ERalpha increases expression and interacts with TERT in cataractous canine lens epithelial cells.

PURPOSE: Estrogen receptor alpha (ERalpha) expression has previously been evaluated in lens epithelial cells (LEC). However, its function in the lens has not been determined. One potential function may be its interaction with the catalytic subunit of telomerase (TERT), which is present in normal LEC and higher in LEC that have undergone epithelial to mesenchymal transition (EMT). ERalpha is known to play a role in EMT, a process that may also involve TERT. METHODS: A commercially available transcription factor array was used to evaluate potential interactions between TERT and other proteins in normal and cataractous LEC. Based on these findings, ERalpha protein and mRNA expressions were measured using western blot analysis, immunohistochemical staining, and quantitative reverse transcription polymerase chain reaction (RT-PCR). Co-immunoprecipitation assays were used to evaluate the interaction of TERT with ERalpha as well as their phosphorylation in normal and cataractous LEC. RESULTS: The transcription factor array suggested that TERT interacted with ERalpha via the estrogen response element (ERE) in cataractous LEC but not in normal LEC. Expression of ERalpha protein and mRNA increased in cataractous LEC compared with normal LEC. Co-immunoprecipitation assays confirmed the interaction of TERT with ERalpha in cataractous LEC while no interaction was found in normal LEC. LEC that have undergone EMT, e.g., cataracts, are rapidly proliferating and migrating along the posterior lens capsule. CONCLUSIONS: ERalpha is known to play a role in EMT, and our data suggests that TERT and phosphorylated protein kinase B (pAkt) may be involved in the regulation of this process in cataractous LEC.

Expression and characterization of the catalytic subunit of telomerase in normal and cataractous canine lens epithelial cells.

PURPOSE: 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.