In the eye, the barrier and transport properties of the RPE are essential for maintaining the health and functional activity of the photoreceptor cells. The vectorial transport of nutrients and metabolites into and out of the retina depends on the specific expression and polarized distribution of metabolic transporters in the apical and basolateral plasma membranes of the RPE. Recent work from our laboratory has demonstrated the importance of MCT3, an RPE-specific lactate transporter, in regulating light-stimulated rod responses and lactate levels in the outer retina. The present studies showed that disruption of the RPE monolayer resulted in the downregulation of MCT3 at the edge of the wound and that MCT3 was reexpressed in cells in the wound area only after re-epithelialization and re-establishment of cell-cell contact.
In the embryonic chick eye, we observed a switch in the expression of MCT isoforms in the RPE during development. MCT4, a lactate transporter primarily expressed in glycolytic tissues, was detected in the neuro-ectoderm, the optic vesicle, and the optic cup in developing chick embryos (Supplementary Fig. S1, top panels). As the RPE cells of the outer layer of the optic cup began to pigment and differentiate into the mature RPE, there was a decrease in MCT4 and an increase in MCT3 labeling (Supplementary Fig. S1, bottom panels). This switch correlated temporally with development of the choroidal vasculature and retinal differentiation. Interestingly, our current studies also revealed that there was a switch in MCT expression when E9 to E12 RPE cells were isolated and grown in culture; there was a downregulation of MCT3 and an upregulation of MCT4 were observed. This finding suggested that factors from the neural retina or the basement membrane, or maintenance of cell-cell contacts, may be essential for directing and maintaining the differentiated properties of the RPE.
23,24
Two RPE culture models were used to examine the regulation of MCT3: chick RPE/choroid explant cultures and hfRPE cells. The chick RPE/choroid explants were cultured while attached to their native basement membrane, whereas the hfRPE cells were plated on filters coated with human extracellular matrix. As observed in RPE in situ, both the chick RPE/choroid explants and the hfRPE cell cultures expressed MCT3 in the basolateral membrane. Scratch-wounding of chick RPE/choroid explants and hfRPE monolayers led to a loss of MCT3 expression in cells at the leading edges of the wounds. After re-epithelialization of the monolayers, the cells redifferentiated, as indicated by hexagonal packing of cells, pigmentation, and expression of MCT3. Although both chick RPE/explant cultures and hfRPE cell cultures were re-epithelialized by 3 days after wounding, the time course for redifferentiation of these RPE cultures was different. MCT3 was detected in re-epithelialized wounds after 5 days after wounding in chick RPE/choroid explant cultures but not until 16 days after wounding in the hfRPE cells. These results are consistent with other studies showing that loss of cell-cell contact led to epithelial-mesenchymal transition and increased migration of RPE cells.
26 In addition, the more rapid differentiation of the chick RPE cells suggests that specific components of the basal lamina may be responsible for directing the differentiation of RPE. Indeed, many studies have demonstrated the importance of the basement membrane in modulating RPE differentiation.
19,27 Overall, these findings suggest that re-expression of MCT3 in RPE cells after wounding was dependent on both the reestablishment of cell-cell contacts and the composition of the basement membrane.
After scratch wounding of the RPE, we also found that MCT4 was expressed in the migrating cells at the leading edge of the wound. MCT4 was not detected after re-differentiation of the RPE monolayer in the chick explant cultures, mimicking the coordinated regulation of MCT3 and MCT4 expression observed during embryonic development (Supplementary Fig. S1). In the hfRPE cultures, we found MCT4 expression in the migrating cells at the edge of the wound; however, MCT4 was also expressed at the basolateral membrane of polarized hfRPE monolayers. These data support our speculation that factors from the basement membrane are also required to modulate MCT expression in the RPE.
The increased expression of MCT4 in cells at the edge of the wound is interesting, because previous reports from our laboratory have shown a role for MCT4 in cell motility in the human RPE cell line ARPE-19. These studies showed that MCT4 interacted with the adhesion receptor β1-integrin and that silencing of MCT4 slowed cell migration.
28 The observation that MCT4 is also increased in chick RPE cells after wounding and is localized to the leading edges of migrating hfRPE cells would indicate a role for MCT4 in RPE cell motility in these models.
Similar to the wounded edges of the RPE cultures, we found that MCT4, but not MCT3, was detected in RPE cells in the periphery of the chick RPE explants and hfRPE cultures, where cells had a free edge. Cells at the free edges in both these culture are like “wounds that cannot heal” and, in that capacity, provide a model for studying RPE cell migration after injury or disease. In vivo, the RPE does not have a free edge but is continuous with the pigmented epithelium of the ciliary body, forming an uninterrupted epithelium around the eye. Therefore, the models of RPE used in this study, though not exact mimics of RPE in vivo, provide interesting insights about RPE biology during trauma or disease, namely, aberrant migration and proliferation. Our previous observations highlighting a role for MCT4 in cell motility support this hypothesis, and understanding whether this transporter plays a role in RPE migration in ocular diseases characterized by aberrant wound healing, such as proliferative vitreoretinopathy, warrants further study.
Along with changes in MCT3 expression after wounding, downregulation of the gap junction protein Cx43 was also observed in hfRPE cells during wound healing (data not shown) but was detected at the lateral cell borders when the wound had redifferentiated. Recently, it was reported that Cx43 contributes to the differentiation of RPE through cAMP signaling.
29 Specifically, it was reported that differentiated RPE cells exhibit increased levels of cAMP, which then increase the expression of Cx43 in the epithelium. RPE cells fail to differentiate without the cAMP-induced expression of Cx43, highlighting the importance of this gap junction protein in regulating the differentiation state of the RPE. The temporal correlation in expression of Cx43 and MCT3 suggests that Cx43 may provide an additional level of regulation that contributes to the differentiation of RPE cells after wounding and re-epithelialization.
Taken together, these studies demonstrated that MCT3 is a specific marker for differentiated RPE and that expression of MCT3 in the RPE is dependent on the maintenance of cell-cell junctions. Our data also highlight the importance of the basement membrane in modulating the speed of differentiation and the ability to turn off expression of MCT4 after redifferentiation of wounded RPE. Understanding the exquisite control of MCT expression in quiescent and migratory RPE is critical for the understanding of RPE biology during diseases such as proliferative vitreoretinopathy and may be useful in designing stem cell and transplant therapies to repair diseased or damaged RPE.