title: Ochratoxin A-induced stimulation of extracellular signal-regulated kinases 1/2 is associated with Madin-Darby canine kidney-C7 cell dedifferentiation.
abstract: The kidneys represent one of the main targets of ochratoxin A (OTA), a secondary fungal metabolite that is produced by certain species of Aspergillus and Penicillium. OTA has the ability to disturb Madin-Darby canine kidney (MDCK) cell pH homeostasis, leading to intracellular alkalinization and morphological alterations resembling those that occur when MDCK cells are exposed to transient alkaline stress. Because alkali-induced epithelial dedifferentiation of MDCK-C7 cells is associated with an increase in the activity of extracellular signal-regulated kinases (ERK), we performed experiments that investigated a possible role for ERK1 and ERK2 as intracellular signaling molecules mediating some of the mycotoxin's effects on renal epithelia. We studied the effects of OTA on ERK1/2 phosphorylation and activation, as well as on cell morphology by using cloned MDCK-C7 and MDCK-C11 cells. In MDCK-C7 cells, but not in MDCK-C11 cells, OTA led to a time-dependent and concentration-dependent increase in ERK1/2 phosphorylation. OTA-induced ERK1/2 phosphorylation in MDCK-C7 cells occurred at concentrations of 500 nM, started after 2 hr and was maximal after 8 hr. Furthermore, after 8 hr of incubation, 500 nM and 1 microM OTA significantly increased ERK1/2 activity in MDCK-C7 but not in MDCK-C11 cells. This OTA-stimulated ERK1/2 phosphorylation and ERK1/2 activation in MDCK-C7 cells was partially inhibited by the synthetic mitogen-activated protein kinase kinase (MKK or MEK) inhibitor PD098059. Transepithelial resistance and lactate dehydrogenase release remained unaltered after incubation in the presence of 1 microM OTA for 8 hr or of 100 nM OTA for 24 hr, so it is unlikely that these OTA effects on ERK1/2 are due to secondary toxic effects of the mycotoxin. Interestingly, OTA-induced long-term activation of ERK1/2 in MDCK-C7 cells was associated with epithelial dedifferentiation, as assessed by analysis of vectorial solute and water transport as well as cell morphology. In contrast, MDCK-C11 cells, which do not show significant increases in ERK1/2 phosphorylation and ERK1/2 activity in response to OTA, retained their epithelial phenotype under identical experimental conditions. Taken together, our data demonstrate an epithelial dedifferentiation of MDCK-C7 cells, but not of MDCK-C11 cells, after long-term incubation in the presence of OTA, a result associated with the ability of this mycotoxin to stimulate ERK1/2 in MDCK-C7 cells but not in MDCK-C11 cells. We conclude that OTA-induced activation of ERK1/2 could be an important intracellular signaling pathway that mediates some of the mycotoxin's effects on renal epithelia.
authors.affiliation: Department of Physiology, University of Innsbruck, Innsbruck, Austria.
authors.forename: H
authors.initials: H
authors.lastname: Schramek
authors.forename: D
authors.initials: D
authors.lastname: Wilflingseder
authors.forename: V
authors.initials: V
authors.lastname: Pollack
authors.forename: R
authors.initials: R
authors.lastname: Freudinger
authors.forename: S
authors.initials: S
authors.lastname: Mildenberger
authors.forename: M
authors.initials: M
authors.lastname: Gekle
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects* / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000187:drug effects* / Q000201:enzymology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009183:Mycotoxins / Q000633:toxicity*; D009793:Ochratoxins / Q000633:toxicity*; D010766:Phosphorylation
chemical_list: D005419:Flavonoids; D009183:Mycotoxins; D009793:Ochratoxins; C025589:ochratoxin A; D004247:DNA; D007770:L-Lactate Dehydrogenase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one