Differential expression and activation of MAP kinases in dedifferentiated MDCK-focus cells

Schramek, Herbert; Schumacher, Marion; Wilflingseder, Doris; Oberleithner, Hans; Pfaller, Walter

doi:10.1152/ajpcell.1997.272.2.c383

Cited by 41 publications

(31 citation statements)

References 22 publications

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“…In contrast, JNK1 activity, which represents one member of the stress-activated protein kinase family of MAPKs, was slightly but consistently decreased in both quiescent and anisomycinstimulated MDCK-C7F cells (22). Furthermore, differential activation of ERK2 and JNK1 was accompanied by an inhibition of serum-induced MDCK-C7F cell proliferation (22). Thus, transient exposure of epithelial MDCK-C7 cells to alkaline stress leads to cell dedifferentiation and growth inhibition associated with increased basal and serum-stimulated ERK2 activation but decreased JNK1 activity.…”

mentioning

confidence: 86%

“…When subsequently cultured in standard medium (pH 7.4) MDCK-C7F cells maintained their altered phenotype (22,23). Utilizing this renal epithelial cell model, we obtained a substantially increased ERK2 activity in quiescent and serum-treated dedifferentiated MDCK-C7F cells as compared with their parental epithelial MDCK-C7 cells (22). In contrast, JNK1 activity, which represents one member of the stress-activated protein kinase family of MAPKs, was slightly but consistently decreased in both quiescent and anisomycinstimulated MDCK-C7F cells (22).…”

mentioning

confidence: 99%

“…We have recently reported a differential regulation of ERK1, ERK2, and c-Jun NH 2 -terminal kinase 1 (JNK1) activity in dedifferentiated MDCKC7Focus (MDCK-C7F) cells as compared with their parental epithelial MDCK-C7 cells (22). Cloned MDCK-C7 cells, when grown for 2 weeks in alkaline (pH 7.7) culture medium, dedifferentiate, exhibit a spindle-shaped morphology with long dendrite-like protrusions, and lack contact inhibition as well as monolayer formation (22,23). When subsequently cultured in standard medium (pH 7.4) MDCK-C7F cells maintained their altered phenotype (22,23).…”

mentioning

confidence: 99%

“…Cloned MDCK-C7 cells, when grown for 2 weeks in alkaline (pH 7.7) culture medium, dedifferentiate, exhibit a spindle-shaped morphology with long dendrite-like protrusions, and lack contact inhibition as well as monolayer formation (22,23). When subsequently cultured in standard medium (pH 7.4) MDCK-C7F cells maintained their altered phenotype (22,23). Utilizing this renal epithelial cell model, we obtained a substantially increased ERK2 activity in quiescent and serum-treated dedifferentiated MDCK-C7F cells as compared with their parental epithelial MDCK-C7 cells (22).…”

mentioning

confidence: 99%

“…Utilizing this renal epithelial cell model, we obtained a substantially increased ERK2 activity in quiescent and serum-treated dedifferentiated MDCK-C7F cells as compared with their parental epithelial MDCK-C7 cells (22). In contrast, JNK1 activity, which represents one member of the stress-activated protein kinase family of MAPKs, was slightly but consistently decreased in both quiescent and anisomycinstimulated MDCK-C7F cells (22). Furthermore, differential activation of ERK2 and JNK1 was accompanied by an inhibition of serum-induced MDCK-C7F cell proliferation (22).…”

mentioning

confidence: 99%

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Constitutively Active Mutant of the Mitogen-activated Protein Kinase Kinase MEK1 Induces Epithelial Dedifferentiation and Growth Inhibition in Madin-Darby Canine Kidney-C7 Cells

Schramek

Feifel

Healy³

et al. 1997

Journal of Biological Chemistry

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Overexpression of a constitutively active mitogen-activated protein kinase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the present study, we used a constitutively active MAPK/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the function of the highly conserved MEK1-ERK2 signaling module in renal epithelial cell differentiation and proliferation. Stable expression of constitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serumstimulated ERK1 and ERK2 phosphorylation as well as ERK2 activation when compared with mock-transfected cells. In both mock-transfected and CA-MEK1-transfected MDCK-C7 cells, basal and serum-stimulated ERK1 and ERK2 phosphorylation was almost abolished by the synthetic MEK inhibitor PD098059. Increased ERK2 activation due to stable expression of CA-MEK1 in MDCK-C7 cells was associated with epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increased vimentin expression. In addition, we obtained a delayed and reduced serum-stimulated cell proliferation in CA-MEK1-transfected cells (4.6-fold increase in cell number/cm 2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm 2 after 5 days). This result was confirmed by flow cytometric DNA analysis showing that stable expression of CA-MEK1 decreased the proportion of MDCK-C7 cells moving from G 0 /G 1 to G 2 /M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serumstimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells. Thus, the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation thereby leading to an attenuation of MDCK-C7 cell proliferation.Extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) 1 represent one subfamily of serine/threonine protein kinases collectively referred to as the mitogen-activated protein kinase (MAPK) family. They have the unique feature of being activated by phosphorylation on threonine and tyrosine residues by an upstream dual-specificity kinase called MAPK kinase (MAPKK or MKK) or MAPK/ERK kinase (MEK) (reviewed in Refs. 1 and 2). The MEKs upstream of ERKs constitute an evolutionary conserved family of protein kinases that includes at least three highly homologous mammalian isoforms, namely MEK1a, MEK1b, and MEK2 (2). They are highly specific for both of their downstream targets ERK1 and ERK2 (2) and are typically activated by serine/threonine phosphorylation catalyzed by three different classes of upstream kinases: the Raf family of serine/threonine kinases, Raf-1, A-Raf, and B-Raf (3-7), the protooncogene product Mos (8, 9), and the MEK kinase 1 (MEKK1) (10). Despite their high degree of similarity, MEK1a and MEK2...

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mentioning

confidence: 86%

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confidence: 99%

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confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Constitutively Active Mutant of the Mitogen-activated Protein Kinase Kinase MEK1 Induces Epithelial Dedifferentiation and Growth Inhibition in Madin-Darby Canine Kidney-C7 Cells

Schramek

Feifel

Healy³

et al. 1997

Journal of Biological Chemistry

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ERK1/2‐driven and MKP‐mediated inhibition of EGF‐induced ERK5 signaling in human proximal tubular cells

Sarközi

Miller

Pollack

et al. 2006

Journal Cellular Physiology

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The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1.

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Use of two-dimensional gel electrophoresis in predictive toxicology: Identification of potential early protein biomarkers in chemically induced hepatocarcinogenesis

Fella

Glückmann²,

Hellmann

et al. 2005

Proteomics

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Our current approach focused on the identification of potential early protein biomarker signatures which are indicative of the carcinogenic processes in rats exposed to 20 mg/kg of the liver carcinogen N-nitrosomorpholine (NNM). Treated liver was investigated at different timepoints. Therefore, proteins were separated by two-dimensional gel electrophoresis as a first step prior to identification of differentially expressed proteins by mass spectrometry. Proteomic analysis of liver samples after one day of exposure revealed significant upregulation of proteins involved in response to cellular stress induced by NNM (superoxide dismutase, heat shock protein 60, peroxiredoxin). Eighteen weeks after withdrawal of NNM, we were able to identify cancer-related proteins in rat liver bearing malignant, transformed cells (caspase-8 precursor, vimentin, Rho GDP dissociation inhibitor). Some of these proteins were already deregulated after three weeks of exposure indicating their potential usefulness as early predictive biomarkers for liver carcinogenicity (annexin A5, fructose-1,6-bisphosphatase). As regulatory toxicology approaches usually include the investigation of carcinogenicity in two-years studies in rodents, especially the detection of early protein biomarker signatures which precede the appearance of neoplasia, demonstrates the high potential of proteomics approaches to substantially reduce the time and costs of carcinogenicity testing.

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Differential expression and activation of MAP kinases in dedifferentiated MDCK-focus cells

Cited by 41 publications

References 22 publications

Constitutively Active Mutant of the Mitogen-activated Protein Kinase Kinase MEK1 Induces Epithelial Dedifferentiation and Growth Inhibition in Madin-Darby Canine Kidney-C7 Cells

Constitutively Active Mutant of the Mitogen-activated Protein Kinase Kinase MEK1 Induces Epithelial Dedifferentiation and Growth Inhibition in Madin-Darby Canine Kidney-C7 Cells

ERK1/2‐driven and MKP‐mediated inhibition of EGF‐induced ERK5 signaling in human proximal tubular cells

Use of two-dimensional gel electrophoresis in predictive toxicology: Identification of potential early protein biomarkers in chemically induced hepatocarcinogenesis

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