PDF(Pubmed)
Abstract：
BMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension.
To get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry.
Large-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45β. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1.
Overall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.
To get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry.
Large-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45β. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1.
Overall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.
摘要：
背景：BMP9和BMP10是血管稳态的两种主要调节因子。这两种配体以高亲和力结合内皮I型激酶受体ALK1，与II型受体，导致SMAD转录因子的直接磷酸化。除了这个典型的途径，鲜为人知。有趣的是,已经在两种罕见的心血管疾病中发现了这种信号通路的突变，遗传性出血性毛细血管扩张症和肺动脉高压。
方法：为了获得内皮细胞中BMP9和BMP10刺激调节的信号通路的概述，我们采用了基于无偏磷酸化蛋白质组的策略.通过蛋白质印迹分析验证鉴定的磷酸位点,并通过RT-qPCR验证调节的靶标。通过流式细胞术分析细胞周期。
结果：大规模的磷酸化蛋白质组学显示，BMP9和BMP10处理诱导了非常相似的磷酸化蛋白质组分布。这些BMP激活了非典型转录SMAD依赖性MAPK途径(MEKK4/P38)。我们能够验证该信号传导途径，并证明该激活需要蛋白质GADD45β的表达。反过来,激活的P38磷酸化热休克蛋白HSP27和内吞蛋白Eps15（EGF受体途径底物），并调节特定基因的表达(E-选择素，透明质酸合酶2和环氧合酶2)。该研究还强调了参与转录调节（内皮转录因子ERG的磷酸化）和细胞周期抑制（CDK4/6途径）的蛋白质磷酸化的调节。因此，我们发现BMP10诱导G1细胞周期停滞，并抑制E2F2，cyclinD1和cyclinA1的mRNA表达。
结论：总体而言，我们的磷酸化蛋白质组筛选鉴定了许多蛋白质，其磷酸化状态受BMP9和BMP10处理的影响，为更好地理解血管疾病中BMP信号调节的分子机制铺平了道路。
方法：为了获得内皮细胞中BMP9和BMP10刺激调节的信号通路的概述，我们采用了基于无偏磷酸化蛋白质组的策略.通过蛋白质印迹分析验证鉴定的磷酸位点,并通过RT-qPCR验证调节的靶标。通过流式细胞术分析细胞周期。
结果：大规模的磷酸化蛋白质组学显示，BMP9和BMP10处理诱导了非常相似的磷酸化蛋白质组分布。这些BMP激活了非典型转录SMAD依赖性MAPK途径(MEKK4/P38)。我们能够验证该信号传导途径，并证明该激活需要蛋白质GADD45β的表达。反过来,激活的P38磷酸化热休克蛋白HSP27和内吞蛋白Eps15（EGF受体途径底物），并调节特定基因的表达(E-选择素，透明质酸合酶2和环氧合酶2)。该研究还强调了参与转录调节（内皮转录因子ERG的磷酸化）和细胞周期抑制（CDK4/6途径）的蛋白质磷酸化的调节。因此，我们发现BMP10诱导G1细胞周期停滞，并抑制E2F2，cyclinD1和cyclinA1的mRNA表达。
结论：总体而言，我们的磷酸化蛋白质组筛选鉴定了许多蛋白质，其磷酸化状态受BMP9和BMP10处理的影响，为更好地理解血管疾病中BMP信号调节的分子机制铺平了道路。
