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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.
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PMID:Insulin-like growth factor-I signaling in smooth muscle cells is regulated by ligand binding to the 177CYDMKTTC184 sequence of the beta3-subunit of alphaVbeta3. 1619 48

The osteogenic potential of mesenchymal stem cells (MSCs) cultured on poly(lactide-co-glycolide) (PLGA) or poly(caprolactone) (PCL), two widely used polymeric biomaterials that have been reported to differentially support osteogenic differentiation, was compared in these studies. Here we report that MSCs cultured in 3-D PLGA scaffolds for up to 5 weeks significantly upregulate osteocalcin gene expression levels. By contrast, osteocalcin expression was markedly downregulated in 3-D PCL-based constructs over the same time course. We hypothesized that differential adsorption of extracellular matrix (ECM) proteins present in serum-containing culture medium and subsequent differences in integrin-mediated adhesion are responsible for these differences, and tested this hypothesis using thin (2-D) polymeric films. Supporting this hypothesis, significant amounts of fibronectin and vitronectin deposited onto both materials in serum-containing osteogenic media, with type-I collagen present in lower amounts. Adhesion-blocking studies revealed that MSCs adhere to PCL primarily via vitronectin, while type-I collagen mediates their attachment to PLGA. These adhesive mechanisms correlated with higher levels of alkaline phosphatase (ALP) activity after 2 weeks of monolayer culture on PLGA versus PCL. These data suggest that the initial adhesion of MSCs to PLGA via type-I collagen fosters osteogenesis while adhesion to PCL via vitronectin does not, and stress the need for an improved molecular understanding of cell-ECM interactions in stem cell-based therapies.
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PMID:Adhesion of mesenchymal stem cells to polymer scaffolds occurs via distinct ECM ligands and controls their osteogenic differentiation. 1660 24

The intracellular signaling events controlling human mesenchymal stem cells (hMSC) differentiation into osteoblasts are not entirely understood. We recently demonstrated that contact with extracellular matrix (ECM) proteins is sufficient to induce osteogenic differentiation of hMSC through an ERK-dependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK1/2 by ECM, and stimulate subsequent phosphorylation of the Runx2/Cbfa-1 transcription factor that controls osteogenic gene expression. We plated hMSC on purified collagen I (COLL-I) and vitronectin (VN) in the presence or absence of FAK-specific siRNA, and assayed for phosphorylation of Runx2/Cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein-2, osteocalcin, alkaline phosphatase, calcium deposition, and spectroscopically determined mineral:matrix ratio). We found that siRNA treatment reduced FAK mRNA levels by >40% and decreased ECM-mediated phosphorylation of FAK Y397 and ERK1/2. Serine phosphorylation of Runx2/Cbfa-1 was significantly reduced after 8 days in treated cells. Finally, FAK inhibition blocked osterix transcriptional activity and the osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results suggest that FAK signaling plays an important role in regulating ECM-induced osteogenic differentiation of hMSC.
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PMID:Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells. 1708 17

EGF and TGF-alpha induce an equipotent stimulation of fibroblast migration and proliferation. In spite of their homologous structure and ligation by the same receptor (EGFR), we report that their respective motogenic activities are mediated by different signal transduction intermediates, with p70(S6K) participating in EGF signalling and phospholipase Cgamma in TGF-alpha signalling. We additionally demonstrate that EGF and TGF-alpha motogenic activities may be resolved into two stages: (a) cell "activation" by a transient exposure to either cytokine, and (b) the subsequent "manifestation" of an enhanced migratory phenotype in the absence of cytokine. The cell activation and manifestation stages for each cytokine are mediated by distinct matrix-dependent mechanisms: motogenetic activation by EGF requires the concomitant functionality of EGFR and the hyaluronan receptor CD44, whereas activation by TGF-alpha requires EGFR and integrin alphavbeta3. Manifestation of elevated migration no longer requires the continued presence of exogenous cytokine and functional EGFR but does require the above mentioned matrix receptors, as well as their respective ligands, i.e., hyaluronan in the case of EGF, and vitronectin in the case of TGF-alpha. In contrast, the mitogenic activities of EGF and TGF-alpha are independent of CD44 and alphavbeta3 functionality. These results demonstrate clear qualitative differences between EGF and TGF-alpha pathways and highlight the importance of the extracellular matrix in regulating cytokine bioactivity.
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PMID:EGF AND TGF-alpha motogenic activities are mediated by the EGF receptor via distinct matrix-dependent mechanisms. 1719 62

IGF-I can bind to the extracellular matrix protein vitronectin (VN) through the involvement of IGF-binding proteins-2, -3, -4, and -5. Because IGF-I and VN have established roles in tumor cell dissemination, we were keen to investigate the functional consequences of the interaction of IGF-I, IGF binding proteins (IGFBPs), and VN in tumor cell biology. Hence, functional responses of MCF-7 breast carcinoma cells and normal nontumorgenic MCF-10A mammary epithelial cells were investigated to allow side-by-side comparisons of these complexes in both cancerous and normal breast cells. We demonstrate that substrate-bound IGF-I-IGFBP-VN complexes stimulate synergistic increases in cellular migration in both cell types. Studies using IGF-I analogs determined this stimulation to be dependent on both heterotrimeric IGF-I-IGFBP-VN complex formation and the involvement of the IGF-I receptor (IGF-IR). Furthermore, the enhanced cellular migration was abolished on incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. Analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway while simultaneously producing a sustained activation of the phosphatidylinositide 3-kinase/AKT pathway. Experiments using pharmacological inhibitors of these pathways determined a requirement for phosphatidylinositide 3-kinase/AKT activation in the observed response. Overexpression of wild type and activated AKT further increases substrate-bound IGF-I-IGFBP-VN-stimulated migration. This study provides the first mechanistic insights into the action of IGF-I-IGFBP-VN complexes and adds further evidence to support the involvement of VN-binding integrins and their cooperativity with the IGF-IR in the promotion of tumor cell migration.
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PMID:Substrate-bound insulin-like growth factor (IGF)-I-IGF binding protein-vitronectin-stimulated breast cell migration is enhanced by coactivation of the phosphatidylinositide 3-Kinase/AKT pathway by alphav-integrins and the IGF-I receptor. 1807 1

The urokinase receptor (uPAR) is upregulated upon tumor cell invasion and correlates with poor lung cancer survival. Although a cis-interaction with integrins has been ascribed to uPAR, whether this interaction alone is critical to urokinase (uPA)- and uPAR-dependent signaling and tumor promotion is unclear. Here we report the functional consequences of point mutations of uPAR (H249A-D262A) that eliminate beta1 integrin interactions but maintain uPA binding, vitronectin attachment and association with alphaV integrins, caveolin and epidermal growth factor receptor. Disruption of uPAR interactions with beta1 integrins recapitulated previously reported findings with beta1-integrin-derived peptides that attenuated matrix-dependent ERK activation, MMP expression and in vitro migration by human lung adenocarcinoma cell lines. The uPAR mutant cells acquired enhanced capacity to adhere to vitronectin via uPAR-alphaVbeta5-integrin, rather than through the uPAR-alpha3beta1-integrin complex and they were unable to initiate uPA signaling to activate ERK, Akt or Stat1. In an orthotopic lung cancer model, uPAR mutant cells exhibited reduced tumor size compared with cells expressing wild-type uPAR. Taken together, the results indicate that uPAR-beta1-integrin interactions are essential to signals induced by integrin matrix ligands or uPA that support lung cancer cell invasion in vitro and progression in vivo.
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PMID:Signaling through urokinase and urokinase receptor in lung cancer cells requires interactions with beta1 integrins. 1894 Sep 13

Metalloproteinase MT1-MMP is induced and Pro-MMP-2 up modulated early in rat preosteoblasts (ROB) set to differentiate. We here show that the induction of MMPs, accompanied by activation of Pro-MMP-2, occurs by 6 h of adhesion on endogenous extracellular matrix (ECM), Fibronectin (FN) and Collagen type I (CI). These events do not occur after adhesion on Collagen III (CIII), Vitronectin (VN) or BSA. Within the first hour on inducing substrata or plastic, FAK is unchanged and ERK(1,2), is activated, but this activation is not sufficient for MT1-MMP induction. The function of p38 MAPK and PTKs is not required for the induction by substrata of MMPs. Six hours after plating preosteoblasts on MMP-inducing substrata, complexes of beta1 integrin with MT1-MMP are formed, that contain integrin dimers specifically engaged by the substratum, alpha4 and alpha5 chains for cells plated on FN, and alpha2 chain for cells plated on CI and ECM. Induction of MT1-MMP and its expression during osteogenesis pleiotropically regulate alkaline phosphatase (AP) expression. During differentiation, variant clones derived from preosteoblasts and MMPs-over-expressing osteoblasts show high MT1-MMP level associated with high AP level both persisting in time, while inhibition of MMPs is accompanied by inhibition of AP. Up or down modulation of AP, transcriptionally or by inhibition of the enzyme activity, has no effect on level or timing of expression of MT1-MMP and Pro-MMP-2. The persistence in expression of MT1-MMP during differentiation, and the associated persistence in expression of AP, as well as their inhibition, both impair the formation of nodules and mineral deposition. A transient pattern of expression of MT1-MMP is required for the establishment of nodules, and MT1-MMP decrease is permissive for nodule mineralization. The expression of AP is required for nodule formation and its level modulates the mineralization. MT1-MMP has multiple functions and is implicated in multiple steps of the differentiation process, acting to regulate homeostasis of the osteogenic differentiation.
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PMID:Role of MT1-MMP in the osteogenic differentiation. 1902 88

Integrins of the beta1 subfamily are highly expressed in the early mouse embryo and are essential for the formation of primitive germ layers from the inner cell mass (ICM). We investigated the mechanisms by which alphabeta1 integrins regulate ICM morphogenesis by using the embryonic-stem-cell-derived embryoid body (EB), a model for peri-implantation development. Ablation of integrin beta1 in EBs resulted in endoderm detachment and in maturation defects, which were manifested by the mislocalization of GATA4 in the cytoplasm and the markedly reduced synthesis of basement membrane (BM) components and the lineage marker disabled homolog 2. The mutant endoderm cells failed to spread on BM substrates, but could spread on vitronectin, which induced upregulation of alphavbeta3 integrin and integrin-dependent GATA4 nuclear translocation. Forced expression of integrin beta3 in the mutant EBs completely rescued endoderm morphogenesis, suggesting that integrin beta3 can substitute for integrin beta1 in the endoderm. Furthermore, the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (ERK1/2) and p38 were activated in endoderm in an integrin-dependent fashion. Pharmacological inhibition of ERK1/2 or p38 MAPK blocked vitronectin-induced GATA4 nuclear translocation and endoderm maturation, whereas expression of a constitutively active ERK kinase (MEK1) or p38 MAPK in the mutant cells rescued endoderm maturation in integrin-beta1-null endoderm cells. Collectively, these results suggest that integrins are required for both the stable adhesion and maturation of visceral endoderm, the latter being mediated through the activation of ERK1/2 and p38 MAPK.
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PMID:Integrins are required for the differentiation of visceral endoderm. 1911 16

It has been proposed that ligand occupancy of integrin alphavbeta3 with extracellular matrix ligands (e.g. vitronectin) plays a critical role in insulin-like growth factor-1 (IGF-1) signaling. We found that expression of alphavbeta3 enhanced IGF-1-induced proliferation of Chinese hamster ovary cells in serum-free conditions (in the absence of vitronectin). We hypothesized that the direct integrin binding to IGF-1 may play a role in IGF-1 signaling. We demonstrated that alphavbeta3 specifically and directly bound to IGF-1 in cell adhesion, enzyme-linked immunosorbent assay-type binding, and surface plasmon resonance studies. We localized the amino acid residues of IGF-1 that are critical for integrin binding by docking simulation and mutagenesis. We found that mutating two Arg residues at positions 36 and 37 in the C-domain of IGF-1 to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, although the mutant still bound to IGF1R, it was defective in inducing IGF1R phosphorylation, AKT and ERK1/2 activation, and cell proliferation. Furthermore wild type IGF-1 mediated co-precipitation of alphavbeta3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between alphavbeta3 and IGF1R. These results suggest that the direct binding to IGF-1 to integrin alphavbeta3 plays a role in IGF-1 signaling through ternary complex formation (alphavbeta3-IGF-IGF1R), and integrin-IGF-1 interaction is a novel target for drug discovery.
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PMID:The direct binding of insulin-like growth factor-1 (IGF-1) to integrin alphavbeta3 is involved in IGF-1 signaling. 1957 19

Carbohydrate structures with a 3'-sulfo betaGal linkage, such as 3'-sulfo-Le(x), can be synthesized by Gal:3-O-sulfotransferase-2 (Gal3ST-2) catalysis, but little is known about their roles in many biological processes. To investigate the role of Gal3ST-2 and its product 3'-sulfo-Le(x), we depleted Gal3ST-2 via siRNA and added exogenous Lewis-x trisaccharide 3'-sulfate sodium salt in human SMMC7721 hepatoma cells. After siRNA transfection, a striking morphological change in SMMC7721 hepatoma cells from polygon to shuttle shape and a significant decrease in the level of adhesion to sL-selectin, HUVEC, fibronectin, vitronectin, and fibrinogen were observed. The expression of integrin subunit alphaV was markedly downregulated, and 3'-sulfated subunit alphaV almost disappeared in the transfectants. The level of cell surface integrin alphaVbeta3 was reduced simultaneously, although total subunit beta3 underwent almost no change. After treatment with exogenous Lewis-x 3'-sulfate, cellular integrin subunit alphaV was upregulated and the level of cell surface integrin alphaVbeta3 was elevated. Interestingly, knockdown of Gal3ST-2 expression effectively inhibited cell proliferation, and the result was significantly correlated with the decrease in the levels of ILK, phosphorylated AKT, and ERK. On the other hand, treatment with Lewis-x trisaccharide 3'-sulfate sodium salt greatly upregulated the phosphorylation of AKT and ERK. Our results also indicated that downregulation of Gal3ST-2 via siRNA transfection was associated with the decrease in the level of expression of anti-apoptotic protein, Bcl-2, with a consequent decrease in the ratios for Bcl-2 to Bax. By exposure to Lewis-x trisaccharide 3'-sulfate sodium salt, the apoptotic response of cells was inhibited. Therefore, Gal3ST-2 and its product, 3'-sulfo-Le(x), were involved in regulation of integrin subunit alphaV and might be associated with cancer cell regulation.
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PMID:3'-Sulfo-Le(x) is important for regulation of integrin subunit alphaV. 2069 81


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