Abstract
The kidneys represent one of the main targets of ochratoxin A (OTA), a secondary fungal metabolite that is produced by certain species ofAspergillus 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 μM 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 μM 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.
Footnotes
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Send reprint requests to: Herbert Schramek, M.D., Department of Physiology, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.
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↵1 This study was supported by the Austrian Science Foundation, Grant P11125-MED (to H.S.) and the Deutsche Forschungsgemeinschaft, Grant SI 170/7-2 (to M.G.).
- Abbreviations:
- MAPK
- mitogen-activated protein kinase
- ERK
- extracellular signal-regulated kinase
- JNK
- c-Jun NH2-terminal kinase
- MKK = MEK
- MAPK kinase = MAPK/ERK kinase
- MEKK
- MEK, kinase
- CA-MEK1
- consititutively active MEK1
- OTA
- ochratoxin A
- PMA
- phorbol 12-myristate 13-acetate
- MBP
- myelin basic protein
- MDCK cells
- Madin-Darby Canine kidney cells
- PC12
- adrenal pheochromyocytoma 12
- FCS
- fetal calf serum, PAGE, polyacrylamide gel electrophoresis
- MEM
- minimal essential medium
- PBS
- phosphate-buffered saline
- BSA
- bovine serum albumin
- LDH
- lactate dehydrogenase
- Received May 28, 1997.
- Accepted August 11, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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