J Am Soc Nephrol 10: 2297–2305, 1999 ATP Depletion Increases Tyrosine Phosphorylation of b-Catenin and Plakoglobin in Renal Tubular Cells JOHN H. SCHWARTZ, THEODORA SHIH, SARAH A. MENZA, and WILFRED LIEBERTHAL Evans Department of Clinical Research, Department of Medicine, Renal Division, Boston Medical Center, Boston, Massachusetts. Abstract. This study examines the hypothesis that the loss of ameliorates the fall in transepithelial resistance induced by integrity of the junctional complex induced by ATP depletion ATP depletion. Also, using immunofluorescence microscopy it is related to alterations in tyrosine phosphorylation of the was demonstrated that ATP depletion results in a marked adherens junction proteins b-catenin and plakoglobin. ATP diminution of E-cadherin staining in the basolateral membrane depletion of cultured mouse proximal tubular (MPT) cells of MPT cells. Vanadate mimics this effect of ATP depletion, induces a marked increase in tyrosine phosphorylation of both whereas genistein ameliorates the reduction in the intensity of b-catenin and plakoglobin. The tyrosine phosphatase inhibitor E-cadherin staining induced by ATP depletion. Because it is vanadate has the same effect in ATP-replete (control) mono- has been well established that hyperphosphorylation of the layers, whereas genistein, a tyrosine kinase inhibitor, reduces catenins leads to dissociation of the adherens junction and to phosphorylation of both proteins in ATP-replete monolayers dysfunction of the junctional complex, it is proposed that the and prevents the hyperphosphorylation of these proteins with increase in tyrosine phosphorylation of catenins observed in ATP depletion. This study also demonstrates that the fall in the MPT cells during ATP depletion contributes to the loss of transepithelial resistance of MPT monolayers induced by ATP function of the junctional complex associated with sublethal depletion can be reproduced by treatment of ATP-replete injury. monolayers with vanadate, whereas genistein substantially Sublethal injury to renal tubular cells, induced by ATP deple- somes (11,12). In mature epithelia, the ZO and adherens junc- tion, leads to the rapid loss of functional integrity of the tight tion both completely circumscribe adjoining epithelial cells at junction (1,2), the loss of cell polarity (3,4), and severe im- the boundary between apical and basolateral membrane do- pairment of the epithelial permeability barrier (2,3,5). After mains and both play a role in maintaining the functional only a few minutes of ATP depletion, the transepithelial elec- integrity of the junctional complex (11,12). Although the ZO is trical resistance (TER) of renal epithelia falls to very low levels the component of the junctional complex that represents the and paracellular permeability increases (2,3,5). These func- barrier to the paracellular flux of molecules and ions across the tional changes are completely reversible if the ATP levels are epithelium (11,12), the adherens junction, which lies immedi- restored before lethal cell injury occurs (1,6). This loss of the ately basal to the ZO, is necessary not only for the formation of renal epithelial permeability barrier associated with sublethal the ZO but also for the maintenance of a functionally normal injury is believed to contribute, at least in part, to the “back- ZO (11,12). leak” of glomerular filtrate, which is believed to be an impor- The adherens junction of renal epithelial cells is comprised tant contributing factor to the profound loss of GFR associated of a number of proteins that include cadherin and the catenins. with acute ischemic renal injury (7–10). E-cadherin is a transmembrane protein that mediates adhesion The molecular events that lead to the rapid loss of function of adjacent cells to one another. The extracellular domain of of the junctional complex after ATP depletion remain uncer- the E-cadherin molecules of adjacent cells bind to one another tain. The junctional complex between epithelial cells is com- in a homophilic, calcium-dependent manner (11,13,14). The prised anatomically of at least three distinct structures: the intracellular domain of E-cadherin is connected to the actin zonula occludens (ZO), the adherens junction, and the desmo- cytoskeleton by a complex of cytosolic proteins called the a catenins (13,14). At least three different catenins ( -catenin, b-catenin and g-catenin [plakoglobin]) mediate the attachment Received January 22, 1999. Accepted May 11, 1999. of E-cadherin to actin by binding to an intermediary actin- Correspondence to Dr. John H. Schwartz, Boston University Medical Center, binding protein, a-actinin. There is now substantial evidence Evans Building Room 401, Boston Medical Center, One Boston Medical that tyrosine phosphorylation of the catenins plays an impor- Center Place, Boston, MA 02118. Phone: 617-638-7321; Fax: 617-638-8281; E-mail: [email protected] tant role in regulating the formation and functional integrity of 1046-6673/1011-2297 the adherens junction (14–16). The maintenance of an intact Journal of the American Society of Nephrology and stable adherens junction depends on maintaining the Copyright © 1999 by the American Society of Nephrology catenins in a dephosphorylated state. Increased tyrosine phos- 2298 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2297–2305, 1999 phorylation of b-catenin and/or plakoglobin results in the onset of the experiment, 1 mg/ml lucifer yellow was added to the structural and functional disruption of the adherens junction apical solution. The entire volume of the basolateral compartment (1 (16,17). ml) was sampled at 30-min intervals and then replaced with fresh m In these studies, we have examined the novel hypothesis that medium. Apical solution samples (1 l) were also obtained at the ATP depletion results in hyperphosphorylation of b-catenin same intervals as for the basolateral solution. The concentration of lucifer yellow in each compartment was determined by fluorescence and plakoglobin. We will also determine whether hyperphos- spectrofluorometry at an excitation wavelength of 425 nm and an phorylation of these cytoskeletal proteins contributes to the emission of 535 nm. The emission intensity of a standard curve was functional disturbance of the junctional complex associated linear over the range of samples tested and exhibited an r value of with ATP depletion. .0.95. Flux rates and membrane permeabilities were determined as described previously (2,6). Materials and Methods Cell Culture Preparation of MPT Lysates for Western Blotting The primary culture of mouse proximal tubule (MPT) cells was Confluent MPT cells were washed three times in cold phosphate- performed as described previously (18). In brief, collagenase digested buffered saline, scraped from the culture dish, and pelleted by cen- fragments were obtained from the renal cortices of mice (Harlan, trifugation at 1000 3 g for 10 min. The pellet was suspended in 4 vol Sprague Dawley, C57BL6) and placed in serum-free medium consist- of ice-cold homogenizing buffer containing 10 mM Tris-HCl, 150 ing of a 1:1 mixture of Dulbecco’s modified Eagle’s medium and mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium Ham’s F-12 medium that contained 2 mM glutamine, 15 mM Hepes, vanadate, 5 mM ethylenediaminetetra-acetic acid, aprotinin (0.5 mg/ 5 mg/ml transferrin, 5 mg/ml insulin, 50 nM hydrocortisone, 500 ml), N-tosyl-L-phenylalanine chloromethyl ketone (2 mg/ml), phenyl- mg/ml penicillin, and 50 mg/ml streptomycin. These cells have been methylsulfonyl fluoride (4 mM), DNAase (5 mg/ml), RNAase (5 identified previously as proximal tubular epithelial cells (18). mg/ml) and 1% Nonidet P-40. The suspended pellet was homogenized Cells were grown either on permeable filter supports (for epithelial by ten 1-s strokes in a Teflon homogenizer. The homogenate was permeability studies), in 6-well dishes (for Western blot analysis), or centrifuged for 10 min at 1000 3 g at 4°C to remove nuclei and on glass slides (for immunofluorescence studies). The filter supports remaining intact cells, and the supernatant was stored at 220°C for used for studies of epithelial permeability were 6.5-mm Transwell Western blotting and immunoprecipitation studies. filter inserts (Costar, Cambridge, MA) consisting of a polycarbonate membrane with 0.4-mm pores coated with rat tail collagen. Immunoprecipitation ATP Depletion MPT lysates were immunoprecipitated using the anti-peptide ATP depletion was induced by chemical anoxia using sodium mouse monoclonal antibodies to b-org-catenin (Becton Dickinson, cyanide (CN) in the absence of glucose. MPT monolayers were rinsed Bedford, MA) according to the following protocol. The homogenate three times with Krebs-Hensleit buffer (KHB) that contained 1 mM was diluted to a protein concentration of 100 mg/ml with the homog- calcium and 1 mM magnesium, pH 7.40, at 37°C to remove residual enizing buffer that also contains 0.5% deoxycholate. Nonimmune substrates in the medium. Cells were then incubated for 1.5 h in serum (2 ml) and a 25% suspension of protein A-Sepharose 4B beads glucose-free KHB containing 5 mM CN. These conditions have been (30 ml) was added to a 900-ml aliquot of the cell lysate. The mixture reported previously by our laboratory to lower ATP content to ,5% was incubated at 4°C for 2 h and then centrifuged at 13,000 rpm in a of the control value (2,18). Control monolayers were incubated in microcentrifuge. The supernatant was incubated with 20 ml of anti-b- CN-free KHB to which 10 mM glucose was added. or anti-g-catenin antibody and 50 ml of a protein A-Sepharose 4B bead suspension that had been prereacted with goat anti-mouse IgG Transcellular Electrical Resistance (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 TER, a sensitive marker of tight junction integrity, was measured adequate to precipitate all of the catenin present in the sample.