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From the 1 Departments of Ophthalmology, and 2 Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee.
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Abstract |
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METHODS. Cultures were maintained at confluence without passage for intervals to
several months. Adherens junction proteins (N-cadherin, E-cadherin,
-catenin, ß-catenin, plakoglobin, and actin) and the proliferation
marker Ki-67 were localized in the cultures by fluorescence microscopy,
and in vitro wound healing was compared. Adherens junctions were
analyzed for protein solubility in detergent buffers and sensitivity to
disruption by treatment with anti-cadherin antibodies and low calcium
conditions.
RESULTS. Compared with cultures in early-confluence (2–3 days), postconfluent cultures (weeks) had more mature adherens junctions characterized by a circumferential (rather than linear) actin organization, and a zonular (rather than punctate) distribution of more detergent resistant cadherin and catenins. Postconfluent cultures also had fewer Ki-67-positive cells and a higher cell packing density. Early-confluence cells migrated into in vitro wounds as dissociated single cells, whereas postconfluent cells moved as contiguous sheets, retaining an intact junction during wound-induced cell migration and proliferation. Mature junctions were not disrupted by treatment of living cells with N-cadherin antibodies, which bound to and remained detectable at junctions for several days. Calcium withdrawal displaced N-cadherin from mature junctions and rendered it more soluble, but the dominant circumferential pattern of actin was stable. Restoration of medium calcium resulted in a rapid (hours) recovery of a nearly complete zonular pattern of insoluble N-cadherin.
CONCLUSIONS. Over long postconfluent periods, cultured RPE cells became more growth quiescent, and intercellular cadherin adhesions became more stable, exhibiting increased resistance to calcium removal and greater retention of junctional integrity during in vitro wound closure. Consideration should be given to whether the behavior of RPE cells in postconfluent cultures, where intercellular adhesions are more mature, more closely simulates RPE cells in situ than cells in early-confluence cultures, which are more commonly used for analysis.
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Introduction |
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Retinal pigment epithelium (RPE) cells differ from most monolayer epithelial cells in that the dominant cadherin is N-cadherin,11 12 13 14 15 and adherens junction development is markedly slower, at least in RPE cell subpopulations selected for their highly epithelioid phenotype.15 16 Rather than forming a mature junction within hours after cell–cell contact, fully dissociated epithelioid RPE cells require weeks at confluence to develop an N-cadherin junction that is completely zonular and stable to detergent extraction.15 E-cadherin is expressed by some epithelioid variants of RPE, but E-cadherin may not contribute to early stages of junction formation because it does not localize to junctions until after the N-cadherin–containing junction is already established.17 Unselected cultures of RPE cells, of the type typically used for experimentation, are also likely to undergo a slow postconfluence junctional maturation. Time at confluence therefore might produce RPE cells with different levels of junction development and therefore different baseline properties in functional assays.
A functional assay that is widely used to analyze the induction of cell growth and migration in epithelial cells, including the RPE,18 19 20 21 22 is in vitro wounding. In wound experiments, cultures are typically grown to confluence to generate contiguous monolayers, then defects are produced by chemical or mechanical treatments. Confluence is used to establish a quiescent baseline state resulting from the long-established contact inhibition of cell growth23 and motility.24 Confluence also triggers the morphogenetic process that produces an epithelial phenotype. Confluent epithelial monolayers are therefore presumed to resemble mature, quiescent cells within the intact tissue and to respond to wounding in a manner that simulates the response of cells in situ. This presumption might be questioned for RPE cells in which adherens junction development is delayed, so that RPE cultures shortly after confluence may not manifest the epithelial properties that have been attributed to them.
In the current study we analyzed RPE cultures in early confluence (within 2–3 days of cell contact), and in late confluence (approximately 8 weeks after confluence) to evaluate the developmental state of the adherens junction and the functional response of cells to in vitro wounding. Wound closure was also examined in a few cultures after several months at confluence. We conclude that maintaining RPE cultures for extended postconfluent periods produces more growth-quiescent cells with stable intercellular adhesions whose behavior during wound closure differs from cells in early-confluence cultures, when most epithelial cells are typically analyzed.
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Methods |
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Fluorescence Microscopy
RPE cultures in chamber slides were fixed for 15 minutes in cold
3% phosphate-buffered paraformaldehyde (pH 7.4) then permeabilized by
incubation for 5 minutes at room temperature in 0.5% Triton X-100 in
phosphate-buffered saline (PBS). To visualize detergent-insoluble,
cytoskeletally linked protein, some cultures were extracted before
fixation with a detergent buffer containing protease inhibitors using a
previously described protocol.26
For immunostaining, fixed monolayers were incubated with primary
antibodies overnight at room temperature, followed by three rinses in
blocking buffer (10 mg/ml bovine serum albumin in PBS) and a 1-hour
incubation in secondary antibodies. The following mouse monoclonal
primary antibodies were used: anti-N-cadherin (clone GC-4; Sigma, St.
Louis, MO), anti-E-cadherin (clone HECD-1; Zymed Laboratories, San
Francisco, CA), anti-/catenin (Becton Dickinson, San Jose, CA),
anti-ß-catenin and anti-
-catenin/plakoglobin (Transduction
Laboratories, Lexington, KY), and anti-nuclear antigen Ki-67 (a
proliferation marker; MIB-1; Immunotech, Westbrook, ME). Monoclonal and
polyclonal pancadherin antibodies (Sigma) were also used. The
pancadherin antibodies, which are directed against an epitope in the
cytoplasmic domain of the cadherins, react primarily with
N-cadherin.17
27
Appropriate fluorescein- or
rhodamine-conjugated secondary antibodies were from Jackson
ImmunoResearch (West Grove, PA). F-actin was visualized by treatment of
fixed cells for 30 minutes at room temperature with fluor-conjugated
phalloidin (Sigma). After staining, coverslips were mounted with
antifade reagent (FluoroGuard; BioRad, Hercules, CA), and specimens
were examined and photographed with a fluorescence microscope.
Western Blot Analysis
The sensitivity to detergent extraction of adherens junction
proteins was examined by preparing detergent-soluble and
-insoluble protein extracts from early- and late-confluence RPE
cultures from three donors. Soluble protein was extracted by the
protocol described, using 50 µl buffer/cm2 of
culture surface, then mixed with an equal volume of
Laemmli28
electrophoresis buffer. The detergent-insoluble
protein that remained in the culture dish was rinsed three times with
fresh detergent buffer and lysed with a 1:1 mixture of detergent buffer
and electrophoresis buffer.
For electrophoresis, reducing agent (5 mM ß-mercaptoethanol) was added to the soluble and insoluble fractions. Samples were boiled for 10 minutes, loaded in equivalent volumes on gels, and electrophoresed using 10% sodium dodecyl sulfate (SDS) separating gels. Proteins were transferred to polyvinylidene (PVDF) membranes by electroblotting.29 Membranes were blocked by gentle agitation for 1 hour in 15% nonfat dry milk and 0.1% Tween-20 in PBS (pH 7.4). Primary and appropriate secondary antibodies (listed in the previous section) were diluted in PBS containing 0.1% Tween-20, and each was incubated with the membrane for periods ranging from 1 hour to overnight. Bands were visualized by the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, Piscataway, NJ).
Antibody Treatment and Calcium Depletion
Several protocols were conducted in which RPE cells were treated
with anti-cadherin antibodies that are directed against the
extracellular domain of the protein and have been reported to block
adhesive function, leading to cell dissociation.30
31
N-cadherin antibodies were used at a concentration of 45 to 200 µg/ml
in complete culture medium (MEM with 10% FBS). In a few experiments,
function-blocking E-cadherin antibodies (4 µg/ml) were also used.
Cultures were incubated with the antibodies for intervals from 6 to 24 hours. Parallel control cultures were exposed to medium containing an amount of nonimmune IgG equal to the antibody. Control cultures were immunostained for N-cadherin, using anti-N-cadherin antibodies and/or pancadherin antibodies, by the protocol described. For treated cultures, antibody that had bound to the living cells was visualized with the fluor-conjugated secondary antibody alone, with or without costaining with pancadherin antibodies. In some experiments on postconfluent cells, paired cultures were treated with E-cadherin function blocking antibodies or with a combination of E-cadherin and N-cadherin antibodies. For these experiments, immunostaining for E-cadherin was also included.
After we observed that N-cadherin antibodies bound to the mature junctions of living cells without disrupting the junctions, we performed experiments to examine retention of bound antibodies. Late-confluence RPE cultures were incubated for 6 hours with N-cadherin antibodies or nonimmune IgG (control), then the medium containing the antibodies was removed and replaced with antibody-free medium. Cultures were harvested at intervals to three days after removal of the antibody, fixed and stained with secondary antibodies alone (to detect antibody pre-bound to living cells), and costained with pancadherin antibodies (to determine total N-cadherin distribution).
The effect of calcium removal on the calcium-dependent cadherin junctions was examined by treating early- and late-confluence RPE cultures for intervals from 1 to 45 minutes with the calcium chelator EGTA (2 mM in complete serum-containing culture medium). EGTA-treated cultures and control cultures incubated in complete medium lacking EGTA were fixed and stained to localize N-cadherin and F-actin as described. In postconfluent cultures, in which 45 minutes of EGTA treatment was necessary to displace N-cadherin from the junctions of most cells, recovery experiments were also performed. Cultures were EGTA-treated for 45 minutes, refed with EGTA-free medium, and harvested 2 hours later. To determine whether recovery of N-cadherin at junctions required protein synthesis, recovery experiments were also conducted on paired cultures in the presence of the inhibitor cycloheximide (CHX, 100 mM). CHX was present during the 45-min EGTA treatment period and the 2-hour recovery interval. Preliminary experiments confirmed that CHX did not affect the displacement of N-cadherin from junctions with EGTA treatment. In some experiments the effect of EGTA treatment on the solubility of N-cadherin and actin in postconfluent cells was examined. EGTA-treated cultures were extracted with detergent buffer followed by examination of detergent-resistant protein by fluorescence microscopy or by western blot analysis of detergent-soluble and -insoluble fractions by using the protocols described.
To examine the effects of prolonged exposure to calcium-depleted medium, postconfluent RPE cultures were incubated in culture medium containing a concentration of calcium (5 µM), below that necessary to support cadherin adhesion.32 33 34 Incubation in low-calcium medium lasted for intervals to 1 month, with biweekly changes of the culture medium. Paired control cultures were similarly maintained in medium containing normal calcium levels (1.8 mM).
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Results |
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The distribution and detergent solubility of the cadherin-linked
protein ß-catenin was similar to that of N-cadherin. In the immature
junctions of early-confluence RPE cultures, ß-catenin distributed in
a punctate pattern at cell borders (Fig. 1E)
that was partially
resistant to detergent extraction (Fig. 1F)
. As junctions matured with
time at confluence, ß-catenin was more completely zonular (Fig. 1K) and largely detergent resistant (Fig. 1L)
. Similar results were
obtained for -catenin and plakoglobin (not shown).
Western blot analysis of detergent-soluble and -insoluble fractions of RPE cells illustrated the increasing resistance to extraction of N-cadherin and all catenins as the junction matured with time at confluence (Fig. 2) . The extent to which the proteins partitioned between soluble and insoluble fractions differed among proteins and varied among cell populations. For all proteins, however, there was a shift to increasing insolubility in late-confluence cultures. E-cadherin was undetectable in early-confluence cultures, but was found in late confluence, as previously reported,17 where it partitioned to both fractions.
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Although the pattern of cell movement into wounds differed, the timing of closure of 1-mm linear wounds was similar, with both early- and late-confluence cultures showing a restored monolayer by 3 to 4 days. Six to 7 days after wound closure (day 10 after wounding), the site of the wound could not be identified in the early-confluence cultures because cells within the wound resembled those in the adjacent monolayer in density and gross phenotype (Fig. 4A ). However, the wounded region of late-confluence cultures remained identifiable on day 10 as a zone of less tightly packed cells that were irregular in shape, often still oriented in the direction of movement into the wound. Cultures that were maintained even longer at confluence before wounding showed delays in the timing of closure. In very-late-confluence cultures (approximately 6 months), wounds were not yet closed at 10 days after wounding (Fig. 4C) . Wounds in very-late-confluence cultures required 3 to 4 weeks for closure, and the site remained identifiable as a zone of less epithelioid cells for at least 2 months after wounding (not shown).
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Discussion |
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The development of an epithelial phenotype is another gradual process that is triggered by cell–cell contact at confluence. The process has been studied in some detail in the kidney epithelial cell line MDCK, which has served as a model of in vitro epithelial morphogenesis. In these cells, early stages of phenotype development occur during the hours after intercellular contact as junctions form and strengthen,1 2 3 4 followed by a more protracted postconfluent maturation.26 35 For RPE cells, however, even initial phenotype development is slow. RPE subpopulations selected for their highly epithelioid phenotype15 16 25 and the less uniformly epithelioid RPE cultures grown by commonly used methods (shown here) require several weeks at confluence to develop epithelial features: an actin cytoskeleton that is largely circumferential and an adherens junction that is largely zonular and detergent stable.
At least two features distinguish postconfluent from early-confluence RPE cultures: greater growth quiescence and a more highly developed adherens junction. Growth state is a well recognized variable affecting cell function, but differences in junctional development are likely to have many functional ramifications as well. Cell–cell junctions are signaling sites, in addition to sites of physical adhesion, and the signaling pathways initiated at junctions converge with pathways regulating growth,36 37 38 39 motility,40 and cell–substrate adhesion.40 Because of the differing developmental states of their adherens junctions, early- and late-confluence RPE cultures may therefore be nearly as functionally divergent as subconfluent and confluent cultures of epithelial cell lines. Indeed, it is likely that cells maintained for extended postconfluent periods have many biologic properties that differ from cells at other stages of in vitro propagation.
To begin to evaluate whether and how time at confluence affects RPE cell function, early- and late-confluence cultures were subjected to a commonly used experimental protocol: in vitro wounding. The response to injury differed, with early-confluence RPE cells migrating largely as single cells, and late-confluence cells moving as a contiguous sheet. The early-confluence movement pattern resembles the pattern shown by others who have examined wounds in RPE cultures shortly after they became confluent.18 19 21 This migration pattern should not be considered characteristic of RPE, however, because simply allowing a longer period at confluence before wounding produced a different outcome. Occasionally, primary cultures of RPE are used for analysis. Cell number is a limitation of this type of culture, but it is presumed that cells recently harvested from eyes function more like cells in situ. In vitro wounds in primary RPE cultures close by movement of contiguous sheets of cells,20 22 similar to the postconfluence cultures shown here. It appears that RPE cells that are propagated in culture by standard methods can be made to reacquire some of the desirable features of primary cultures if a long postconfluence interval is allowed for redevelopment of phenotype.
Time at confluence for RPE cells affected other events in the wound repair process aside from migration pattern, including phenotype recovery in cells that filled the defect. As might be expected, in early-confluence RPE cultures, cells within newly closed wounds were almost immediately indistinguishable from those in the adjacent monolayer, where cells were only a few days ahead in phenotype development. Cells within late-confluence wounds, however, required several additional days to reacquire the appearance and density of cells outside the wounds. After very long postconfluence periods (months), phenotype recovery was even more protracted and possibly indefinite. In this regard, postconfluent cultures may resemble RPE cells within eyes, which exhibit an irregular phenotype at sites of a retinal detachment,41 suggesting that cell shape may not be restored after damage to the monolayer in situ.
Although postconfluent cultures cannot be considered identical with the RPE monolayer within eyes, their slower growth and better developed junctions may make late-confluence cultures a more relevant model for RPE functional analyses than cultures in early confluence.
The Mature Cadherin Junction of Postconfluent Cells
As indicated previously, the development of the adherens junction
has been studied in some detail, especially in epithelial cell lines
that express E-cadherin. However, less has been done to probe the
properties of the adhesion after it has formed. In cells with nascent
E-cadherin or N-cadherin adhesions, anti-cadherin antibodies with
function blocking properties have been used to induce cell
dissociation.30
31
The mechanism whereby this occurs has
not been explored in detail, but the presumption is that antibodies
directed against the extracellular cadherin domain compete with the
cadherin on adjacent cells and inhibit binding of the newly delivered
cadherin. Antibody treatment would therefore disrupt formed junctions
as they turn over. Early-confluence cells showed partial junctional
loss with function-blocking N-cadherin antibody treatment, but antibody
treatment had no effect on junctional integrity in postconfluent cells.
On the assumption that even low levels of E-cadherin in postconfluent
cells17
might stabilize the junction, function-blocking
E-cadherin antibodies were also used, and still no junction disassembly
was observed. It appears that the cadherin adhesion of RPE cells,
although slow to form, may be become stable with a low turnover. This
conclusion is supported by the observation that N-cadherin antibodies
that were bound to the zonular junctions of postconfluent cells
remained detectable for several days after antibody removal.
Increasing junctional stability in RPE cells with time at confluence was also indicated by an increasing resistance of junctional proteins to detergent extraction. Junctional proteins are not alone, however, in showing a time-dependent increase in detergent resistance. The amount of total cellular protein that is detergent stable increases after confluence at a rate that is similar to junctional proteins, so that the relative amount of cadherin and catenins remains nearly constant in detergent resistant protein extracts.26 This observation was interpreted to suggest that junctions are among the protein compartments of epithelial cells that form increasingly stable cytoskeletal associations during postconfluence maturation.26
Another indicator of the stability of the formed junction of RPE cells was its maintenance during wound closure, despite the upregulation of cell movement and growth. In postconfluent RPE cultures, cells moving into the wound often spread dramatically and, although N-cadherin junctions separated into segments, cell–cell contact was retained. For E-cadherin junctions in other cells, adhesions first form as puncta that later coalesce into linear segments, then complete zonular arrays.1 2 3 Similar punctate and segmented accumulations of N-cadherin are seen during the postconfluence formation of the zonular RPE junction.15 With migration into the wound from a postconfluent monolayer, the N-cadherin junction again becomes segmented, suggesting that the mature zonular junction consists of discrete segmental units that give the junction plasticity, allowing cells to spread without losing intercellular contact.
Yet another indicator of the stability of the formed RPE junction was its resistance to disruption by calcium depletion. Longer EGTA treatments were required to displace N-cadherin from mature junctions in postconfluent cells than immature junctions in early-confluence cultures. An unexpected feature of the cadherin junction in postconfluent RPE cells was the stability of the circumferential actin bundle, even when cadherin adhesion was inhibited for weeks by low-calcium treatment. As junctions form, the circumferential actin bundle develops in parallel with the zonular cadherin adhesion15 to which it is linked. It might be expected that actin would lose its circumferential distribution when cadherin adhesion is blocked for extended periods. However, although the actin bundle became less compact and partially displaced from lateral cell borders, the circumferential actin array was maintained with a prolonged low-calcium treatment that rendered N-cadherin nonjunctional. All cadherin-mediated adhesion would be blocked by this protocol, including the dominant RPE cadherin (N-cadherin), less abundant RPE cadherins (E- and P-cadherin17 ), and other unidentified cadherins, should they exist. Perhaps other intercellular adhesion proteins in the RPE15 help maintain the formed circumferential actin bundle in the absence of cadherin linkage.
With EGTA treatment, the actin in some RPE cells contracted within minutes into a smaller ring, presumably because the disruption of cadherin adhesion by rapid calcium removal produced a relatively abrupt release of tension on the circumferential actin bundle. N-cadherin remained transitorily associated with the contracted actin ring, then developed a diffuse extrajunctional distribution. The diffuse N-cadherin may be less tightly associated with the cytoskeleton after EGTA treatment, because treatment renders N-cadherin more soluble in detergent buffer. Nonetheless, restoration of calcium levels to cells in which cadherin had become largely diffuse produced a rapid recovery of the zonular N-cadherin distribution and of detergent insolubility of the protein. Junctional recovery was accompanied by an increased stability of the actin cytoskeleton as well, which also became more detergent resistant. Because the junction reformed rapidly and in the presence of a protein synthesis inhibitor, it appears that the junction is reassembled from preexisting rather than newly synthesized cadherin. How this is accomplished is not yet determined. Little is currently known about the turnover of mature cadherin adhesions, although a recent investigation of the E-cadherin junction of MDCK cells indicated that E-cadherin is continuously removed from and shuttled back to the cell surface in endocytic vesicles and that calcium chelation can induce cadherin internalization.42 It remains to be determined whether the N-cadherin of RPE cells is handled in the same way.
The uncoupling of the zonular distribution of cadherin and actin by calcium removal may provide a clue to why the adherens junction in RPE cells is slow to form. When the junction forms de novo in RPE cells that were fully dissociated, actin often has a stress fiber–type organization and the process of developing a zonular organization of both N-cadherin and actin requires weeks at confluence. However, the reformation of a zonular junction in cells in which actin is already circumferential, as after calcium removal and restoration, is rapid, requiring only hours. This time differential suggests that reorganizing the dominant actin network from linear to circumferential arrays may be the step in the process that retards the de novo formation of a zonular junction. The importance of the organization of actin filaments to the development of an epithelial-type adherens junction was previously recognized.43 Indeed, most cells that express N-cadherin as their dominant cadherin are not epithelial cells, and determining how RPE cells organize actin into a circumferential epithelial pattern may therefore be critical for understanding RPE morphogenesis.
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Footnotes |
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Supported by National Institutes of Health Grants R01-EY10832 (JMB) and P30-EY01931, and by unrestricted grants from the Posner Foundation (Milwaukee, Wisconsin) and Research to Prevent Blindness. JMB is a recipient of a Senior Scientific Investigator Award from Research to Prevent Blindness.
Submitted for publication November 5, 1999; revised April 24, 2000; accepted May 22, 2000.
Commercial relationships policy: N.
Corresponding author: Janice M. Burke, The Eye Institute, 925 North 87th Street, Milwaukee, WI 53226-4812. jburke{at}mcw.edu
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renes, VA, Slingerland, JM, Kerbel, RS (1998) E-cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27KIP1 J Cell Biol 142,557-571This article has been cited by other articles:
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