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

Patterns of TR regulation.

<p>Pattern types (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052407#pone-0052407-g009" target="_blank">fig. 9</a>) and numbers of genes that conform to each pattern in HepG2 and HeLa with different TRs. Overlaps between genes are shown.</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

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

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

Hypothetical patterns of TR regulation.

<p>Genes with statistically significant responses to T₃ or unliganded TRs were assigned into categories according to net repression (R), induction (I) or no change (O) represented in the schematic heat map. Numbers of genes in each category and overlaps between genes that respond to TRα or TRβ in this manner are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052407#pone-0052407-t001" target="_blank">Table 1</a>.</p

Cells that express TRs.

<p><b>A</b>. Equal expression of exogenously expressed TRs. Upper panel, western blot of extracts of HepG2 parental cells (1), HepG2 cells infected with control lentivirus (2) or cells infected with lentivirus expressing TRα (3) or TRβ (4) and blotted with anti-flag antibody. Inset beneath shows the same extracts blotted with a β-actin antibody as a loading control. Lower panel; western blot of HeLa-TR extracts after +/− doxycycline withdrawal to induce TRs and blotted with anti-myc. <b>B</b>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052407#s3" target="_blank">Results</a> of T₃ binding assays performed on extracts of HeLa-TR cells after 24 hrs doxycycline withdrawal; figures in panels represent deduced affinities of expressed TRs for T₃. <b>C</b>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052407#s3" target="_blank">Results</a> of luciferase assays performed upon HeLa-TR cells transfected with standard TRE-driven reporters, DR-4 Luc and IP-6 (F2)-Luc after doxyclycline withdrawal to induce TR expression. <b>D</b>. Western blot of HepG2-TR extracts at various times after initial T₃ treatment.</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

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

Unusual ligand-independent TR gene-regulation patterns.

<p>qPCR verification of genes that display hormone-independent repression by both TRs in HepG2. <b>A</b>, mst1, <b>B</b>, hel308.</p