phosphorylation of p44(Erk1) and p42(Erk2) between 2 and 24h was unaffected by addition of BMP6. PC12 cells transfected with the SBE(4x)-luc reporter showed that BMP4 significantly increased receptor-activated Smad activity. Expression of constitutively active BMP receptor ALK2 activating Smad1 and Smad5 resulted in a strong increase in the SBE(4x)-luc reporter response. Adding the inhibitory Smad7 drastically reduced this signal. In contrast to wild-type (wt) Smad5, a Smad5 variant lacking five Erk phosphorylation sites in the linker region (designated Smad5/5SA) showed a strong background transcriptional activity. A fusion construct (Gal4-Smad5/5SA) was also highly transcriptionally active. Addition of the MEK inhibitor U0126 to PC12 cells expressing Gal4-Smad5/wt did not increase background transcriptional activity. However, upon activation by constitutively active ALK2 both Gal4-Smad5/wt and Gal4-Smad5/5SA strongly stimulated transcription. The data show that serine residues of the linker region of Smad5 reduce spontaneous transcriptional activity and that NGF-activated Erk does not antagonise BMP signalling at this site. Hence, NGF and BMP signals are likely to interact further downstream at the transcriptional level in neuronal differentiation of the PC12 cells.
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PMID:Bone morphogenetic protein signalling in NGF-stimulated PC12 cells. 1289 70


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