Introduction

Abstract Introduction Materials & Methods Results Discussion References

OTA is a secondary fungal metabolite produced by certain species of Aspergillus and Penicillium. The kidneys represent one of the main targets of this toxin, which has been detected in improperly stored animal chows and human food and thus in up to 80% of human blood samples of several Western countries (for recent reviews, see Gekle and Silbernagl, 1996; Simon, 1996). Epidemiologic studies revealed that in areas where high OTA levels are reached in food and in the blood of the population, a high incidence of nephropathy and renal tumors exists (Simon, 1996). OTA seems to be involved in the pathogenesis of Balkan endemic nephropathy (Petkova-Bocharova and Castegnaro, 1985; Kuiper-Goodman and Scott, 1989), certain forms of interstitial nephritis (Mihatsch et al., 1979; Maaroufi et al., 1995a; Maaroufi et al., 1995b; Spoendlin et al., 1995; Godin et al., 1995) and acute renal failure (Di Paolo et al., 1994). In addition, this toxic fungal metabolite has been reported to increase the incidence of renal carcinomas and adenomas in rats (Bendele et al., 1985; Boorman, 1989), and there is increasing evidence that Balkan endemic nephropathy is associated with a dramatic increase in urinary tract tumors (Castegnaro et al., 1989). Thus, the hazard that OTA poses to human health arises from both its widespread occurrence and the resulting high risk of exposure.

Depending on the duration and concentration of exposure, OTA affects different parts of the nephron, from glomeruli to collecting ducts (Gekle and Silbernagl, 1996). Investigations on the interaction of OTA with the growth of proximal tubular cells in primary culture revealed a rapid proliferative effect of the toxin under certain conditions (Gekle et al., 1995). However, the proximal tubule is only one among several renal targets, such as postproximal parts of the nephron, which seem to be affected mainly during short-term exposure (Gekle et al., 1993). In MDCK cells, which resemble collecting duct cells (Valentich, 1981), for example, OTA has been shown to inhibit the synthesis of macromolecules at concentrations of 10 µM or higher (Creppy et al., 1986). At nanomolar concentrations, OTA has been reported to inhibit dome formation of MDCK cell monolayers as early as 2 hr after administration of the toxin (Gekle et al., 1993). Moreover, OTA has the ability to disturb MDCK cell pH homeostasis via the inhibition of an anion conductance, leading to intracellular alkalinization associated with morphological alterations resembling those that occur when MDCK cells are exposed to transient alkaline stress (Gekle et al., 1994b).

MAPKs are serine/threonine kinases activated by dual phosphorylation on both a tyrosine and a threonine (for recent reviews, see Seger and Krebs, 1995; Cobb and Goldsmith, 1995; Bokemeyer et al., 1996; Robinson and Cobb, 1997). These enzymes have emerged as components of one of the most important membrane-to-nucleus signaling pathways in eukaryotes. A MAPK cascade consists of a module of at least three kinases: a MAPK kinase kinase (MAPKKK), which phosphorylates and activates a dual-specificity MAPK kinase (MAPKK), which in turn phosphorylates and activates a MAPK. The classical and best-studied MAPK cascade consists of Raf kinase (as a MAPKKK), MEK1 or MEK2 (as MAPKK) and ERK1 or ERK2 (the respective MAPK). The Raf/MEK/ERK signaling cascade is activated in response to a variety of mitogenic stimuli operating through different mechanisms, e.g., receptor tyrosine kinases, certain G protein-coupled receptors or cytokine receptors (Cobb and Goldsmith, 1995). Recently, several additional MAPK cascades have been characterized (for review, see Robinson and Cobb, 1997). One of them consists of MEKK, MKK4 and JNK. Unlike the classical MAPK pathway, the MEKK/MKK4/JNK module is only modestly activated by growth factors and phorbol esters but is instead strongly activated by cellular stresses, including heat shock, UV irradiation, protein synthesis inhibitors and inflammatory cytokines (Kyriakis et al., 1994).

We have recently reported a differential regulation of ERK1, ERK2 and JNK1 activity in dedifferentiated MDCK-C7Focus (MDCK-C7F) cells as compared with their parental epithelial MDCK-C7 cells and have suggested that MAPK could be involved in the regulation of renal epithelial MDCK cell phenotype (Schramek et al., 1997a). Utilizing this renal epithelial cell model, we obtained a substantially increased ERK2 activity but a slightly decreased JNK1 activity in both quiescent and agonist-treated dedifferentiated MDCK-C7F cells as compared with epithelial MDCK-C7 cells (Schramek et al., 1997a). Differential activation of ERK2 and JNK1 was accompanied by an inhibition of serum-induced MDCK-C7F cell proliferation (Schramek et al., 1997a). Furthermore, overexpression of constitutively active MEK1 in epithelial MDCK-C7 cells resulted in cell dedifferentiation and growth inhibition (Schramek et al., 1997b).

Despite of all the evidence pointing toward an important pathophysiological role of OTA in kidneys, studies elucidating possible intracellular signaling mechanisms involved in these effects have yet to be performed. On the basis of observation that OTA induces morphological alterations resembling those that occur when MDCK cells are exposed to transient alkaline stress (Gekle et al., 1994b), and on the basis of evidence suggesting a negative regulatory function of the MEK1-ERK2 signaling module in epithelial differentiation (Schramek et al., 1997a; Schramek et al., 1997b), we hypothesized a role for extracellular signal-regulated protein kinases as intracellular signaling molecules that mediate some of the mycotoxin's effects on renal epithelia. In the present study, we therefore investigated the effects of OTA on ERK1/2 phosphorylation, on ERK1/2 activation and on cell morphology, utilizing cloned MDCK-C7 and MDCK-C11 cells. Here we demonstrate an association of OTA-induced ERK1/2 activation with epithelial dedifferentiation after long-term incubation of MDCK-C7 cells in the presence of this mycotoxin. OTA-induced stimulation of ERK1/2 could thus be an important intracellular signaling pathway mediating some of the mycotoxin's effects on renal epithelia.

	Materials and Methods

Abstract Introduction Materials & Methods Results Discussion References

Cell culture. Experiments were carried out using cloned MDCK-C7 cells and MDCK-C11 cells (American Type Culture Collection) grown on plastic Petri dishes in MEM (Sigma) with Earl's salts, nonessential amino acids and L-glutamine at a pH of 7.4 (Gekle et al., 1994a; Schramek et al., 1997a). MEM was supplemented with 10% FCS and 26 mM NaHCO₃ (Sigma). Cells were grown at 37°C in a 5% CO₂-95% air humidified atmosphere and split in a ratio 1:10, two times a week (MDCK-C7) or once a week (MDCK-C11). For measurement of ERK1/2 phosphorylation and ERK1/2 activation, cells were grown to a subconfluent state in the presence of 10% FCS, then washed once with FCS-free media and finally made quiescent by 24 hr of incubation in FCS-free media before they were used for stimulation experiments. For analysis of [³H]thymidine incorporation, LDH release, transepithelial resistance, dome formation and cell counts, confluent MDCK-C7 and MDCK-C11 cell monolayers were used. Again, stimulation with OTA was performed in the absence of FCS.

Western blot analysis. MDCK-C7 and MDCK-C11 cells were washed three times with ice-cold PBS and lysed in ice-cold Triton X-100 lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM -glycerophosphate, 200 µM sodium-orthovanadate, 0.1 mM phenylmethyl-sulfonyl fluoride, 1 µg/ml leupeptin, 1 µM pepstatin A, 1% (Triton X-100) for 25 min at 4°C. Insoluble material was removed by centrifugation at 12,000 × g for 15 min at 4°C. The protein content was determined using a micro-bicinchononic acid assay (Pierce, Rockford, IL) with BSA as standard. Cell lysates were matched for protein, separated on SDS-12%PAGE and transferred to a polyvinylidene difluoride (PVDF) microporous membrane. Subsequently, membranes were blotted with an anti-P-ERK1,2 antibody, which detects phosphorylated tyrosine 204 of both ERK1 and ERK2 (New England Biolabs, Beverly, MA) (Schramek et al., 1996). The primary antibody was detected using alkaline phosphatase-conjugated goat anti-rabbit IgG visualized by Phototope Chemiluminescent Western Detection System (New England Biolabs).

Immunoprecipitation. MDCK-C7 and MDCK-C11 cells were washed three times with ice-cold PBS and lysed in ice-cold Triton X-100 lysis buffer for 25 min at 4°C. Insoluble material was removed by centrifugation at 12,000 × g for 15 min at 4°C. The protein content was determined using a micro-bicinchononic acid assay with BSA as standard. Cell lysates were matched for protein and precleared with 2 µl of preimmune serum preadsorbed to 50 µl of protein A-Sepharose-coated beads for 1 hr at 4°C. The precleared supernates were further incubated overnight with 2 µl of a polyclonal antibody recognizing ERK1 and ERK2 (generous gift of M. J. Dunn, Milwaukee) (Wang et al., 1992) preadsorbed to protein A-sepharose. Immunocomplexes were then used to measure ERK1/2 activity.

ERK1/2 activity assay. For measurement of ERK1/2 activity, the respective immunocomplexes were collected by centrifugation, were washed four times with a washing buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EGTA, 0.5% Triton X-100) and once with kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl₂, 1 mM dithiothreitol, and 10 mM p-nitrophenylphosphate) and were resuspended in a final volume of 40 µl kinase buffer containing 0.25 mg/ml MBP, 50 µM ATP and 10 µCi [-³²P]ATP. The reaction was initiated by incubation at 30°C and continued for 10 min. Thereafter, 40 µl of 2 × Laemmli sample buffer was added to terminate the reaction. Samples were then boiled for 3 min and subjected to SDS-12%PAGE. The gels were stained in Coomassie brilliant blue, dried and exposed for 1 to 2 hr to Amersham Hyperfilm MP at 70°C with intensifying screens. In addition, kinase activity was determined by cutting the MBP bands and measuring the radioactivity in a liquid scintillation counter (TRI-CARB 2700TR, Packard, Downers Grove, IL).

[³H]Thymidine incorporation and cell counts. Tritiated thymidine incorporation was used as an index of DNA synthesis. The cells were exposed to 0.15 MBq of [methyl-³H]thymidine (37 MBq/ml, 185 GBq/mmol; Amersham, Buckinghamshire, UK) for 20 hr in serum-free media with or without OTA. At the end of the labeling period, the monolayers were rapidly rinsed three times with ice-cold PBS. Cells were solubilized in 2% sodium dodecyl sulfate and precipitated with 10% ice-cold trichloroacetic acid. The pellets were placed in scintillation fluid and counted in a liquid scintillation counter (1215 Rackbeta II, LKB Wallac, Turku, Finland). After dislodgement with trypsin, the cell number was determined in a standard hemacytometer.

LDH release. LDH activity in the media and in the cell lysate was determined according to Schmitt (1974) at room temperature in a photometer (UV VIS Spektrol, Zeiss, Jena, Germany). LDH release represents the percentage of LDH activity in the media as compared to total LDH activity.

Transepithelial resistance and dome formation. For the investigation of transepithelial resistance (R_te), cells were seeded near density on permeable supports (Falcon, Heidelberg, Germany) with an area of 4.9 cm² as described previously (Gekle et al., 1994a). R_te was measured directly in the 6-well dishes using a voltohmmeter (EVOM, WPI, Sarasota, CA). The measurements were performed immediately after removing the cells from the incubator. Input resistance of the filter alone was 100 . Dome formation was evaluated as described previously (Gekle et al., 1993). Briefly, dome formation is due to vectorial transport of salt and water from the apical to the basolateral side of the monolayer and serves as an indirect measure for transepithelial reabsorption. Because vectorial transport is an unique feature of differentiated epithelial cells, determination of dome formation and its comparison with both R_te and LDH release yield a valuable measure for the degree of cell differentiation/dedifferentiation.

Measurements of intracellular pH. Intracellular pH of single cells was determined using the pH-sensitive dye BCECF (27-µbis-(2-carboxyethyl)-5,6-carboxyfluorescein) as described elsewhere (Gekle et al., 1994b). In brief, cells were incubated with MEM containing BCECF in a final concentration of 3 µmol/l for 15 min. Thereafter, the coverslips were rinsed four times with superfusion solution to remove the BCECF-containing medium and transferred to the stage of an inverted IM 100 Zeiss microscope (magnification ×400, oil immersion). The excitation light source was a 100-W mercury lamp. The excitation wavelengths were 436 nm and 488 nm, and the emitted light was filtered through a bandpass-filter (515-565 nm). The data acquisition rate was one fluorescence intensity ratio (488/436 nm) every 2 sec. Images were digitized on-line using video-imaging software (Attofluor, Zeiss, Oberkochen, Germany). After background subtraction, fluorescence intensity ratios (488/436 nm) were calculated. Calibration was performed after each experiment by the nigericin technique, using at least three calibration solutions in the range from pH 6.8 to 7.8 (Thomas et al., 1979). The superfusion solution during measurements of intracellular pH was composed of (mmol/l): 100 NaCl, 24 NaHCO₃, 5.4 KCl, 1.2 CaCl₂, 1 NaH₂PO₄/Na₂HPO₄, 0.8 MgCl₂, 5.5 D-glucose; gassed with 5% CO₂ to a pH of 7.4 at 37°C.

Materials. The following drugs and chemicals were used in this study: leupeptin and pepstatin A (Peptide Institute Inc., Osaka, Japan), PD098059 (Calbiochem-Novabiochem, Nottingham, UK), phorbol 12-myristate 13-acetate (PMA) (GIBCO/BRL, Gaithersburg, MD), cell culture media (Sigma Chemical Co., St. Louis, MO), [-³²P]ATP (5500 Ci/mmol) (New England Nuclear, Vienna, Austria), OTA, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), nigericin, phenylmethylsulfonylfluoride (PMSF), sodium orthovanadate, dithiothreitol (DTT), MBP, EDTA, EGTA and all other reagents (Sigma Chemical Co., St. Louis, MO), protein A-sepharose (Pharmacia Biotech. Inc., Piscataway, NJ) and BCECF (Molecular Probes, Eugene, OR).

Statistics. Statistical data are presented as mean ± S.E.M. An unpaired Student's t test was used to test for statistical significance. A probability of .05 or less was deemed statistically significant.

	Results

Abstract Introduction Materials & Methods Results Discussion References

OTA-induced ERK1/2 phosphorylation and ERK1/2 activation in MDCK-C7 as compared with MDCK-C11 cells. Utilizing an antibody that detects only phosphorylated tyrosine 204 of both ERK1 and ERK2, we first investigated the phosphorylation of both kinases after stimulation with OTA in the C7 subtype and the C11 subtype of MDCK cells, which resemble principal cells (PC) and intercalated cells (IC) of the renal collecting duct, respectively. As depicted in figure 1, OTA (1 µM) time-dependently increased ERK1/2 phosphorylation in PC-like MDCK-C7 cells but not in IC-like MDCK-C11 cells. OTA-induced ERK1/2 phosphorylation in MDCK-C7 cells started after 2 hr, was maximal after 8 hr and remained elevated for at least 16 hr (fig. 1A). This OTA-induced phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2 in MDCK-C7 cells was concentration-dependent (fig. 1B). Although concentrations of up to 100 nM OTA did not alter ERK1/2 phosphorylation after 8 hr of incubation, both 500 nM and 1 µM OTA led to a clear increase in the phosphorylation of both ERK1 and ERK2. After short-term incubation, 1 µM OTA did not stimulate ERK1/2 phosphorylation in either MDCK-C7 cells (fig. 1C) or MDCK-C11 cells (data not shown). ERK1/2 enzymatic activity measurements in MDCK-C7 cells revealed a 2.2-fold stimulation of ERK1/2 activity at a concentration of 500 nM OTA (after 8 hr of incubation) and a 3.2-fold ERK1/2 stimulation at a concentration of 1 µM OTA (fig. 2). This OTA-induced ERK1/2 activation after 8 hr was comparable in size to that obtained after incubation of MDCK-C7 cells for 10 min in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA) (3.1-fold, fig. 2, lane 5). Concentrations of 10 nM OTA (data not shown) and 100 nM OTA (fig. 2), when administered for 8 hr to quiescent MDCK-C7 cells, had no effect on ERK1/2 enzymatic activity when compared with untreated controls. In MDCK-C11 cells, on the other hand, OTA did not stimulate ERK1/2 after 8 hr of stimulation at any of the concentrations used (fig. 2). Interestingly, 100 nM PMA, when incubated for 10 min, did not activate ERK1/2 in these cells either, which suggests that these cells lack OTA- and PMA-inducible ERK1/2.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Time-dependent and concentration-dependent OTA-induced ERK1/2 phosphorylation in MDCK-C7 cells. Subconfluent MDCK-C7 and MDCK-C11 cells were made quiescent for 24 hr as described in "Materials and Methods" and stimulated with 1 µM OTA for different periods of time (2 hr, 4 hr, 8 hr and 16 hr) (panel A), with different OTA concentrations (10 nM, 50 nM, 100 nM, 500 nM and 1 µM) for 8 hr (panel B) or with 1 µM OTA for 10 min, 20 min or 40 min (panel C) and compared with untreated controls. Phosphorylation of ERKs was determined by Western blot analysis using an anti-P-ERK antibody, which detects phosphorylated tyrosine 204 of both ERK1 and ERK2 (for details, see "Materials and Methods"). Unphosphorylated ERK2 protein served as a negative control, and phosphorylated ERK2 protein was utilized as a positive control (data not shown). Asterisks indicate the phosphorylated ERK isoforms (ERK1* and ERK2*, respectively). One representative Western blot of four separate experiments is shown.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent OTA-induced ERK1/2 activation in MDCK-C7 cells. Subconfluent MDCK-C7 and MDCK-C11 cells were made quiescent for 24 hr as described in "Materials and Methods" and stimulated with different OTA concentrations (100 nM, 500 nM and 1 µM) for 8 hr or with 100 nM PMA for 10 min and compared with untreated controls. ERK1/2 activity was determined in these cell lysates by immunoprecipating ERK1/2 and measuring its ability to phosphorylate myelin basic protein (MBP) as a substrate. In addition to the autoradiogram (upper panel), kinase activity has been quantified by cutting the MBP bands and measuring the radioactivity in a liquid scintillation counter (lower panel). The results from one representative ERK1/2 activity assay of four separate experiments are shown.

Effect of PD098059 on OTA-induced ERK1/2 phosphorylation and ERK1/2 activation. PD098059, a synthetic inhibitor of the mitogen-activated protein kinase cascade, has been reported to inhibit selectively the ERK activator MEK, without significant inhibitory activity on ERK itself (Dudley et al., 1995). Inhibition of MEK by PD098059 prevented activation of ERK and subsequent phosphorylation of ERK substrates both in vitro and in intact Swiss 3T3 fibroblasts (Dudley et al., 1995). In addition, PD098059 completely blocked NGF-induced neurite formation in PC-12 cells without altering cell viability (Pang et al., 1995). As shown in figure 3A, preincubation of MDCK-C7 cells for 30 min with 50 µM PD098059 reduced basal as well as OTA-induced ERK1/2 phosphorylation when compared with untreated controls. A partial inhibition of OTA-stimulated ERK1/2 phosphorylation was also observed when PD098059 was used at concentrations as high as 100 µM (data not shown). Furthermore, a reduction of OTA-induced ERK1/2 activation was obtained when MDCK-C7 cells were stimulated with the mycotoxin in the presence of 50 µM PD098059 (30 min of preincubation) (fig. 3B).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   OTA-stimulated ERK1/2 phosphorylation (panel A) and ERK1/2 activation (panel B) in the absence and presence of the synthetic MEK inhibitor PD098059. A) Subconfluent MDCK-C7 cells were made quiescent for 24 hr as described in "Materials and Methods" and stimulated for 8 hr with 1 µM OTA, either in the absence (lane 2) or in the presence of 50 µM PD098059 (lane 4) and compared with unstimulated controls (lane 1). Lane 3 shows basal ERK1/2 phosphorylation from quiescent MDCK-C7 cells, which were treated for 8 hr with 50 µM PD098059 only. Phosphorylation of ERKs was determined by Western blot analysis using an anti-P-ERK antibody, which detects phosphorylated tyrosine 204 of both ERK1 and ERK2 (for details, see "Materials and Methods"). Unphosphorylated ERK2 protein served as a negative control, and phosphorylated ERK2 protein was utilized as a positive control (data not shown). Asterisks indicate the phosphorylated ERK isoforms (ERK1* and ERK2*, respectively). One representative Western blot of four separate experiments is shown. B) Subconfluent MDCK-C7 cells were made quiescent for 24 hr as described in "Materials and Methods" and stimulated for 8 hr with 1 µM OTA, either in the absence (lane 2) or in the presence of 50 µM PD098059 (lane 3) and compared with unstimulated controls (lane 1). ERK1/2 activity was determined in these cell lysates by immunoprecipitating ERK1/2 and measuring its ability to phosphorylate myelin basic protein (MBP) as a substrate. The results from one representative ERK1/2 activity assay of three separate experiments are shown.

Effects of OTA on transepithelial resistance, LDH release and dome formation. Measurements of transepithelial resistance (R_te) in both MDCK-C7 and MDCK-C11 cells revealed that OTA, when used at a concentration of 1 µM, did not alter R_te during the first 8 hr of incubation (data not shown). Incubation of either MDCK-C7 or MDCK-C11 cells in the presence of 1 µM OTA for 24 hr, however, led to a significant decrease in R_te (fig. 4A). In contrast, nanomolar OTA concentrations did not alter R_te for up to 24 hr. Thus in both cell types, monolayer integrity is reduced neither after 8 hr nor after 24 hr of incubation in the presence of micromolar and nanomolar OTA concentrations, respectively. After 24 hr of incubation, LDH release, which represents a general measure of cell damage, was increased in MDCK-C7 and MDCK-C11 cells at 1 µM OTA only, whereas nanomolar OTA concentrations did not have any effect (fig. 4B). However, the increase in LDH release induced by 1 µM OTA was significantly higher in MDCK-C7 cells (32 ± 2%; n = 8) than in MDCK-C11 cells (10 ± 1%; n = 8). Moreover, in MDCK-C7 cells but not in MDCK-C11 cells, OTA significantly reduced the number of domes at concentrations as low as 100 nM (fig. 4C). Although 100 nM OTA had no effect on the number of domes in MDCK-C11 monolayers, 1 µM OTA significantly reduced dome formation after 24 hr to 10 ± 2% of control (40 domes/cm²; n = 6). In contrast to MDCK-C7 cells, where reduction of dome number was obtained as early as 2 hr after addition of 100 nM OTA (fig. 4D), even 1 µM OTA did not affect dome number for as long as 8 hr in MDCK-C11 cells (data not shown). Because neither LDH release nor R_te was altered at 100 nM OTA (fig. 4, A and B), this OTA effect on dome formation in MDCK-C7 cells cannot be explained by cell damage. Furthermore, comparison of the effect of 1 µM OTA on R_te and dome number in MDCK-C7 cells revealed that the reduction in vectorial transport cannot be explained by loss of epithelial tightness. Whereas dome formation was virtually abolished, R_te was still in the range of extremely tight epithelia (3000  × cm²) (fig. 4, A and B).

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Effect of 24-h exposure to different OTA concentrations on transepithelial resistance (Rte) (panel A), LDH release (panel B) and number of domes per square centimeter (panel C) in MDCK-C7 and MDCK-C11 monolayers. D) Time course of the effect of OTA exposure on the number of domes in MDCK-C7 monolayers. The experiments were performed in serum-free media to prevent a reduction of the free OTA concentration as a result of binding to serum proteins. All values given are arithmetic means ± S.E. of n = 8 independent experiments. * Significantly different from untreated controls (= 0 µmol/l OTA) (P < .05).

Cell number and DNA synthesis. In both MDCK-C7 and MDCK-C11 cells, long-term incubation of OTA (24 hr) affected neither cell number nor [³H]thymidine incorporation when OTA was used at concentrations of 100 nM or lower (fig. 5). In both cell types, however, cell number (fig. 5A) and DNA synthesis (fig. 5B) were significantly reduced when 1 µM OTA was administered for 24 hr.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effect of 24 hr exposure to different OTA concentrations on cell number (panel A) and DNA synthesis ([3H]thymidine incorporation) (panel B) in MDCK-C7 and MDCK-C11 monolayers. The experiments were performed in serum-free media to prevent a reduction of the free OTA concentration as a result of binding to serum proteins. All values given are arithmetic means ± S.E. of n = 8 independent experiments. * Significantly different from untreated controls (= 0 µmol/l OTA) (P < .05).

Differential effect of OTA on MDCK-C7 and MDCK-C11 cell morphology. Figure 6 shows the differential effect of 1 µM OTA after long-term incubation (24 hr) of MDCK-C7 cells and MDCK-C11 cells. Although both cell types showed typical epithelial morphology in the absence of OTA (fig. 6, A and C), 24-hr exposure of MDCK-C7 cells to 1 µM OTA induced phenotypical alterations (fig. 6B) resembling those seen after transient exposure to alkaline stress (Wünsch et al., 1995). Much like alkali-dedifferentiated MDCK-C7F cells (Wünsch et al., 1995), OTA-treated MDCK-C7 cells are pleiomorphic and exhibit a spindle-shaped morphology (fig. 6B). In contrast to MDCK-C7 cells, however, incubation of MDCK-C11 cells for 24 hr in the presence of 1 µM OTA did not alter the morphology of the C11 cell clone (fig. 6D), although desintegration of the monolayers with cell detachment due to toxic cell death was observed in certain areas.

View larger version (153K): [in this window] [in a new window]   Fig. 6.   Photomicrographs of MDCK-C7 cells (panels A, B). and MDCK-C11 cells (panels C, D) under control conditions (panels A, C) and after 24-hr exposure to 106 mol/l OTA (panels B, C). OTA-treated MDCK-C7 cells show phenotypical alterations, which are typical for dedifferentiation of renal epithelial cells and comparable to those seen in MDCK-C7 cells transiently exposed to alkaline stress as well as in CA-MEK1-transfected MDCK-C7 cells (for details, see "Results").

Effect of OTA on intracellular pH in MDCK-C7 and MDCK-C11 cells. Measurements of intracellular pH (pH_i) in MDCK-C7 and MDCK-C11 cell clones revealed overall control pH_i values of 7.23 ± 0.01 (n = 174) and 7.38 ± 0.01 (n = 130; P < .05 vs. MDCK-C11 cells), respectively. MDCK-C11 cells responded to 1 µM OTA with a significant cytoplasmic alkalinization of +0.11 ± 0.02 pH units (n = 100). The onset of alkalinization after addition of OTA was within 30 to 60 sec. The new plateau value was reached after 3 to 4 min and remained constant during the period of observation (20 min). In contrast, administration of 1 µM OTA to MDCK-C7 cells led to only a slight acidification of 0.03 ± 0.01 pH-units (n = 130; P < .05). In addition, utilizing 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 100 µM), a well-known inhibitor of HCO₃/Cl exchange, we tested the sensitivity of cellular pH in both MDCK cell clones. MDCK-C11 cells responded to DIDS with significant larger pH_i changes (+0.13 ± 0.02 pH units, n = 49) than did the MDCK-C7 subtype (+0.04 ± 0.01 pH units, n = 40), which suggests that the HCO₃/Cl exchange activity was higher in the MDCK-C11 cells than in the MDCK-C7 cells.

	Discussion

Abstract Introduction Materials & Methods Results Discussion References

Despite much evidence pointing toward an important role for OTA in the pathogenesis of certain forms of nephropathy and renal tumors, studies elucidating possible intracellular signaling mechanisms involved in these effects have not yet been performed. Accordingly, we investigated two members of the MAPK family of protein kinases, ERK1 and ERK2, as potential intracellular signaling molecules mediating some of the mycotoxin's cellular effects. Our present study provides evidence that long-term incubation (2 hr) of cloned MDCK-C7 cells, but not MDCK-C11 cells, in the presence of OTA induces a time- and concentration-dependent phosphorylation and activation of both ERK1 and ERK2. After short-term incubation, however, OTA phosphorylates ERK1/2 in neither MDCK-C7 nor MDCK-C11 cells. Furthermore, OTA-induced long-term activation of ERK1/2 in MDCK-C7 cells is associated with epithelial dedifferentiation, whereas MDCK-C11 cells, which do not show increases in ERK1/2 phosphorylation or ERK1/2 activity in response to OTA, retain their epithelial phenotype under identical experimental conditions.

Incubation of wild-type MDCK cells for 3 to 5 days in the presence of OTA has been reported to induce an irreversible alteration of cell morphology, which resembles the dedifferentiation obtained in cloned MDCK-C7 cells after a transient alkaline stress (Gekle et al., 1994b). MDCK-C7 cells, when grown for two weeks in alkaline (pH 7.7) culture media, dedifferentiate, exhibit a spindle-shaped morphology with long, dendrite-like protrusions and lack contract inhibition and monolayer formation (Wünsch et al., 1995). When subsequently cultured in standard medium (pH 7.4), dedifferentiated MDCK-C7 cells (now called MDCK-C7Focus or MDCK-C7F cells) maintained their altered phenotype (Wünsch et al., 1995). Utilizing this renal epithelial cell model, we recently reported a substantially increased ERK2 activity in quiescent and serum-treated dedifferentiated MDCK-C7F cells as compared with their parental epithelial MDCK-C7 cells (Schramek et al., 1997a). In contrast to ERK2 activity, that of JNK1, which represents one member of the stress-activated protein kinase family of MAPK, was slightly but consistently decreased in both quiescent and anisomycin-stimulated MDCK-C7F cells (Schramek et al., 1997a). Differential activation of ERK2 and JNK1 was associated not only with epithelial dedifferentiation but also with an inhibition of serum-induced MDCK-C7F cell proliferation, which suggests that ERKs and JNKs could be involved in the regulation of renal epithelial MDCK cell phenotype and growth (Schramek et al., 1997a). Constitutively active as well as dominant negative MEK mutants have been utilized by different laboratories to study the function of the highly conserved MEK-ERK module in cell proliferation and differentiation: expression of constitutively active MEK mutants, generated by substitution of the Raf1-dependent regulatory phosphorylation sites serine 218 and serine 222 by aspartic acid or glutamic acid, has been reported to transform fibroblasts and to induce tumor formation in nude mice (Brunet et al., 1994; Cowley et al., 1994; Mansour et al., 1994). In contrast to these experiments performed in fibroblasts, a constitutively active MEK1 mutant stimulated PC12 cell differentiation, whereas interfering mutants of MEK1 inhibited ligand-induced neurite outgrowth (Cowley et al., 1994). Thus, constitutive activation of the only known ERK activator MEK leads to transformation of fibroblasts but to differentiation of PC12 cells, which suggests that cell type-specific differences in the function of the MEK-ERK signaling module exist. Using a constitutively active MEK1 (CA-MEK1) mutant and a stable transfection approach, we were recently able to demonstrate an association of increased basal and serum-stimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells (Schramek et al., 1997b). These results support the idea that the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation in these cells, thereby leading to an attenuation of MDCK-C7 cell proliferation (Schramek et al., 1997b).

In accordance with these data, we now show that, in parallel to its stimulating effects on ERK1/2, the mycotoxin OTA is able to induce phenotypic alterations in MDCK-C7 cells (fig. 6). At 24 hr after administration of 1 µM OTA, MDCK-C7 cells show a pleiomorphic phenotype and exhibit a spindle-shaped morphology (fig. 6B). These phenotypic alterations are typical for renal epithelial cell dedifferentiation and resemble those that have been obtained in MDCK wild-type cells and MDCK-C7 cells transiently exposed to alkaline stress or OTA (Oberleithner et al., 1991; Gekle et al., 1994b; Wünsch et al., 1995) as well as in MDCK-C7 cells that express CA-MEK1 (Schramek et al., 1997b). In contrast, MDCK-C11 cells, which did not show an OTA-induced ERK1/2 activation, did not dedifferentiate in response to OTA administration (fig. 6, C and 6D). The fact that nanomolar OTA concentrations did not affect dome formation in MDCK-C11 cells further indicates the lack of an OTA-induced effect on cell dedifferentiation in the C11 cell clone. Dome (hemicyst) formation is known to be due to vectorial transport of salt and water from the apical to the basolateral side of the epithelial monolayer and to depend on both epithelial tightness and transport activity (Gekle et al., 1993). If the number of domes decreases without measurable changes in R_te or LDH release, then it is unlikely that this effect is due to toxic cell damage rather than due to a reduction in vectorial solute transport across the epithelial monolayer. Because vectorial solute transport is a typical feature of differentiated epithelial cells, a decrease in vectorial transport (expressed by reduced dome number) in the absence of toxic cell damage can be used as a measure of renal epithelial cell dedifferentiation. Furthermore, it is unlikely that the effects of OTA on ERK1/2 in MDCK-C7 cells represent secondary toxic effects of the mycotoxin, because 1) neither ERK1/2 phosphorylation nor ERK1/2 activity was affected in MDCK-C11 cells and 2) ERK1/2 activity was increased at time-points and OTA concentrations that did not lead to cell damage. Moreover, OTA-induced increases in LDH release were significantly higher in MDCK-C7 cells (30%) than in MDCK-C11 cells (10%). With respect to the effects of OTA on the intracellular pH of both MDCK cell clones, we found that MDCK-C7 cells displayed only a slight acidification after the application of OTA, whereas in MDCK-C11 cells, in which OTA is not able to increase ERK1/2 activity, the mycotoxin led to a clear intracellular alkalinization. Thus it seems likely that the OTA-induced stimulation of ERK1/2 in MDCK-C7 cells does not depend on an OTA-induced intracellular alkalinization.

Besides its effects on ERK1/2 activity and cell differentiation in MDCK-C7 cells, it is an interesting observation that the synthetic MEK inhibitor PD098059 only partially inhibited both OTA-induced ERK1/2 phosphorylation and ERK activation after long-term incubation in these cells. In this respect, it is important to note that no upstream activator of ERK1/2 other than MEK1/2 is yet known. Inhibition of MEK by PD098059 prevented activation of ERK and subsequent phosphorylation of ERK substrates both in vitro and in intact Swiss 3T3 fibroblasts (Dudley et al., 1995). In addition, PD098059 completely blocked nerve growth factor induced neurite formation in PC-12 cells without altering cell viability (Pang et al., 1995) and almost completely abolished basal and serum-stimulated ERK1/2 phosphorylation in both mock-transfected and CA-MEK1-transfected MDCK-C7 cells (Schramek et al., 1997b). Although a MEK-independent activation of MAPK has been reported recently in bisperoxovanadium 1,10-phenanthroline-treated rat hepatocytes, peroxovanadium compounds are known to be strong inhibitors of tyrosine phosphatases. They may inhibit specific tyrosine/threonine phosphatases involved in the negative regulation of MAPK and thereby substantially increase MAPK activity (Band and Posner, 1997). Similar experiments performed in glomerular mesangial cells have revealed that agonist-specific and time-dependent stimulation of distinct ERK2-regulating protein phosphatases is critical for the duration of ERK2 activation (Schramek et al., 1996). Whether or not the partial inhibition of OTA-induced ERK1/2 activation by PD098059 in MDCK-C7 cells after long-term stimulation indeed reflects the stimulation of an additional ERK activator in OTA-stimulated MDCK-C7 cells remains to be investigated.

In summary, we recently described dramatic alterations in the activation of certain MAPK in alkali-dedifferentiated MDCK-C7F cells as compared with their parental MDCK-C7 cells (Schramek et al., 1997a), as well as an association of CA-MEK1 expression with renal epithelial MDCK-C7 cell differentiation (Schramek et al., 1997b). Our present results offer evidence for a role of ERK1/2 in OTA-induced MDCK-C7 cell dedifferentiation. These described effects of OTA on ERK1/2 phosphorylation, on ERK1/2 activity and on renal epithelial cell differentiation appear to be cell-specific and might thus explain why certain epithelial cell types along the nephron differ in susceptibility.