We previously observed that mice with deletion of peroxisome proliferator-activated receptor γ (PPARγ) in endothelial cells (EC) (EC-Pparγ-/-) do not reverse pulmonary hypertension (PH) during recovery in room air after three weeks of chronic hypoxia. Impaired PPARγ interaction with ß-catenin was linked to pulmonary arterial (PA) EC dysfunction in the pathogenesis of PH. To gain further insight into PPARγ function, important in reversing PH, we applied affinity purification-mass spectrometry in HEK293T cells to analyse the PPARγ interactome. Analyses revealed a network highly enriched in DNA damage response (DDR) proteins, including the MRE11, RAD50 and NBN (MRN) heterotrimeric complex. MRN senses damaged DNA and activates the ATM/ATR pathway essential for DNA repair. Using co-immmunoprecipitation in PAECs, we confirmed the PPARγ:MRN interaction via the PPARγ ligand binding domain. To determine the function of this novel interaction, we depleted PPARγ with siRNA and induced DNA damage with doxorubicin (DOX) or hydroxyurea (HU). PPARγ depletion impaired DDR activation after DOX/HU, with reduced levels of pATM and its substrates, pCHK1, pCHK2 and γH2AX. To test whether these abnormalities were related to reduced DNA repair, we assessed damaged DNA 24h after removal of DOX/HU using the COMET assay. PPARγ depleted PAECs showed longer comet tails reflective of unrepaired DNA. To investigate if persistent PH in EC-Pparγ-/- mice after chronic hypoxia correlated with impaired DDR, we evaluated both γH2AX (sensor) and Brca1 (repair) foci formation in the mouse lung tissues using confocal microscopy. Consistent with impaired DNA repair, Brca1 foci formation was significantly increased in ECs of EC-Pparγ-/- mice compared to the wildtype littermates. Moreover, reduced PPARγ previously described in PAECs from PH patients relative to controls was also associated with unrepaired DNA, judged by longer comet tails and by persistent γH2AX foci as assessed by confocal microscopy in tissue sections. In summary we report a novel interaction between PPARγ and the MRN that is necessary in DNA damage sensing and repair. We suggest that persistent DNA damage associated with PPARγ deficiency in PAECs could cause dysfunction or transformation related to the pathology of PH.
Mechanistic link between DNA damage sensing, repairing and signaling factors and immune signaling