The BMP-PPARy Axis and Pulmonary Hypertension

 描述（由申请人提供）：在上一个资助期间，我们确定BMPR 2-PPARγ轴通过调节肺动脉（PA）内皮细胞（EC）和平滑肌细胞（SMC）中的不同基因来防止肺动脉高压（PAH）的发展。我们假设这种细胞特异性基因调控是由与PPARγ相互作用的核蛋白的差异引起的。为了通过亲和纯化和质谱鉴定与PPARγ相互作用的蛋白，我们用FLAG标记的PPARγ转染HEK 293细胞。正如预期的那样，我们发现了已知与PPARγ相关的蛋白质，但令人惊讶的是，有多种新的相互作用蛋白，与DNA损伤传感和修复有关。此外，在多柔比星（Dox）的DNA损伤作用下，这些蛋白与PPARγ的相互作用尤为突出。我们现在打算研究其中两种相互作用的功能意义：一种是PPARγ与DNA损伤感应复合物MRN（MRE 11，Rad 50和NBS 1）之间的相互作用，另一种是PPARγ与p53之间的相互作用，这在细胞周期阻滞，DNA修复和线粒体代谢中很重要。在特定目的I中，我们证实了我们使用PPARγ siRNA的研究，以表明减少PPARγ-MRN复合物的形成会降低ATM、γ H2 AX和pCHK 1/2的活化，并导致未修复的DNA。然后，我们确定当BMPR 2缺失导致PPARγ减少时，是否也观察到DNA损伤的感知和修复受损。此外，我们还研究了当PPARγ或BMPR 2减少时，其他DNA损伤刺激，如缺氧和再氧合以及TNF-α，是否会导致未修复的DNA。研究表明PAH患者PA EC中未修复的DNA增加，因为我们确定DNA是否可以通过FK 506（一种在受体缺陷时“拯救”BMPR 2信号传导的试剂）或NO2-FA（一种内源性PPARγ激动剂）修复。在Specific Aim II中，我们确定BMPR 2减少（通过减少PPARγ）是否会损害DNA损伤刺激时PPARγ和p53之间的相互作用。我们还研究了PAH患者（包括BMPR 2突变患者）的PA EC中是否存在PPARγ-p53复合物缺陷。基于初步数据，我们探讨了PPARγ-p53相互作用的功能意义，这些数据表明：（i）PPARγ影响p53的转录活性，（ii）PPARγ-p53复合物 调节影响PA EC存活、细胞周期、DNA修复以及线粒体生物发生和DNA合成的基因。采用无偏ChIPSeq方法寻找新的PPARγ-p53介导的基因调控靶点。在特定目标III中，我们研究了EC中BMPR 2或DNA损伤传感器MRE 11出生后缺失的小鼠，有两个目标：（i）将肺动脉高压（PH）的发展与未修复的DNA联系起来，以及（ii）确定使用FK 506改善BMPR 2信号传导或诱导PPARγ-p53与nutlin- 3a的相互作用是否可以挽救DNA修复并预防或逆转PH。

 DESCRIPTION (provided by applicant): During the previous funding period we determined that the BMPR2-PPARγ axis protects against the development of pulmonary arterial hypertension (PAH) by regulating different genes in pulmonary arterial (PA) endothelial (EC) and smooth muscle cells (SMC). We hypothesized that this cell-specific gene regulation resulted from differences in nuclear proteins that interacted with PPARγ. To identify PPARγ interacting proteins by affinity purification and mass spectrometry, we transfected HEK293 cells with FLAG-tagged PPARγ. As expected, we found proteins known to associate with PPARγ, but, surprisingly, there were multiple novel interacting proteins, related to DNA damage sensing and repair. Moreover, in PA EC exposed to the DNA damaging effects of doxorubicin (Dox), the interaction between PPARγand these proteins was particularly prominent. We now propose to pursue the functional significance of two of these interactions: the one between PPARγ and the DNA damage sensing complex MRN (MRE11, Rad50 and NBS1) and the one between PPARγ and p53, that is important in cell cycle arrest, DNA repair and mitochondrial metabolism. In Specific Aim I, we confirm our studies using PPARγ siRNA, to show that reduced formation of the PPARγ-MRN complex decreases activation of ATM, γH2AX and pCHK1/2 and results in unrepaired DNA. We then determine whether impaired sensing and repair of DNA damage is also observed when PPARγ is reduced as a consequence of loss of BMPR2. In addition, we investigate whether other DNA damaging stimuli, such as hypoxia and reoxygenation and TNF-α, result in unrepaired DNA when PPARγ or BMPR2 are reduced. Studies showing an increase in unrepaired DNA in PA EC of PAH patients are pursued, as we determine whether DNA can be repaired by FK506, an agent that 'rescues' BMPR2 signaling when the receptor is deficient, or by NO2-FA, an endogenous PPARγ agonist. In Specific Aim II, we determine whether reduced BMPR2 (via decreased PPARγ) impairs the interaction between PPARγ and p53 in response to DNA damaging stimuli. We also investigate whether the PPARγ-p53 complex is deficient in PA EC, from PAH patients including those with a BMPR2 mutation. The functional significance of the PPARγ-p53 interaction is pursued, based upon preliminary data indicating that (i) PPARγ influences the transcriptional activity of p53 and (ii) that the PPARγ-p53 complex regulates genes that influence PA EC survival, cell cycle, DNA repair, and mitochondrial biogenesis and DNA synthesis. An unbiased ChIPSeq approach is applied to find novel targets of PPARγ-p53 mediated gene regulation. In Specific Aim III, we study mice with postnatal deletion of BMPR2 or of the DNA damage sensor MRE11 in EC, with two goals: (i) to relate the development of pulmonary hypertension (PH) to unrepaired DNA and (ii) to determine if improving BMPR2 signaling with FK506 or inducing a PPARγ-p53 interaction with nutlin- 3a salvages DNA repair and prevents or reverses PH.