Gene Specific Actions of Thyroid Hormone Receptor Subtypes

<div><p>There are two homologous thyroid hormone (TH) receptors (TRs α and β), which are members of the nuclear hormone receptor (NR) family. While TRs regulate different processes <em>in vivo</em> and other highly related NRs regulate distinct gene sets, initial studies of TR action revealed near complete overlaps in their actions at the level of individual genes. Here, we assessed the extent that TRα and TRβ differ in target gene regulation by comparing effects of equal levels of stably expressed exogenous TRs +/− T₃ in two cell backgrounds (HepG2 and HeLa). We find that hundreds of genes respond to T₃ or to unliganded TRs in both cell types, but were not able to detect verifiable examples of completely TR subtype-specific gene regulation. TR actions are, however, far from identical and we detect TR subtype-specific effects on global T₃ response kinetics in HepG2 cells and many examples of TR subtype specificity at the level of individual genes, including effects on magnitude of response to TR +/− T₃, TR regulation patterns and T₃ dose response. Cycloheximide (CHX) treatment confirms that at least some differential effects involve verifiable direct TR target genes. TR subtype/gene-specific effects emerge in the context of widespread variation in target gene response and we suggest that gene-selective effects on mechanism of TR action highlight differences in TR subtype function that emerge in the environment of specific genes. We propose that differential TR actions could influence physiologic and pharmacologic responses to THs and selective TR modulators (STRMs).</p> </div

Verification of different T₃ response patterns in HepG2 cells.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052407#s3" target="_blank">Results</a> of qPCR analysis of representative gene expression changes at various times after T₃ induction in HepG2-TRα and HepG2-TRβ cells. <b>A</b>, PCK1, similar with both TRs, <b>B</b>, SLCA16A, similar with both TRs at most times, <b>C</b>, HIF2A, TRβ preference at both early and late times, <b>D</b>, Myh6, TRα preference at early and late times.</p

Gene expression changes with unliganded TRs.

<p><b>A</b>. Numbers of genes that meet fold cutoffs for activation/repression and statistical significance in response to unliganded TR expression in HepG2 cells, TRα and TRβ expressing cells were compared to parental. <b>B</b>. HeLa cells, TRα and TRβ expressing cells after doxycycline withdrawal to induce TRs versus doxycyclin treated cells. Similar results were obtained in comparisons with parental HeLa cells (not shown). <b>C</b>. Plots of fold induction/repression by TRβ (y-axis) versus TRα (x-axis) in HepG2 cells. <b>D</b>. Plots of fold induction/repression by TRβ (y-axis) versus TRα (x-axis) in HeLa cells.</p

T₃ Response in HepG2 cells.

<p><b>A–D</b>. Numbers of genes that meet cut-offs for fold induction and statistical significance in parental HepG2, HepG2-TRα or HepG2-TRβ at each time point, <b>A</b>, 3 hr, <b>B</b>, 6 hr, <b>C</b>, 24 hr, <b>D</b>, all three time points combined. T₃ induced genes are represented in upper panels (red) and T₃ repressed genes in lower panels (blue), note the difference in scale which means that many more genes are positively regulated than negatively regulated. <b>E–H</b>. Plots of fold induction/repression by T₃ in the presence of TRβ (y-axis) versus TRα (x-axis). <b>E</b>, 3 hr blue, <b>F</b>, 6 hr green, <b>G</b>, 24 hr red, <b>H</b>, all three time points.</p

T₃ Induced Genes are Direct TR Targets.

<p><b>A</b>. Bar graph representing numbers of T₃ induced (upper panels) and repressed (lower panels) genes at 3 hrs in HepG2-TRα and HepG2-TRβ cells that persist with CHX pre-treatment (upper panel, red, lower panel, blue). <b>B</b>. Heat map representing gene expression changes at 3 hrs timepoint in HepG2-TRα and HepG2-TRβ cells with T₃, CHX and T₃ + CHX. Note that most target genes retain their T₃ responses with CHX. Examples of genes with unusual responses are marked by lower case letters: a = stronger T₃ responses with TRα, b = amplification of weak T₃ responses in the presence of TRβ with CHX, c = selective CHX-dependent gene induction in the presence of TRα, d = selective CHX-dependent gene induction in the presence of TRβ.</p

T₃ Responses in HeLa cells.

<p><b>A</b>. Numbers of genes that meet cutoffs for fold T₃ activation (upper panel, blue) or repression (lower panel, red) in HeLa-TRα and Hela-TRβ cells at 24 hrs treatment, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052407#pone-0052407-g002" target="_blank">Fig. 2</a>. <b>B</b>. Plots of fold induction/repression by T₃ in the presence of TRβ (y-axis) versus TRα (x-axis) in HeLa cells. <b>C–D</b>. Representative qPCR analysis showing examples of different gene regulation patterns with the two TRs. <b>C</b>, pck1, <b>D</b>, thrsp.</p

Verification of TRβ preference of late responding genes.

<p>qPCR analysis of T₃ induction of two genes (<b>A</b>, G6Pc and <b>B</b>, GSTA1) identified as preferential late TRβ responders. Note that TRα-responses are weak and that TRβ preference persists across all timepoints.</p

Heatmap to Illustrate Patterns of T₃ Response in HepG2.

<p><b>A</b>. Representation of changes in all T₃-dependent genes that meet cutoffs for fold induction/statistical significance in HepG2 parental cells and HepG2-TRα or HepG2-TRβ cells at each time point. Red, upregulated, green, downregulated, black, no change. Genes were clustered according to similarities in response patterns as described in Methods. <b>B</b>. As for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052407#pone-0052407-g003" target="_blank">Fig. 3A</a>, with a section of the heatmap expanded to reveal one of the clusters of late emerging TRβ-preferential T₃ responses.</p

Verification of TR subtype preferences in gene regulation pattern.

<p><b>A</b>, Myh6 <b>B</b>, furin. Both genes display the same pattern of response to unliganded TRs and T₃, despite preferential T₃ induction of Myh6 with TRα. <b>C</b>, ALPI, display exclusively ligand-dependent induction with TRα and ligand-dependent induction with TRβ coupled to a strong ligand-independent component. <b>D</b>, HIF2A, displays the opposite profile to ALPI in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052407#pone-0052407-g012" target="_blank">Fig. 12C</a>.</p