The human ubiquitin conjugating enzyme, UBE2E3, is required for proliferation of retinal pigment epithelial cells.
OBJECTIVE
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 p27(Kip1) 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 beta-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 p27(Kip1), 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.
Plafker
Kendra S
KS
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
Farjo
Krysten M
KM
Wiechmann
Allan F
AF
Plafker
Scott M
SM
D000818:Animals; D015153:Blotting, Western; D002452:Cell Count; D002453:Cell Cycle; D049109:Cell Proliferation; D048429:Cell Size; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015536:Down-Regulation; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000201:enzymology*; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*
C436291:CDKN1B protein, human; D047908:Intracellular Signaling Peptides and Proteins; D019394:Ki-67 Antigen; D034741:RNA, Small Interfering; D050760:Cyclin-Dependent Kinase Inhibitor p27; C495210:Ube2e1 protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases