Expression and characterization of the catalytic subunit of telomerase in normal and cataractous canine lens epithelial cells.
OBJECTIVE
To determine whether the catalytic subunit of telomerase reverse transcriptase (TERT) is regionally distributed in canine lens epithelial cells (LEC), compare TERT and the RNA subunit of telomerase (TR) mRNA expression and TERT protein expression in normal and cataractous LEC, and to evaluate whether telomerase activity is present in the cytoplasm and nucleus from normal LEC. Finally, the expression of p23 and heat shock protein 90 (hsp90), coactivators of TERT in neoplastic cells, were evaluated in normal and cataractous LEC.


METHODS
TERT protein was detected by imunohistochemical staining and western immunoblotting in normal and cataractous LEC. Quantitative RT-PCR (qRT-PCR) was used to measure TERT and TR expression. Separated cytoplasmic and nuclear extracts from primary cultures of normal canine LEC were evaluated for TERT protein expression and telomerase activity. Western immunoblotting was performed on normal and cataractous LEC for p23 and hsp90, and coimmunoprecipitation was used to determine whether p23 and hsp90 were interacting with TERT in LEC.


RESULTS
TERT expression in normal lens capsule whole mounts varied by region in normal LEC. All cataractous LEC demonstrated more intense TERT immunostaining in both the nucleus and cytoplasm when compared to normal LEC. Normal LEC expressed less TERT protein and less TERT and TR mRNA than cataractous LEC. Normal LEC expressed hsp90 while cataractous LEC did not; p23 was not significantly expressed in either normal or cataractous LEC. Neither hsp90 nor p23 interacted with TERT.


CONCLUSIONS
The localization of TERT in normal LEC corresponded with the LEC's regional functions. There was more cytoplasmic TERT in the central region that corresponds with the need for inhibited apoptosis and for proliferative capabilities; there was more nuclear TERT in the germinal and equatorial regions corresponding with the need for proliferative capabilities. In addition, cataractous LEC demonstrated increased TERT protein and increased TERT and TR mRNA expression than normal LEC corresponding with their increased proliferative potential. However, the telomerase coactivators, p23 and hsp90, are not overexpressed and do not associate with TERT in cataractous LEC, suggesting that telomerase regulation in cataractous LEC, a somatic cell type, differs from that in neoplastic cells.
Colitz
Carmen Maria Helena
CM
Department of Veterinary Clinical Science, The Ohio State University, Columbus, OH, USA. ccolitz@gmail.com
Barden
Curtis Andrew
CA
Lu
Ping
P
Chandler
Heather Lynn
HL
D000818:Animals; D015153:Blotting, Western; D020134:Catalytic Domain; D002386:Cataract / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D004285:Dogs / Q000378:metabolism*; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation; D007908:Lens, Crystalline / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D013194:Staining and Labeling; D013347:Subcellular Fractions / Q000201:enzymology; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution
D018841:HSP90 Heat-Shock Proteins; D012333:RNA, Messenger; D019098:Telomerase