Dact2 Represses PITX2 Transcriptional Activation and Cell Proliferation through Wnt/beta-Catenin Signaling during Odontogenesis

<div><p>Dact proteins belong to the Dapper/Frodo protein family and function as cytoplasmic attenuators in Wnt and TGFβ signaling. Previous studies show that Dact1 is a potent Wnt signaling inhibitor by promoting degradation of β-catenin. We report a new mechanism for Dact2 function as an inhibitor of the canonical Wnt signaling pathway by interacting with PITX2. PITX2 is a downstream transcription factor in Wnt/β-catenin signaling, and PITX2 synergizes with Lef-1 to activate downstream genes. Immunohistochemistry verified the expression of <em>Dact2</em> in the tooth epithelium, which correlated with <em>Pitx2</em> epithelial expression. <em>Dact2</em> loss of function and <em>PITX2</em> gain of function studies reveal a feedback mechanism for controlling Dact2 expression. Pitx2 endogenously activates Dact2 expression and Dact2 feeds back to repress Pitx2 transcriptional activity. A Topflash reporter system was employed showing PITX2 activation of Wnt signaling, which is attenuated by Dact2. Transient transfections demonstrate the inhibitory effect of Dact2 on critical dental epithelial differentiation factors during tooth development. Dact2 significantly inhibits PITX2 activation of the Dlx2 and amelogenin promoters. Multiple lines of evidence conclude the inhibition is achieved by the physical interaction between Dact2 and Pitx2 proteins. The loss of function of Dact2 also reveals increased cell proliferation due to up-regulated Wnt downstream genes, cyclinD1 and cyclinD2. In summary, we have identified a novel role for Dact2 as an inhibitor of the canonical Wnt pathway in embryonic tooth development through its regulation of cell proliferation and differentiation.</p> </div

<i>Dact2</i> expression in dental and oral epithelia.

<p>(<b>A–F</b>) Endogenous Dact2 protein was stained using a Dact2 antibody and secondary FITC conjugated antibody and nuclei were stained by DAPI on wild type mouse embryos. (<b>A</b>) E12.5 upper molar tooth germ, (<b>B</b>) E14.5 upper molar tooth germ, (<b>C</b>) E16.5 oral epithelium, (<b>D</b>) E12.5 lower molar tooth germ, (<b>E</b>) E14.5 lower molar tooth germ, and (<b>F</b>) E16.5 first lower molar all shows epithelial expression of Dact2. (<b>G</b> and <b>H</b>) molar germs at E14.5 stained without Dact2 primary antibody as negative controls. (<b>I</b>) LacZ staining with eosin counter staining on E14.5 <i>Pitx2^cre/+X Rosa26^+/−</i> mice showed Pitx2 highly expressed cell linages in the upper molar bud, indicating overlapping expression of Dact2 with Pitx2 at the same developmental stages. White dotted lines indicate the mesenchyme-epithelium boundaries. Scale bar represents 100 μm.</p

Knock down of <i>Dact2</i> activates endogenous Pitx2 target genes.

<p>(<b>A</b>) Knocking down of endogenous Dact2 in LS-8 cells by shRNA was shown by Western Blot. Negative control shRNA transfected cells show no change. GAPDH was probed as loading controls. Protein band intensities were quantified and shown as relative value ±SEM. (<b>B</b>) mRNAs were extracted from LS-8 cells transfected with <i>Dact2</i> shRNA or NC shRNA, and subjected to RT-PCRs and real-time PCRs. Relative expression levels of <i>Dlx2</i> and <i>Amelx</i> were analyzed and correlated with <i>Dact2</i> expression level. All Real-time PCRs were performed in triplicates and repeated six times.</p

Dact2 represses cell proliferation.

<p>(<b>A</b>) 96-hour cell proliferation assays were performed with <i>Dact2^−/−, Dact2^+/−</i> and <i>Dact2^+/+</i> MEF cells at passage 3. All cell counting were performed in triplicate. (<b>B</b>) Microscopic photos of seeded MEF cells at the beginning and end of the proliferation assay.</p

Model for the mechanism of Dact2.

<p><i>Dact2</i> is a direct downstream target gene of Pitx2 and Wnt signaling. Dact2 negatively feeds back and represses the transcriptional activity of Pitx2, and in turn inhibits Wnt/β-catenin signaling responsive proliferation. Dact2 also inhibits Wnt signaling responsive cell proliferation.</p

β-catenin cellular localization is changed in the dental epithelium of <i>Dact2</i> null mice.

<p>(<b>A</b> and <b>E</b>) E18.5 WT and <i>Dact2^−/−</i> lower incisors were examined by immunohistochemistry for β-catenin expression. Boxed region were examined under higher magnification. (<b>B</b> and <b>F</b>) Detailed views of the labial dental epithelium were shown. β-catenin was labeled with FITC. (<b>C</b> and <b>G</b>) Nuclei were stained with DAPI. (<b>D</b> and <b>H</b>) Merged signals of FITC and DAPI are shown. Arrowheads indicate the differentially localized β-catenin. MES, mesenchyme; OD, odontoblasts; AB, ameloblast; SI, stratum intermedium.</p

Dact2 and Pitx2 physically interact.

<p>(<b>A</b>) LS-8 cells were used for endogenous immunoprecipitation assays. Western Blots shows the high expression levels of Dact2 and Pitx2A in the 10 times diluted input LS-8 cell lysate. The right lane of the IP Western Blot shows Dact2 protein pulled down by Pitx2 antibody. Left lane shows the parallel IP performed using normal rabbit IgG as negative control. (<b>B</b>) Schematics of the gene structures of PITX2A truncations used in GST pull down assay. (<b>C</b>) Coomassie blue staining of the purified PITX2A truncated proteins fused with GST tag (bands with the correct sizes marked by *). (<b>D</b>) Dact2 Western blot of the GST pull-down assay. Dact2 pure protein was incubated with different truncated PITX2A in lane 1, 3, 5 and 7. Incubation of corresponding truncated PITX2A only controls were in lane 2, 4, 6 and 8. Lane 9 contains 10% Dact2 pure protein input. Results indicated Dact2 binds PITX2A through the homeodomain.</p

PITX2 activates Dact2 expression.

<p>(<b>A</b>) CHO cells were co-transfected with <i>CMV-PITX2A</i> expression plasmids and luciferase reporter driven by <i>Dact2</i> 10 kb promoter. Empty <i>CMV-PITX2A</i> expression plasmids were transfected in parallel as a negative control. All transfections included the SV-40-β-galactosidase reporter to control the transfection efficiency. Cells were incubated for 24 hrs and then assayed for luciferase and β-galactosidase activities. (<b>B</b>) Luciferase reporters driven by a duplicated 66 bp DNA segment of <i>Dact2</i> promoter flanking the Pitx2 binding site in Fig. 2A at (−6172 to −6106) was co-transfected with or without <i>CMV-PITX2A</i> overexpression plasmid in CHO cells. A similar reporter with the mutated Pitx2 binding motif was transfected in parallel as control. All luciferase activities are shown as mean-fold activation compared with the <i>Dact2</i> promoter plasmid co-transfected with empty CMV expression plasmid (±SEM from five independent experiments). (<b>C</b>) E14.5 Embryos from <i>Pitx2^−/−, Pitx2</i> transgenic and wild type mice were harvested to generate MEF cells. These MEFs were lysed and analyzed by Western blots to show endogenous Dact2 expression levels. GAPDH expression was probed as loading controls. Protein band intensities were quantified and shown as relative value ±SEM.</p

Endogenous Pitx2 binds to a conservative region on the <i>Dact2</i> promoter.

<p>(<b>A</b>) Schematic of <i>Dact2</i> 10 kb promoter with six PITX2 binding motifs (TAATCC) indicated by arrowheads. The red arrowhead indicates the site verified by ChIP assay. The location of the sense primer and the antisense primer are shown for amplification of the immunoprecipitated chromatin. Blue arrowheads are putative binding sites with less conservation. The white arrowhead indicate a non-conserved Pitx2 binding motif that we tested in ChIP experiment as negative control shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054868#pone.0054868.s003" target="_blank">Figure S3</a>. (<b>B</b>) Endogenous ChIP assay was performed in LS-8 cells. Lane 1 contains the PCR marker. Lane 2 shows the <i>Dact2</i> primers-only control. Lane 3 is the immunoprecipitation using normal rabbit IgG and <i>Dact2</i> primers. Lane 4 is the Pitx2 immunoprecipitated chromatin amplified using the specific <i>Dact2</i> promoter primers. Lane 5 is the chromatin input amplified using the Dact2 primers. Lane 6 shows the <i>Msx2</i> promoter primers-only control. Lane 7 is the immunoprecipitation using normal rabbit IgG and <i>Msx2</i> primers. Lane 8 is the Pitx2 immunoprecipitated chromatin amplified using the specific <i>Msx2</i> promoter primers. Lane 9 is the chromatin input amplified using the <i>Msx2</i> primers. The amplified region of <i>Msx2</i> promoter is −632 to −359 bp relative to transcription start site. All PCR products were sequenced to confirm their identity. (<b>C</b>) The PITX2 binding element on mouse <i>Dact2</i> promoter verified by ChIP was mapped to a highly conserved (>70%) region among Mouse, Human, Chimpanzee, Rhesus macaque and Rat. The blue box indicates the PCR amplified region on Dact2 promoter in (<b>B</b>).</p

Dact2 attenuates PITX2 transcription activity.

<p>(<b>A</b>) CHO cells were transfected with <i>CMV-PITX2A, CMV-Dact2</i> and luciferase reporter driven by <i>Amelx</i> 2.2 kb promoter. Empty CMV expression plasmids were transfected in parallel as a negative control. (<b>B</b>) CHO cells were transfected with combinations of <i>CMV-PITX2A, CMV-β-catenin, CMV-Dact2</i> and luciferase reporter driven by <i>Dlx2</i> 3.2 kb promoter. Empty CMV expression plasmids were transfected in parallel as a negative control. The titration gradient of transfected <i>Dact2</i> expression plasmids are from 0.5 μg to 8 μg in 2-fold increment. The luciferase activities were normalized by co-transfected β-galactosidase and ±SEM was from five independent experiments. (<b>C</b>) Dlx2 was transfected instead of PITX2A to show Dact2 attenuation is specific to PITX2 transcription activity. All luciferase activities were normalized by co-transfected β-galactosidase and ±SEM were from at least three independent experiments. (<b>D</b>) Dact2and PITX2A transfected CHO cells were lysed and analyzed by Western blot, probing for transfected PITX2A. β-catenin expression is shown by Western blot in transfected cells. β-tublin was probed as loading control.</p