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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 …