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)

A prolonged ouabain blockade of the Na(+),K(+)-ATPase detaches cells from each other and from the substrate. This suggests the existence of a link between pump (P) and attachment (A). In the present work, we report that MDCK-W cells treated with ouabain increase tyrosine phosphorylation and content of active MAP kinase, redistribute molecules involved in cell attachment (occludin, ZO-1, desmoplakin, cytokeratin, alpha-actinin, vinculin and actin), and detach. Genistein and UO126, inhibitors of protein tyrosine kinase and of MAP kinase kinase, respectively, block this detachment. The content of P190(Rho-GAP), a GTPase activating protein of the Rho small G-protein subfamily, is increased by ouabain, suggesting that both the Rho/Rac and MAPK pathways are involved. Another clone of MDCK cells whose Na(+),K(+)-ATPase has a negligible affinity for the drug, show none of the effects described for MDCK-W and remain attached. Ma104 cells, a line that has a high affinity for ouabain and stops pumping, fail to modify phosphorylation, as well as the pattern of distribution of attaching molecules, and remain in the monolayer. Taken together, these results suggest that there is a mechanism (P-->A) that transduces a blockade of the pump in a detachment of the cell from neighbors and substrate, in which Ma104 cells are faulty.
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PMID:Relationship between Na(+),K(+)-ATPase and cell attachment. 1056 41

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.
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PMID:Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells. 1071 4

Enhancement of tumor cell growth and invasiveness by transforming growth factor-beta (TGF-beta) requires constitutive activation of the ras/MAPK pathway. Here we have investigated how MEK activation by epidermal growth factor (EGF) influences the response of fully differentiated and growth-arrested pig thyroid epithelial cells in primary culture to TGF-beta1. The epithelial tightness was maintained after single stimulation with EGF or TGF-beta1 (both 10 ng/ml) for 48 hours. In contrast, co-stimulation abolished the transepithelial resistance and increased the paracellular flux of [(3)H]inulin within 24 hours. Reduced levels of the tight junction proteins claudin-1 and occludin accompanied the loss of barrier function. N-cadherin, expressed only in few cells of untreated or single-stimulated cultures, was at the same time increased 30-fold and co-localised with E-cadherin at adherens junctions in all cells. After 48 hours of co-stimulation, both E- and N-cadherin were downregulated and the cells attained a fibroblast-like morphology and formed multilayers. TGF-beta1 only partially inhibited EGF-induced Erk phosphorylation. The MEK inhibitor U0126 prevented residual Erk phosphorylation and abrogated the synergistic responses to TGF-beta1 and EGF. The observations indicate that concomitant growth factor-induced MEK activation is necessary for TGF-beta1 to convert normal thyroid epithelial cells to a mesenchymal phenotype.
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PMID:Transforming growth factor-beta and epidermal growth factor synergistically stimulate epithelial to mesenchymal transition (EMT) through a MEK-dependent mechanism in primary cultured pig thyrocytes. 1237 55

Earlier studies have implicated the significance of transforming growth factor-beta3 (TGFbeta3) in the regulation of Sertoli cell tight junction (TJ) dynamics, possibly via its inhibitory effects on the expression of occludin, claudin-11, and zonula occludens-1 (ZO-1). Yet the mechanism by which TGFbeta3 regulates the Sertoli cell TJ-permeability barrier is not known. Using techniques of semiquantitative reverse transcription-PCR (RT-PCR), immunoblotting, immunohistochemistry, and inhibitors against different kinases coupled with physiological techniques to assess the Sertoli cell TJ barrier function, it was shown that this TGFbeta3-induced effect on Sertoli cell TJ dynamics is mediated via the p38 mitogen-activated protein (MAP) kinase pathway. First, the assembly of the Sertoli cell-TJ barrier was shown to be associated with a transient but significant decline in both the TGFbeta3 production and expression by Sertoli cells. Furthermore, addition of TGFbeta3 to Sertoli cell cultures during TJ assembly indeed perturbed the TJ barrier with an IC50 at approximately 9 pM. Second, the TGFbeta3-induced disruption of the TJ barrier was associated with a transient induction in MEKK2 but not the other upstream signaling molecules that mediate TGFbeta3 action, such as Smad2, Cdc42, Rac2, and N-Ras, suggesting this effect might be mediated via the p38 MAP kinase pathway. This postulate was confirmed by the observation that TGFbeta3 also induced the protein level of the activated and phosphorylated form of p38 MAP kinase at the time the TJ barrier was perturbed. Third, and perhaps the most important of all, this TGFbeta3-mediated inhibitory effect on the TJ barrier and the TGFbeta3-induced p-p38 MAP kinase production could be blocked by SB202190, a specific p38 MAP kinase inhibitor, but not U0126, a specific MEK1/2 kinase inhibitor. These results thus unequivocally demonstrate that TGFbeta3 utilizes the p38 MAP kinase pathway to regulate Sertoli cell TJ dynamics.
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PMID:Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway. 1260 50

Constitutive activation of Ras or Ras-mediated signaling pathways is one of the initial steps during tumorigenesis that promotes neoplastic transformation. Recently it was reported that in Ha-Ras overexpressing MDCK cells the tight junction proteins claudin-1, occludin and ZO-1 were absent at cell-cell contact sites but present in the cytoplasm. Inhibition of MEK1 activity recruited all three proteins to the cell membrane leading to a restoration of the tight junction barrier function in MDCK cells. In order to evaluate the relevance of the MEK1 pathway in tight junction regulation in breast cancer cells, we investigated the effect ofMEK1 inhibition on expression of claudin-1, occludin and ZO-1 in natively claudin-1 expressing T47-D cells (low Ras activity), claudin-1 negative MCF-7 cells (elevated Ras activity) as well as two retroviral claudin-1 transduced MCF-7 daughter cell lines with prominent membrane and cytoplasmic claudin-1 dominant homing, respectively. Although we effectively blocked phosphorylation of MAPKs ERK-1 and ERK-2 using the selective MEK1 inhibitor PD98059, no quantitative changes of mRNA or protein levels of claudin-1, occludin and ZO-1 could be detected in all cell lines investigated. Furthermore, immnfluorescence analysis of claudin-1 revealed that inhibition of the MAPK pathway did not alter th e subcellular cytoplasmic distribution of claudin-1 to be more membrane specific. Finally, the diffusion barrier properties of tight junctions as analyzed by transepithelial resistance (TER) or paracellular flux analysis of 3 and 40 kDa dextran of tight junctions were not altered in the claudin-1 positive T47-D and the MCF-7 cell lines. Our findings indicate that the proposed involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated tight junction formation in breast tumor cells and indicates that elevated activity of Ras might not be of general importance for the disruption of tight junction structures in breast tumors.
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PMID:Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the Ras-MEK1 pathway. 1283 32

Activation of protein kinase C (PKC) by exposure of cultured human corneal epithelial cells to phorbol 12-myristate 13-acetate (PMA) resulted in an increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance (TER). A change in the membrane distribution of the tight junction protein ZO-1 was also observed in the PMA-treated cells. In contrast, when the cells were treated with PMA in the presence of PD98059, a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, all barrier characteristics were preserved, suggesting that PKC induces tight junction disruption through the activation of MAPK. The role of this signaling pathway in the regulation of epithelial permeability was further elucidated by the use of corneal epithelial-derived cell lines expressing constitutively activated (ca) or dominant-negative (dn) mutants of MAPK kinase-1 (MEK1). Transfectants of caMEK1, when compared to parental cells, had higher levels of phosphorylated extracellular regulated protein kinase (ERK), altered distribution of ZO-1 and occludin, and much reduced TER. On the other hand, dnMEK1 transfectants had lower but detectable levels of ERK phosphorylation, more flattened morphology, and, most importantly, significantly higher TER when compared to parental cells. Our study demonstrates that activation of PKC causes the disruption of tight junctions through activation of MAP kinase and that the MAP kinase signaling pathway plays a key role in the regulation of epithelial cell morphology and barrier function in the cornea.
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PMID:Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells. 1466 34

Mitogen-activated protein kinase kinase 2 (MEK2) was detected as an invasion-metastasis related factor between highly invasive (PC-1.0) and weakly invasive (PC-1) pancreatic cancer cell lines in our previous study. On the other hand, tight junction (TJ) was found to be correlated with carcino-genesis and tumor development. In this study, the expressions and correlation of TJ transmembrane protein occludin and MEK/extracellular signal-regulated kinase (ERK) signaling pathway were analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and human (AsPC-1 and CAPAN-2) pancreatic cancer cell lines were analyzed immunocytochemically with anti-occludin, phosphorylated MEK1/2 (p-MEK1/2), phosphorylated ERK1/2 (p-ERK1/2) antibodies. MEK1/2 inhibitor U0126 significantly induced the expression of occludin at the cell-cell junction and substantially suppressed the p-MEK1/2 and p-ERK1/2 expressions in PC-1.0 and AsPC-1 cells. In contrast, dissociation factor (DF) treatment obviously disrupted the occludin expressions at the sites of cell-cell junction and markedly induced the p-MEK1/2 and p-ERK1/2 expressions in PC-1 and CAPAN-2 cells. In addition, occludin expressions at cell-cell junction were restored and p-MEK1/2 and p-ERK1/2 expressions were suppressed by subsequent U0126-treatment in DF treated PC-1 and CAPAN-2 cells. Correspondingly, light microscopic images showed that DF induced the dissociation of cell island-like colonies in PC-1 and CAPAN-2 cells, and U0126-treatment induced cell aggregation in these pancreatic cancer cells. Occludin is involved in the cell dissociation in pancreatic cancer cells. Moreover, MEK/ERK signaling pathway probably regulates the cell dissociation status of pancreatic cancer through influencing the intracellular localization and expression of occludin.
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PMID:Analysis of invasion-metastasis mechanism in pancreatic cancer: involvement of tight junction transmembrane protein occludin and MEK/ERK signal transduction pathway in cancer cell dissociation. 1506 37

The epithelial to mesenchymal transition (EMT) is considered to be an important event during malignant tumor progression and metastasis. Although Raf/MEK/ERK signaling causes EMT, the mechanisms, including the signaling pathways, are as yet unclear. In the present study we have examined the effects of signal transduction pathways on oncogenic Raf-1-induced EMT, using an immortalized mouse hepatic cell line. Oncogenic Raf-1-induced EMT is characterized by down-regulation of adherens and tight junctions and the reorganization of actin. An active Raf-1 gene was introduced into a mouse hepatic cell line which was then treated with the MAP kinase inhibitor PD98059, the p38 MAP kinase inhibitor SB203580, the PI3 kinase inhibitor LY294002 or the c-Src tyrosine kinase inhibitor PP2. The expression and localization of the adherens and tight junction proteins E-cadherin, occludin, ZO-1, claudin-1 and claudin-2 were determined by western blotting, RT-PCR and immunocytochemistry. The barrier function of tight junctions was assessed by measurements of transepithelial electric resistance (TER) and permeability in terms of fluxes of [(14)C]mannitol and [(14)C]inulin. In Raf-1-transfected cells expression of occludin and claudin-2 was markedly down-regulated at the protein and mRNA levels and the TER value was decreased, while the permeability was increased. The distribution of ZO-1, pancadherin and F-actin was changed from linear to zipper-like structures at cell borders. In Raf-1-transfected cells treated with PD98059 and SB203580, but not LY294002, expression and localization of claudin-2, but not occludin, recovered, together with barrier function, measured as the TER value. The distributions of ZO-1, pancadherin and F-actin also recovered on treatment with PD98059 and SB203580, but not LY294002. Expression and localization of occludin recovered slightly on treatment with PP2. Thus, oncogenic Raf-1 regulates EMT via distinct MAP kinase, p38 MAP kinase and c-Src tyrosine kinase signal pathways in the mouse hepatic cell line.
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PMID:Oncogenic Raf-1 regulates epithelial to mesenchymal transition via distinct signal transduction pathways in an immortalized mouse hepatic cell line. 1530 85

Tight junctions as an epithelial barrier against paracellular diffusion have mainly been investigated on the protein level with particular respect to subcellular localization. In this study, real-time PCR has been established to investigate the influence of protein kinase C (PKC) modulation on the transcription of tight junction elements occludin and ZO-1 in the cell line T84. Activation of PKC by the phorbol ester TPA induced ZO-1 and occludin transcription, whereas PKC inhibition lead to decreased expression levels. Activation of PKC exerted its effect on transcript level directly. PKC signal was partially transduced via MEK1/MEK2 but depended strongly on MAPK independent pathways probably involving nuclear localized PKC, whereas p38 signaling was not implicated. TPA induced loss of function concomitant with a dislocation of ZO-1 and occludin could be prevented by inhibition of MEK1 by PD98059. Overall ZO-1 and occludin seem to be identically regulated in colonic epithelium on the transcript level.
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PMID:Influence of protein kinase C on transcription of the tight junction elements ZO-1 and occludin. 1562 22

Recent studies have shown that transforming growth factor (TGF)-beta3 regulates blood-testis barrier (BTB) dynamics in vivo, plausibly by determining the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB site via the p38 MAP kinase signaling pathway. Since BTB is composed of coexisting TJs and basal ectoplasmic specializations [ES, a testis-specific adherens junction (AJ) type] in the seminiferous epithelium of the rat testis, we sought to examine if TGF-beta3 would also regulate anchoring junction dynamics. Using an in vivo model in which rats were treated with AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb Sertoli-germ cell AJs without affecting the integrity of TJs at the BTB, it was noted that the event of germ cell loss from the epithelium was associated with a transient surge in TGF-beta3. Furthermore, it was also associated with a surge in the protein levels of Ras, p-ERK, and the intrinsic activity of ERK, illustrating TGF-beta3 apparently regulates Sertoli-germ cell ES function via the Ras/MEK/ERK signaling pathway. Indeed, pretreatment of rats with TbetaRII/Fc chimera, a TGF-beta antagonist, or U0126, a specific MEK inhibitor, could significantly delay and partially block the disruptive effects of AF-2364 in depleting germ cells from the epithelium. While the protein levels of the cadherin/catenin complex were significantly induced during AF-2364-mediated germ cell loss, perhaps being used to retain germ cells in the epithelium, this increase failed to reverse the loss of adhesion function between Sertoli and germ cells because of a loss of protein-protein interactions between cadherins and catenins. Collectively, these results illustrate that the testis has a novel mechanism in place in which an agent that primarily disrupts TJs can induce secondary loss of AJ function, leading to germ cell loss from the seminiferous epithelium. Yet an agent that selectively disrupts AJs (e.g., AF-2364) can limit its effects exclusively at the Sertoli-germ cell adhesive site without perturbing the Sertoli-Sertoli TJs.
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PMID:TGF-beta3 regulates anchoring junction dynamics in the seminiferous epithelium of the rat testis via the Ras/ERK signaling pathway: An in vivo study. 1588 76


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