ATP Depletion Increases Tyrosine Phosphorylation of β-Catenin and Plakoglobin in Renal Tubular Cells

Cell Culture

The primary culture of mouse proximal tubule (MPT) cells was performed as described previously (18). In brief, collagenase digested fragments were obtained from the renal cortices of mice (Harlan, Sprague Dawley, C57BL6) and placed in serum-free medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium that contained 2 mM glutamine, 15 mM Hepes, 5 μg/ml transferrin, 5 μg/ml insulin, 50 nM hydrocortisone, 500 μg/ml penicillin, and 50 μg/ml streptomycin. These cells have been identified previously as proximal tubular epithelial cells (18).

Cells were grown either on permeable filter supports (for epithelial permeability studies), in 6-well dishes (for Western blot analysis), or on glass slides (for immunofluorescence studies). The filter supports used for studies of epithelial permeability were 6.5-mm Transwell filter inserts (Costar, Cambridge, MA) consisting of a polycarbonate membrane with 0.4-μm pores coated with rat tail collagen.

ATP Depletion

ATP depletion was induced by chemical anoxia using sodium cyanide (CN) in the absence of glucose. MPT monolayers were rinsed three times with Krebs-Hensleit buffer (KHB) that contained 1 mM calcium and 1 mM magnesium, pH 7.40, at 37°C to remove residual substrates in the medium. Cells were then incubated for 1.5 h in glucose-free KHB containing 5 mM CN. These conditions have been reported previously by our laboratory to lower ATP content to <5% of the control value (2,18). Control monolayers were incubated in CN-free KHB to which 10 mM glucose was added.

Transcellular Electrical Resistance

TER, a sensitive marker of tight junction integrity, was measured with an epithelial Voltohmeter (EVOM; World Precision Instruments, New Haven, CT) in confluent cell monolayers grown on permeable filters. To control for intrinsic resistance of the filters, measurements of electrical resistance were obtained across cell-free inserts equilibrated in KHB at 37°C. The electrical resistance of the cell-free filter insert was then subtracted from all subsequent measurements. The electrodes were sterilized with 95% ethanol and were washed and equilibrated in sterile phosphate-buffered saline before use. To reduce the variability of the determination, the electrodes were placed to the same depth in the solutions bathing the cultured monolayer with a micromanipulator. Monolayers that did not have an initial resistance of at least 175 Ω · cm² were not used in this study.

Transcellular Permeability

Another measure of the “gate” function of the tight junction is the barrier to the passage of molecules between epithelial cells. To examine permeability, we measured the unidirectional flux across confluent MPT monolayers grown on permeable supports of lucifer yellow, a fluorescence, fluid-phase marker (MW = 482) (3). At the onset of the experiment, 1 mg/ml lucifer yellow was added to the apical solution. The entire volume of the basolateral compartment (1 ml) was sampled at 30-min intervals and then replaced with fresh medium. Apical solution samples (1 μ1) were also obtained at the same intervals as for the basolateral solution. The concentration of lucifer yellow in each compartment was determined by fluorescence spectrofluorometry at an excitation wavelength of 425 nm and an emission of 535 nm. The emission intensity of a standard curve was linear over the range of samples tested and exhibited an r value of >0.95. Flux rates and membrane permeabilities were determined as described previously (2,6).

Preparation of MPT Lysates for Western Blotting

Confluent MPT cells were washed three times in cold phosphate-buffered saline, scraped from the culture dish, and pelleted by centrifugation at 1000 × g for 10 min. The pellet was suspended in 4 vol of ice-cold homogenizing buffer containing 10 mM Tris-HCl, 150 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium vanadate, 5 mM ethylenediaminetetra-acetic acid, aprotinin (0.5 μg/μl), N-tosyl-L-phenylalanine chloromethyl ketone (2 μg/μl), phenylmethylsulfonyl fluoride (4 mM), DNAse (5 μg/ml), RNAase (5 μg/ml) and 1% Nonidet P-40. The suspended pellet was homogenized by ten 1-s strokes in a Teflon homogenizer. The homogenate was centrifuged for 10 min at 1000 × g at 4°C to remove nuclei and remaining intact cells, and the supernatant was stored at -20°C for Western blotting and immunoprecipitation studies.

Immunoprecipitation

MPT lysates were immunoprecipitated using the anti-peptide mouse monoclonal antibodies to β- or γ-catenin (Becton Dickinson, Bedford, MA) according to the following protocol. The homogenate was diluted to a protein concentration of 100 μg/ml with the homogenizing buffer that also contains 0.5% deoxycholate. Nonimmune serum (2 μl) and a 25% suspension of protein A-Sepharose 4B beads (30 μl) was added to a 900-μl aliquot of the cell lysate. The mixture was incubated at 4°C for 2 h and then centrifuged at 13,000 rpm in a microcentrifuge. The supernatant was incubated with 20 μl of anti-β- or anti-γ-catenin antibody and 50 μl of a protein A-Sepharose 4B bead suspension that had been prereacted with goat anti-mouse IgG (Sigma, St. Louis, MO) for 12 h at 4°C. In preliminary studies, we determined that the quantity of primary antibody used was more than adequate to precipitate all of the catenin present in the sample. The beads were pelleted by centrifugation and washed three times and suspended in sodium dodecyl sulfate sample buffer.

Immunoblot

Whole cell homogenates and immunoprecipitated samples, prepared as described above, were heated at 65 or 100°C for 5 min, respectively, before loading on a 7% polyacrylamide sodium dodecyl sulfate gel and run under reducing conditions as described previously (6). Proteins were then electrophoretically transferred to nitrocellulose filters. After transfer, the filters were washed in 150 mM NaCl, 100 mM Tris-HCl, pH 7.5, and 0.05% Tween 20 (TBST), and blocked for 1 h in TBST containing 5% wt/vol nonfat powdered milk (TBSTM) before incubation with the anti-phosphotyrosine antibody PY20 (Transduction Laboratories, Lexington, KY) at a dilution of 1:1000 in TBSTM at 4°C overnight. The filters were then washed three times with TBST and incubated in secondary horseradish peroxidase-labeled goat anti-mouse, 1:2000 in TBSTM for 2 h at room temperature. After three additional washes with TBST, bound secondary antibody was detected using the enhanced chemiluminescence (ECL) system (Amersham).

Immunofluorescence Studies

MPT cells grown on glass coverslips were fixed in 3.7% paraformaldehyde and permeabilized with 0.2% Triton X-100. Detection of E-cadherin in these cells was performed by indirect immunofluorescence using methods we have described previously (2). The anti-E-cadherin antibody (rat monoclonal antibody, clone DECMA-1; Sigma) was used at a dilution of 1:100. The secondary antibody was labeled with CY-3 (Molecular Probes, Eugene, OR) and used at a dilution of 1:500. The monolayers were photographed using a Nikon epifluorescence microscope, and the same exposure time, optimized for the control, was used for all coverslips to depict differences in staining intensity.

Phosphatase and Kinase Inhibitors

Stock solution of vanadate (100 mM) and the sodium salt of okadaic acid (5 mM) were prepared in KHB. Genistein (1 mM), calyculin A (0.5 mM), H7 (1 mM), and H8 (10 mM) were dissolved as stock solutions in DMSO. The final concentration of DMSO in the incubation media never exceeded 1.0%. All of these inhibitors were obtained from Calbiochem (La Jolla, CA).

Protein Assay

Protein concentrations were determined from a colorimetric dye binding assay (Bio-Rad) and expressed in milligrams per milliliter.

Statistical Analyses

Data are expressed as mean ± SEM. Protocols that required comparisons between three or more groups were compared with ANOVA and Fisher post hoc test. When two groups were compared, analysis was performed with an unpaired, two-tailed t test. A P value of <0.05 was defined as significant.

Transepithelial Resistance

We have documented in previous studies that ATP depletion induced by incubating MPT cell monolayers in a glucose-free medium containing CN results in a rapid fall in TER and a rise in permeability to fluid phase markers (2,6). To evaluate the possible role of changes in tyrosine phosphorylation in the alteration in tight junction function, we first examined the effects of phosphotyrosine protein kinase and phosphotyrosine phosphatase inhibitors on transepithelial resistance. Treatment of control, dextrose-treated (ATP-replete) MPT monolayers with the phosphotyrosine phosphatase inhibitor vanadate at 1 mM, a concentration shown in prior studies to increase tyrosine phosphorylation in intact cells (19), resulted in a decline in the transepithelial resistance from 281.2 ± 25.2 to 170.8 ± 18.2 Ω · cm² (n = 6; P < 0.05) in 20 min and a further decline to 143 ± 12.2 Ω · cm² in 60 min (Figure 1). Although the initial rate of decline in TER induced by vanadate was similar to that induced by ATP depletion, the reduction in TER after 60 min was somewhat less in the vanadate-treated group than in the ATP-depleted monolayers (Figure 1). The combination of vanadate and ATP depletion resulted in a more rapid decline in TER than with either maneuver alone, but the minimum value obtained after 2 h was no different from that obtained with ATP depletion alone. Similar functional effects of vanadate were observed when the permeability of the epithelium was measured using a fluid phase marker (lucifer yellow) in parallel experiments. Epithelial permeability to lucifer yellow increased 1 h after the addition of vanadate from a control value of 3.9 ± 0.3 × 10^-5 to 24.3 ± 0.2 × 10^-5 cm/s (n = 5; P < 0.05). This increase in permeability was less than the increase observed with CN-induced ATP depletion (54.7 ± 0.4 × 10^-5 cm/s) (n = 6; P < 0.05) or the combined effects of both vanadate and CN (65.3 ± 0.5 × 10 to 5 cm/s) (n = 5; P < 0.05).

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Figure 1.

Effect of vanadate on transepithelial resistance of ATP-replete (control) and ATP-depleted (cyanide [CN]-treated) mouse proximal tubular (MPT) monolayers grown on permeable supports. □, control; ⋄, vanadate alone; ▵, CN and vanadate; ○, CN. All values are means ± SEM of six studies. ^*P < 0.05 comparing vanadate versus control; +P < 0.05 comparing vanadate versus vanadate + CN.

Because vanadate can potentially alter the activity of other enzymes in addition to phosphotyrosine phosphatases, its functional effects cannot be definitively attributable to phosphotyrosine phosphatase inhibition alone. We therefore examined the effect of other phosphatase inhibitors that have a different spectrum of action and specificity (Figure 2). Dephostatin (20 μM), another phosphotyrosine phosphatase inhibitor, reduced TER in ATP-replete MPT monolayers by 61 ± 9% (n = 5; P < 0.05), an effect similar to that of vanadate. In contrast, two different serine/threonine phosphatase inhibitors, okadaic acid (20 nM) and calyculin A (10 nM), had no effect on TER of ATP-replete MPT monolayers (Figure 2).

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Figure 2.

Comparison of the effect of protein tyrosine phosphatase inhibitors with protein phosphatase 1 and 2 inhibitors on transepithelial resistance of ATP-replete monolayers grown on permeable supports. Values are means ± SEM of six studies obtained 60 min after the addition of either diluent (Control) or 1 mM vanadate, 20 μM dephostatin, 20 nM okadaic acid, or 10 nM calyculin A. ^*P < 0.05 compared with control.

The phosphotyrosine protein kinase inhibitor genistein (19) at a concentration of 25 μM had no effect on TER in control, ATP-replete MPT monolayers (Figure 3). However, genistein substantially ameliorated the reduction in TER induced by ATP depletion (Figures 3 and 4). As depicted in Figures 3 and 4, the decline in TER after 60 min in genistein-treated ATP-depleted monolayers (32 ± 7%) was significantly less than after ATP depletion alone (69 ± 11%, P < 0.05, n = 6). This protective effect of genistein on the reduction in TER induced by ATP depletion could not be reproduced by other types of kinase inhibitors (20). Neither the protein kinase C inhibitor H7 (25 μM) nor the protein kinase A inhibitor H8 (25 μM) affected the reduction in TER induced by ATP depletion (Figure 4).

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Figure 3.

Effect of genistein on transepithelial resistance of ATP-replete and ATP-depleted MPT monolayers grown on permeable supports. □, control; ○, genistein alone; ▵, CN and genistein; ⋄, CN alone. All values are means ± SEM of six studies. ^*P < 0.05 comparing CN and genistein or CN alone with control; †P < 0.05 comparing CN and genistein versus CN alone.

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Figure 4.

Comparison of the effect of protein tyrosine kinase inhibitor genistein with protein kinase A and protein kinase C inhibitors on ATP depletion-induced reduction of transepithelial resistance MPT monolayers grown on permeable supports. Values are means ± SEM of six studies obtained 60 min after the addition of either diluent (Control) or 5 mM cyanide or a combination of cyanide plus 25 μM genistein or 25 μM H7 or 25 μM H8. ^*P < 0.05 comparing CN with control; †P < 0.05 comparing vanadate versus vanadate plus genistein.

If the vanadate-induced reduction in TER is primarily dependent on tyrosine hyperphosphorylation and not by other mechanisms, then one would predict that its action should be antagonized by a tyrosine kinase inhibitor. To verify this assertion, we investigated whether the reduction in TER induced by vanadate could be ameliorated by the tyrosine kinase inhibitor genistein. In Figure 5, we demonstrate that 1 mM vanadate reduces TER after 1 h by 149 ± 12 Ω · cm². However, with the simultaneous addition of both 1 mM vanadate and 25 μM genistein, the reduction in TER was reduced by one-third, to only 49 ± 5 Ω · cm². Thus, the effect of vanadate must be dependent on enhanced tyrosine phosphorylation.

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Figure 5.

Reversal in the vanadate-induced reduction of transepithelial resistance by genistein. □, control; ⋄, vanadate alone; ○, genistein alone; ▵, vanadate and genistein. All values are means ± SEM of six studies. ^*P < 0.05 comparing vanadate with control; †P < 0.05 CN versus CN plus genistein.

Phosphorylation of Proteins in Whole Cell Homogenates of MPT Cells

Based on the above observations, we would predict that ATP depletion induces changes in the function of cell junction via tyrosine phosphorylation of proteins. To evaluate this possibility, we first analyzed whole cell lysates of MPT cells by immunoblot analysis to assess the degree of protein tyrosine phosphorylation and to determine the general effects of ATP depletion, vanadate, and genistein on tyrosine phosphorylation of cell proteins (Figure 6). As expected, lysates of MPT cells incubated with 1 mM vanadate for 1 h demonstrated a generalized increase in the immunodetectable degree of tyrosine phosphorylation (Figure 3, lane 2) compared to control cells (lane 1), whereas 25 μM genistein reduced phosphorylation of all protein bands. (Figure 3, lane 3). Most protein bands from MPT cells treated with CN for 1 h (Figure 3, lane 4) demonstrated a decrease in tyrosine phosphorylation. However, several protein bands (one at 50 kD and one at 160 kD) were hyperphosphorylated compared with control MPT cells. There also was a marked increase in the tyrosine phosphorylation of proteins compared to either control or only vanadate treatment when MPT cells were treated with both CN and vanadate (Figure 3, lane 5). This latter change indicates that neither ATP depletion nor kinase activity is rate-limiting for protein tyrosine phosphorylation. In addition, the combination of genistein and ATP depletion (Figure 3, lane 6) reduced phosphorylation more than any one of these maneuvers alone. These observations are consistent with the hypothesis that ATP depletion is associated with alterations in the activity of both tyrosine kinases and phosphatase with resultant dephosphorylation of some proteins and hyperphosphorylation of others.

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Figure 6.

Effect of tyrosine kinase inhibitors, tyrosine phosphatase inhibitors, and ATP depletion on tyrosine phosphorylation of proteins in MPT cells. An immunoblot of whole cell homogenates (50 μg protein/lane) from monolayers treated with vehicle (control) (lane 1), vanadate (lane 2), genistein (lane 3), CN (lane 4), CN and vanadate (lane 5), and CN and genistein (lane 6) and probed with an antityrosine phosphate antibody. The blot shown is representative of four experiments.

Phosphorylation of β-Catenin and Plakoglobin

Since an intact zonula adherens (ZA) is required to maintain a functional ZO and phosphorylation of catenins is known to regulate the stability of the ZA (E-cadherin complexes), we examined the effect of ATP depletion on catenin phosphorylation. In these studies, β-catenin and plakoglobin were immunoprecipitated from whole cell homogenates of MPT cells. The immunoprecipitates were then subjected to immunoblot analysis and probed first with an anti-tyrosine phosphate antibody and then with an anti-catenin antibody. The amount of β-catenin and plakoglobin expressed was not measurably changed by ATP depletion or exposure to either vanadate or genistein (Figure 7B). The treatment of ATP-replete (control) monolayers with vanadate (Figure 7, lane 2) enhanced tyrosine phosphorylation of β-catenin (upper band) by 398 ± 42% and plakoglobin (lower band) by 283 ± 23% above control (lane 1). Genistein treatment of ATP-replete monolayers reduced the tyrosine phosphorylation of β-catenin by 30 ± 9% and plakoglobin by 35 ± 6% below control (Figure 7, lane 3). ATP depletion with CN increased phosphorylation of β-catenin by 348 ± 37% and plakoglobin by 355 ± 23% (Figure 7, lane 4). The effect of ATP depletion on phosphorylation of both proteins was comparable to that of vanadate alone. The combination of genistein and CN (Figure 7, lane 5) reduced phosphorylation of β-catenin and plakoglobin by 50 ± 11% and 65% ± 10% below control, respectively. Thus, genistein prevented the hyperphosphorylation of both β-catenin and plakoglobin induced by ATP depletion (Figure 7, lane 4). The combination of vanadate and CN (Figure 7, lane 6) resulted in hyperphosphorylation of both β-catenin (428 ± 33%) and plakoglobin (393 ± 53%), an effect somewhat greater than that induced by vanadate (Figure 7, lane 2) or ATP depletion alone (Figure 4, lane 4).

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Figure 7.

Phosphotyrosine phosphorylation of β-catenin (92 kD) and plakoglobin (82 kD). Whole cell homogenates from monolayers treated with vehicle (control) (lane 1), vanadate (lane 2), genistein (lane 3), CN (lane 4), CN and genistein (lane 5), and CN and vanadate (lane 6) were immunoprecipitated with an antibody directed against both β-catenin and plakoglobin and then analyzed by Western blot for tyrosine phosphorylation (A) or β-catenin and plakoglobin (B). The blot shown is representative of four experiments.

In addition to determining the effect of ATP depletion on the phosphorylation of ZA proteins, we also examined the effect of ATP depletion on two ZO proteins: occludin and ZO-1. These proteins were immunoprecipitated from whole cell homogenates with either an antibody to ZO-1 or to occludin. The immunoprecipitates obtained (Figure 8) were then subjected to immunoblot analysis, and the blots were probed first with an antibody to either ZO-1 or to occludin and then with an anti-tyrosine phosphate antibody (PY20). Neither the amount of ZO-1 (Figure 8, lanes 1 to 2) and occludin (Figure 8, lanes 3, 4) expressed nor the degree of tyrosine phosphorylation of these proteins was affected by ATP depletion (Figure 8, lanes 2, 4, 6, and 8).

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Figure 8.

Effect of ATP depletion on expression and tyrosine phosphorylation of occludin (68 kD) and ZO-1 (225 kD). ZO-1 and occludin were immunoprecipitated from whole cell homogenate of control (lanes 1 and 3) and ATP-depleted (lanes 2 and 4) MPT monolayers. The immunoprecipitate was analyzed by Western blot for the amount of proteins immunoprecipitated (A) and for the degree of tyrosine phosphorylation of these proteins (B).

Immunofluorescence of E-Cadherin

Tyrosine phosphorylation of β-catenin and/or plakoglobin has been associated with the withdrawal of E-cadherin complexes from the lateral membrane. Immunohistochemical studies were performed to determine whether the maneuvers that induce tyrosine hyperphosphorylation also induce withdrawal of E-cadherin from the lateral membrane. Immunofluorescence microscopy demonstrated that the distribution of E-cadherin in MPT cells is comparable to that described in other epithelial monolayers (11,16). E-cadherin is present primarily at cell-cell borders and in the basolateral membrane (Figure 9a). One hour after treatment of MPT monolayers with 5 mM CN, there is a marked decline in the intensity of basolateral E-cadherin staining (Figure 9b). Treatment of MPT monolayers with 1 mM vanadate resulted in a generalized diminution in E-cadherin staining in a pattern similar to that seen after CN (Figure 9c). E-cadherin staining in MPT monolayers treated with the combination of CN and genistein was similar to that in control monolayers (Figure 9d), suggesting that genistein ameliorates the effect of CN on the redistribution of E-cadherin. Genistein alone had no effect on E-cadherin staining (data not shown).

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Figure 9.

Immunohistochemical distribution of E-cadherin in MPT cells subjected to ATP depletion. Photomicrographs of MPT cells grown on glass coverslips stained for E-cadherin with a rat monoclonal antibody for mouse E-cadherin. (a) Control monolayer. ATP-replete MPT cells demonstrate the typical basolateral distribution of E-cadherin with predominant staining at cell-cell borders. (b) CN-treated monolayers. After 1 h exposure to 5 mM CN, there is a marked reduction in the intensity of the E-cadherin staining. (c) Vanadate-treated monolayer. MPT cells treated with 1 mM vanadate for 1 h demonstrate a reduction in the intensity of E-cadherin staining, an effect similar to that induced by CN. (d) Genistein and CN. The CN-induced reduction in E-cadherin staining (b) can be ameliorated by the simultaneous incubation with 1 mM genistein. To facilitate an assessment of the relative degree of E-cadherin staining, the exposure time for each photomicrograph was identical. Magnification: ×400.