Variable RXR requirements for thyroid hormone responsiveness of endogenous genes

Abstract

Thyroid hormone receptors heterodimerize with retinoid X receptors in vitro and it is widely assumed that these heterodimers mediate the T3 induction of target genes. However, the importance of RXR for the T3 induction of endogenous genes has not been assessed. We used cDNA microarrays to identify 54 genes induced by T3 in Neuro2a cells that express thyroid hormone receptor beta. RNA interference-mediated knock down of endogenous RXRs showed that these genes vary from being highly dependent on RXR for T3 induction to being independent of RXR. Thus, the availability of RXR may differentially regulate the T3 induction of subsets of genes within a cell. Furthermore, coregulatory proteins that preferentially interact with TR homodimers or RXR-TR heterodimers may further expand the range of T3 response for genes within the same cell.

Fig. 1

siRNA-mediated knock down of RXR in N2a/TRβ cells. The cells were transfected with siRNAs as described in Methods. The cells in (A) and (B) received siRNAs directed against RXRβ (or control siRNA), and the cells in (C) received siRNAs directed against RXRα (or control siRNA). RNA expression was measured by Northern blot and protein expression by Western blot.

Fig. 2

Functional validation of RXR knock down. N2a/TRβ cells were transfected with siRNAs directed against RXRs α and β (or control siRNA), as well as the T3-responsive reporter plasmid MHC-Luc and a control Renilla luciferase plasmid. For the right two bars, the cells also received an RXRγ expression plasmid. The cells were cultured ±T3. Fold T3 induction is firefly luciferase/Renilla luciferase for cells treated +T3, divided by firefly luciferase/Renilla luciferase for cells treated -T3.

Fig. 3

Correlation of gene expression measured by microarray and real time reverse transcription PCR. Ten genes were selected from the microarray data based upon their T3 inductions spanning the range of RXR dependence. The expression levels of these genes were then measured by real time RTPCR. (A) The data represent the fold T3 inductions in cells treated with RXR siRNAs divided by the fold T3 inductions in cells treated with control siRNA. The x-axis plots these ratios measured by real time RTPCR (qRTPCR), and the y-axis plots these ratios measured by microarray. (B) The data represent the expression levels +T3 in cells treated with RXR siRNAs divided by those in cells treated with control siRNA. The x-axis plots these ratios measured by real time RTPCR, and the y-axis plots these ratios measured by microarray.