The BMP-PPARy Axis and Pulmonary Hypertension

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 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.