Coordinated Expression of Tristetraprolin Post-Transcriptionally Attenuates Mitogenic Induction of the Oncogenic Ser/Thr Kinase Pim-1

<div><p>The serine/threonine kinase Pim-1 directs selected signaling events that promote cell growth and survival and is overexpressed in diverse human cancers. Pim-1 expression is tightly controlled through multiple mechanisms, including regulation of mRNA turnover. In several cultured cell models, mitogenic stimulation rapidly induced and stabilized <em>PIM1</em> mRNA, however, vigorous destabilization 4–6 hours later helped restore basal expression levels. Acceleration of <em>PIM1</em> mRNA turnover coincided with accumulation of tristetraprolin (TTP), an mRNA-destabilizing protein that targets transcripts containing AU-rich elements. TTP binds <em>PIM1</em> mRNA in cells, and suppresses its expression by accelerating mRNA decay. Reporter mRNA decay assays localized the TTP-regulated mRNA decay element to a discrete AU-rich sequence in the distal 3′-untranslated region that binds TTP. These data suggest that coordinated stimulation of TTP and <em>PIM1</em> expression limits the magnitude and duration of <em>PIM1</em> mRNA accumulation by accelerating its degradation as TTP protein levels increase. Consistent with this model, <em>PIM1</em> and TTP mRNA levels were well correlated across selected human tissue panels, and <em>PIM1</em> mRNA was induced to significantly higher levels in mitogen-stimulated fibroblasts from TTP-deficient mice. Together, these data support a model whereby induction of TTP mediates a negative feedback circuit to limit expression of selected mitogen-activated genes.</p> </div

Control of <i>PIM1</i> mRNA turnover in mitogen-stimulated HeLa cells.

<p>The decay kinetics of <i>PIM1</i> mRNA were measured in serum-starved HeLa cells (uninduced) or at selected times after stimulation with serum+TPA using actD time course assays. For each experiment, the fraction of <i>PIM1</i> mRNA remaining was plotted as a function of time following inhibition of transcription by actD, and <i>PIM1</i> mRNA decay constants resolved by nonlinear regression to a first-order decay model (<i>lines</i>). Average decay constants measured across replicate independent experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#pone-0033194-t001" target="_blank">Table 1</a>.</p

Localization of TTP-responsive elements to an ARE-like sequence in the <i>PIM1</i> mRNA 3′UTR.

<p>(A) Schematic of the 3′-end of the <i>PIM1</i> mRNA coding sequence (<i>black box</i>) and complete 3′UTR, including the ARE domain (<i>white box</i>). The scale bar (top) is relative to the translational initiation codon. The positions of biotin-labeled riboprobes corresponding to the <i>PIM1</i> ARE and coding sequence fragment (CDS) are shown as black bars above the mRNA schematic. Bars below delineate <i>PIM1</i> 3′UTR domains that were subcloned downstream of the translational termination codon of the βG gene for reporter mRNA decay assays. At the bottom is the sequence at the extreme 3′-end of the ARE domain that contains known high affinity TTP-binding motifs. In the βG-<i>PIM1</i> AREmut reporter mRNA and biotin-labeled AREmut RNA probe, these motifs were disrupted by mutating underlined uridylate residues to cytidines. (B) Decay rates of βG-<i>PIM1</i> chimeric reporter mRNAs were resolved by Dox time course assays in HeLa/Tet-Off cells co-transfected with an empty vector (pcDNA; <i>solid circles</i>, <i>solid lines</i>) or vectors expressing FLAG-TTPwt (<i>open circles</i>, <i>dashed lines</i>) or FLAG-TTP C147R (<i>triangles</i>, <i>dotted lines</i>) as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#s2" target="_blank">Materials and Methods</a>”. mRNA half-lives resolved from multiple independent experiments are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#pone-0033194-t002" target="_blank">Table 2</a>. (C) Western blots probed with indicated antibodies (Ab) show levels of FLAG-TTP wt and C147R mutant proteins (top panel) and GAPDH (second panel) in crude cytoplasmic extracts prepared from untransfected HeLa/Tet-Off cells (ut) or stable clonal lines expressing each FLAG-TTP variant. Samples of each lysate were fractionated using biotin-RNA pull-down assays programmed with riboprobes encoding a <i>PIM1</i> coding sequence fragment (CDS), the <i>PIM1</i> ARE or the ARE mutant containing the U→C substitutions specified above (AREmut). FLAG-TTP proteins co-purifying with each riboprobe were detected by Western blot (bottom panels). The positions of molecular weight markers (in kDa) are shown to the left of each Western blot panel.</p

Decay kinetics of βG-<i>PIM1</i> chimeric mRNAs in transfected HeLa cells.

a<p>First-order constants (<i>k</i>) describing the decay kinetics of indicated βG-chimeric mRNAs were measured in HeLa/Tet-Off cells co-transfected with indicated expression plasmids by Dox time course assays as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#s2" target="_blank">Materials and Methods</a>” and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#pone-0033194-g005" target="_blank">Figure 5</a>. mRNA half-lives were calculated using <i>t</i>_1/2 = ln2/<i>k</i>. Quoted values represent the mean ± SD for <i>n</i> independent experiments.</p

<i>PIM1</i> mRNA decay kinetics during mitogenic stimulation of cancer cell lines.

a<p>Cultures were incubated for 16–20 h in medium containing 0.5% serum prior to each experiment. Where indicated, cells were stimulated by adding medium containing serum (10%) and TPA (100 nM) for indicated periods prior to inhibition of transcription with actD.</p>b<p>First-order mRNA decay constants (<i>k</i>) were resolved for each cell population by actD time course assay as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#s2" target="_blank">Materials and Methods</a>”. mRNA half-lives were then calculated using <i>t</i>_1/2 = ln2/<i>k</i>. Quoted values represent the mean ± SD across <i>n</i> independent time course experiments.</p

Transient induction of endogenous <i>PIM1</i> mRNA by mitogenic stimuli in cancer cell lines.

<p>Total RNA was isolated from HeLa, HepG2, and MDA-MB-231 cells at selected times following stimulation with serum+TPA as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#s2" target="_blank">Materials and Methods</a>”. Bars represent the relative levels of <i>PIM1</i> mRNA determined by qRT-PCR and normalized to GAPDH mRNA (mean ± SD of quadruplicate qRT-PCR reactions). Independent replicate experiments yielded similar results.</p

Regulation of <i>PIM1</i> mRNA induction by TTP following mitogenic stimulation in MEF models.

<p>(A) Whole cell lysates were prepared from MEFs derived from TTP knockout mice (TTP^−/−) and wild-type littermates (TTP^+/+) following serum-starvation and stimulation with serum+TPA as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#s2" target="_blank">Materials and Methods</a>”. Expression of TTP and GAPDH were assessed at selected time points by Western blot, with the positions of molecular weight markers (in kDa) shown at left. (B) Total RNA was isolated from MEF cultures stimulated as described in (A). Bars show the relative level of <i>PIM1</i> mRNA in TTP^+/+ (<i>solid bars</i>) and TTP^−/− (<i>open bars</i>) MEFs at indicated times following mitogenic stimulation as determined by qRT-PCR and normalized to GAPDH mRNA (mean ± SD of quadruplicate qRT-PCR reactions, *<i>p</i><0.01 <i>versus</i> TTP^+/+). Independent replicate experiments yielded similar results. (C) ActD was added to MEF cultures 2 hours after stimulation with serum+TPA. <i>PIM1</i> mRNA decay rates were then measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033194#pone-0033194-g002" target="_blank">Figure 2</a>, and yielded half-lives of 0.42±0.11 h (<i>n</i> = 4) for TTP^+/+ cells <i>versus</i> 1.56±0.12 h (<i>n</i> = 3) for TTP^−/− (<i>p</i><0.0001 <i>versus</i> TTP^+/+).</p

Induction of the ARE-binding protein TTP in mitogen-stimulated cancer cell lines.

<p>(A) Schematic of <i>PIM1</i> mRNA showing a putative ARE sequence within the distal 3′UTR. Sequences corresponding to consensus TTP-binding sites are underlined. (B) Western blots showing the induction of TTP protein at selected times following mitogenic stimulation of cancer cell lines. Whole cell lysates were prepared at indicated time points following addition of serum+TPA to serum-starved cultures. GAPDH levels were used to normalize protein loading. The positions of molecular weight markers (in kDa) are indicated left of each blot.</p

Coordinated expression of <i>PIM1</i> and TTP mRNAs in selected human tissues.

<p>Levels of <i>PIM1</i> and TTP mRNAs were extracted from gene array datasets derived from a panel of human non-transformed (normal) and tumorous prostate tissues (Gene Expression Omnibus accession no. GSE6919), breast tumors (Gene Expression Omnibus accession no. GSE2294), and CD138+ cells purified from bone marrow of multiple myeloma (MM) patients (Gene Expression Omnibus accession no. GSE2912). Correlation between levels of <i>PIM1</i> and TTP mRNAs was analyzed using the Spearman nonparametric test. Correlation coefficients (<i>r</i>) and associated <i>p</i> values are listed in each panel. The dotted lines indicate the 95% confidence intervals of each regression solution.</p