Treatment of A431 Cells with Dexamethasone Induces Dramatic Morphological Changes
Confluent monolayers of A431 cells had the cobblestone appearance that is typical of squamous epithelial cells. Treatment of A431 cultures with 10−7 M dexamethasone resulted in the development of small islands of cells that showed distinct morphological changes from the parent cells, beginning at ~14 d after addition of steroid. Specifically, the affected cells displayed a more fibroblastic morphology along with marked plasma membrane blebbing (Fig. ). We found that we could enrich for the affected subpopulation by mild trypsinization, since these cells were detached much more readily by trypsin than the normal-appearing ones. Several rounds of such selective trypsinization, followed by further incubation and growth, led to the development of cultures that consisted almost entirely of the abnormal-appearing cells, which we renamed A431D cells to distinguish them from the parent A431 cell line. To characterize the A431D cells, several clones, all of which exhibited a similar phenotype, were isolated by limiting dilution. Aside from their fibroblastic morphology and membrane blebbing, another characteristic that distinguished the A431D cells from the parent A431 line was their growth pattern. Confluent A431 cultures exhibited several vertical layers with desmosomal attachments between layers; however, the A431D grew as a monolayer of cells without desmosomal attachments to one another (Fig. ). Although both types of cultures, when confluent, contained numerous floating cells, they exhibited very different properties. The floating cells of the parent A431 cultures were not viable, as evidenced by their inability to attach and proliferate when transferred to a new tissue culture plate; however, the floating cells of the A431D cultures rapidly reattached to a fresh culture plate and continued to grow (data not shown).
Figure 1 A431D cells have fibroblast-like morphology. Living A431 cells (A and C) and A431D cells (B and D) were photographed using the 40× objective (A and B) or the 100× objective (C and D). Note the fibroblast-like morphology and the minimal (more ...)
Figure 2 A431D cells grow as a monolayer without desmosomal contacts. A431 cells (A and B) and A431D cells (C) were plated on filters and processed for electron microscopy. A431 cells formed several layers with extensive desmosomal contacts. A431D cells grew as (more ...)
Both the morphological appearance as well as the unusual growth properties of the A431D cultures suggested to us that the adhesive properties of the cells were altered. We therefore compared the expression of adherens junction and desmosome components in the parent and derived cell lines.
Stable Expression of Most Adherens Junction Components Is Depressed in A431D Cells
In the first series of experiments, we immunocytochemically stained A431 cultures that had been treated with dexamethasone for ~14 d and therefore were comprised of areas with normal-appearing cells as well as foci with altered morphology. The areas of normal morphology consistently and intensely stained with antibodies to E-cadherin, P-cadherin, α-catenin, and β-catenin, with concentration along the cell–cell borders, as expected for an epithelial cell line. In marked contrast, the foci of altered morphology did not stain with antibodies to constituents of the adherens junction (Fig. ). Cells that lacked expression of E- and P-cadherin initially appeared very flat and developed noticeable surface blebs with even fewer cell–cell contacts, as can be noted in Fig. , A and C. To analyze this population biochemically, we cloned out cadherin-negative cells from the population; all clones exhibited the more fibroblast-like appearance seen in Fig. , which appears to represent the final stage of the morphological changes induced by longterm culture in dexamethasone.
Figure 3 A431 cells show loss of E-cadherin staining as they convert to A431D cells. A431 cells were grown in 10-7 M dexamethasone for 2 wk, transferred to glass coverslips, and processed for immunofluorescence. Phase microscopy (A and C) depicts the altered (more ...)
Several clones of A431D cells were then analyzed by immunoblotting and immunofluorescence for their levels of adherens junctional and desmosomal proteins. Since there was minimal variation, the results from one clone are presented. As shown in Fig. , there was no detectable E- or P-cadherin in the A431D cells, although comparable amounts of cell lysate protein from the A431 parent cells contained readily detectable amounts of these cadherins. When A431D cell extracts were immunoblotted with a monoclonal antibody against human N-cadherin or with a polyclonal anti–pan-cadherin antibody, the results were totally negative, indicating that the A431D cells did not express any classical cadherins (data not shown). Immunoblotting and immunofluorescence with antibodies to the catenins revealed that greatly decreased levels of both α- and β-catenins were present in the A431D cells compared to the A431 parent line. To accurately compare A431D cells with A431 cells in immunofluorescence, timed exposures were taken (Fig. ). Under conditions in which intense staining was found in A431 cells, α-catenin, β-catenin, and plakoglobin were barely visible in A431D cells. When longer exposures were taken, however, all three catenins were detectable (Fig. and not shown). In contrast, neither E-cadherin nor P-cadherin was detectable even after prolonged exposures (data not shown).
Figure 4 A431D cells have decreased levels of cadherins and catenins. (A) A431 cells (odd numbered lanes) and A431D cells (even numbered lanes) were extracted with NP-40, and equal amounts of protein from each extract were resolved by 7% SDSPAGE, transblotted (more ...)
Figure 5 Expression of adherens junction and desmosome proteins is abnormal in A431D cells. A431 cells (A, C, E, G, and I) or A431D cells (B, D, F, H, and J) were grown on glass coverslips and processed for immunofluorescence. (A and B) The localization of E-cadherin (more ...)
β-Catenin and plakoglobin are expressed at low levels by A431D cells. When automatic exposures were made of cells stained for β-catenin (A; β-cat) or plakoglobin (B; pg), each protein was detectable. Bar, 30 μm.
To compare the synthetic capacity of A431D and A431 cells for cadherins and catenins, both types of cultures were biosynthetically labeled with 35S[Methionine/Cysteine] for 1 h. Extracts were prepared from equal numbers of cells, subjected to immunoprecipitation with antibodies against each of these proteins, resolved with SDS-PAGE, and visualized with autoradiography. When immunoprecipitated with antibodies to either E- or P-cadherin, A431D extracts revealed no bands, although intense bands were present in the A431 parent extracts (data not shown). These data are consistent with the immunofluorescence and immunoblotting results, all of which demonstrate that A431D cells do not make detectable levels of E- or P-cadherin.
In contrast, when immunoprecipitation was performed with antibodies to the catenins, it became clear that A431D cells synthesized close to normal levels of both α- and β-catenin (Fig. A
). However, anticatenin antibodies did not coimmunoprecipitate other catenins or cadherins from the A431D extracts, although catenins, cadherins, and plakoglobin did coimmunoprecipitate in the A431 parent cell extracts. Hence, although A431D cells synthesize α- and β-catenins, they do not form a complex with other cellular proteins. To examine the stability of the catenins, we performed pulse chase labeling experiments (Fig. B
). After a 1-h chase, β-catenin had almost completely disappeared in A431D cells. By contrast, in the parent A431 cells after a 2-h chase, β-catenin was still present at almost the same level as in the 0-h control. Interestingly, α-catenin was stably expressed by A431D cells, at least for a 2-h chase period. The catenin biosynthetic labeling results, coupled with the evidence for reduced total levels of these proteins as observed in immunoblots and via immunofluorescence, suggest that β-catenin is expressed but then rapidly degraded in A431D cells. Given the total lack of E- and P-cadherins in the A431D cells, the instability of β-catenin is not surprising since in L cells, stable catenin expression has been shown to be dependent upon cadherin expression (Nagafuchi and Takeichi 1988
; Ozawa et al., 1989
). It was surprising, however, that α-catenin was stably expressed. The level of expression, as shown by immunoblot analysis (see Fig. ), was lower than in A431 cells, but the turnover rate was not as rapid as that for β-catenin. Consistent with α- and β-catenins, plakoglobin was expressed at a lower level in A431D cells; its turnover rate in A431D cells and A431 cells appeared to be similar, presumably due to its association with desmosomal cadherins. Thus, our data concerning adherens junction proteins demonstrate that A431D cells have no detectable classical cadherins and decreased levels of α-catenin, β-catenin, and plakoglobin.
Figure 7 Catenins do not form normal complexes in A431D cells. (A) A431 cells and A431D cells were labeled with [35S][Methionine/Cysteine] for 1 h and extracted with NP-40. Cell extracts were immunoprecipitated with monoclonal anti–α-catenin (more ...)
Expression of Desmosomal Components Is Not Severely Affected in A431D Cells
Extracts of A431D or A431 cells were compared by immunoblot analysis for expression of the desmosomal cadherins (desmoglein and desmocollin). A431D cells showed approximately equal levels of desmocollin (data not shown) and desmoglein when compared with the parent A431 cells (Fig. ).
Immunofluorescence staining with antibodies against the desmosomal proteins similarly revealed the presence of these proteins in A4341D cells (Figs. and ). However, the pattern of staining was distinct in the A431D and A431 cells. When A431 cells were examined at a higher magnification, it was clear that both desmoglein and desmoplakin were present in a punctate pattern along the cell–cell borders, indicative of desmosome organization (Figs. , A and C). In contrast, staining in the A431D cells was much more diffuse; desmoplakin was completely cytosolic (Fig. D), and desmoglein appeared to be diffuse but with some indication of membrane staining. However, it was not present in a punctate pattern (Fig. B). To rule out the possibility that A431D cells had lost the ability to either synthesize or properly organize keratin filaments, we compared staining patterns of A431 cells and A431D cells (Fig. ). Cells were plated sparsely so that the A431 cells would have minimal cell–cell contact. Keratin filaments were expressed in A431D cells and were organized in a pattern similar, but not identical, to that seen in A431 cells. Thus, the data we have presented indicate that, although A431D cells synthesize the proteins necessary for desmosome formation, they are not able to organize these constituents into a functional structure (Figs. , , , and ).
Figure 8 A431D cells show abnormal localization of desmosomal proteins. A431 cells (A and C) and A431D cells (B and D) were grown on glass coverslips, processed for immunofluorescence with antibodies against desmoglein (A and B; dg) or desmoplakin (C and D; (more ...)
The pattern of keratin staining is similar in A431 cells and A431D cells. A431 cells (A) or A431D cells (B) were grown on glass coverslips and processed for immunofluorescence with antibodies against keratin. Bar, 30 μm.
Transfection of E- or P-Cadherin into A431D Cells Restores Some but Not All Cadherin-related Functions
In previous studies using human keratinocytes and other epithelial cells, we and others have demonstrated that either E- or P-cadherin function is required for normal desmosome organization, as determined by redistribution of the desmosomal components to a punctate pattern along cell–cell borders and by ultrastructural analysis (Hodivala and Watt, 1994
; Lewis et al., 1994
; Amagai et al., 1995
; Jensen et al., 1996). We therefore hypothesized that the lack of desmosomes in A431D cells was secondary to the loss of E- and P-cadherin expression. To test this hypothesis, we reexpressed a cadherin in the A431D cells by transfecting the entire cDNA for either E- or P-cadherin, using a plasmid that also conferred G418 resistance. Resistant colonies were screened for the expression of E- or P-cadherin by immunofluorescence and positive clones were selected. Clones of A431D cells transfected with E-cadherin (A431DE cells) or P-cadherin (A431DP) that expressed levels of cadherin nearly equivalent to that of the parent A431 cells were selected for further analysis (Fig. ). As anticipated, A431DE cells expressed only E-cadherin and not P-cadherin, while A431DP cells expressed only P-cadherin and not E-cadherin. Thus, transfection of one cadherin cDNA did not lead to expression of the other cadherin by activation of the endogenous genes.
Figure 10 Transfection of E- or P-cadherin into A431D cells stabilizes the expression of α- and β-catenin. (A) A431 cells (lane 1), A431D cells (lane 2), A431D cells transfected with E-cadherin (A431DE; lane 3), and A431D cells transfected with (more ...)
Stable expression of both α- and β-catenin increased in the A431D cells transfected with either E- or P-cadherin (Fig. ). These results confirm our hypothesis that the lower levels of α- and β-catenin in A431D cells were due to instability because of a lack of association with cadherin rather than to a direct effect of dexamethasone treatment.
Immunofluorescence localization of either E-cadherin in the A431DE cells (Fig. A) or P-cadherin in the A431DP cells (data not shown) showed that the cadherin was found at cell–cell borders and was not diffusely distributed in the cytoplasm, suggesting that the cadherin was organized into junctional structures. The desmosomal protein desmoglein was also localized at cell–cell borders, but it did not exhibit the punctate pattern that is indicative of desmosome formation (compare Fig. B with 8 A). Furthermore, desmoplakin remained in a diffuse cytoplasmic pattern (Fig. C) similar to that of A431D cells (Fig. D). These data indicate that, despite the expression and cell–cell border localization of the classical cadherin in these cells, the desmosomal proteins were not being organized into punctate structures indicative of desmosomes. Throughout the remainder of this paper, only the A431DE cells will be discussed, although identical experiments were carried out for the A431DP cells, and similar results were obtained.
Figure 11 A431D cells transfected with E-cadherin do not regain the ability to organize desmosomal proteins at cell–cell borders. A431D cells transfected with E-cadherin (A431DE) were grown on glass coverslips and processed for immunofluorescence. (A) (more ...)
To evaluate in another way the functionality of the transfected cadherin, we tested its ability to form complexes with the cellular catenins. Whole cell extracts of the A431DE cells were immunoprecipitated with anti–Ecadherin antibodies, followed by immunoblot analysis to look for the presence of coimmunoprecipitated β-catenin or plakoglobin. As shown in Fig. A, β-catenin coimmunoprecipitated with E-cadherin to an equivalent extent in A431 and A431DE cells. In contrast, only very small amounts of plakoglobin coimmunoprecipitated with the transfected E-cadherin when compared with that normally seen in A431 cells (Fig. A).
Figure 12 Plakoglobin is not associated with E-cadherin in A431DE cells. (A) A431 cells (lane 1) or A431D cells transfected with E-cadherin (A431DE; lane 2) were extracted with NP-40 and equal amounts of protein from each extract were immunoprecipitated (IP (more ...)
To test the possibility that plakoglobin expressed by the A431DE cells was altered such that it could not associate with any cadherins, we immunoprecipitated desmoglein from A431, A431D, and A431DE cells and then compared the levels of plakoglobin coimmunoprecipitated with this cadherin in each cell line (Fig. B
). In all three cell lines, plakoglobin was found to be complexed with desmoglein to a similar (although not identical) extent. Similarly, plakoglobin was also associated with desmocollin in all the tested cell lines (data not shown). The domain of plakoglobin that associates with the classical cadherins overlaps the domain that associates with the desmosomal cadherins (Chitaev et al., 1996
; Wahl et al., 1996
; Witcher et al., 1996
). Thus, the inherent ability of plakoglobin to associate with desmosomal (and presumably classical) cadherins did not seem to be completely disrupted in A431D cells.
Plakoglobin Association with E-Cadherin Is Necessary for Desmosome Organization
As described in the previous section, transfected E-cadherin in A431DE cells appeared in many ways to be functional in that it associated with α- and β-catenins and was localized along cell–cell borders. Surprisingly, it did not complex with cellular plakoglobin, and it apparently could not mediate desmosome organization. Based on these observations, we hypothesized that the inability of the A431DE cells to form desmosomes might be related to a lack of E-cadherin–plakoglobin interaction. To address this hypothesis, we attempted to increase the amount of E-cadherin–plakoglobin complex within the A431DE cells by overexpressing plakoglobin. A431D cells were therefore cotransfected with full-length plakoglobin cDNA as well as E-cadherin cDNA, and coexpressing clones were isolated (A431DEpg cells).
To determine whether restoration of the plakoglobin level led to restoration of the ability to form desmosomes, we performed immunofluorescence and electron microscopic analyses. Staining of A431DEpg cells with antibody to desmoplakin revealed a punctate pattern along the cell– cell borders (Fig. B), strongly suggesting formation of desmosomes. This was confirmed by ultrastructural analysis which demonstrated the presence of desmosomes in A431DEpg cells (Fig. , C and D).
Figure 13 Cotransfection of A431D cells with E-cadherin and plakoglobin results in formation of desmosomes. A431D cells transfected with E-cadherin (A431DE; A) or cotransfected with E-cadherin and plakoglobin (A431DEpg; B) were grown on glass coverslips (more ...)
To demonstrate that we had in fact increased the level of classical cadherin–plakoglobin complex in the double transfectants, we semiquantified the relative levels of plakoglobin in different compartments of the parent and transfected cell lines using immunoblots. We prepared three sequential cell fractions: (a) an aqueous soluble fraction prepared by homogenizing the cells in the absence of detergent, (b) a nonionic detergent-soluble fraction prepared by NP-40 extraction of the pellet recovered from the first step, and (c) an insoluble or cytoskeletal fraction prepared by solubilizing the pellet from the second step in SDS. Fraction b was further fractionated into plakoglobin that was associated with classical cadherins and plakoglobin that was associated with desmosomal cadherins by exhaustive immunoprecipitation. As shown in Fig. , the amount of plakoglobin in each fraction was very similar in A431 and A431DEpg cells. Furthermore, the amount of plakoglobin complexed to cadherins (in the NP-40 soluble fraction) was also very similar in the two cell lines. All of these data thus demonstrate that the amount and distribution of plakoglobin in the A431DEpg transfectant is very close to that observed in the parent A431 cells.
Figure 14 Relative amount and distribution of plakoglobin are similar in A431 and A431DEpg cells. Cells were fractionated into soluble (no detergent), NP-40–soluble, and NP-40–insoluble fractions. Equal volumes of each fraction was resolved by (more ...)
An important control for this experiment was the demonstration that transfection of plakoglobin alone into A431D cells was not sufficient for desmosomal organization. This is demonstrated in Fig. , which shows the staining pattern for desmoplakin in A431D cells that were overexpressing plakoglobin in the absence of a classical cadherin. Desmoplakin was localized throughout the cytoplasm in a diffuse pattern; no desmoplakin was detected at the cell– cell borders. Even though these clones were expressing as much plakoglobin as the parent A431 cells (as demonstrated by immunoblotting, not shown), they could not organize desmoplakin in the absence of a classical cadherin.
Figure 15 Overexpression of plakoglobin alone does not result in organization of desmoplakin at cell–cell borders. A431D cells (A and C) and A431D cells transfected with plakoglobin (pg; B and D) were grown on glass coverslips and processed for immunofluorescence (more ...)
The most straightforward interpretation of all these data is that desmosome organization requires a sufficient level of plakoglobin to permit complex formation both with desmosomal cadherins and with classical cadherins. Hence, we hypothesize that the complex between classical cadherins and plakoglobin has signal-transducing capacity that initiates desmosome organization.
A Plakoglobin–E-Cadherin Chimera Can Restore Desmosome Organization
To test further our hypothesis that a plakoglobin–classical cadherin complex is required to initiate desmosome organization, we constructed cDNA for an E-cadherin–plakoglobin chimeric molecule (Fig. B
) consisting of: (a
) the entire extracellular and transmembrane domains of E-cadherin as well as the first 61 amino acids of its cytoplasmic domain, excluding the region shown to associate with β-catenin (Stappert and Kemler, 1994
) a 9–amino acid spacer, and (c
) amino acids 19–745 of plakoglobin, which includes the domain that is necessary for association with α-catenin (Sacco et al., 1995
). This chimeric cDNA was transfected into A431D cells. Clones expressing the chimeric protein (A431DchiE/pg) were verified by immunoblotting with anti–E-cadherin and antiplakoglobin antibodies, both of which reacted with a protein at 150 kD, the expected molecular mass (not shown). Immunoprecipitations of the chimeric protein with anti–E-cadherin antibodies showed that it was capable of associating with α-catenin, as expected (data not shown). Localization of desmoplakin in the A431DchiE/pg cells revealed a punctate pattern concentrated along the cell–cell borders, highly indicative of desmosome organization and indistinguishable from the parent A431 cells (Fig. ).
Figure 16 Transfection of an E-cadherin–plakoglobin chimeric protein restores the ability of A431D cells to organize desmoplakin at cell–cell borders. (A) A431 cells (A) and A431D cells transfected with the chimera (A431DchiE/pg; B) were grown (more ...)
Thus, our data with the chimeric transfectants verify and extend our findings with the cotransfected lines expressing both E-cadherin and plakoglobin. An association between a classical cadherin and plakoglobin appears to be essential for the formation of desmosomes. Plakoglobin appears to be a central molecule in desmosomal organization, not only because of its presumed structural role but also because of a regulatory function in conjunction with the classical cadherins.