Arkadia Is Required for Efficient P-Smad2/3 Target Gene Transcription and Super-Activates Signaling in <i>Akd−/−</i> ES Cells

<div><p>(A) Relative luciferase activity from three different wild-type and three different <i>Akd−/−</i> ES cell lines transfected with the <i>Pitx2-luc</i> reporter. All values are expressed as a percentage relative to wild-type cell line 3 (3) represented as 0%. <i>Akd−/−</i> ES cell lines have lower activity compared to wild-type, indicative of reduced target gene expression. Average differences (Av) between wild-type and <i>Akd−/−</i> ES cells are shown.</p> <p>(B) <i>Nodal</i> real-time PCR performed on two different wild-type and <i>Akd−/−</i> ES cell lines (1 and 2). The abundance of endogenous <i>Nodal</i> transcripts was quantified and normalized against two different housekeeping control genes, <i>YWHAZ</i> and <i>GAPDH,</i> in quadruplicate reactions (<i>n</i> = 4 for each primer pair). Both <i>Akd−/−</i> ES cell lines show reduced levels of endogenous <i>Nodal,</i> a known target gene of P-Smad2/FoxHI compared to wild-type.</p> <p>(C) Percentage change in Nodal/TGF-β signaling as quantified by <i>Pitx2-luc</i> assays upon transient transfection of GFP or GAkd plasmids in three wild-type and three <i>Akd</i>−/− ES cell lines. GAkd expression doubles <i>Pitx2-luc</i> reporter activity in <i>Akd−/−</i> ES cells and the enhancement is 50% higher than the average level (Av) of wild-type ES cells (horizontal line). The values are normalized against GFP-transfected <i>Akd</i>−/− cell line 3, which has the lowest signaling.</p> <p>(D) Percent (%) increase in luciferase activity generated by the transiently transfected Smad3 specific <i>9xCAGA</i>-<i>luc</i> reporter in the presence of GFP- or GAkd-expressing plasmids in <i>Akd</i>−/− and wild-type ES cells showing that Arkadia super-activates Smad3-dependent transcription. The enhancement is 100% higher than that in wild-type ES cells (horizontal line). The luciferase values are expressed relative to that of <i>Akd−/−</i> cells transfected with GFP taken as 0%.</p> <p>(E) Super-activation is abolished when mutant Arkadias, GAkdR*, and GAkdNRG* are transfected, indicating that the enhancement requires Arkadia ubiquitin ligase activity and an interaction with P-Smad2/3.</p> <p>(F) Time course of super-activation shows that it is an early but transient phenomenon as quantified by the <i>Pitx2</i>-<i>luc</i> reporter. Relative luciferase activity of <i>Akd−/−</i> and wild-type ES cells are expressed as percent increase over the GFP control and compared at time points (h, hours) taken after transfection of GAkd or GFP. WT, wild-type.</p>