Department of Medicine, Indiana University Medical Center, Indianapolis, Indiana 46202-5116
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ABSTRACT |
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Rho family GTPase signaling regulates actin cytoskeleton and junctional complex assembly. Our previous work showed that RhoA signaling protects tight junctions from damage during ATP depletion. Here, we examined whether RhoA GTPase signaling protects adherens junction assembly during ATP depletion. Despite specific RhoA signaling- and ATP depletion-induced effects on adherens junction assembly, RhoA signaling did not alter adherens junction disassembly rates during ATP depletion. This shows that RhoA signaling specifically protects tight junctions from damage during ATP depletion. Rac1 GTPase signaling also regulates adherens junction assembly and therefore may regulate adherens junction assembly during ATP depletion. Indeed, we found that Rac1 signaling protects adherens junctions from damage during ATP depletion. Adherens junctions are regulated by various GTPases, including RhoA and Rac1, but adherens junctions are specifically protected by Rac1 signaling.
ischemia; cadherin; catenin
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INTRODUCTION |
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CADHERINS ARE A FAMILY of cell-cell adhesion molecules that nucleate the assembly of adherens junctions (56). Normal cadherin function requires association with a set of membrane-cytoskeletal proteins that include the catenins. The cadherin-catenin complex links the adherens junction to the actin cytoskeleton. Dynamic regulation of cell-cell adhesion is necessary for normal cellular processes (59). Important regulators of adherens junction assembly are the Rho family GTPases (5, 14, 15, 20, 24, 25, 51).
Rho family GTPases are members of the Ras GTPase superfamily of proteins that switch between GTP-bound active and GDP-bound inactive conformations (31). The Rho family includes Rho, Rac, and Cdc42. These signaling proteins control cytoskeleton and junctional complex assembly in response to various extracellular signals, including growth factors (20, 31). RhoA signaling induces stress fiber and focal adhesion assembly; Rac1 signaling induces actin polymerization at the cell periphery and lamellipodia formation; and Cdc42 signaling induces filopodia formation (20). In epithelial cells, RhoA and Rac1 regulate tight junction and adherens junction assembly and function and Cdc42 regulates adherens junction assembly (5, 14, 15, 20, 24, 25, 37, 51).
Protein phosphorylation pathways that regulate cell adhesion and
junctional complex assembly have been studied extensively, but the
specific mechanisms remain obscure (12, 62). Loss of cell
adhesion and disruption of adherens junction assembly have been
correlated with tyrosine phosphorylation of -catenin and
p120ctn (1, 35, 41-43, 50). However,
Tsukita and colleagues (52), using E-cadherin/
-catenin
fusion proteins, showed that the transition from a strong cadherin
adhesion state to a weak cadherin adhesion state was independent of the
presence of
-catenin in the cadherin complex. The role of
serine/threonine phosphorylation of cadherin-catenin complex components
in the regulation of cell adhesion has been studied less. However,
detailed information comes from studies of
-catenin serine
phosphorylation and dephosphorylation in the Wnt and adenomatous
polyposis coli (APC) tumor suppressor signaling pathways
(39).
Renal ischemia is a consequence of numerous disease processes, and it results in significant morbidity and mortality (53). ATP content in epithelial cells is rapidly depleted during renal ischemia. Cellular consequences of renal ischemia have been investigated in human biopsy tissue samples and animal models (30). Hallmark features of ischemic injury include epithelial cell damage in the proximal tubule that alters epithelial cell polarity and actin-associated cellular structures (13, 30). Many of the consequences to renal epithelial cells in vivo are recapitulated in cultured epithelial cells that are subjected to ATP depletion (13). Tight junctions and adherens junctions are among the actin-associated structures that are disrupted in response to ATP depletion in the kidney (7, 28, 29, 36) and in cultured epithelial cell models (28, 32, 33, 57). Mechanistic details of the injury processes are still largely unknown.
We have hypothesized (18) that Rho family GTPase signaling is inhibited during ATP depletion of cultured epithelial cells. We found (18) that activating RhoA signaling protects tight junctions from disassembly during ATP depletion and inhibiting RhoA accelerates tight junction disassembly during ATP depletion. Cadherin adhesion (adherens junction) and tight junction assembly are coupled (19, 54, 55). In this study, we examined adherens junction disassembly during ATP depletion. RhoA and Rac1 signaling regulates adherens junction assembly (4, 5, 24, 51). ATP depletion also alters assembly and phosphorylation state of cadherin-catenin complex components. However, RhoA signaling did not alter adherens junction disassembly rates during ATP depletion. This indicates that the protective role of RhoA signaling on tight junctions and actin structures during ATP depletion of epithelial cells is limited and selective (18, 40). In contrast, activation of Rac1 signaling protects adherens junctions from disassembly during ATP depletion, and inhibiting Rac1 accelerates adherens junction disassembly during ATP depletion. These data suggest that Rac1 signaling selectively controls adherens junction assembly state during cellular injury and that Rho family GTPase signaling could provide general protective roles for epithelial cell architecture.
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MATERIALS AND METHODS |
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Cell culture, antibodies, and reagents. Madin-Darby canine kidney (MDCK) type II cells were maintained in DMEM (GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum with penicillin, streptomycin, and glutamine (GIBCO BRL). Chemicals were purchased from Sigma (St. Louis, MO) or Midwest Scientific (St. Louis, MO) unless otherwise indicated.
One monoclonal antibody against E-cadherin (DECMA-1) was purchased from Sigma. Another monoclonal antibody against E-cadherin (rr-1) was purchased from the Developmental Studies Hybridoma Bank (maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA, under contract from the National Institute of Child Health and Human Development). The polyclonal antibody against E-cadherin was previously described (34). Anti-peptide polyclonal antibody againstATP depletion. MDCK cells were plated at a density of 2.0 × 105 per 35-mm culture dish. Three- to five-day cultures were rinsed with prewarmed depletion medium and ATP depleted for different times by incubating cells with depletion medium containing 0.1 µM antimycin A (7). For transfected cells, ATP depletion was performed at 30 h after transfection. ATP levels were assayed as described previously (7).
Immunoblotting.
MDCK cells were plated at a density of 2 × 105 cells
per 35-mm dish. Three days later cells were ATP depleted for 30 or 60 min or left untreated in control dishes. Cultures were then 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
collected. Samples were heated at 100°C for 5-10 min and
sonicated. Samples were cleared by centrifugation. Protein assays on
supernatants were performed with a bicinchoninic acid (BCA) protein
assay kit (Pierce Chemical, Rockford, IL). Thirty micrograms of protein were separated on 7.5% SDS polyacrylamide gels. Gels were transferred to nitrocellulose filters (Bio-Rad, Hercules, CA) and blocked in
Tris-buffered saline containing Tween 20 (TBST; in mM: 10 Tris · HCl, pH 7.5, 100 NaCl, and 0.1% Tween 20) containing
milk (5% nonfat dry milk). Filters were then incubated in
primary antibody (E-cadherin 1:10,000 and -catenin 1:1,000) diluted
in blocking solution for 1 h at room temperature, and blots were
then washed in TBST for 1 h with several changes. Filters were
then incubated for 1 h at room temperature with species-matched
HRP-conjugated secondary antibody (Amersham, Arlington Heights, IL)
diluted 1:5,000 in blocking solution. Filters were again washed, and
signal was detected by enhanced chemiluminescence (ECL kit; Amersham,
Arlington Heights, IL) and exposed to film (Kodak Bio-Max ML; Eastman
Kodak, Rochester, NY).
Transient transfection. MDCK cells were plated at 2.5 × 105 per 35-mm culture dish. Cells were transfected 24 h later with 1.5-2 µg each of control vector or plasmids with RhoA-V14, RhoA-N19, Rac1-V12, or Rac1-N17 cDNAs expressed from SV40 promoters (generously provided by Dr. Marc Symons, Picower Institute for Medical Research, Manhasset, NY) with Lipofectamine plus according to manufacturer's protocol (GIBCO BRL). Cells were incubated with the transfection mixture for 3 h. This mixture was then replaced with normal growth medium. Cells were analyzed at various times, but the experiments shown were analyzed 30 h after transfection.
Immunoprecipitation.
Cultures were rinsed with ice-cold phosphate-buffered saline (PBS; in
mM: 2.7 KCl, 1.5 KH2PO4, 137 NaCl, 8.1 Na2HPO4, and 0.45 CaCl2 with 0.5 M
MgCl2). Cells were extracted with CSK buffer [in mM: 50 NaCl, 300 sucrose, 3 MgCl2, 1 phenylmethylsulfonyl fluoride
(PMSF), 0.5% Triton X-100, and 10 PIPES, pH 6.8] for 15 min on ice.
Monolayers were scraped, and lysates were collected. Extracts were
cleared by centrifugation in a microfuge at 13,000 rpm for 5 min at
4°C. Primary antibody (E-cadherin or -catenin polyclonal antibody)
was added to supernatants, and tubes were rotated at 4°C for 1 h. Immune complexes were collected with protein A-Sepharose beads
(Pharmacia, Piscataway, NJ) and washed three times with CSK buffer.
Beads were resuspended in SDS-PAGE sample buffer for analysis, and
separated on 7.5% SDS polyacrylamide gels. Immunoblotting was
performed as described in Immunoblotting except that TBST
containing 1% BSA was used for phosphotyrosine blotting.
Confocal microscopy: immunofluorescence, image acquisition, and
image analysis.
MDCK cells were plated and transfected as described in Transient
transfection. At 30 h after transfection, cells were left untreated or ATP depleted as described in ATP depletion.
Cells were fixed in PBS containing 3.7% paraformaldehyde for 10 min at
room temperature. Cells were washed in PBS and then permeabilized in
PBS containing 0.5% Triton X-100 for 10 min at room temperature. Cells
were then blocked in PBS containing 0.2% BSA and 2% goat serum for 30 min at room temperature. The first primary antibody diluted in blocking
buffer [DECMA-1 (1:500) or -catenin polyclonal antibody (1:300)]
was incubated for 45 min at room temperature. Cells were washed in PBS
containing 0.2% BSA and incubated with mouse anti-myc
antibody (9E10) for 45 min at room temperature. Coverslips were again
washed and incubated with Cy5-conjugated goat anti-rat or
Cy5-conjugated goat anti-rabbit antibody (diluted 1:100 in blocking
buffer) and FITC-conjugated goat anti-mouse antibody (1:100; Jackson
Labs, Bar Harbor, ME) 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 (DABCO).
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Two-photon microscopy.
MDCK cells (2.5 million) were plated on six-well polycarbonate filter
inserts (Corning-Costar, Kennebunk, ME) and cultured for 4 days. Cells
were ATP depleted for 30 or 60 min with 0.1 µM antimycin A or left
untreated in the control filters. Filters were then fixed with 4%
paraformaldehyde for 10 min at room temperature and processed for
indirect immunofluorescence with antibodies against E-cadherin and
-catenin. FITC-conjugated secondary antibodies were purchased from
Jackson Labs. Samples were mounted in PBS containing DABCO, and
two-photon microscopy was performed with the use of a Bio-Rad MRC1024
confocal/two-photon system fitted to a Nikon Eclipse inverted
microscope (Melville, NY) with a ×60 water-immersion, NA 1.2 objective. Illumination for the multiphoton fluorescence excitation was
provided by a Spectra-Physics (Mountain View, CA) Tsunami Lite
titanium-sapphire laser. Data sets were collected as
z-series of ~100 images with a spacing of 0.1 µm. Acquired images were first processed for background subtraction by
applying a 3 × 3 low-pass filter with Metamorph version 4.1.7 image-processing software. The digital contrast of the stacks was
manipulated, and a 256 × 256 subsection from each stack was chosen for further analysis. Data stacks were rendered in three dimensions with volume-visualization software (Voxx). 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.
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RESULTS |
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ATP depletion alters adherens junction assembly.
Adherens junctions are disrupted during ATP depletion by unknown
disassembly mechanisms (33). To study the effects of ATP depletion on adherens junction component distribution in greater detail, distributions of E-cadherin and -catenin were examined in
three-dimensional reconstructions of immunofluorescence images collected with two-photon microscopy (Fig.
1A). The two-photon microscopy
minimizes photobleaching during the acquisition of ~100 images used
for three-dimensional data analysis. Data sets were processed with a
voxel rendering software program (Voxx) developed in the Indiana
University Imaging Facility (11). Please refer to the
Supplementary Material1 for
this article (published online at the American Journal of Physiology-Cell Physiology web site) to view the movies of
rotating volumes for each image in Fig. 1A.
ATP depletion decreased cadherin-catenin complex phosphorylation. Adherens junction disassembly that occurs during ATP depletion (Fig. 1; Ref. 33) may be a consequence of protein phosphorylation changes that regulate protein complex assembly. Protein kinases are generally inactivated during ATP depletion, resulting in a general decrease in protein phosphorylation in epithelial cells after injury (27). Therefore, we examined the consequences of ATP depletion on cadherin-catenin complex phosphorylation.
MDCK cells were ATP depleted for various times. Cells were extracted with Triton X-100-containing buffer, and cadherin-catenin complexes were immunoprecipitated with E-cadherin-specific antibodies. These immunoprecipitates were separated on SDS polyacrylamide gels, transferred to nitrocellulose, and immunoblotted to detect phosphotyrosine or phosphoserine/phosphothreonine. Immunoprecipitates were also blotted with E-cadherin antibodies to show that comparable amounts of E-cadherin were immunoprecipitated.RhoA signaling did not protect adherens junctions from disassembly during ATP depletion. Previous work showed that tight junctions, stress fibers, and the cortical actin cytoskeleton are protected from disassembly during ATP depletion by constitutive RhoA signaling (18, 40). We examined whether RhoA signaling protects adherens junctions. MDCK cells were transfected with plasmids encoding RhoA mutant proteins, and these transiently transfected monolayers were ATP depleted with antimycin A.
Effects of RhoA GTPase signaling and ATP depletion were assayed with confocal microscopy and quantitative image analysis. MDCK monolayers transiently transfected with plasmids encoding either RhoA-N19 or RhoA-V14 were fixed and processed for double-label immunofluorescence to detect myc-tagged mutant RhoA proteins and adherens junction components (E-cadherin orRac1 signaling protects adherens junctions from disassembly during
ATP depletion.
Because RhoA GTPase signaling did not protect adherens junctions from
disassembly during ATP depletion, we sought to determine whether
another Rho family member may have a protective role. Rac1 GTPase
signaling also regulates adherens junction assembly, and consequently,
Rac1 may protect adherens junctions from disassembly during ATP
depletion. MDCK monolayers transiently transfected with plasmids
encoding either Rac1-N17 or Rac1-V12 were fixed and processed for
double-label immunofluorescence to detect myc-tagged mutant
Rac1 proteins and adherens junction components (E-cadherin or
-catenin). Fluorescence intensity ratio of the intensity
measurements from junctions between two transfected cells divided by
the intensity measurements from junctions between two nontransfected
cells was calculated as in the RhoA experiments.
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DISCUSSION |
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Regulation of junctional complex assembly and function is incompletely understood, but insight comes from the finding that Rho family GTPases regulate epithelial junctional complexes (5, 14, 18, 20, 22, 24, 25, 37, 51). Rho family GTPases regulate intracellular membrane trafficking and epithelial cell polarity (44). Cadherin adhesion helps maintain epithelial monolayer integrity and provides spatial cues that determine epithelial cell polarity (61). Cadherin assembly also controls assembly of the tight junction (which separates membrane domains in epithelial cells) (3, 8, 16, 17, 19, 54, 55, 60).
Adherens junctions are disrupted during ATP depletion by unknown disassembly mechanisms that lead to E-cadherin redistribution (Refs. 6, 33; Fig. 1). We and others (6, 33) found that E-cadherin was internalized during ATP depletion. Appearance of cytoplasmic vesicular structures was coincident with a reduction in E-cadherin biotin labeling on the cell surface (33). These findings suggest that endocytosis mechanisms lead to cadherin-catenin complex internalization during ATP depletion. Endocytosis trafficking of plasma membrane proteins requires ATP. Therefore, direct experiments will be required to determine the specific pathways utilized for E-cadherin internalization during ATP depletion. Reduced cell surface E-cadherin may decrease cell adhesion between epithelial cells, which is consistent with findings that tubular epithelial cells are shed during the ischemic event (30, 53).
We investigated the consequences of ATP depletion on the phosphorylation of cadherin-catenin complexes. Many proteins are dephosphorylated during ATP depletion because ATP levels are reduced below the Km for protein kinases, yet protein phosphatases remain active (27). Our previous studies (18) and those of other investigators (2, 9, 10, 27, 49, 58) showed that membrane-cytoskeletal and junctional complex components were dephosphorylated during ATP depletion. In this study, we found that cadherin-catenin complex components were rapidly dephosphorylated during cellular injury and that the normal phosphorylation state returned when cells were allowed to recover from ATP depletion. These dephosphorylation events that occur during ATP depletion may contribute directly to junctional complex disassembly and cadherin redistribution.
A transient increase in -catenin tyrosine phosphorylation was
observed at short times after ATP depletion. Schwartz and colleagues (48) found that
-catenin tyrosine phosphorylation
showed a sustained increase during cyanide-induced ATP depletion of
mouse proximal tubule primary culture cells. We ATP-depleted MDCK cells with cyanide, and a sustained increase in
-catenin tyrosine
phosphorylation was not observed. It is unclear whether there is a
relationship between the transient increase that we observed in the
MDCK cell model and the sustained increase found in the mouse proximal
tubule primary culture model (48).
We have examined whether the effects of cellular injury on epithelial junctional complex assembly are regulated by Rho GTPase signaling. Previously, we found (18) that RhoA signaling protects tight junctions from disassembly during ATP depletion. Because RhoA also controls adherens junction assembly and adherens junction assembly controls tight junction assembly, we wanted to determine whether RhoA signaling protection of tight junctions was an indirect consequence of RhoA signaling protection of adherens junction assembly. In this study, we show that RhoA does not protect cadherin complexes from disassembly during ATP depletion. RhoA protection of tight junction but not adherens junction disassembly during ATP depletion may indicate that RhoA regulation of tight junction assembly is a more proximal effect than RhoA regulation of adherens junction assembly. RhoA protection of cellular architecture is not limited to tight junctions (18). Cortical actin and stress fibers were also protected by RhoA signaling during ATP depletion (40). Together, these findings indicate that the protective effects of RhoA signaling during cellular injury are selective and specific for a subset of cellular structures and processes.
Our finding that RhoA signaling does not protect adherens junctions from disassembly during ATP depletion raised the question of whether another Rho family member may selectively protect adherens junctions from disassembly during cellular injury. Indeed, our current studies show that, like the effect of RhoA on tight junctions, inhibition of Rac1 signaling accelerates ATP-depletion-induced adherens junction disassembly mechanisms and activation of Rac1 signaling protects adherens junctions from disassembly during ATP depletion.
Previous studies described effects of Rac1 signaling on adherens
junction development (5, 14, 15, 20, 24, 25, 51).
Increased adherens junction assembly is found in cells expressing
constitutively active Rac1. Downstream Rac1 signaling effectors that
control adherens junction assembly are being studied. For example,
IQGAP1 is a target of Rac1 and Cdc42 signaling that binds -catenin
and dissociates
-catenin from the cadherin-catenin complex. Future
studies will address how inhibition of Rac1 signaling by ATP depletion
may cause cadherin-catenin complex disassembly. In addition, it will be
of interest to determine Rac1 effectors that are responsible for
protecting adherens junctions during cell injury by ATP depletion.
Actin and associated structures are disrupted by ATP depletion
(13, 30). Indeed, the actin-binding proteins vinculin and -catenin, which are associated with the adherens junction, are also
redistributed during ATP depletion (6, 40). RhoA signaling protects actin stress fibers during ATP depletion, but vinculin assembly in focal adhesions was not protected by constitutive RhoA
signaling (40). Perhaps differential effects of Rho family GTPase signaling on vinculin within the adherens junction during ATP
depletion could explain the differential protective effects of RhoA and
Rac1. Experiments are under way that examine this issue.
Several Ras superfamily members control adherens junction assembly and cadherin function. Ras itself downregulates cadherin adhesion and increases epithelial cell migration (21, 26, 45, 47). As discussed above, Rho family GTPases control cadherin adhesion and adherens junction assembly (5, 14, 15, 20, 24, 25, 51). ARF6, another Ras superfamily of GTPase, also regulates cadherin function by controlling intracellular membrane trafficking of cadherin molecules (38). ARF6 signaling also activates Rac1 signaling (46), and this connection may partially explain how ARF6 control adherens junction assembly mechanisms. There are potentially numerous points of control for cadherin function, including cytoskeletal assembly, catenin binding, vesicle trafficking, and protein turnover, that are regulated by small GTPases, and many or all these processes may be disrupted by ATP and GTP depletion during ischemia. A more complete understanding of these regulatory and pathophysiological processes will help us determine how epithelial cells are injured during ischemia, which may indicate new therapeutic approaches to reverse this damage.
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ACKNOWLEDGEMENTS |
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We thank Dr. Sherry Babb and Jeff Clendenon (Renal Epithelial Biology Laboratory Imaging Facility, Indiana University) for expert assistance with image acquisition and analysis. We also thank Dr. Marc Symons (Picower Institute for Medical Research, Manhasset, NY) for providing mutant RhoA expression plasmids. We thank Drs. Bruce Molitoris and Simon Atkinson for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported by a fellowship from the American Heart Association, Indiana Affiliate, Inc. to S. Gopalakrishnan. K. W. Dunn was supported by grants from the Indiana University Strategic Directions Initiative, and J. A. Marrs was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54518 and DK-53465. This work was also supported by a grant (InGen) from the Lilly Foundation to the Indiana University School of Medicine.
Address for reprint requests and other correspondence: J. A. Marrs, Dept. of Medicine, Indiana Univ. Medical Center, Fesler Hall 115, 1120 South Dr., Indianapolis, IN 46202-5116 (E-mail: jmarrs{at}iupui.edu).
1 Supplemental material to this article is available online at http://ajpcell.physiology.org/cpi/content/full/283/1/C261/DC1.
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.
10.1152/ajpcell.00604.2001
Received 19 December 2001; accepted in final form 6 March 2002.
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