OCHRATOXIN A-INDUCED STIMULATION OF EXTRACELLULAR-SIGNAL-REGULATED-KINASES-1 2 IS ASSOCIATED WITH MADIN-DARBY CANINE KIDNEY-C7 CELL DEDIFFERENTIATION/

The kidneys represent one of the main targets of ochratoxin A (OTA), a secondary fungal metabolite that is produced by certain species of As pergillus and Penicillium. OTA has the ability to disturb Madin-Darby canine kidney (MDCK) cell pH homeostasis, leading to intracellular alk alinization and morphological alterations resembling those that occur when MDCK cells are exposed to transient alkaline stress. Because alka li-induced epithelial dedifferentiation of MDCK-C7 cells is associated with an increase in the activity of extracellular signal-regulated ki nases (ERK), we performed experiments that investigated a possible rol e for ERK1 and ERK2 as intracellular signaling molecules mediating som e of the mycotoxin's effects on renal epithelia. We studied the effect s of OTA on ERK1/2 phosphorylation and activation, as well as on cell morphology by using cloned MDCK-CT and MDCK-CII cells, In MDCK-C7 cell s, but not in MDCK-C11 cells, OTA led to a time-dependent and concentr ation-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 mu M OTA significantly increased ERK1/2 activity in MDCK-C7 but not in MDCK-C11 cells. This OTA-stimulated ER K1/2 phosphorylation and ERK1/2 activation in MDCK-C7 cells was partia lly inhibited by the synthetic mitogen-activated protein kinase kinase (MKK or MEK) inhibitor PD098059. Transepithelial resistance and lacta te dehydrogenase release remained unaltered after incubation in the pr esence of ? mu M OTA for 8 hr or of 100 nM OTA for 24 hr, so ii is unl ikely that these OTA effects on ERK1/2 are due to secondary toxic effe cts of the mycotoxin. Interestingly, OTA-induced longterm activation o f ERK1/2 in MDCK-C7 cells was associated with epithelial dedifferentia tion, 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 activi ty in response to OTA, retained their epithelial phenotype under ident ical experimental conditions. Taken together, our data demonstrate an epithelial dedifferentiation of MDCK-C7 cells, but not of MDCK-CII cel ls, after long-term incubation in the presence of OTA, a result associ ated 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 activat ion of ERK1/2 could be an important intracellular signaling pathway th at mediates some of the mycotoxin's effects on renal epithelia.