Abstract
Abstract: :
Purpose: Deletion of the gene encoding thyroid hormone receptor ß–2 (TRß2) in mice caused selective loss of M (middle–wave–sensitive) cone photoreceptors and concomitant increase of short–wave–sensitive cones (Ng et al, 2001, Nature Genetics 27:94–98). However, whether TRß2 directly induces M opsin or it exerts its action at an earlier stage of M–cone commitment is unknown. The goal of this study was to examine whether the human L– (long–wave–sensitive) and M–cone pigment genes are directly induced by thyroid hormone and 9–cis retinoic acid (RA) in the human retinoblastoma cell line WERI which expresses low levels of both L– and M– pigment mRNAs. Methods: WERI cells were grown in RPMI 1640 medium supplemented with a defined serum substitute (2% B27) and treated with triiodothyronine (T3), the agonist for thyroid hormone receptor, RA, the agonist for retinoid X receptor (RXR), and a combination of T3 and cyclohexamide, a protein synthesis inhibitor. Total cellular RNA was extracted and used as template in quantitative real–time RT–PCR to measure the levels of L/M pigment mRNAs. Semi–quantitative RT–PCR analysis was also performed to determine if the levels of TR and RXR mRNAs in WERI cells. Results: Treatment with 10 nM T3 for 48 hrs dramatically increased (about 20 fold) the levels of both L and M pigment mRNAs. The induction was rapid (few hours), dose–dependent and did not require new protein synthesis. This suggests that induction of the L and M pigment genes occurred directly, i.e. did not involve activation of intermediate upstream genes. Since TR and RXR activate transcription by binding as heterodimers to regulatory DNA sequences, we investigated the contribution of RA to induction of the L/M pigment genes by T3. RA induced expression (12–fold) by itself and additively (30–fold) with T3. WERI cells express both TRß2 and RXRγ mRNAs. Conclusions: This report presents evidence that TRß2/RXRγ heterodimers and their agonists play a major role in directly activating transcription of the human L/M gene locus during L/M cone differentiation.
Keywords: photoreceptors • color pigments and opsins • gene/expression