Honors & Awards
Stanford University Mass Spectrometry Seed Funding Grant, Stanford University (2016)
Keystone Symposia Scholarship, Nuclear Receptors: Biological Networks, Genome Dynamics and Disease, NM, USA (2014)
Claude McCarthy Fellowship, the New Zealand Vice-Chancellor’s Committee (2008)
Dunedin School of Medicine Finishing Your PhD Grants-in-Aid, University of Otago, New Zealand (2008)
International Doctoral Scholarship, University of Otago, New Zealand (2005)
PhD Scholarship, the Health Research Council of New Zealand (2005)
Professional Affiliations and Activities
Member, New Zealand Society for Biochemistry and Molecular Biology (2004 - 2005)
Member, The American Association for the Advancement of Science (AAAS) (2011 - Present)
Member, The Human Proteome Organisation (HUPO) (2011 - Present)
Member, Association for Women in Science (AWIS) (2012 - Present)
Regulation of the BCR signalosome by the class II peptide editor, H2-M, affects the development and repertoire of innate-like B cells.
1800; 38 (4): 110200
The non-classical Major Histocompatibility Complex class II (MHCII) protein, H2-M, edits peptides bound to conventional MHCII in favor of stable peptide/MHCII (p/MHCII) complexes. Here, we show that H2-M deficiency affects B-1 cell survival, reduces cell renewal capacity, and alters immunoglobulin repertoire, allowing for the selection of cells specific for highly abundant epitopes, but not low-frequency epitopes. H2-M-deficient B-1 cells have shorter CDR3 length, higher content of positively charged amino acids, shorter junctional regions, less mutation frequency, and a skewed clonal distribution. Mechanistically, H2-M loss reduces plasma membrane p/MHCII association with B cell receptors (BCR) on B-1 cells and diminishes integrated BCR signal strength, a key determinant of B-1 cell selection, maturation, and maintenance. Thus, H2-M:MHCII interaction serves as a cell-intrinsic regulator of BCR signaling and influences the selection of the B-1 cell clonal repertoire.
View details for DOI 10.1016/j.celrep.2021.110200
View details for PubMedID 35081339
C3aR Signaling Inhibits NK-cell Infiltration into the Tumor Microenvironment in Mouse Models.
Cancer immunology research
Many solid tumors have low levels of cytotoxic CD56dim NK cells, suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells, but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK cell infiltration, we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin, LFA-1, orchestrates the migration of activated NK cells, we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1, which led to a high affinity LFA-1 conformation, decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME, through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1, represents a new strategy to improve the efficiency of immunotherapy for cancer treatment.
View details for DOI 10.1158/2326-6066.CIR-21-0435
View details for PubMedID 34819308
- Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination & ndash;deficient cancer models JOURNAL OF CLINICAL INVESTIGATION 2021; 131 (11)
ALDH1A3 Coordinates Metabolism with Gene Regulation in Pulmonary Arterial Hypertension.
Background: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC. Methods: RNA Sequencing was applied to pulmonary arterial (PA) SMC from PAH patients with and without a BMPR2 mutation vs. control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 (ALDH1A3), an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC. Results: ALDH1A3 mRNA and protein were increased in PAH vs control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced PA muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl-CoA to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at nuclear factor Y (NFY)A binding sites via the acetyltransferase KAT2B and permitted NFY mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was beta-catenin dependent. Conclusions: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not EC, is an important therapeutic consideration.
View details for DOI 10.1161/CIRCULATIONAHA.120.048845
View details for PubMedID 33764154
The HIF target MAFF promotes tumor invasion and metastasis through IL11 and STAT3 signaling.
2021; 12 (1): 4308
Hypoxia plays a critical role in tumor progression including invasion and metastasis. To determine critical genes regulated by hypoxia that promote invasion and metastasis, we screen fifty hypoxia inducible genes for their effects on invasion. In this study, we identify v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (MAFF) as a potent regulator of tumor invasion without affecting cell viability. MAFF expression is elevated in metastatic breast cancer patients and is specifically correlated with hypoxic tumors. Combined ChIP- and RNA-sequencing identifies IL11 as a direct transcriptional target of the heterodimer between MAFF and BACH1, which leads to activation of STAT3 signaling. Inhibition of IL11 results in similar levels of metastatic suppression as inhibition of MAFF. This study demonstrates the oncogenic role of MAFF as an activator of the IL11/STAT3 pathways in breast cancer.
View details for DOI 10.1038/s41467-021-24631-6
View details for PubMedID 34262028
Intracellular C4BPA Levels Regulate NF-kappaB-Dependent Apoptosis.
2020; 23 (10): 101594
The importance of innate immunity in cancer is increasingly being recognized with recent reports suggesting tumor cell-intrinsic intracellular functions for innate immunity proteins. However, such functions are often poorly understood, and it is unclear whether these are affected by patient-specific mutations. Here, we show that C4b-binding protein alpha chain (C4BPA), typically thought to reside in the extracellular space, is expressed intracellularly in cancer cells, where it interacts with the NF-kappaB family member RelA and regulates apoptosis. Interestingly, intracellular C4BPA expression is regulated in a stress- and mutation-dependent manner and C4BPA mutations are associated with improved cancer survival outcome. Using cell lines harboring patient-specific C4BPA mutations, we show that increasing intracellular C4BPA levels correlate with sensitivity to oxaliplatin-induced apoptosis invitro and invivo. Mechanistically, sensitive C4BPA mutants display increased IkappaBalpha expression and increased inhibitory IkappaBalpha-RelA complex stability. These data suggest a non-canonical intracellular role for C4BPA in regulating NF-kappaB-dependent apoptosis.
View details for DOI 10.1016/j.isci.2020.101594
View details for PubMedID 33205012
PPARγ-p53-Mediated Vasculoregenerative Program to Reverse Pulmonary Hypertension.
Rationale: In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of bone morphogenetic protein type 2 receptor (BMPR2) via two downstream transcription factors, PPARγ and p53. Objective: We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ- p53 transcription factor complex in the pulmonary endothelium. Methods and Results: In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial (PA) and lung microvascular (MV) endothelial cells (EC) in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation (ChIP) sequencing (seq) and RNA-seq established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung EC survival, angiogenesis and DNA repair including, EPHA2, FHL2, JAG1, SULF2 and TIGAR. Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in PAEC subjected to oxidative stress. In EC-Bmpr2-knockout mice unable to stabilize p53 in ECs under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAEC from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis. Conclusions: We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAEC downstream of dysfunctional BMPR2 to rehabilitate PAH PAEC, regenerate pulmonary microvessels and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.
View details for DOI 10.1161/CIRCRESAHA.119.316339
View details for PubMedID 33322916
PPARgamma Interaction with UBR5/ATMIN Promotes DNA Repair to Maintain Endothelial Homeostasis.
2019; 26 (5): 1333
Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor gamma (PPARgamma) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARgamma promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARgamma 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 PPARgamma DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARgamma signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARgamma-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARgamma DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH.
View details for DOI 10.1016/j.celrep.2019.01.013
View details for PubMedID 30699358
- Smooth Muscle Contact Drives Endothelial Regeneration by BMPR2-Notch1-Mediated Metabolic and Epigenetic Changes CIRCULATION RESEARCH 2019; 124 (2): 211–24
SMAD proteins directly suppress PAX2 transcription downstream of transforming growth factor-beta 1 (TGF-β1) signalling in renal cell carcinoma.
2018; 9 (42): 26852-26867
Canonical TGF-β1 signalling promotes tumor progression by facilitating invasion and metastasis, whereby release of TGF-β1, by (for example) infiltrating immune cells, induces epithelial to mesenchymal transition (EMT). PAX2, a member of the Paired box family of transcriptional regulators, is normally expressed during embryonic development, including in the kidney, where it promotes mesenchymal to epithelial transition (MET). PAX2 expression is silenced in many normal adult tissues. However, in contrast, PAX2 is expressed in several cancer types, including kidney, prostate, breast, and ovarian cancer. While multiple studies have implicated TGF-β superfamily members in modulating expression of Pax genes during embryonic development, few have investigated direct regulation of Pax gene expression by TGF-β1. Here we have investigated direct regulation of PAX2 expression by TGF-β1 in clear cell renal cell carcinoma (CC-RCC) cell lines. Treatment of PAX2-expressing 786-O and A498 CC-RCC cell lines with TGF-β1 resulted in inhibition of endogenous PAX2 mRNA and protein expression, as well as expression from transiently transfected PAX2 promoter constructs; this inhibition was abolished in the presence of expression of the inhibitory SMAD, SMAD7. Using ChIP-PCR we showed TGF-β1 treatment induced SMAD3 protein phosphorylation in 786-O cells, and direct SMAD3 binding to the human PAX2 promoter, which was inhibited by SMAD7 over-expression. Overall, these data suggest that canonical TGF-β signalling suppresses PAX2 transcription in CC-RCC cells due to the direct binding of SMAD proteins to the PAX2 promoter. These studies improve our understanding of tumor progression and epithelial to mesenchyme transition (EMT) in CC-RCC and in other PAX2-expressing cancer types.
View details for DOI 10.18632/oncotarget.25516
View details for PubMedID 29928489
View details for PubMedCentralID PMC6003550
- Induced Pluripotent Stem Cell Model of Pulmonary Arterial Hypertension Reveals Novel Gene Expression and Patient Specificity AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 2017; 195 (7): 930-941
Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension.
2017; 2 (2)
Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by γH2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1α, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome c oxidase 4 isoform switch. Mitochondrial oxidative phosphorylation is inappropriately enhanced and, as a result of impaired electron transport and mitochondrial ROS increase, caspase-3 is activated and DNA damage is induced. In mice given binge doses of METH followed by hypoxia, HIF1α is suppressed and pulmonary artery DNA damage foci are associated with worse pulmonary vascular remodeling. Thus, chronic AMPH/METH can induce DNA damage associated with vascular disease by subverting the adaptive responses to oxidative stress.
View details for DOI 10.1172/jci.insight.90427
View details for PubMedID 28138562
Upregulation of HERV-K is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.
Background -Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue and elevated cytokines have been related to PAH pathogenesis but without clear understanding of how these abnormalities are initiated, perpetuated and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. Methods -Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry (LCMS), confirmed by ELISA, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next generation sequencing, functional studies in cultured monocytes and endothelial cells (EC) and hemodynamic and lung studies in a rat. Results -SAM domain and HD1 domain-containing protein (SAMHD1), an innate immune factor that suppresses HIV replication was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH vs. 12 control lungs. Elevated SAMHD1 was localized to endothelial cells (EC), perivascular dendritic cells and macrophages and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH vs. control lungs (n=4 each). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) mRNAs were elevated in PAH vs. control lungs (n=10) and proteins were localized to macrophages. HERV-K dUTPase induced SAMHD1 and pro-inflammatory cytokines (e.g., IL6, IL1β and TNFα) in circulating monocytes and pulmonary arterial (PA) EC, and activated B cells. Vulnerability of PAEC to apoptosis was increased by HERV-K dUTPase in an IL6 independent manner. Furthermore, three weekly injections of HERV-K dUTPase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8), and elevated IL6. Conclusions -Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.
View details for PubMedID 28935667
Loss of PPAR gamma in endothelial cells leads to impaired angiogenesis
JOURNAL OF CELL SCIENCE
2016; 129 (4): 693-705
Tie2 promoter-mediated loss of peroxisome proliferator-activated receptor gamma (PPARγ) in mice leads to osteopetrosis and pulmonary arterial hypertension. Vascular disease is associated with loss of PPARγ in pulmonary microvascular endothelial cells (PMVEC), we evaluated the role of PPARγ in PMVEC functions, such as angiogenesis and migration. The role of PPARγ in angiogenesis was evaluated in Tie2CrePPARγ(flox/flox) and wild type (WT) mice, and in mouse and human PMVECs. RNA-sequencing and bioinformatic approaches were utilized to reveal angiogenesis-associated targets for PPARγ. Tie2CrePPARγ(flox/flox) mice showed an impaired angiogenic capacity. Analysis of endothelial progenitor-like cells using bone marrow transplantation combined with evaluation of isolated PMVECs revealed that loss of PPARγ attenuates the migration and angiogenic capacity of mature PMVECs. PPARγ-deficient human PMVECs showed a similar migration defect in culture. Bioinformatic and experimental analyses revealed E2F1 as a novel target of PPARγ in the regulation of PMVEC migration. Disruption of the PPARγ-E2F1 axis was associated with a dysregulated Wnt pathway related to the GSK3β interaction protein. In conclusion, PPARγ plays an important role in sustaining angiogenic potential in mature PMVECs through E2F1-mediated gene regulation.
View details for DOI 10.1242/jcs.169011
View details for Web of Science ID 000370240900006
RNA Sequencing Analysis Detection of a Novel Pathway of Endothelial Dysfunction in Pulmonary Arterial Hypertension
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
2015; 192 (3): 356-366
Pulmonary arterial hypertension is characterized by endothelial dysregulation, but global changes in gene expression have not been related to perturbations in function.RNA sequencing was utilized to discriminate changes in transcriptomes of endothelial cells cultured from lungs of patients with idiopathic pulmonary arterial hypertension vs. controls and to assess the functional significance of major differentially expressed transcripts.The endothelial transcriptomes from seven control and six idiopathic pulmonary arterial hypertension patients' lungs were analyzed. Differentially expressed genes were related to BMPR2 signaling. Those downregulated were assessed for function in cultured cells, and in a transgenic mouse.Fold-differences in ten genes were significant (p<0.05), four increased and six decreased in patients vs.No patient was mutant for BMPR2. However, knockdown of BMPR2 by siRNA in control pulmonary arterial endothelial cells recapitulated six/ten patient-related gene changes, including decreased collagen IV (COL4A1, COL4A2) and ephrinA1 (EFNA1). Reduction of BMPR2 regulated transcripts was related to decreased β-catenin. Reducing COL4A1, COL4A2 and EFNA1 by siRNA inhibited pulmonary endothelial adhesion, migration and tube formation. In mice null for the EFNA1 receptor, EphA2, vs. controls, VEGF receptor blockade and hypoxia caused more severe pulmonary hypertension, judged by elevated right ventricular systolic pressure, right ventricular hypertrophy and loss of small arteries.The novel relationship between BMPR2 dysfunction and reduced expression of endothelial COL4 and EFNA1 may underlie vulnerability to injury in pulmonary arterial hypertension.
View details for DOI 10.1164/rccm.201408-1528OC
View details for PubMedID 26030479
Elafin Reverses Pulmonary Hypertension via Caveolin-1-Dependent Bone Morphogenetic Protein Signaling
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
2015; 191 (11): 1273-1286
Pulmonary arterial hypertension is characterized by endothelial cell dysfunction, impaired BMPR2 signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates the development of hypoxic pulmonary hypertension in mice, but its potential to improve endothelial cell function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown.To assess elafin-mediated regression of pulmonary vascular pathology in rats with pulmonary hypertension induced by VEGF receptor blockade and hypoxia (Sugen/Hypoxia), and in lung explants from pulmonary hypertension patients. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells from controls and patients, and to elucidate the underlying mechanism. Methods, Measurements and Main Results: In Sugen/Hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a product of BMPR2 signaling. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and in lung organ culture elafin decreased neointimal lesions. In normal and patient pulmonary artery endothelial cells, elafin enhanced survival and promoted angiogenesis by increasing pSMAD dependent and independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via elafin-mediated stabilization of caveolin-1 on endothelial surfaces.Elafin reverses obliterative changes in rat and human pulmonary arteries via elastase inhibition and caveolin-1 dependent amplification of BMPR2 signaling.
View details for DOI 10.1164/rccm.201412-2291OC
View details for Web of Science ID 000356105000014
View details for PubMedID 25853696
BMPR2 Preserves Mitochondrial Function and DNA during Reoxygenation to Promote Endothelial Cell Survival and Reverse Pulmonary Hypertension
2015; 21 (4): 596-608
Mitochondrial dysfunction, inflammation, and mutant bone morphogenetic protein receptor 2 (BMPR2) are associated with pulmonary arterial hypertension (PAH), an incurable disease characterized by pulmonary arterial (PA) endothelial cell (EC) apoptosis, decreased microvessels, and occlusive vascular remodeling. We hypothesized that reduced BMPR2 induces PAEC mitochondrial dysfunction, promoting a pro-inflammatory or pro-apoptotic state. Mice with EC deletion of BMPR2 develop hypoxia-induced pulmonary hypertension that, in contrast to non-transgenic littermates, does not reverse upon reoxygenation and is associated with reduced PA microvessels and lung EC p53, PGC1α and TFAM, regulators of mitochondrial biogenesis, and mitochondrial DNA. Decreasing PAEC BMPR2 by siRNA during reoxygenation represses p53, PGC1α, NRF2, TFAM, mitochondrial membrane potential, and ATP and induces mitochondrial DNA deletion and apoptosis. Reducing PAEC BMPR2 in normoxia increases p53, PGC1α, TFAM, mitochondrial membrane potential, ATP production, and glycolysis, and induces mitochondrial fission and a pro-inflammatory state. These features are recapitulated in PAECs from PAH patients with mutant BMPR2.
View details for DOI 10.1016/j.cmet.2015.03.010
View details for Web of Science ID 000352500800014
View details for PubMedID 25863249
Whole-Exome Sequencing Reveals TopBP1 as a Novel Gene in Idiopathic Pulmonary Arterial Hypertension
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
2014; 189 (10): 1260-1272
Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disorder characterized by progressive loss of pulmonary microvessels. Although mutations in the bone morphogenetic receptor 2 (BMPR2) are found in 80% of heritable and ∼15% of patients with IPAH, their low penetrance (∼20%) suggests that other unidentified genetic modifiers are required for manifestation of the disease phenotype. Use of whole-exome sequencing (WES) has recently led to the discovery of novel susceptibility genes in heritable PAH, but whether WES can also accelerate gene discovery in IPAH remains unknown.To determine whether WES can help identify novel gene modifiers in patients with IPAH.Exome capture and sequencing was performed on genomic DNA isolated from 12 unrelated patients with IPAH lacking BMPR2 mutations. Observed genetic variants were prioritized according to their pathogenic potential using ANNOVAR.A total of nine genes were identified as high-priority candidates. Our top hit was topoisomerase DNA binding II binding protein 1 (TopBP1), a gene involved in the response to DNA damage and replication stress. We found that TopBP1 expression was reduced in vascular lesions and pulmonary endothelial cells isolated from patients with IPAH. Although TopBP1 deficiency made endothelial cells susceptible to DNA damage and apoptosis in response to hydroxyurea, its restoration resulted in less DNA damage and improved cell survival.WES led to the discovery of TopBP1, a gene whose deficiency may increase susceptibility to small vessel loss in IPAH. We predict that use of WES will help identify gene modifiers that influence an individual's risk of developing IPAH.
View details for DOI 10.1164/rccm.201310-17490C
View details for Web of Science ID 000336017200018
View details for PubMedID 24702692
View details for PubMedCentralID PMC4225850
FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension.
journal of clinical investigation
2013; 123 (8): 3600-3613
Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.
View details for DOI 10.1172/JCI65592
View details for PubMedID 23867624
Loss of adenomatous poliposis coli-a3 integrin interaction promotes endothelial apoptosis in mice and humans.
2012; 111 (12): 1551-1564
Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins promote pulmonary angiogenesis by recruiting the Wnt/β-catenin pathway, we proposed that β-catenin activation could reduce loss and induce regeneration of small pulmonary arteries (PAs) and attenuate PH.This study aims to establish the role of β-catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis after injury.To assess the impact of β-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (Apc(Min/+)) mouse, where reduced APC causes constitutive β-catenin elevation. Surprisingly, hypoxic Apc(Min/+) mice displayed greater PH and small PA loss compared with control C57Bl6J littermates. PA endothelial cells isolated from Apc(Min/+) demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the α3 integrin, to stabilize focal adhesions and activate integrin-linked kinase-1 and phospho Akt. We found that PA endothelial cells from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin, and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC.We show that APC is integral to PA endothelial cells adhesion and survival and is reduced in PA endothelial cells from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.
View details for DOI 10.1161/CIRCRESAHA.112.267849
View details for PubMedID 23011394
View details for PubMedCentralID PMC3821702
PAX Genes in Cancer; Friends or Foes?
Frontiers in genetics
2012; 3: 6-?
PAX genes have been shown to be critically required for the development of specific tissues and organs during embryogenesis. In addition, PAX genes are expressed in a handful of adult tissues where they are thought to play important roles, usually different from those in embryogenesis. A common theme in adult tissues is a requirement for PAX gene expression in adult stem cell maintenance or tissue regeneration. The connections between adult stem cell PAX gene expression and cancer are intriguing, and the literature is replete with examples of PAX gene expression in either situation. Here we systematically review the literature and present an overview of postnatal PAX gene expression in normal and cancerous tissue. We discuss the potential link between PAX gene expression in adult tissue and cancer. In addition, we discuss whether persistent PAX gene expression in cancer is favorable or unfavorable.
View details for DOI 10.3389/fgene.2012.00006
View details for PubMedID 22303411
View details for PubMedCentralID PMC3269002
PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein
2011; 30 (48): 4824-4834
The retinoblastoma protein (RB)-E2F1 pathway has a central role in regulating the cell cycle. Several PAX proteins (tissue-specific developmental regulators), including PAX8, interact with the RB protein, and thus regulate the cell cycle directly or indirectly. Here, we report that PAX8 expression is frequent in renal cell carcinoma, bladder, ovarian and thyroid cancer cell lines, and that silencing of PAX8 in cancer cell lines leads to a striking reduction in the expression of E2F1 and its target genes, as well as a proteasome-dependent destabilization of RB protein, with the RB1 mRNA level remaining unaffected. Cancer cells expressing PAX8 undergo a G(1)/S arrest and eventually senesce following PAX8 silencing. We demonstrate that PAX8 transcriptionally regulates the E2F1 promoter directly, and E2F1 transcription is enhanced after RB depletion. RB is recruited to the PAX8-binding site, and is involved in PAX8-mediated E2F1 transcription in cancer cells. Therefore, our results suggest that, in cancer, frequent and persistent expression of PAX8 is required for cell growth control through transcriptional activation of E2F1 expression and upregulation of the RB-E2F1 pathway.
View details for DOI 10.1038/onc.2011.190
View details for Web of Science ID 000298134700006
View details for PubMedID 21602887
PAX3 knockdown in metastatic melanoma cell lines does not reduce MITF expression.
PAX3 and MITF are important transcriptional activators in the melanocyte lineage and PAX3 is thought to control MITF expression during normal melanocyte differentiation. However, it is not clear whether this is still true in melanoma and whether the effects of knockdown of PAX3 on the inhibition of melanoma growth or survival are by its regulation of MITF. By western blot and quantitative real-time reverse transcription-PCR, we investigated the relationship between PAX3 and MITF expression in 27 metastatic melanoma and one immortalized melanocyte cell lines. All lines were found to express both PAX3 and MITF proteins but levels varied by 15 fold and more than 100 fold, respectively. The expression of PAX3 protein was correlated with that of MITF (r=0.75; P<0.001) but the expression of PAX3 protein and MITF mRNA was not. Immunofluorescence microscopy showed that individual cells expressed widely differing relative amounts of PAX3 and MITF protein. By MTT cell proliferation and flow cytometry assays, both MITF and PAX3 proteins seemed to be functional, as knockdown with siRNA led to reduced proliferation and induction of apoptosis. However, knockdown of PAX3 with small interfering RNA did not decrease MITF expression and vice versa. In one cell line (NZM15), silencing of PAX3 induced terminal differentiation whereas silencing of MITF induced expression of FOXD3, a repressor of melanogenesis. The results suggest that the melanoma lines used in this study show considerable phenotypic variation of expression of these two transcriptional activators and reflect a deregulation of the developmental process operating in the genesis of the melanocyte lineage, and that they probably function independently to enhance the survival of melanoma cells.
View details for DOI 10.1097/CMR.0b013e328341c7e0
View details for PubMedID 21164369