Class III β-tubulin in human retinal pigment epithelial cells in culture and in epiretinal membranes

The class III β-tubulin isotype (β_III) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in 1 day-old primary cultures; however, β_III is present in RPE cells in 5-day primary cultures and in passaged RPE cells grown in monolayer cultures as determined by immunohistochemistry and Western blotting. β_III-positivity in cultured RPE cells is not affected by cell density or hydroxyurea- or retinoic-acid-mediated growth inhibition, but only a few cells weakly express β_III in cyclohexamide-treated cultures and RPE cells maintained in serum-free medium fail to produce β_III. When monolayer-cultured RPE cells grown in normal, serum-containing medium, are transferred to irradiated bovine vitreous, β_III is undetectable in most cells. Cultured RPE cells coexpress β_III with keratin and cellular retinaldehyde-binding protein (both RPE cell markers), but not with glial fibrillary acidic protein. Some cultured RPE cells also express neuron-specific (γ) enolase, which is neuron-associated but not neuron-specific, and occasional cells in confluent or super-confluent cultures contain the 200-kDa neurofilament protein. Retinal glia, fibroblasts, endothelial cells, and smooth muscle cells do not express β_III under the same culture conditions. We have detected β_III in 45 of 56 epiretinal membranes, frequently in cells with a bipolar or dedifferentiated morphology, where its expression coincides with other RPE cell-associated antigens. Cells with morphological features resembling normal RPE cells in epiretinal membranes are usually negative for β_III, but RPE cells appearing to be in the early stages of dedifferentiation express the isotype weakly. Electron microscopic immunocytochemistry localizes β_III to microtubules, ribosomes and cytoplasm. β_III may be a useful marker for recognizing the fraction of RPE cells in epiretinal membranes that are no longer identifiable by morphological criteria or other RPE cell markers. These findings demonstrate that mature human RPE cells have the capacity to express a neuron-associated gene in response to conditions that promote dedifferentiation.