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EXTRACELLULAR MATRIX, CELL INTERACTIONS
Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, Indiana
Submitted 3 March 2006 ; accepted in final form 16 August 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, and mLgl-1 redistribution from sites of cell-cell contact, which was restored following recovery from ATP depletion. Zonula occludens (ZO)-1 and Par3 phosphorylation was reduced and association of aPKC with its substrates Par3 and mLgl-1 was stabilized in ATP-depleted Madin-Darby canine kidney cells. ATP depletion also induced a stable association of Par3 with Tiam-1, a Rac GTPase exchange factor, which explains how aPKC and Rac activities were suppressed. Experimental inhibition of aPKC during recovery from ATP depletion interfered with reassembly of ZO-1 and Par3 at cell junctions. These data indicate that aPKC signaling is impaired during ATP depletion, participates in tight junction disassembly during cell injury and is important for tight junction reassembly during recovery.
ischemia; atypical PKC; Par3; zonula occludens-1; mLgl-1
Tight junction assembly is regulated by an evolutionarily conserved protein complex that includes atypical protein kinase C (aPKC) and two homologs of Caenorhabditis elegans partitioning defective gene products, Par3 and Par6 (reviewed in Ref. 41). Successful assembly and activation of the aPKC-Par3-Par6 complex is a critical step in tight junction formation and epithelial polarization (26, 46). Par6 interacts directly with activated forms of Cdc42 and Rac, but not Rho GTPase (24, 36), and this association regulates the serine/threonine kinase activity of aPKC (39, 53). Par3 and mLgl (a member of the evolutionarily conserved tight junction regulatory Scribble complex) have been identified as kinase targets of aPKC (34, 38). aPKC forms independent complexes with mLgl and Par3 during tight junction assembly and epithelial polarization (52).
Ischemic injury is the major cause of acute renal failure in patients with various forms of renal disease and is associated with epithelial cell injury caused by depletion of cellular ATP levels (47). A commonly used, powerful, and reversible model of epithelial cell injury during renal ischemia is ATP depletion of cultured epithelial cells using inhibitors of oxidative phosphorylation. This model recapitulates many pathological effects on epithelial cells associated with renal ischemia in animal models and human patients (10), including disruption of the epithelial tight junction barrier, which is accompanied by breakdown of plasma membrane polarity.
How ischemia disrupts signaling pathways that regulate tight junctions is unclear. ATP depletion is accompanied by a concomitant reduction in cellular GTP levels (9) and Rho GTPases were shown to be differentially sensitive to nucleotide depletion in LLC-PK cells (17). Activation of Rho and Rac GTPase signaling protects tight junctions from disassembly during ATP depletion (Ref. 15 and S. Gopalakrishnan and J. A. Marrs, unpublished observations). As mentioned previously, Rac GTPase associates with the aPKC-Par6 complex (24, 36) and activates aPKC (39). In the present study, we examined the aPKC signaling pathway in the ATP-depletion model of ischemic epithelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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pseudosubstrate (PS) inhibitor was purchased from EMD Biosciences (La Jolla, CA). Other chemicals were purchased from Sigma (St. Louis, MO) or Midwest Scientific (St. Louis, MO).
Antibodies against ZO-1, occludin, Par3, phosphothreonine, and phosphoserine were from Zymed (San Francisco, CA); monoclonal antibodies against Rac1 and Cdc42, and polyclonal antibody against phosphorylated aPKC were from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal aPKC antibody was from Upstate (Charlottesville, VA); Par3 antibody was a gift from Ian Macara (University of Virginia, Charlottesville, VA), and mLgl-1 antibody was a gift from Patrick Brennwald (Cornell University, Ithaca, NY). Fluorophore-conjugated antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Horseradish peroxidase-conjugated goat anti-rabbit antibody was from Amersham-Pharmacia (Piscataway, NJ).
ATP depletion and repletion.
MDCK cultures were rinsed in prewarmed depletion medium and ATP depleted for 60 min by incubating cells with depletion medium containing 0.1 µM antimycin A (5). For ATP repletion, depletion medium was replaced with DMEM in the presence or absence of 25 µM aPKC pseudosubstrate inhibitor.
Calcium switch assay. The calcium switch assay was previously developed by Cereijido and colleagues (13). MDCK cells were washed once in low-calcium medium (DMEM with 5 µM CaCl2) and incubated with low calcium medium plus 2.5% FBS extensively dialyzed against Tris saline for 3 h to disassemble junctions (48). Cells were then processed as described in Immunoprecipitation and immunoblotting.
Immunofluorescence and image analysis. MDCK cells (2.5 million) were plated on 6-well polycarbonate filter supports (Corning-Costar, Kennebunk, ME) and cultured for 5–7 days (for two-photon microscopy) or cells were plated on coverslips and grown for 3 days. Cells were ATP depleted for 45 or 60 min with 0.1 µM antimycin A or left untreated in the control filters and coverslips. Cells were then fixed in methanol-acetone (1:1) or PBS containing 4% paraformaldehyde for 10 min at room temperature. Cells were washed in PBS and then permeabilized in PBS containing 0.5% Triton X-100 for 5 min at room temperature. Cells were then blocked in PBS containing 0.2% BSA and 2% goat serum (blocking solution) for 30 min at room temperature, followed by incubation with primary antibody diluted in blocking solution for 45 min at room temperature. Cells were washed in PBS containing 0.2% BSA and incubated with secondary antibodies diluted in blocking solution for 45 min at room temperature. Coverslips were washed and mounted in PBS containing 50% glycerol, 0.1% sodium azide, and 100 mg/ml 1,4-diazabicyclo(2.2.2)octane.
For two-photon microscopy, samples were viewed with a confocal/two-photon system (model MRC 1024; Bio-Rad, Hercules, CA) fitted to a Nikon Eclipse inverted microscope (Melville, NY) with a x60 water immersion, 1.2 numerical aperture objective. Data sets were collected as a z-series of 
100 two-photon images with a spacing of 0.1 µm. Acquired images were first processed for background subtraction by applying a 3 x 3 low-pass filter with Metamorph version 4.1.7 image-processing software. The digital contrast of the stacks was manipulated, and a 256 x 256 subsection from each stack was chosen for further analysis. Data stacks were rendered in three dimensions with volume-visualization software (Voxx) (8). Transparency of the volume was manipulated by varying image opacity coefficients with Voxx. Data collection and image analysis for all control and ATP-depleted cells were manipulated identically.
For laser scanning confocal microscopy, individual planes through the entire cell volume were collected at 0.5 µm intervals. A projection image was generated by summing the z-series images through the entire cell volume into one 16-bit image.
Immunoprecipitation and immunoblotting.
Control and ATP-depleted cultures were rinsed in ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (0.15 M NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 0.05 M Tris·HCl, pH 8.0) for ZO-1, and in Triton X-100-containing lysis buffer for aPKC and Par3 (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml leupeptin, 1 mM PMSF, 1.8 µg/ml aprotinin, 1% Triton X-100, 0.1% deoxycholate, and 0.1% SDS) (22) for 15 min on ice. Monolayers were scraped, and lysates were collected. Extracts were cleared by centrifugation at 15,000 g for 5 min at 4°C. Primary antibody was added to supernatants and tubes were rotated at 4°C for 1 h. Immune complexes were collected with protein A-Sepharose beads (Amersham Pharmacia, Piscataway, NJ), and washed three times in lysis buffers. Beads were resuspended in SDS-PAGE sample buffer, and separated on 7.5% SDS polyacrylamide gels. For analysis of total protein levels of ZO-1 and occludin (Fig. 1B), control and ATP-depleted MDCK cells were extracted in SDS-containing buffer (1% SDS, 10 mM Tris·HCl, pH 7.5, and 2 mM EDTA) at 100°C. Cells were scraped, and lysates were heated at 100°C for 5–10 min and sonicated. Samples were clarified by centrifugation. Protein assays on supernatants were performed with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Thirty micrograms of protein were separated by SDS-PAGE. Samples separated by SDS-PAGE were transferred to nitrocellulose filters (Bio-Rad), blocked in Tris-buffered saline containing Tween 20 (TBST; 10 mM Tris·HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20) containing 3% BSA and 5% nonfat dry milk (ZO-1 and occludin), and in TBST containing 3% BSA for aPKC, Par3, mLgl-1, and phosphoserine/threonine. Filters were incubated with primary antibody diluted in blocking solution for 1 h at room temperature. Filters were washed in TBST for 1 h. Filters were then incubated with species-matched horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia) diluted in blocking solution. Filters were again washed in TBST, and signal was detected by enhanced chemiluminescence (ECL kit; Amersham Pharmacia) and exposed to film (Bio-Max ML; Eastman Kodak, Rochester, NY).
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-D-glucopyranoside, and [1 mM PMSF, 1 mM benzamidine, 1 µg/ml pepstatin A, 40 µg/ml bestatin, 5 µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µM butyrated hydroxy toluene]) for 10 min on ice. Lysates were cleared by centrifugation for 5 s at 15,000 g. Twenty-five microliters of lysate were then incubated with 15 µl of GST p21-associated kinase (Pak) binding domain (GST PAK-BD) for 1 h at 4°C with rotation. Beads were washed three times in buffer B (25 mM Tris·HCl, pH 7.5, 150 mM K acetate, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, and 10% glycerol). The unbound fractions from each wash were pooled and TCA precipitated. Beads were dried using a Speedvac (Savant Instruments, Holbrook, NY). Beads and TCA precipitates were resuspended in 50 µl x 2 SDS sample buffer (100 mM Tris·HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 6 M urea) and heated to 70°C for 5 min. Equal volumes from each fraction were analyzed on a 15% SDS Polyacrylamide gel, transferred to Immobilon PSQ (Millipore, Bedford, MA) and analyzed by Western blotting with an anti-Rac1 or anti-Cdc42 antibody. Immunoblots were quantified using a Fluor-S MultiImager (Bio-Rad), and the results presented as a ratio of active (bound) to inactive (unbound) Rac1-GTPase. |
RESULTS |
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MDCK cells were ATP depleted for 60 min, or left untreated in control cells. Cells were processed by immunofluorescence using antibodies against occludin and ZO-1. In control cells both occludin and ZO-1 were concentrated at the apical region of the lateral membrane (Fig. 1A). Distinct breaks were observed in the lateral membrane staining for occludin at 60-min ATP depletion, suggesting that tight junction integrity was disrupted. The fluorescence intensity of ZO-1 at the tight junction was also severely reduced following 60 min of ATP depletion. However, immunoblotting experiments showed that the amounts of the two proteins were unchanged in control and ATP-depleted cells (Fig. 1B), showing that there was redistribution of occludin and ZO-1 without significant changes in ZO-1 or occludin levels by 60 min of ATP depletion (15, 49).
Evidence that ATP depletion downregulates the aPKC signaling pathway in MDCK cells.
aPKC and Par3 localize to tight junctions in epithelial cells (22), and stable association of Par3 with junctions requires aPKC activity (45). mLgl, the other known substrate of aPKC, associates with the lateral membrane, and phosphorylation of mLgl by aPKC is required to restrict the protein to the lateral membrane and exclude it from the apical membrane (33, 52). We examined the distribution of aPKC, Par3, and mLgl-1 in MDCK cells following ATP depletion and recovery. ATP depletion caused ATP levels to drop rapidly to 5% of control within 15 min following antimycin A treatment, and ATP levels recover to 50% of control levels 60 min after cells are returned to normal growth medium (15). Cells were ATP depleted for 45 min, ATP depleted for 45 min and allowed to recover for 45 min, or untreated in controls, and cell monolayers were processed for indirect immunofluorescence. A z-series of x-y images through the entire cell volume of the monolayer was collected with a laser scanning confocal microscope and combined into a single projection image to avoid missing fluorescent signal. ATP depletion caused a redistribution of aPKC, Par3, and mLgl-1 from cell junctions (Fig. 2). Wash out of antimycin A and allowing recovery of ATP levels led to reassembly and redistribution of all three proteins back into cell junctions (Fig. 2).
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on Thr410 is essential for aPKC activation (43). We examined aPKC phosphorylation in MDCK cells by immunoprecipitating total aPKC and immunoblotting the immunoprecipitate using a Thr410 phosphorylation-specific aPKC antibody (Fig. 5A). ATP depletion reduced Thr410 phosphorylation levels of aPKC (Fig. 5B), suggesting that aPKC was inhibited by ATP depletion. In addition, its activity could be reduced due to reduced ATP levels during ATP depletion.
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is critical for tight junction assembly, and Ser827 phosphorylated Par3 is the predominant form found in mature tight junctions (20). We analyzed the phosphoserine/threonine content of ZO-1 and Par3 in MDCK cells subjected to ATP depletion. Cells were ATP depleted for 60 min or ATP depleted and allowed to recover for 60 min. ZO-1 or Par3 was immunoprecipitated from cell extracts, and the phosphoserine/threonine content was examined by immunoblotting these immunoprecipitates using anti-phosphoserine and anti-phosphothreonine antibodies. ZO-1 phosphoserine/threonine content was significantly reduced in ATP-depleted cells compared with control cells (Fig. 6). Both the 180 and 150 kDa forms of Par3 were detected in MDCK cells (22). The 180 kDa form of Par3 was serine/threonine phosphorylated in control cells, and a significant reduction in Par3 serine/threonine phosphorylation was observed following 60 min of ATP depletion (Fig. 6). After 60 min of ATP repletion, the phosphorylation levels of both ZO-1 and Par3 recovered to nearly that of control levels. Par3 phosphorylation was reduced slightly in MDCK cells switched to low-calcium medium, whereas ZO-1 phosphorylation was not affected (see online supplemental Fig. S2), indicating effects of ATP depletion were not replicated merely by inducing tight junction disassembly.
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and these substrates in MDCK cells during ATP depletion and recovery. aPKC was immunoprecipitated from cell extracts and immune complexes were probed with antibodies against Par3 and mLgl-1. ATP depletion increased the amount of Par3 and mLgl-1 that coimmunoprecipitated with aPKC (Fig. 7). Perhaps these interactions are stabilized because aPKC binds but cannot phosphorylate its substrates due to reduced Thr410 phopshorylation and reduced levels of ATP. As a control, treatment of MDCK cells with low calcium medium did not alter the interaction of aPKC with mLgl-1 (see online supplemental Fig. S1B).
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during recovery from ATP depletion interferes with tight junction assembly.
Successful assembly and activation of the aPKC-Par3-Par6 complex is critical for tight junction formation and epithelial polarization. Overexpression of a kinase negative aPKC mutant blocks the formation of tight junctions in a calcium switch assay (46). To better understand the role of aPKC in tight junction assembly in ischemic injury, we tested the effect of a pseudosubstrate inhibitor of aPKC during recovery from ATP depletion. MDCK cells were established on coverslips for 3 days and ATP depleted for 45 min. ATP-depleted MDCK cells were allowed to recover in the presence of a cell-permeable, myristoylated pseudosubstrate inhibitor of aPKC, which directly inhibits aPKC autophosphorylation and transactivation (43), or allowed to recover without pseudosubstrate inhibitor in controls. Distribution of ZO-1 and Par3 was examined by immunofluorescence. Pseudosubstrate inhibitor treatment interfered with the reassembly of ZO-1 and Par3 at cell junctions during recovery from ATP depletion (Fig. 8), indicating that aPKC activity is important for tight junction reassembly during recovery from cell injury.
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pseudosubstrate inhibitor for 45 or 90 min. Pseudosubstrate treatment of confluent MDCK cells did not dramatically affect the junctional distribution of Par3 (see online supplemental Fig. S3), supporting the conclusion that the pseudosubstrate inhibitor specifically inhibits tight junction reassembly during recovery from ATP depletion. Together, the above results indicate that aPKC signaling pathway participates in tight junction recovery after ischemia. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Cdc42 and Rac activation induces aPKC activity by regulating the assembly and activity of the aPKC-Par complex. In turn, activation of aPKC-Par complex stimulates tight junction assembly. Here, we found that ATP depletion of MDCK cells downregulated Rac GTPase activity, and we present evidence that sequestration of the Rac exchange factor Tiam-1 by forming a stable complex with Par3 contributes to the inhibition of Rac activity in ATP-depleted MDCK cells. Association of Par3 with Tiam-1 was not stabilized in MDCK cells treated with low-calcium medium to disrupt tight junctions, indicating that effects of ATP depletion are specific and not a general response to junction disassembly. Interestingly, Cdc42 remained active even after 60 min of ATP depletion (data not shown). Similar differential sensitivity of Rac and Cdc42-GTPases to ATP depletion was also observed in LLC-PK cells (17). Inhibition of aPKC activity, even in the presence of active Cdc42, suggests the possibility that active Cdc42 may be spatially separated from aPKC-Par6 in ATP-depleted MDCK cells and is unable to activate aPKC.
Activity of aPKC is dependent on phosphorylation events. Phosphorylation of aPKC on Thr410 is a prerequisite for its complete activation (43). ATP depletion caused a decrease in Thr410 phosphorylation of aPKC. Par3 and mLgl have been identified as substrates for aPKC (34, 38). aPKC was also shown to phopshorylate occludin, claudin, and ZO-1 in vitro (37). We found that the phosphoserine/threonine content of ZO-1 was reduced following ATP depletion, but not after incubation in low-calcium medium. However, in previous studies (21), a decrease in phosphorylation of ZO-1 was observed in MDCK cells that were treated with low-calcium medium for 16 h. Par3 phosphorylation was also significantly reduced in ATP-depleted MDCK cells, and this correlated with a loss of Par3 from cell-cell junctions during ATP depletion. A decrease in Par3 phosphorylation was also observed in MDCK cells incubated in low-calcium medium, consistent with the finding that Par3 phosphorylated at Ser827 accumulates in assembled tight junctions (20).
ATP depletion also caused redistribution of mLgl-1, another substrate of aPKC, from cell junctions to a diffuse cytosolic distribution. Distribution of mLgl-1 at the basolateral membrane is dependent on its phosphorylation (33). ATP depletion may induce dephosphorylation of mLgl-1, similar to that of Par3, but we were unable to detect any serine phosphorylation of mLgl-1 in control MDCK cells using phosphoserine antibody. A previous report (52) that a phosphomimicking mutant of mLgl failed to bind aPKC
supports the idea that mLgl-1 is dephosporylated during ATP depletion, since we found that there was increased association of mLgl-1 with aPKC in ATP-depleted cells compared with control cells. Furthermore, Yamanaka et al. (52), have shown that in MDCK cells expressing a kinase-deficient mutant, aPKC kn, mLgl colocalized with Par6 at the apical region of the cell and suppressed tight junction formation. It is possible that in ATP-depleted MDCK cells, mLgl-1 sequesters aPKC in an inactive complex. ATP-depletion also enhanced the association between aPKC and Par3. Again, this is consistent with the observation that a nonphosphorylatable mutant of Par3, S827A, formed a stable complex with aPKC, and suppressed tight junction formation, whereas S827E (a phosphomimicking mutant) was unable to associate with aPKC (34).
Inhibition of aPKC with a pseudosubstrate inhibitor during recovery from ATP depletion interfered with reassembly of ZO-1 and Par3 at cell junctions strongly implicating that aPKC activity participates in tight junction assembly dynamics during cell injury and recovery. This is consistent with studies that have shown that expression of a kinase-deficient aPKC mutant inhibited assembly of ZO-1 and Par3 at tight junctions in calcium switch experiments (46).
Our results have led us to propose a working model for tight junction disassembly in ATP-depleted cells (Fig. 9). In normal cells, Rac GTPase is activated by Tiam-1. Rac-GTP activates aPKC, which phosphorylates mLgl-1. Phosphorylated mLgl-1 dissociates from aPKC and distributes to the lateral membrane. aPKC interacts with and phosphorylates Par3, which then associates with tight junctions. How does ATP depletion lead to tight junction disruption? In ATP-depleted cells, various kinase activities are reduced due to reduced ATP substrate concentrations. Phosphatases remain active, which dephosphorylate junctional Par3 and other tight junction proteins. Nonjunctional Par3 associates with Tiam-1 and inhibits Rac activation. Rac-GDP fails to activate aPKC. This, coupled with low cellular ATP levels, interferes with the ability of aPKC to phosphorylate its substrates thereby affecting aPKC protein interactions, because the association of aPKC with Par3 and mLgl is regulated by phosphorylation. In the absence of aPKC-mediated phosphorylation, Par3 and mLgl-1 form "inactive" stable complexes with aPKC in ATP-depleted cells, and fail to distribute to cell-cell junctions. This prevents Par3 and mLgl-1 from participating in normal junction assembly processes, which may include associations with other as-yet unidentified binding partners at the junctions. Furthermore, ATP depletion could interfere with phosphorylation of additional proteins that may be required for tight junction stabilization. These could include proteins of the Crumbs-Pals1-PATJ polarity complex, which also participates in tight junction formation (40).
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Taken together, the disruption of tight junctions during ATP depletion could involve the synergistic dysfunction of signaling pathways involving aPKC, Crumbs polarity complex, and E-cadherin. A more detailed understanding of these signaling pathways will help identify novel therapeutic targets for protecting renal epithelial cells from ischemic cellular damage.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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