Expression of ubiquitin carboxy-terminal hydrolase-l1 in photocoagulated human retinal pigment epithelial cells.
OBJECTIVE
To date, the exact mechanisms involved in laser-induced remission of ocular neovascular disorders are still poorly understood. Recent studies suggest that the expression of various antiangiogenic cytokines is upregulated after thermal photocoagulation. In the current study, we sought to identify novel laser-regulated proteins in cultured human retinal pigment epithelial (HRPE) cells.


METHODS
Protein extracts from photocoagulated HRPE cells were subjected to 2D-gel electrophoresis. Potentially regulated protein spots were identified by mass spectroscopy. Regulation of protein and mRNA was determined by Western blot analysis and reverse transcription-polymerase chain reaction, respectively.


RESULTS
2D-Gel electrophoresis of HRPE whole-cell extracts exposed to photocoagulation revealed a reproducible increase in a protein band identified as ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) compared with untreated controls. Protein levels showed a time-dependent upregulation over 24 hr. UCH-L1 mRNA was maximally increased after 8 hr.


CONCLUSIONS
Our findings indicate that the ubiquitin-proteasome system contributes to the effects seen clinically after thermal photocoagulation in eyes with neovascular diseases of the retina or choroid. Because ubiquitin carboxy-terminal hydrolase-L1 has been shown to be involved in the regulation of cell cycle proteins, it may be speculated that deubiquitinating enzymes have a role in the regeneration and proliferation of retinal pigment epithelial cells.
Schacke
Wolfgang
W
Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, Connecticut, USA.
Beck
Karl-Friedrich
KF
Pfeilschifter
Josef
J
Hattenbach
Lars-Olof
LO
D015153:Blotting, Western; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D017075:Laser Coagulation; D007834:Lasers; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000201:enzymology* / Q000528:radiation effects; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D007527:Isoenzymes; D012333:RNA, Messenger; D043222:Ubiquitin Thiolesterase