S98 BMP AND ACTIVIN TYPE-II RECEPTORS BALANCE BMP9 SIGNALLING THROUGH ALK1 IN HUMAN PULMONARY ARTERY ENDOTHELIAL CELLS

¹PD Upton, ¹RJ Davies, ²RC Trembath, ¹NW Morrell. ¹University of Cambridge, Cambridge, UK, ²King’s College London, London, UK

Background: Mutations in ALK1 cause hereditary haemorrhagic telangiectasia (HHT), whereas mutations in the bone morphogenetic protein type II receptor (BMPR-II) underlie familial pulmonary arterial hypertension (PAH). ALK1 may function as a transforming growth factor receptor in endothelial cells, although when it complexes with BMPR-II, a BMP9 receptor results. BMPR-II may also complex with other type I receptors, conferring selectivity for different BMP ligands. We sought to determine how functional differences in endogenous ALK1 and BMPR-II responses in endothelial cells may explain the different pathologies of HHT and PAH.

Methods: Human pulmonary artery endothelial cells (HPAEC) were stimulated with a range of BMP for 1 h for Western blot analysis of Smads and 1, 4 and 8 h for RNA extraction and qPCR analysis. In subsequent experiments, HPAEC were transfected with specific siRNA (siALK1, siALK5, siEndoglin, siActR-II, siBMPR-II, siSmad2, siSmad3, siSmad4) to examine endogenous signalling. After 48 h, cells were then stimulated with BMP9 for 1 h for protein analysis or 8 h for qPCR analysis.

Results: BMP9 selectively induced phosphorylation of Smad1/5, Smad2 and Smad3. In addition, BMP9 induced expression of the Id1 and Id2 genes at 4 and 8 h and IL-8 and E-selectin at 8 h. siALK1, but not siALK5 or siEndoglin, abrogated the Smad responses and gene response to BMP9. siBMPR-II reduced the BMP9-mediated Smad1/5 response to a greater extent than siActR-II. Conversely, siActR-II caused a greater abrogation than siBMPR-II of the Smad2 response. Only cotransfection of siActR-II and siBMPRII abolished the Smad 3 response. In addition, cotransfection of siActRII and siBMPR-II was required to abrogate Id gene induction by BMP9. In contrast, siBMPR-II potently abrogated BMP9-mediated induction of IL-8 and E-selectin, whereas …