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Thyroxine regulates the apoptosis genes for Bax and ATF2 in ARPE‐19 cells
Author(s) -
Getz Rita,
Yoon Christopher,
Lubinski Alice,
Schiavoni Laura
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1461-a
Subject(s) - apoptosis , euthyroid , microbiology and biotechnology , blot , chemistry , hormone , thyroid , polyclonal antibodies , endocrinology , western blot , medicine , gene , antibody , biology , immunology , biochemistry
Purpose Previous work has shown thyroid hormone regulation as well as apoptosis in human retinal pigment epithelium (HRPE). The aim of this study was to investigate the role of varying thyroid hormone levels in the regulation of apoptosis in HRPE, specifically Bax and Activated Transcription Factor 2 (ATF2). Bax accelerates apoptosis and inhibition of ATF2 promotes apoptosis. Methods Gene microarray analysis (Superarray) was performed on confluent monolayers of ARPE‐19 cells (ATCC) incubated in MEM supplemented with thyroxine (T4, Sigma) at free‐T4 plasma levels mimicking hypo‐, eu‐ and hyperthyroid conditions. Fold changes > 2.0 between hypo‐ and hyperthyroid conditions were considered for further testing. For SDS‐PAGE/immunoblotting and RT‐PCR, cells were incubated in the T4 conditions for 72 hours. Blots were incubated with either monoclonal Bax (NeoMarkers) or polyclonal ATF2 (Active Motif) antibodies. RT‐PCR (Invitrogen) used gene‐specific forward and reverse primers. Results For Western blots, Bax showed a band at 20–21 kD (expected Mr; 21 kD) for each thyroid condition. Density of the band was lightest in hypothyroid and darkest in hyperthyroid with intermediate density in the euthyroid samples. Densitometric analysis (Photoshop) showed significant differences (p<0.008) between each condition. ATF2 showed a band at 58 kD (expected Mr; 60 kD) for each thyroid condition. Density of the band was darkest in hypothyroid and lightest in hyperthyroid with intermediate density in the euthyroid samples. Densitometric analysis showed significant differences (p<0.016) between each condition. RT‐PCR confirmed these results. Conclusion Increased expression of Bax and decreased expression of ATF2 in the hyperthyroid condition suggests that maintaining euthyroid status may inhibit apoptosis in HRPE. Grant support‐1R15EY13954‐01 to RG