Abstract
BackgroundBone morphogenetic proteins (BMPs) and their receptors, such as bone morphogenetic protein receptor (BMPR) II, have been implicated in a wide variety of disorders including pulmonary arterial hypertension (PAH). Similarly, endothelin-1 (ET-1), a mitogen and vasoconstrictor, is upregulated in PAH and endothelin receptor antagonists are used in its treatment. We sought to determine whether there is crosstalk between BMP signalling and the ET-1 axis in human pulmonary artery endothelial cells (HPAECs), possible mechanisms involved in such crosstalk and functional consequences thereof.Methodology/Principal FindingUsing western blot, real time RT-PCR, ELISA and small RNA interference methods we provide evidence that in HPAECs BMP-9, but not BMP-2, -4 and -6 significantly stimulated ET-1 release under physiological concentrations. This release is mediated by both Smad1 and p38 MAPK and is independent of the canonical Smad4 pathway. Moreover, knocking down the ALK1 receptor or BMPR II attenuates BMP-9 stimulated ET-1 release, whilst causing a significant increase in prepro ET-1 mRNA transcription and mature peptide release. Finally, BMP-9 induced ET-1 release is involved in both inhibition of endothelial cell migration and promotion of tubule formation.Conclusions/SignificanceAlthough our data does not support an important role for BMP-9 as a source of increased endothelial ET-1 production seen in human PAH, BMP-9 stimulated ET-1 production is likely to be important in angiogenesis and vascular stability. However, increased ET-1 production by endothelial cells as a consequence of BMPR II dysfunction may be clinically relevant in the pathogenesis of PAH.
Highlights
Bone morphogenetic proteins (BMPs) are the largest subgroup of signalling molecules in the transforming growth factor (TGF)-b superfamily
To investigate whether Smad independent signalling pathways may be involved in BMP-9 induced ET-1 release from human pulmonary artery endothelial cells (HPAECs) we looked at phosphorylation of ERK1/2, JNK and p38 MAPK at baseline and following BMP-9 stimulation
The main findings of this study are: (1) BMP-9 induced prepro ET-1 mRNA transcription and mature peptide release in HPAECs are dependent on Smad1 signalling but independent of co-Smad4; (2) p38 MAPK signalling is necessary for BMP-9 induced ET-1 release; (3) BMP-9 stimulated ET-1 release is only partially mediated through bone morphogenetic protein receptor (BMPR) II and ALK1; (4) BMPR II knock-down increases basal prepro ET-1 mRNA transcription and mature peptide release and (5) BMP-9 induced endogenous ET-1 release is involved in BMP-9 stimulated HPAEC tubule formation and inhibition of HPAEC migration
Summary
Bone morphogenetic proteins (BMPs) are the largest subgroup of signalling molecules in the transforming growth factor (TGF)-b superfamily. Ligands for BMPRs classically signal through receptor-mediated Smads (R-Smads) 1, 5 and 8, whereas TGF-b typically signals through Smad and 3, via the ALK5 receptor Both utilise a common partner (co)-Smad, Smad, to form a complex that translocates to the nucleus to alter gene expression [2]. In addition to Smad signalling, BMPs may act via their receptors through ‘‘Smad independent’’ signalling pathways, including p38 MAPK (mitogen activated protein kinase), ERK1/2 (extracellular signal related kinase) and JNK (c-Jun N-terminal kinase) [3]. The activation of such additional pathways appears to be cell context specific. We sought to determine whether there is crosstalk between BMP signalling and the ET-1 axis in human pulmonary artery endothelial cells (HPAECs), possible mechanisms involved in such crosstalk and functional consequences thereof
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