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P. Lu, C.A. Barden, H.L. Chandler, Y. Sugimoto, C.M. H. Colitz; Overexpression of ER alpha in Canine Lens Epithelial Cells Results in Up–Regulation of TERT and TR . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2894.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Estrogen has been shown to protect the lens from cataract formation via estrogen receptor alpha (ERa). ERa can bind the catalytic subunit of telomerase (TERT) promoter and up–regulate telomerase activity in estrogen dependent cells. Our laboratory has found that ERa and TERT interact in cataractous LEC but not in normal LEC. We intend to determine if ERa overexpression leads to increased transcription of the telomerase components, TERT and TR. Methods: Canine ERa was cloned by RT–PCR. The canine ERa gene was sub–cloned into the eukaryotic expression vector pIRES–hrGFP–2a for transfection experiments. Primary cultures of canine LEC were transfected with FuGene 6 Reagent and ERa/pIRES–hrGFP plasmid DNA for 48 hr according to the manufacturers protocol. Estrogen was added to the culture 16 hr before cells were harvested. RNAs extracted from lens tissues and LEC were used for the first strand cDNA synthesis. Relative quantitative real time RT–PCR was performed in the Mx3000p System (95C for 10 minutes, then 40 cycles of 94C for 30 seconds, 60C for 30 seconds, and 72C for 30 seconds) using QuantiTect SYBR Green PCR kit. The relative quantification of TR, TERT, and ERa, normalized to HPRT (housekeeping control), are calculated using the LinRegPCR software. The primers to amplify sequences of the TR, TERT, ERa, and HPRT genes were designed based on the sequence data obtained in our laboratory. Results: ERa, TR, and TERT expression levels are higher in cataractous LEC than in normal LEC by relative quantitative real time RT–PCR. Preliminary data found ERa to be up–regulated in the presence and absence of estrogen after overexpression. In addition, TERT and TR were up–regulated in an estrogen–dependent in response to ERa overexpression. Conclusions: This data supports that ERa expression regulates the transcription of TERT and TR in LEC in an estrogen dependent manner. The future experiments are planned to check whether the proteins, ERa, TERT, and TR, are also increased in ERa transfected LEC.
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