Introduction BMP4 signals via the Smad pathway to induce the expression of the ID dominant-negative basic helix-loop-helix transcription factors (ID1–4) that regulate cell differentiation. We have shown that ID induction is blunted in human pulmonary artery smooth muscle cells (HPASMCs) from pulmonary arterial hypertension (PAH) patients with mutations in the bone morphogenetic type-II receptor (BMPR-II). TGFβ1 is implicated in the pathogenesis of PAH. We therefore examined whether TGFβ1 and BMP4 signalling directly interact in HPASMCs.
Methods Explant-derived HPASMCs from unaffected donors or PAH patients with identified BMPR-II mutations were studied. The transcriptional responses of ID1, ID2, PAI1 and CTGF to BMP4, TGFβ1 or co-treatment were examined by qPCR In some experiments, cells were pre-incubated with cycloheximide or pharmacological inhibitors of ALK5 (SD208), MAP kinase (U0126), TAK1 or p38 MAP kinase (SB203580). The roles of Smad2, Smad3, Smad6 and Smad7 were investigated using siRNAs. Smad-dependent transcription was examined using the BMP-responsive luciferase reporter (BRE-luc) and the TGF-responsive luciferase reporter, CAGA12-luc. Protein lysates collected at 1 and 4hrs were immunoblotted for phosphorylated and total Smads and candidate kinases. In some experiments, nuclear and cytoplasmic fractions were prepared and immunoblotted.
Results BMP4 induced ID1 and ID2 expression at 1, 4 and 24h whereas TGFβ1 induced ID1/2 at 1h and repressed them at 4h and 24h. TGFβ1, but not BMP4, induced PAI1 and CTGF expression. BMP4 did not alter TGFβ1-mediated transcriptional responses. In contrast, TGFβ1 attenuated BMP4-mediated ID1/2 induction and the BRE-luc response in donor HPASMCs. Moreover, TGFβ1 abolished BMP4 responses in PAH PASMCs with BMPR-II mutations. This was reversed by the ALK5 inhibitor, SD208, but not by cycloheximide or a TAK1 inhibitor. BMP-Smad phosphorylation and nuclear translocation did not differ between co-treatment and treatment with individual ligands. Smad3 siRNA, but not Smad2, Smad6 or Smad7, reversed the inhibitory effect of TGFβ1.
Conclusions TGFβ1 negatively regulates BMP4-mediated ID genes transcription by interfering with BMP-Smad signalling via Smad3, abolishing the BMP signals in cells with BMPR-II mutations. These data reinforce the notion that TGFβ1 plays a pathogenic role in PAH, via direct inhibition of the attenuated BMP response caused by BMPR-II insufficiency.
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