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• W2913014410 abstract "•PPARγ proteomics identifies interactions with the MRE11-RAD50-NBS1 complex and UBR5•Upon DNA damage, PPARγ promotes UBR5-mediated ATMIN degradation to activate ATM•PPARγ-UBR5 interaction is disrupted in endothelial cells isolated from PAH patients•Depleting ATMIN in PAH endothelial cells restores ATM signaling upon DNA damage Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor γ (PPARγ) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARγ promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARγ depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARγ DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARγ signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARγ-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARγ DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH. Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor γ (PPARγ) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARγ promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARγ depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARγ DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARγ signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARγ-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARγ DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH. Peroxisome proliferator activated receptor γ (PPARγ) is a member of the nuclear receptor family that interacts with canonical retinoic acid receptors (RXR) (Chandra et al., 2008Chandra V. Huang P. Hamuro Y. Raghuram S. Wang Y. Burris T.P. Rastinejad F. Structure of the intact PPAR-gamma-RXR- nuclear receptor complex on DNA.Nature. 2008; 456: 350-356Crossref PubMed Scopus (590) Google Scholar) and other co-factors as a transcription factor complex in multiple cell types, including vascular cells (Alastalo et al., 2011Alastalo T.P. Li M. Perez Vde.J. Pham D. Sawada H. Wang J.K. Koskenvuo M. Wang L. Freeman B.A. Chang H.Y. Rabinovitch M. Disruption of PPARγ/β-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival.J. Clin. Invest. 2011; 121: 3735-3746Crossref PubMed Scopus (201) Google Scholar). Aberrant PPARγ-mediated transcription has been implicated in disease conditions, including obesity, diabetes, cancer, inflammation, and vascular disorders (Ahmadian et al., 2013Ahmadian M. Suh J.M. Hah N. Liddle C. Atkins A.R. Downes M. Evans R.M. PPARγ signaling and metabolism: the good, the bad and the future.Nat. Med. 2013; 19: 557-566Crossref PubMed Scopus (1242) Google Scholar, Rabinovitch, 2010Rabinovitch M. PPARgamma and the pathobiology of pulmonary arterial hypertension.Adv. Exp. Med. Biol. 2010; 661: 447-458Crossref PubMed Scopus (55) Google Scholar) that include atherosclerosis (Duval et al., 2002Duval C. Chinetti G. Trottein F. Fruchart J.C. Staels B. The role of PPARs in atherosclerosis.Trends Mol. Med. 2002; 8: 422-430Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar), aortic aneurysm (Hamblin et al., 2010Hamblin M. Chang L. Zhang H. Yang K. Zhang J. Chen Y.E. Vascular smooth muscle cell peroxisome proliferator-activated receptor-γ deletion promotes abdominal aortic aneurysms.J. Vasc. Surg. 2010; 52: 984-993Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and pulmonary arterial hypertension (PAH) (Rabinovitch, 2010Rabinovitch M. PPARgamma and the pathobiology of pulmonary arterial hypertension.Adv. Exp. Med. Biol. 2010; 661: 447-458Crossref PubMed Scopus (55) Google Scholar). Endothelial dysfunction is a feature of all these vascular diseases, and in PAH, it is associated with the obliteration and loss of microvessels that increase resistance to pulmonary blood flow and can culminate in heart failure and the need for a lung transplant (Rabinovitch, 2012Rabinovitch M. Molecular pathogenesis of pulmonary arterial hypertension.J. Clin. Invest. 2012; 122: 4306-4313Crossref PubMed Scopus (488) Google Scholar). Mice with PPARγ deleted in endothelial cells (ECs) (Tie2-Pparγ−/−) develop pulmonary hypertension that persists upon re-exposure to room air after hypoxia (Guignabert et al., 2009Guignabert C. Alvira C.M. Alastalo T.P. Sawada H. Hansmann G. Zhao M. Wang L. El-Bizri N. Rabinovitch M. Tie2-mediated loss of peroxisome proliferator-activated receptor-gamma in mice causes PDGF receptor-beta-dependent pulmonary arterial muscularization.Am. J. Physiol. Lung Cell. Mol. Physiol. 2009; 297: L1082-L1090Crossref PubMed Scopus (123) Google Scholar). In human pulmonary arterial ECs (PAECs), an interaction between PPARγ and β-catenin co-regulates the gene expression of apelin, a major factor that promotes PAEC survival and suppresses smooth muscle cell proliferation (Alastalo et al., 2011Alastalo T.P. Li M. Perez Vde.J. Pham D. Sawada H. Wang J.K. Koskenvuo M. Wang L. Freeman B.A. Chang H.Y. Rabinovitch M. Disruption of PPARγ/β-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival.J. Clin. Invest. 2011; 121: 3735-3746Crossref PubMed Scopus (201) Google Scholar). This interaction is disrupted by rosiglitazone, an agonist previously used to treat type II diabetes (Alastalo et al., 2011Alastalo T.P. Li M. Perez Vde.J. Pham D. Sawada H. Wang J.K. Koskenvuo M. Wang L. Freeman B.A. Chang H.Y. Rabinovitch M. Disruption of PPARγ/β-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival.J. Clin. Invest. 2011; 121: 3735-3746Crossref PubMed Scopus (201) Google Scholar). These observations reinforce the need to discover interactions between PPARγ and other proteins that are perturbed in PAH and other vascular disorders and have pharmacologic relevance. Here, we report the results of a proteomic approach using affinity purification with mass spectrometry (AP-MS) to identify PPARγ nuclear interacting proteins. These studies uncovered PPARγ interactions with the DNA damage sensor MRN (MRE11-RAD50-NBS1) and the E3 ubiquitin ligase UBR5 and a role for PPARγ in the DNA damage response (DDR) pathway. We showed that PPARγ promotes UBR5 ubiquitin ligase activity and regulates ATM interactor (ATMIN) levels, thereby permitting efficient ATM phosphorylation and the initiation of DNA repair upon DNA damage. Perturbation of this axis is observed in PAH and can account for unresolved DNA damage that is associated with impaired endothelial functions (de Jesus Perez et al., 2014de Jesus Perez V.A. Yuan K. Lyuksyutova M.A. Dewey F. Orcholski M.E. Shuffle E.M. Mathur M. Yancy Jr., L. Rojas V. Li C.G. et al.Whole-exome sequencing reveals TopBP1 as a novel gene in idiopathic pulmonary arterial hypertension.Am. J. Respir. Crit. Care Med. 2014; 189: 1260-1272Crossref PubMed Scopus (63) Google Scholar, Diebold et al., 2015Diebold I. Hennigs J.K. Miyagawa K. Li C.G. Nickel N.P. Kaschwich M. Cao A. Wang L. Reddy S. Chen P.I. et al.BMPR2 preserves mitochondrial function and DNA during reoxygenation to promote endothelial cell survival and reverse pulmonary hypertension.Cell Metab. 2015; 21: 596-608Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). We transiently transfected 293T cells with a FLAG-tagged PPARγ1 construct and isolated nuclear extracts in the presence of micrococcal nuclease for affinity purification using a FLAG antibody. We used 293T cells for their high transfection efficiency that permitted efficient pull-down of FLAG-PPARγ and detection of interactors. The quadruplicate AP-MS screen revealed 352 proteins that co-purified with FLAG-PPARγ with a log2 fold change (Log2FC) of >1.5 and an adjusted p value (adj. P) ≤ 0.05 (Figure S1A). Not surprisingly, we detected known PPARγ interactors, such as mediator of RNA polymerase II transcription subunit 1 and 24 (MED1 and MED24, respectively), promyelocytic leukemia protein (PML), p53, and others. We ranked 87 proteins as high-confidence PPARγ-interacting proteins, and those included the canonical partners RXRα and β (Figure S1A; Table S1). Using databases of published physical and functional interactions, we constructed and analyzed networks of high confidence proteins for enriched biological functions. In addition to cellular metabolism, we observed DDR and DNA replication among the most enriched functions (Figure 1A; Table S2). From the DDR network, four interactions were verified by co-immunoprecipitation, i.e., the components of the DNA damage sensing complex MRN (MRE11-RAD50-NBS1) and p53 (Figure S1B). MRN initiates the DDR pathway using NBS1 to recruit proteins necessary for DNA repair (Reinhardt and Yaffe, 2013Reinhardt H.C. Yaffe M.B. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.Nat. Rev. Mol. Cell Biol. 2013; 14: 563-580Crossref PubMed Scopus (192) Google Scholar). We hypothesize that PPARγ binds to MRN via NBS1. To test this, we used tandem affinity purification (TAP) of PPARγ-2x Streptavidin (PPARγ-2xStrep) and FLAG-NBS1 in 293T cells, and the crosslinking agent bis(sulfosuccinimidyl)suberate (BS3) was added on beads before elution. The crosslinked immunocomplexes were analyzed by mass spectrometry (XL-MS) (Figure S1C). XL-MS identified three PPARγ peptides crosslinked to NBS1 (Figure S1D), demonstrating a direct interaction. Using structural mapping based on PPARγ crystal structure (Chandra et al., 2008Chandra V. Huang P. Hamuro Y. Raghuram S. Wang Y. Burris T.P. Rastinejad F. Structure of the intact PPAR-gamma-RXR- nuclear receptor complex on DNA.Nature. 2008; 456: 350-356Crossref PubMed Scopus (590) Google Scholar), we located two of the three peptides in the zinc-finger motif within the PPARγ DNA-binding domain (DBD) and one in the ligand-binding domain (LBD) (Figures S1E and S1F). These data suggest that NBS1 binding might interfere with PPARγ transcription factor function. We used size-exclusion chromatography of nuclear extracts overexpressing PPARγ-2xStrep and FLAG-NBS1 and showed that PPARγ exists in multiple pools: a higher molecular weight (MW, approximated >1,500 kDa) pool, a lower MW (approximated 67–440 kDa) pool, and a monomeric pool (from overexpression, <67 kDa). NBS1 and RXRα reside in the high and low MW PPARγ pools, respectively, supporting mutually exclusive PPARγ interactions with NBS1 or RXRα (Figure S2A). In the absence of NBS1, we also found that PPARγ and three out of the seven PPARγ target genes were upregulated (Figure S2B). The requirement of PPARγ-LBD for MRN interactions was confirmed using mutagenesis (Figure S2C). These data suggest that upon MRN binding, PPARγ undergoes structural changes, which can interfere with its transcription factor property, implicating an independent function for PPARγ. To investigate PPARγ functions in relation to MRN binding, we performed initial silver staining of the TAP elution from unperturbed cell lysates and identified all components of MRN but not RXRα (Figure 1B), supporting our XL-MS and size-exclusion chromatography results. Silver-stained gel fragments from the TAP elution also identified TR150 (thyroid hormone receptor-associated protein 3, encoded by THRAP3) and the ubiquitin ligase UBR5 co-purifying with the PPARγ-MRN complex (Figure 1B). Under conditions of DNA damage induced by hydroxyurea (HU), TAP-MS revealed associations of UBR5 and TR150 with the PPARγ-MRN complex (Figure 1C; Tables S3 and S4). We performed nuclear co-immunoprecipitation (co-IP) of endogenous UBR5 and NBS1 and showed that both UBR5 and NBS1 bind strongly to PPARγ but weakly to each other (Figure S2D). This was confirmed by co-IP of UBR5 and PPARγ in the absence of NBS1 (Figure S2E). To verify the specificity of these PPARγ interactions, we altered PPARγ conformations by using the pharmacological modulator SR10221, which destabilizes helix 12 in the PPARγ LBD (Marciano et al., 2015Marciano D.P. Kuruvilla D.S. Boregowda S.V. Asteian A. Hughes T.S. Garcia-Ordonez R. Corzo C.A. Khan T.M. Novick S.J. Park H. et al.Pharmacological repression of PPARγ promotes osteogenesis.Nat. Commun. 2015; 6: 7443Crossref PubMed Scopus (81) Google Scholar). SR10221 disrupted PPARγ interactions with MRN and UBR5, which were restored by pre-treatment with GW9662, which blocks the SR10221 target site (Figure 1D). Our proteomic and biochemical data suggest that PPARγ interactions with MRN and UBR5 implicate a potential role for PPARγ in the DDR pathway. The MRN complex (Lee and Paull, 2004Lee J.H. Paull T.T. Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex.Science. 2004; 304: 93-96Crossref PubMed Scopus (584) Google Scholar) and UBR5 (Zhang et al., 2014Zhang T. Cronshaw J. Kanu N. Snijders A.P. Behrens A. UBR5-mediated ubiquitination of ATMIN is required for ionizing radiation-induced ATM signaling and function.Proc. Natl. Acad. Sci. USA. 2014; 111: 12091-12096Crossref PubMed Scopus (40) Google Scholar) are required for ATM activity, which is necessary for DNA repair induced by genotoxic agents. In this study, we activated ATM signaling using doxorubicin (DoxR), which intercalates DNA and generates double-strand breaks (Kurz et al., 2004Kurz E.U. Douglas P. Lees-Miller S.P. Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species.J. Biol. Chem. 2004; 279: 53272-53281Crossref PubMed Scopus (209) Google Scholar), and HU, which induces replication fork collapse and a progressive accumulation of double-strand breaks (Cuadrado et al., 2006Cuadrado M. Martinez-Pastor B. Murga M. Toledo L.I. Gutierrez-Martinez P. Lopez E. Fernandez-Capetillo O. ATM regulates ATR chromatin loading in response to DNA double-strand breaks.J. Exp. Med. 2006; 203: 297-303Crossref PubMed Scopus (190) Google Scholar). We first verified endogenous nuclear PPARγ interactions with UBR5 and MRN at baseline and in response to DoxR or HU (Figure 2A). To determine if PPARγ is necessary for ATM activation, we depleted PPARγ using small interfering RNA (siRNA) and induced damage using HU and DoxR. The loss of PPARγ and UBR5 reduced HU-mediated ATM phosphorylation (pATM, Ser1981) and its targets KAP1 (Ser824) (Ziv et al., 2006Ziv Y. Bielopolski D. Galanty Y. Lukas C. Taya Y. Schultz D.C. Lukas J. Bekker-Jensen S. Bartek J. Shiloh Y. Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway.Nat. Cell Biol. 2006; 8: 870-876Crossref PubMed Scopus (547) Google Scholar), γH2AX (Ser139) (Burma et al., 2001Burma S. Chen B.P. Murphy M. Kurimasa A. Chen D.J. ATM phosphorylates histone H2AX in response to DNA double-strand breaks.J. Biol. Chem. 2001; 276: 42462-42467Crossref PubMed Scopus (1473) Google Scholar), and SMC1 (Ser966) (Yazdi et al., 2002Yazdi P.T. Wang Y. Zhao S. Patel N. Lee E.Y. Qin J. SMC1 is a downstream effector in the ATM/NBS1 branch of the human S-phase checkpoint.Genes Dev. 2002; 16: 571-582Crossref PubMed Scopus (410) Google Scholar) (which was not affected by siUBR5) (Figure 2B; densitometry in Figure S3A). PPARγ/UBR5-dependent ATM signaling was also evident in response to DoxR treatment (Figure S3B). We further investigated the role of PPARγ in HU-induced DNA damage because replication stress damage is relevant to PAH (de Jesus Perez et al., 2014de Jesus Perez V.A. Yuan K. Lyuksyutova M.A. Dewey F. Orcholski M.E. Shuffle E.M. Mathur M. Yancy Jr., L. Rojas V. Li C.G. et al.Whole-exome sequencing reveals TopBP1 as a novel gene in idiopathic pulmonary arterial hypertension.Am. J. Respir. Crit. Care Med. 2014; 189: 1260-1272Crossref PubMed Scopus (63) Google Scholar). To understand how PPARγ and UBR5 regulate ATM signaling, we determined whether PPARγ is required for UBR5 E3 ubiquitin ligase activity. Indeed, PPARγ depletion inhibited UBR5-mediated ubiquitination, judging by a decrease in ubiquitinated proteins immunoprecipitated with UBR5 (Figure 2C). We further investigated whether PPARγ depletion affects ATMIN levels, an UBR5 substrate that regulates ATM phosphorylation. Previous studies indicated that UBR5 ubiquitinates ATMIN upon ionizing radiation to release and allow ATM activation (Zhang et al., 2014Zhang T. Cronshaw J. Kanu N. Snijders A.P. Behrens A. UBR5-mediated ubiquitination of ATMIN is required for ionizing radiation-induced ATM signaling and function.Proc. Natl. Acad. Sci. USA. 2014; 111: 12091-12096Crossref PubMed Scopus (40) Google Scholar, Zhang et al., 2012Zhang T. Penicud K. Bruhn C. Loizou J.I. Kanu N. Wang Z.Q. Behrens A. Competition between NBS1 and ATMIN controls ATM signaling pathway choice.Cell Rep. 2012; 2: 1498-1504Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In contrast, other studies have shown the opposite with replication stress, i.e., that loss of ATMIN suppresses ATM activation (Schmidt et al., 2014Schmidt L. Wiedner M. Velimezi G. Prochazkova J. Owusu M. Bauer S. Loizou J.I. ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress.DNA Repair (Amst.). 2014; 24: 122-130Crossref PubMed Scopus (24) Google Scholar). Here, we observed that upon depletion of PPARγ or UBR5, ATMIN levels were elevated both at baseline and in response to HU in association with the suppression of the ATM target pRPA2 (Ser4/8) (Liu et al., 2012Liu S. Opiyo S.O. Manthey K. Glanzer J.G. Ashley A.K. Amerin C. Troksa K. Shrivastav M. Nickoloff J.A. Oakley G.G. Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress.Nucleic Acids Res. 2012; 40: 10780-10794Crossref PubMed Scopus (163) Google Scholar) (Figures 2D and 2E; densitometry in Figures S3C and S3D). Consistent with the function for PPARγ related to UBR5 ubiquitin ligase activity, elevated ATMIN protein in the absence of PPARγ or UBR5 was accompanied by a decrease in its ubiquitination (Figure 2F). Moreover, ubiquitination of ATMIN was associated with its degradation since the proteasome inhibitor MG132 maintains ATMIN protein levels (Figure 2F, input panel). In the absence of UBR5, PPARγ remained bound to the truncated FLAG-ATMIN (aa1-354), supporting UBR5 as downstream of PPARγ in ATMIN regulation (Figure S3E). In addition, both UBR5 and PPARγ bind to FLAG-ATMIN with and without HU, with UBR5 binding more sustained upon HU treatment (Figure S3F). The effects of PPARγ depletion on protein degradation was further evident judging by the reduced cellular lysine (K)48-linked ubiquitins, which represent protein degradative signals (Glickman and Ciechanover, 2002Glickman M.H. Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction.Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3341) Google Scholar). This reduction was restored by overexpressing siRNA-resistant PPARγ (siResPPARγ) (Figure S3G). Since PPARγ is a transcription factor, we confirmed that ATMIN mRNA levels were not significantly altered by the depletion of PPARγ or of UBR5 (Figure 2G). Taken together, our data indicate that the loss of PPARγ alters cellular protein degradative signals and, specifically, it increases ATMIN levels by suppressing UBR5-mediated ubiquitination, and that this function is not related to PPARγ-mediated transcription. We and others showed that PPARγ promotes endothelial survival and regeneration (Alastalo et al., 2011Alastalo T.P. Li M. Perez Vde.J. Pham D. Sawada H. Wang J.K. Koskenvuo M. Wang L. Freeman B.A. Chang H.Y. Rabinovitch M. Disruption of PPARγ/β-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival.J. Clin. Invest. 2011; 121: 3735-3746Crossref PubMed Scopus (201) Google Scholar, Vattulainen-Collanus et al., 2016Vattulainen-Collanus S. Akinrinade O. Li M. Koskenvuo M. Li C.G. Rao S.P. de Jesus Perez V. Yuan K. Sawada H. Koskenvuo J.W. et al.Loss of PPARγ in endothelial cells leads to impaired angiogenesis.J. Cell Sci. 2016; 129: 693-705Crossref PubMed Scopus (33) Google Scholar). In a transgenic mouse with deficient endothelial PPARγ, pulmonary hypertension and adverse vascular remodeling did not reverse following re-exposure to room air after chronic hypoxia (Guignabert et al., 2009Guignabert C. Alvira C.M. Alastalo T.P. Sawada H. Hansmann G. Zhao M. Wang L. El-Bizri N. Rabinovitch M. Tie2-mediated loss of peroxisome proliferator-activated receptor-gamma in mice causes PDGF receptor-beta-dependent pulmonary arterial muscularization.Am. J. Physiol. Lung Cell. Mol. Physiol. 2009; 297: L1082-L1090Crossref PubMed Scopus (123) Google Scholar). As impaired PPARγ function and chromosomal instability related to persistent DNA damage are features of PAECs from patients with PAH (Aldred et al., 2010Aldred M.A. Comhair S.A. Varella-Garcia M. Asosingh K. Xu W. Noon G.P. Thistlethwaite P.A. Tuder R.M. Erzurum S.C. Geraci M.W. Coldren C.D. Somatic chromosome abnormalities in the lungs of patients with pulmonary arterial hypertension.Am. J. Respir. Crit. Care Med. 2010; 182: 1153-1160Crossref PubMed Scopus (121) Google Scholar), we determined if PPARγ functions in DDR are compromised in PAH and could contribute to the loss of vascular homeostasis. We first verified nuclear PPARγ and UBR5 interactions in primary human PAECs (Figure 3A). Consistent with our findings in 293T cells, PPARγ depletion in PAECs also led to reduced pATM, pRPA2, and γH2AX upon prolonged HU treatment (Figure 3B; densitometry, Figures S4A and S4B). To confirm the specificity of PPARγ-ATM signaling, we restored pATM in human umbilical venous ECs (HUVECs) by overexpressing siResPPARγ (Figure 3C; densitometry, Figure S4C). HUVECs were used to withstand the cytotoxicity from DNA and siRNA sequential transfections. Verifying ATMIN regulation of PPARγ-dependent ATM signaling in ECs, we depleted ATMIN in addition to PPARγ and observed that this restored pATM and its target pKAP1 (Figure 3D; densitometry, Figure S4D). Although ATMIN regulation of ATM signaling is highly context dependent (Leszczynska et al., 2016Leszczynska K.B. Göttgens E.L. Biasoli D. Olcina M.M. Ient J. Anbalagan S. Bernhardt S. Giaccia A.J. Hammond E.M. Mechanisms and consequences of ATMIN repression in hypoxic conditions: roles for p53 and HIF-1.Sci. Rep. 2016; 6: 21698Crossref PubMed Scopus (17) Google Scholar, Schmidt et al., 2014Schmidt L. Wiedner M. Velimezi G. Prochazkova J. Owusu M. Bauer S. Loizou J.I. ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress.DNA Repair (Amst.). 2014; 24: 122-130Crossref PubMed Scopus (24) Google Scholar, Zhang et al., 2014Zhang T. Cronshaw J. Kanu N. Snijders A.P. Behrens A. UBR5-mediated ubiquitination of ATMIN is required for ionizing radiation-induced ATM signaling and function.Proc. Natl. Acad. Sci. USA. 2014; 111: 12091-12096Crossref PubMed Scopus (40) Google Scholar), our results demonstrate that in the absence of PPARγ, abnormal accumulation of ATMIN suppresses ATM activation in response to DNA damage. We also verified the inhibitory effects of siPPARγ on pATM and γH2AX foci by using immunofluorescence in PAEC (Figures S4E and S4F). This response was replicated with three individual siRNAs targeting PPARγ (Figure S4G). Importantly, the reduced ATM signaling upon PPARγ depletion was not due to altered cell cycle progression (Figure S4H). Since elevated oxidative stress has been implicated in PAH pathogenesis (Diebold et al., 2015Diebold I. Hennigs J.K. Miyagawa K. Li C.G. Nickel N.P. Kaschwich M. Cao A. Wang L. Reddy S. Chen P.I. et al.BMPR2 preserves mitochondrial function and DNA during reoxygenation to promote endothelial cell survival and reverse pulmonary hypertension.Cell Metab. 2015; 21: 596-608Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) and ATM signaling is activated by oxidative stress (Hammond et al., 2003Hammond E.M. Dorie M.J. Giaccia A.J. ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation.J. Biol. Chem. 2003; 278: 12207-12213Crossref PubMed Scopus (239) Google Scholar), we investigated if PPARγ also promotes ATM signaling upon oxidant injury. By exposing PAECs to hypoxia (<0.1% O2, 24 h) and reoxygenation (10 min), we detected the presence of 8-oxo-2′-deoxyguanosine (8-oxo-dG) foci (S4I), a marker for oxidative damage DNA (Cheng et al., 1992Cheng K.C. Cahill D.S. Kasai H. Nishimura S. Loeb L.A. 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions.J. Biol. Chem. 1992; 267: 166-172Abstract Full Text PDF PubMed Google Scholar). We showed that PPARγ depletion also suppressed oxidative stress-induced pATM (Figure 3E; replicates, Figure S4J). We now showed that PPARγ is necessary to initiate the DDR, and we hypothesize that it is also important for DNA repair. We used the comet assay and demonstrated that PPARγ depletion did not affect the magnitude of DNA damage, as judged by comet tails assessed after a 6-h exposure to HU (Figure 4A; replicates, Figure S5A), but the capacity to repair DNA was reduced, as judged by persistent comet tails after a 24-h recovery period. We also examined levels of pRPA2 and γH2AX damage foci during recovery (24–72 h), as evidence of unrepaired DNA lesions. These foci were resolved in the control cells but were sustained in PPARγ-depleted PAECs (Figure 4B; replicates, Figure S5B). We validated that ATMIN also functions in PPARγ-dependent DNA repair by demonstrating that depletion of ATMIN in addition to PPARγ resolved pRPA2 foci during recovery (Figures 4C and 4D; densitometry and replicates, Figures S5C and S5D). We then determined whether unresolved DNA damage accompanied the pulmonary hypertension that did not reverse in mice with PPARγ depleted in ECs (Tie2-Pparγ−/−) that were re-exposed to room air after chronic hypoxia (Guignabert et al., 2009Guignabert C. Alvira C.M. Alastalo T.P. Sawada H. Hansmann G. Zhao M. Wang L. El-Bizri N. Rabinovitch M. Tie2-mediated loss of peroxisome proliferator-activated receptor-gamma in mice causes PDGF receptor-beta-dependent pulmonary arterial muscularization.Am. J. Physiol. Lung Cell. Mol. Physiol. 2009; 297: L1082-L1090Crossref PubMed Scopus (123) Google Scholar). Lung sections from Tie2-Pparγ−/− mice and wild-type littermates were co-stained with von Willebrand factor (vWF) antibody to detect ECs and γH2AX antibody. Confocal microscopy revealed increased γH2AX in the ECs of the mutant versus control mice previously studied following re-exposure to room air (Figure 4E). These data further supported our mechanistic studies in cultured PAECs that link PPARγ to regulation of DNA damage sensing and repair. The loss of genome integrity and an increased propensity for apoptosis and transformation are key features of PAECs from PAH patients (PAH-PAECs) (Aldred et al., 2010Aldred M.A. Comhair S.A. Varella-Garcia M. Asosingh K. Xu W. Noon G.P. Thistlethwaite P.A. Tuder R.M. Erzurum S.C. Geraci M.W. Coldren C.D. Somatic chromosome abnormalities in the lungs of patients with pulmonary arterial hypertension.Am. J. Respir. Crit. Care Med. 2010; 182: 1153-1160Crossref PubMed Scopus (121) Google Scholar, Hopper et al., 2016Hopper R.K. Moonen J.R. Diebold I. Cao A. Rhodes C.J. Tojais N.F. Hennigs J.K. Gu M. Wang L. Rabinovitch M. In pulmonary arterial hypertension, reduced BMPR2 promotes endothelial-to-mesenchymal transition via HMGA1 and its target slug.Circulation. 2016; 133: 1783-1794Crossref PubMed Scopus (151) Google Scholar, Ranchoux et al., 2015Ranchoux B. Antigny F. Rucker-Martin C. Hautefort A. Péchoux C. Bogaard H.J. Dorfmüller P. Remy S. Lecerf F. Planté S. et al.Endothelial-to-mesenchymal transition in pulmonary hypertension.Circulation. 2015; 131: 1006-1018Crossref PubMed Scopus (353) Google Scholar, Sa et al., 2016Sa S. Gu M. Chappell J. Shao N.Y. Ameen M. Elliott K.A. Li D. Grubert F. Li C.G. Taylor S. et al.iPSC model of pulmonary arterial hypertension reveals novel gene expression and patient specificity.Am. J. Respir. Crit. Care Med. 2016; 195: 930-941Crossref Scopus (60) Google Scholar). We, therefore, assessed evidence of unrepaired DNA damage in PAH versus unused donor control lung sections and in cultured PAECs harvested from explanted PAH lungs and from control lungs. Demographic information related to controls (unused donor) and PAH-PAECs is provided in Table S5. Representative cell images indicating healthy, actively proliferating primary PAEC cultures are shown in Figure S6A. Increased γH2AX foci were evident in PAH versus control PAECs in lung tissue sections (Figure 5A), and in cell cultures, there were more extended comet tails (Figure 5B) in PAH-PAECs compared with control-PAECs. Upon HU treatment, PAH-PAECs showed reduced pATM foci compared with control-PAECs (Figure 5C; replicates, Figure S6B)." @default.
• W2913014410 created "2019-02-21" @default.
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