Abstract
Cushion formation is the initial step for the development of valvuloseptal structures in mammalian hearts. TGFβ 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 TGFβ regulatory target genes through microarray analysis. Expression of ~1350 genes was significantly altered by TGFβ1 treatment. Subsequent bioinformatic analysis of TGFβ 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 TGFβ1 stimulation in tsA58-AVM cells and in primary atrioventricular cushion cells. We focused on examining regulation of Pdgfrb by TGFβ1, which encodes a tyrosine kinase receptor for PDGF-BB. We found that the ~150bp Pdgfrb promoter can respond to TGFβ 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 TGFβ-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 TGFβ stimulation, SP1 recruits SMAD2 to the promoter of Pdgfrb to up-regulate its expression and thus Pdgfrb is a direct downstream target of the TGFβ/SMAD2 signaling.