Thesis presented October 24, 2023

Abstract:
aBMP9 and BMP10 are key factors in vascular homeostasis, acting via high-affinity receptors (ALK1, BMPRII, and Endoglin) on endothelial cells (ECs). Mutations in this signaling pathway are associated with two rare vascular diseases, hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH), with no currently identified cures. Molecular mechanisms driving the pathogenesis of these diseases is not yet clear. So far, only the canonical SMAD signaling pathway is well studied in response to BMPs, including BMP9 and BMP10, while the activation of alternative pathways by these ligands remains poorly understood, particularly in ECs.
The aim of this work is to address early phosphoproteomic changes in ECs in response to short-term stimulation with BMP9 and BMP10. Discovering new phosphorylated targets and pathways activated by these ligands would enhance our understanding of the molecular mechanisms governing their roles in vascular homeostasis, while identifying potential therapeutic targets for vascular diseases.
I performed mass spectrometry phosphoproteomic analysis of human umbilical vein endothelial cells (HUVECs), stimulated or not with BMP9 or BMP10 for 30 min. To identify downstream-regulated pathways, differentially regulated phosphosites were analysed using different bioinformatics tools. Treatment with BMP9 and BMP10 induced a highly similar phosphoproteomic profile, allowing the identification 289 differentially phosphorylated proteins. Among these, we identified phosphorylation of the activating residues of SMAD1, direct substrate of ALK1, thus validating our phosphoproteomic approach. Bioinformatic analyses highlighted the activation of MAPK signaling in response to BMP9 and BMP10. We identified a central role for the MAPK pathway (MEKK4/P38) leading to the phosphorylation of Eps15-S796 and HSP27-S78/82. Moreover, I showed that BMP10-induced activation of P38 plays an important role in the regulation of a subset of BMP10 target genes. BMP10-induced activation of P38 required a transcriptional step through expression of GADD45β. I also found that BMP10 regulated the phosphorylation of the endothelial transcription factor ERG. Our bioinformatic analyses also highlighted reduced CDK4/6 activity and cell cycle regulation. In line with these data, I showed that BMP10 induced a G1 cell cycle arrest in ECs, reinforcing its role as a vascular quiescence factor. Moreover, our data highlighted a crosstalk between BMP9/10 and JAK/STAT3 signaling pathways through phosphorylation of gp130, a co-receptor of IL-6 family of cytokines. I found that BMP9 and BMP10 attenuated IL-6-induced activation of JAK/STAT pathway via SMAD-induced expression of SOCS3. Preliminary data from mouse models with impaired Bmp9/10 signaling suggests a possible immunomodulatory role of this pathway.
Overall, this work shows that BMP9/10 can activate both direct and indirect (transcription-dependent) pathways in ECs. The direct pathway involves rapid activation of SMADs, essential for inducing BMP target genes. Among these, I identified GADD45β which leads to indirect P38 activation, and SOCS3, which inhibits IL-6/JAK/STAT3 signaling. Given the important role of IL-6 in inflammation, this study provides potential therapeutic approaches in the context for HHT and PAH pathogenesis, where inflammation has been associated with both diseases. While I was able to validate and decipher the mechanisms for several important hits and pathways, I could not study many other intriguing targets due to the lack of specific tools, such as particular phospho-specific antibodies. However, with the development of such tools, I hope that the vascular biology community will further benefit from this unique high-throughput phosphoproteomic study of BMP9/10-mediated signaling, which would aid in characterizing the roles of these ligands not only under physiological conditions but also in pathological vascular diseases, such as HHT and PAH. bstract.

Keywords:
BMP, ALK1, phosphoproteomics, HHT, PAH​