Pdgfrb is a direct regulatory target of TGFβ signaling in atrioventricular cushion mesenchymal cells

Cushion formation is the initial step for the development of valvuloseptal structures in mammalian hearts. TGF beta signaling plays critical roles in multiple steps of cushion morphogenesis. We used a newly developed conditional immortal atrioventricular cushion mesenchymal cell line, tsA58-AVM, to identify the TGF beta regulatory target genes through microarray analysis. Expression of similar to 1350 genes was significantly altered by TGF beta 1 treatment. Subsequent bioinformatic analysis of TGF beta activated genes revealed that PDGF-BB signaling is the top hit as the potential upstream regulator. Among the 37 target molecules, 10 genes known to be involved in valve development and hemostasis were selected for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Our results confirmed that they are all upregulated by TGF beta 1 stimulation in tsA58-AVM cells and in primary atrioventricular cushion cells. We focused on examining regulation of Pdgfrb by TGF beta 1, which encodes a tyrosine kinase receptor for PDGF-BB. We found that the similar to 150bp Pdgfrb promoter can respond to TGF beta stimulation and that this response relies on the two SP1 binding sites within the promoter. Co-immunoprecipitation analysis confirmed SP1 interacts with SMAD2 in a TGF beta-dependent fashion. Furthermore, SMAD2 is associated with the Pdgfrb promoter and this association is diminished by knocking down expression of Sp1. Our data therefore collectively suggest that upon TGF beta stimulation, SP1 recruits SMAD2 to the promoter of Pdgfrb to up-regulate its expression and thus Pdgfrb is a direct downstream target of the TGF beta/SMAD2 signaling.