IOVS Clinical and Diagnostic Laboratory Immunology
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Originally published In Press as doi:10.1167/iovs.08-1698 on July 9, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:5611-5618.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.08-1698
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The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells

Kendra S. Plafker, Krysten M. Farjo, Allan F. Wiechmann, and Scott M. Plafker

From the Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.

PURPOSE. Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation.

METHODS. UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27Kip1 were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for β-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells.

RESULTS. UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27Kip1, and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse.

CONCLUSIONS. UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation.






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