In the current study, we have identified two truncation mutants of ZO-1 (ZO-11–422 and ZO-11–794) that mediated a dramatic change in cell morphology 4–6 wk after transfection into corneal epithelial cells. These two N-terminal mutants of ZO-1 uniquely overlapped in the region between PDZ2 and -3, which was absent in the other truncation mutants that had no effect on cell morphology. After a 4- to 6-wk latent period, the cells transfected with ZO-11–422 and ZO-11–794 displayed a fibroblast-like, elongated appearance unlike either parental cells or clones transfected with ZO-11–1745, ZO-1Δ615–812, or ZO-11–263, which continued to show an epithelial cobblestone morphology. Upon observation of a phenotypic change in cells expressing ZO-11–422, constructs ZO-11–263 and ZO-1263–422 were generated to determine the smallest domain of ZO-1 that could induce this morphological change. As previously described, ZO-11–263 maintained an epithelial morphology, whereas attempts to stably express ZO-1263–422 were unsuccessful, because cells transfected with this construct died during selection. This suggests the possibility that this region of ZO-1, between the PDZ2 and -3 domains, may be responsible for inducing this mesenchymal-like transformation.
To characterize the phenotypic change from an epithelial to a fibroblast-like morphology caused by ZO-11–422
, these cells were examined for their expression of various epithelial and mesenchymal markers. The transformation observed was a partial transformation, as evidenced by the up-regulation of mesenchymal markers such as vimentin and smooth muscle actin and the inconsistent, variable expression of cytokeratins (Savagner et al., 1997
). Cells expressing ZO-11–422
lacked tight junctions and had no measurable transepithelial resistance compared with mock transfected parental cells and ZO-11–1745
(128.1 ± 3.8 Ω/cm2
Tight junction-associated proteins such as ZO-1, ZO-2, and occludin no longer localized at cell borders but were found throughout the cytoplasm and were down-regulated in their expression. To examine whether endogenous ZO-1 in these transformed cells was altered, the transformed cells were metabolically labeled and immunoprecipitated with ZO-1 antibodies. As seen in Figure , immunoprecipitation of endogenous ZO-1 in cells expressing ZO-11–422
demonstrates coprecipitation of a novel band of ~55 kDa. This suggests the possibility that endogenous ZO-1 interacted with a novel protein as a result of this transformation, which may have played a role in the induction of this mesenchymal-like phenotype. It is also of interest that the immunoprecipitated ZO-1 doublet had differing intensities compared with that of the mock-transfected cells. The lower band was more intense in the transformed cells, suggesting the possibility of less-phosphorylated ZO-1 in these cells. This is consistent with studies demonstrating that Madin–Darby canine kidney cells maintained in low calcium with no tight junctions have an altered distribution of ZO-1 as well as lower total phosphorylation of ZO-1 (Howarth et al., 1994
both caused a partial mesenchymal transformation, the morphologies of these transfected cells were different from each other. Cells transformed with ZO-11–422
were more elongated and spindle-like and lacked adherens junctions, as judged by immunostaining with a pan-cadherin antibody. However ZO-11–794
-expressing cells, although fibroblast-like, were more rounded and demonstrated punctate cadherin expression, as has been shown in cultured fibroblasts (Itoh et al., 1991
). Endogenous ZO-1 appeared to be colocalized with cadherins in the cells transfected with ZO-11–794
The most puzzling aspect of this dramatic phenotypic change was the length of time necessary for the phenomenon to occur. After isolating single clones from stable transfections, the cells were cultured for ~4–6 wk with passage twice weekly before they underwent their morphological change. This time frame was not sensitive to subcloning, in that transfectants that were allowed to expand in culture before subcloning required a proportionately shorter time to affect the phenotypic change.
The 4- to 6-wk time frame complicates an exploration into the mechanisms of action of the mutants. Transforming growth factor β3 (TGFβ3) has been demonstrated to promote a transformation from epithelium to mesenchyme of progenitor cells of the heart valves, which arise from the endothelial cells in the atrioventricular canal (Potts et al., 1991
; Runyan et al., 1992
). Similarly, TGFβ3 also promotes EMT of rodent (Kaartinen et al., 1997
) and chicken palate medial edge epithelia within 24–72 h after addition to cultured palates (Sun et al., 1998
). To investigate whether the transformed clones had become more susceptible to TGFβ3, the time course of TGFβ3-induced EMT was studied in the corneal cells by placing the parental cells in 100 ng/ml TGFβ3. We observed that the parental cells underwent EMT within 5 d of TGFβ3 application, indicating that this growth factor operated at a much more rapid time scale than that shown by the ZO-1 mutants (our unpublished data). However, when 0.1–10 ng/ml TGF β3 was added to cells immediately after transfection with ZO-11–422
, the cells did not undergo the morphological change within the 7 d they were treated (our unpublished data). These data imply that stable expression of ZO-11–422
did not cause the cells to become more susceptible to the growth factor. Conditioned medium, harvested from ZO-11–422
cells in culture for >1 mo, and okadaic acid, a potent phosphatase 2A inhibitor, did not cause EMT in the parental cells (our unpublished data), indicating that these agents were not active in inducing EMT in this cell type.
Another possible model to explain the delay in partial mesenchymal transformation was the necessity for the levels of endogenous ZO-1 to be greatly reduced before ZO-11–422 and ZO-11–794 were able to exert their effect. However, comparison of parental cells with stably transfected cells before and after the epithelial–mesenchymal transformation revealed no significant differences in endogenous ZO-1 levels (our unpublished data).
ZO-11–422 and ZO-11–794 cells were cocultured with parental corneal epithelial cells at a 4:1 ratio. The ZO-11–422 and ZO-11–794 cells grew on top of the parental cells and clumped together, whereas the parental cells proliferated rapidly and formed a confluent monolayer under the clumped cells after 4 d in culture. There was no reversion of the ZO-11–422 and ZO-11–794 cells back to an epithelial-like morphology, nor did they cause the parental cells to undergo a similar mesenchymal transformation (our unpublished data).
Epithelial cells transfected with constitutively active Ras become elongated and fibroblast-like but revert back to an epithelial cell morphology when the Ras pathway is inhibited (Y.-H. Chen, personal communication). An inhibitor of the Ras signaling pathway, PD 098059 (Alessi et al., 1995
), was added to ZO-11–422
cells to investigate whether the Ras pathway was involved in this morphological change and whether these cells would revert back to an epithelial cell phenotype. However, PD 098059, which acts within 24 h on Ras-transformed Madin–Darby canine kidney epithelial cells (Y.H. Chen, personal communication), had no detectable effect on ZO-11–422
cells after 3 d in culture (our unpublished data). Similarly, okadaic acid and TGFβ3 did not cause a phenotypic reversion.
Although the length of time necessary between expression of ZO-11–422
and that of ZO-11–794
in corneal epithelial cells and the appearance of a transformed phenotype suggested a complex signaling pathway, the effect was specific to these two ZO-1 N-terminal mutants. The morphological change was not due to expression levels of the mutant proteins, because independent clones of ZO-11–422
varied in their levels of expression up to threefold. ZO-11–422
lacked the proline-rich C terminus of ZO-1 that has been shown to bind actin filaments (Itoh et al., 1997
; Fanning et al., 1998
). Without this cytoskeletal anchor, the mutants may have been free to interact with proteins normally not accessible to full-length ZO-1. Further studies will be necessary to elucidate the binding partners and the signaling pathways activated by these ZO-1 truncation mutants.