Brg1/PRMT5 nuclear complex epigenetically regulates FOXO1 in IPF mesenchymal progenitor cells.

We have discovered intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the human idiopathic pulmonary fibrosis (IPF) lung. IPF MPCs display a durably distinct transcriptome, suggesting that they have undergone epigenetic modifications. Prior studies indicate that the chromatin remodeler Brg1 associates with the arginine methyltransferase PRMT5 to epigenetically regulate transcription factors. We hypothesize that a Brg1/PRMT5 nuclear complex epigenetically regulates critical nodes in IPF MPC self-renewal signaling networks. IPF and control MPCs were isolated from primary mesenchymal cell lines established from IPF and control patients. RNA-sequencing identified increased expression of the FOXO1 transcription factor in IPF MPCs compared with controls, a result we confirmed by Q-PCR and Western blot analysis. Immunoprecipitation identified a CD44/Brg1/PRMT5 nuclear complex in IPF MPCs. Chromatin immunoprecipitation assays showed that PRMT5 and its methylation mark H3R2me2 are enriched on the FOXO1 promoter. We show that loss of Brg1 and PRMT5 function decreases FOXO1 expression and impairs IPF MPC self-renewal, and that loss of FOXO1 function decreases IPF MPC self-renewal and expression of the SOX2 and OCT4 stemness markers. Our findings indicate that the FOXO1 gene is overexpressed in IPF MPCs in a CD44/Brg1/PRMT5 nuclear complex-dependent manner. Our data suggest that Brg1 alters chromatin accessibility, enriching PRMT5 occupancy on the FOXO1 promoter, and PRMT5 methylates histone H3 arginine 2 (H3R2) on the FOXO1 promoter, increasing its expression. Our data are in accord with the concept that this coordinated interplay is responsible for promoting IPF MPC self-renewal and maintaining a critical pool of fibrogenic MPCs that drive IPF progression.NEW & NOTEWORTHY Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

IL-8 concurrently promotes idiopathic pulmonary fibrosis mesenchymal progenitor cell senescence and PD-L1 expression enabling escape from immune cell surveillance.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease. We discovered fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that display cell-autonomous fibrogenicity and drive fibrotic progression. In a study of the IPF MPC nuclear proteome, we identified DNA damage as one of the most altered functions in IPF MPCs. In prior work we found that IL-8 drives IPF MPC self-renewal. IL-8 can promote replicative stress and DNA damage and induce senescence through the CXCR2 receptor. We hypothesized that IL-8 promotes DNA damage-mediated senescence in IPF MPCs. We show that IL-8 induces DNA damage and promotes IPF MPC senescence. We discovered that IL-8 concurrently promotes senescence and upregulation of the programmed death ligand 1 (PD-L1) in a CXCR2-dependent manner. Disruption of programmed cell death protein-1 (PD-1)-PD-L1 interaction promotes natural killer (NK) cell killing of IPF MPCs in vitro and arrests IPF MPC-mediated experimental lung fibrosis in vivo. Immunohistochemical (IHC) analysis of IPF lung tissue identified PD-L1-expressing IPF MPCs codistributing with NK cells and ß-galactosidase-positive cells. Our data indicate that IL-8 simultaneously promotes IPF MPC DNA damage-induced senescence and high PD-L1 expression, enabling IPF MPCs to elude immune cell-targeted removal. Disruption of PD-1-PD-L1 interaction may limit IPF MPC-mediated fibrotic progression.NEW & NOTEWORTHY Here we show that IL-8 concurrently promotes senescence and upregulation of PD-L1 in IPF MPCs. IHC analysis identifies the presence of senescent IPF MPCs intermingled with NK cells in the fibroblastic focus, suggesting that senescent MPCs elude immune cell surveillance. We demonstrate that disruption of PD-1/PD-L1 interaction promotes NK cell killing of IPF MPCs and arrests IPF MPC-mediated experimental lung fibrosis. Disruption of PD-1/PD-L1 interaction may be one means to limit fibrotic progression.

Hyaluronan/CD44 axis regulates S100A4-mediated mesenchymal progenitor cell fibrogenicity in idiopathic pulmonary fibrosis.

Despite modest improvement in patient outcomes from recent advances in pharmacotherapy targeting fibrogenic signaling pathways, idiopathic pulmonary fibrosis (IPF) remains a major unsolved clinical problem. One reason for this is that available antifibrotic agents slow down but do not arrest fibrotic progression. To arrest fibrotic progression, its obligatory drivers need to be identified. We previously discovered that fibrogenic mesenchymal progenitor cells (MPCs) are key drivers of fibrotic progression in IPF, serving as cells of origin for disease-mediating myofibroblasts. IPF MPCs have high levels of nuclear S100A4, which interacts with the proteasome to promote p53 degradation and self-renewal. However, the mechanism underlying S100A4 accumulation in the nucleus of IPF MPCs remains unknown. Here we show that hyaluronan (HA) is present in the fibroblastic focus together with CD44-expressing MPCs and that ligation of CD44 by HA triggers S100A4 nuclear translocation to support IPF MPC self-renewal. The mechanism involves HA-mediated formation of a CD44/S100A4/transportin 1 complex, which promotes S100A4 nuclear import. In a humanized mouse model of pulmonary fibrosis, IPF MPC fibrogenicity was significantly attenuated by 1) knockdown of CD44 or 2) introduction of an S100A4 mutant construct that prevents S100A4 nuclear import. These data indicate that signaling through the HA/CD44/S100A4 axis is an integral component of IPF MPC fibrogenicity.

Distinct Cancer-Promoting Stromal Gene Expression Depending on Lung Function.

Rationale: Chronic obstructive pulmonary disease is an independent risk factor for lung cancer, but the underlying molecular mechanisms are unknown. We hypothesized that lung stromal cells activate pathological gene expression programs that support oncogenesis.Objectives: To identify molecular mechanisms operating in the lung stroma that support the development of lung cancer.Methods: The study included subjects with and without lung cancer across a spectrum of lung-function values. We conducted a multiomics analysis of nonmalignant lung tissue to quantify the transcriptome, translatome, and proteome.Measurements and Main Results: Cancer-associated gene expression changes predominantly manifested as alterations in the efficiency of mRNA translation modulating protein levels in the absence of corresponding changes in mRNA levels. The molecular mechanisms that drove these cancer-associated translation programs differed based on lung function. In subjects with normal to mildly impaired lung function, the mammalian target of rapamycin (mTOR) pathway served as an upstream driver, whereas in subjects with severe airflow obstruction, pathways downstream of pathological extracellular matrix emerged. Consistent with a role during cancer initiation, both the mTOR and extracellular matrix gene expression programs paralleled the activation of previously identified procancer secretomes. Furthermore, an in situ examination of lung tissue showed that stromal fibroblasts expressed cancer-associated proteins from two procancer secretomes: one that included IL-6 (in cases of mild or no airflow obstruction), and one that included BMP1 (in cases of severe airflow obstruction).Conclusions: Two distinct stromal gene expression programs that promote cancer initiation are activated in patients with lung cancer depending on lung function. Our work has implications both for screening strategies and for personalized approaches to cancer treatment.

Dicer1 Deficiency in the Idiopathic Pulmonary Fibrosis Fibroblastic Focus Promotes Fibrosis by Suppressing MicroRNA Biogenesis.

RATIONALE: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore, we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. OBJECTIVES: To determine how IPF-ECM negatively regulates miR-29. METHODS: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNA-binding protein assays, and xenograft models. MEASUREMENTS AND MAIN RESULTS: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing of miR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. CONCLUSIONS: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.

IL-8 mediates idiopathic pulmonary fibrosis mesenchymal progenitor cell fibrogenicity.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease, but the mechanisms driving progression remain incompletely defined. We previously reported that the IPF lung harbors fibrogenic mesenchymal progenitor cells (MPCs), which serve as a cell of origin for IPF fibroblasts. Proliferating IPF MPCs are located at the periphery of fibroblastic foci in an active cellular front at the interface between the myofibroblast-rich focus core and adjacent normal alveolar structures. Among a large set of genes that distinguish IPF MPCs from their control counterparts, we identified IL-8 as a candidate mediator of IPF MPC fibrogenicity and driver of fibrotic progression. IPF MPCs and their progeny displayed increased steady-state levels of IL-8 and its cognate receptor CXCR1 and secreted more IL-8 than did controls. IL-8 functioned in an autocrine manner promoting IPF MPC self-renewal and the proliferation and motility of IPF MPC progeny. Secreted IL-8 also functioned in a paracrine manner stimulating macrophage migration. Analysis of IPF lung tissue demonstrated codistribution of IPF MPCs with activated macrophages in the active cellular front of the fibroblastic focus. These findings indicate that IPF MPC-derived IL-8 is capable of expanding the mesenchymal cell population and recruiting activated macrophages cells to actively evolving fibrotic lesions.

Multi-omic molecular profiling of lung cancer in COPD.

Chronic obstructive pulmonary disease (COPD) is a known risk factor for developing lung cancer but the underlying mechanisms remain unknown. We hypothesise that the COPD stroma contains molecular mechanisms supporting tumourigenesis.We conducted an unbiased multi-omic analysis to identify gene expression patterns that distinguish COPD stroma in patients with or without lung cancer. We obtained lung tissue from patients with COPD and lung cancer (tumour and adjacent non-malignant tissue) and those with COPD without lung cancer for profiling of proteomic and mRNA (both cytoplasmic and polyribosomal). We used the Joint and Individual Variation Explained (JIVE) method to integrate and analyse across the three datasets.JIVE identified eight latent patterns that robustly distinguished and separated the three groups of tissue samples (tumour, adjacent and control). Predictive variables that associated with the tumour, compared to adjacent stroma, were mainly represented in the transcriptomic data, whereas predictive variables associated with adjacent tissue, compared to controls, were represented at the translatomic level. Pathway analysis revealed extracellular matrix and phosphatidylinositol-4,5-bisphosphate 3-kinase-protein kinase B signalling pathways as important signals in the tumour adjacent stroma.The multi-omic approach distinguishes tumour adjacent stroma in lung cancer and reveals two stromal expression patterns associated with cancer.

eIF4E-mediated translational control of cancer incidence.

Mitogen activated translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when deregulated and overexpressed. It remains unknown, how activated eIF4E directs such distinct biological outputs. Our experimental data provide evidence that distinct threshold levels of eIF4E govern its biological output in lactating mammary glands and that eIF4E overexpression in the context of cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints caused by hyperproliferative oncogenic stimuli. These findings point at the cellular level of eIF4E as an important sensor for normal or pro-neoplastic propagation of cells. Here, we describe a model that links the pro-neoplastic function of eIF4F to its ability to disable oncogene-activated tumor surveillance programs; and propose a novel therapeutic strategy for cancer prevention based upon targeting aberrant eIF4E with safe doses of small-molecule antagonists to ensure the maintenance of eIF4E levels below the pro-neoplastic threshold. This article is part of a Special Issue entitled: Translation and Cancer.

Proteome Profiling in Lung Injury after Hematopoietic Stem Cell Transplantation.

Pulmonary complications due to infection and idiopathic pneumonia syndrome (IPS), a noninfectious lung injury in hematopoietic stem cell transplant (HSCT) recipients, are frequent causes of transplantation-related mortality and morbidity. Our objective was to characterize the global bronchoalveolar lavage fluid (BALF) protein expression of IPS to identify proteins and pathways that differentiate IPS from infectious lung injury after HSCT. We studied 30 BALF samples from patients who developed lung injury within 180 days of HSCT or cellular therapy transfusion (natural killer cell transfusion). Adult subjects were classified as having IPS or infectious lung injury by the criteria outlined in the 2011 American Thoracic Society statement. BALF was depleted of hemoglobin and 14 high-abundance proteins, treated with trypsin, and labeled with isobaric tagging for relative and absolute quantification (iTRAQ) 8-plex reagent for two-dimensional capillary liquid chromatography (LC) and data dependent peptide tandem mass spectrometry (MS) on an Orbitrap Velos system in higher-energy collision-induced dissociation activation mode. Protein identification employed a target-decoy strategy using ProteinPilot within Galaxy P. The relative protein abundance was determined with reference to a global internal standard consisting of pooled BALF from patients with respiratory failure and no history of HSCT. A variance weighted t-test controlling for a false discovery rate of ≤5% was used to identify proteins that showed differential expression between IPS and infectious lung injury. The biological relevance of these proteins was determined by using gene ontology enrichment analysis and Ingenuity Pathway Analysis. We characterized 12 IPS and 18 infectious lung injury BALF samples. In the 5 iTRAQ LC-MS/MS experiments 845, 735, 532, 615, and 594 proteins were identified for a total of 1125 unique proteins and 368 common proteins across all 5 LC-MS/MS experiments. When comparing IPS to infectious lung injury, 96 proteins were differentially expressed. Gene ontology enrichment analysis showed that these proteins participate in biological processes involved in the development of lung injury after HSCT. These include acute phase response signaling, complement system, coagulation system, liver X receptor (LXR)/retinoid X receptor (RXR), and farsenoid X receptor (FXR)/RXR modulation. We identified 2 canonical pathways modulated by TNF-α, FXR/RXR activation, and IL2 signaling in macrophages. The proteins also mapped to blood coagulation, fibrinolysis, and wound healing-processes that participate in organ repair. Cell movement was identified as significantly over-represented by proteins with differential expression between IPS and infection. In conclusion, the BALF protein expression in IPS differed significantly from infectious lung injury in HSCT recipients. These differences provide insights into mechanisms that are activated in lung injury in HSCT recipients and suggest potential therapeutic targets to augment lung repair.

Reemergence of hedgehog mediates epithelial-mesenchymal crosstalk in pulmonary fibrosis.

Hedgehog signaling plays important roles in cell development and differentiation. In this study, the ability of Sonic Hedgehog (SHH) to induce myofibroblast differentiation was analyzed in isolated human lung fibroblasts, and its in vivo significance was evaluated in rodent bleomycin-induced pulmonary fibrosis. The results showed that SHH could induce myofibroblast differentiation in human lung fibroblasts in a Smo- and Gli1-dependent manner. Gel shift analysis, chromatin immunoprecipitation assay, and site-directed mutagenesis revealed that a Gli1 binding consensus in the α-SMA gene promoter was important for mediating SHH-induced myofibroblast differentiation. Analysis of Hedgehog reemergence in vivo revealed that of all three Hedgehog isoforms, only SHH was significantly induced in bleomycin-injured lung along with Gli1. The induction of SHH was only noted in epithelial cells, and its expression was undetectable in lung fibroblasts or macrophages. transforming growth factor (TGF)-ß induced SHH significantly in cultured alveolar epithelial cells, whereas SHH induced TGF-ß in lung fibroblasts. Pulmonary fibrosis and α-smooth muscle actin (α-SMA) expression were significantly reduced in mice that were Smo deficient only in type I collagen-expressing cells. Thus, the reemergence of SHH in epithelial cells could result in induction of myofibroblast differentiation in a Smo-dependent manner and subsequent Gli1 activation of the α-SMA promoter.

Pathologic Regulation of Collagen I by an Aberrant Protein Phosphatase 2A/Histone Deacetylase C4/MicroRNA-29 Signal Axis in Idiopathic Pulmonary Fibrosis Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is characterized by the relentless expansion of fibroblasts depositing type I collagen within the alveolar wall and obliterating the alveolar airspace. MicroRNA (miR)-29 is a potent regulator of collagen expression. In IPF, miR-29 levels are low, whereas type I collagen expression is high. However, the mechanism for suppression of miR-29 and increased type I collagen expression in IPF remains unclear. Here we show that when IPF fibroblasts are seeded on polymerized type I collagen, miR-29c levels are suppressed and type I collagen expression is high. In contrast, miR-29c is high and type I collagen expression is low in control fibroblasts. We demonstrate that the mechanism for suppression of miR-29 during IPF fibroblast interaction with polymerized collagen involves inappropriately low protein phosphatase (PP) 2A function, leading to histone deacetylase (HDA) C4 phosphorylation and decreased nuclear translocation of HDAC4. We demonstrate that overexpression of HDAC4 in IPF fibroblasts restored miR-29c levels and decreased type I collagen expression, whereas knocking down HDAC4 in control fibroblasts suppressed miR-29c levels and increased type I collagen expression. Our data indicate that IPF fibroblast interaction with polymerized type I collagen results in an aberrant PP2A/HDAC4 axis, which suppresses miR-29, causing a pathologic increase in type I collagen expression.

Double-stranded RNA-dependent protein kinase deficiency protects the heart from systolic overload-induced congestive heart failure.

BACKGROUND: Double-stranded RNA-dependent protein kinase (PKR) is a eukaryotic initiation factor 2α kinase that inhibits mRNA translation under stress conditions. PKR also mediates inflammatory and apoptotic signaling independently of translational regulation. Congestive heart failure is associated with cardiomyocyte hypertrophy, inflammation, and apoptosis, but the role of PKR in left ventricular hypertrophy and the development of congestive heart failure has not been examined. METHODS AND RESULTS: We observed increased myocardial PKR expression and translocation of PKR into the nucleus in humans and mice with congestive heart failure. To determine the impact of PKR on the development of congestive heart failure, PKR knockout and wild-type mice were exposed to pressure overload produced by transverse aortic constriction. Although heart size increased similarly in wild-type and PKR knockout mice after transverse aortic constriction, PKR knockout mice exhibited very little pulmonary congestion, well-preserved left ventricular ejection fraction and contractility, and significantly less myocardial fibrosis compared with wild-type mice. Bone marrow-derived cells from wild-type mice did not abolish the cardiac protective effect observed in PKR knockout mice, whereas bone marrow-derived cells from PKR knockout mice had no cardiac protective effect in wild-type mice. Mechanistically, PKR knockout attenuated transverse aortic constriction-induced tumor necrosis factor-α expression and leukocyte infiltration and lowered cardiac expression of proapoptotic factors (Bax and caspase-3), so that PKR knockout hearts were more resistant to transverse aortic constriction-induced cardiomyocyte apoptosis. PKR depletion in isolated cardiomyocytes also conferred protection against tumor necrosis factor-α- or lipopolysaccharide-induced apoptosis. CONCLUSION: PKR is a maladaptive factor upregulated in hemodynamic overload that contributes to myocardial inflammation, cardiomyocyte apoptosis, and the development of congestive heart failure.

Identification of a cell-of-origin for fibroblasts comprising the fibrotic reticulum in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the middle aged and elderly with a prevalence of one million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli, creating a reticular network that leads to death by asphyxiation. Lung fibroblasts from patients with IPF have phenotypic hallmarks, distinguishing them from their normal counterparts: pathologically activated Akt signaling axis, increased collagen and α-smooth muscle actin expression, distinct gene expression profile, and ability to form fibrotic lesions in model organisms. Despite the centrality of these fibroblasts in disease pathogenesis, their origin remains uncertain. Here, we report the identification of cells in the lungs of patients with IPF with the properties of mesenchymal progenitors. In contrast to progenitors isolated from nonfibrotic lungs, IPF mesenchymal progenitor cells produce daughter cells manifesting the full spectrum of IPF hallmarks, including the ability to form fibrotic lesions in zebrafish embryos and mouse lungs, and a transcriptional profile reflecting these properties. Morphological analysis of IPF lung tissue revealed that mesenchymal progenitor cells and cells with the characteristics of their progeny comprised the fibrotic reticulum. These data establish that the lungs of patients with IPF contain pathological mesenchymal progenitor cells that are cells of origin for fibrosis-mediating fibroblasts. These fibrogenic mesenchymal progenitors and their progeny represent an unexplored target for novel therapies to interdict fibrosis.

Future directions in idiopathic pulmonary fibrosis research. An NHLBI workshop report.

The median survival of patients with idiopathic pulmonary fibrosis (IPF) continues to be approximately 3 years from the time of diagnosis, underscoring the lack of effective medical therapies for this disease. In the United States alone, approximately 40,000 patients die of this disease annually. In November 2012, the NHLBI held a workshop aimed at coordinating research efforts and accelerating the development of IPF therapies. Basic, translational, and clinical researchers gathered with representatives from the NHLBI, patient advocacy groups, pharmaceutical companies, and the U.S. Food and Drug Administration to review the current state of IPF research and identify priority areas, opportunities for collaborations, and directions for future research. The workshop was organized into groups that were tasked with assessing and making recommendations to promote progress in one of the following six critical areas of research: (1) biology of alveolar epithelial injury and aberrant repair; (2) role of extracellular matrix; (3) preclinical modeling; (4) role of inflammation and immunity; (5) genetic, epigenetic, and environmental determinants; (6) translation of discoveries into diagnostics and therapeutics. The workshop recommendations provide a basis for directing future research and strategic planning by scientific, professional, and patient communities and the NHLBI.

miR-210 promotes IPF fibroblast proliferation in response to hypoxia.

Idiopathic pulmonary fibrosis (IPF) is characterized by the relentless spread of fibroblasts from scarred alveoli into adjacent alveolar units, resulting in progressive hypoxia and death by asphyxiation. Although hypoxia is a prominent clinical feature of IPF, the role of hypoxia as a driver of the progressive fibrotic nature of the disease has not been explored. Here, we demonstrate that hypoxia robustly stimulates the proliferation of IPF fibroblasts. We found that miR-210 expression markedly increases in IPF fibroblasts in response to hypoxia and that knockdown of miR-210 decreases hypoxia-induced IPF fibroblast proliferation. Silencing hypoxia-inducible factor (HIF)-2α inhibits the hypoxia-mediated increase in miR-210 expression and blocks IPF fibroblast proliferation, indicating that HIF-2α is upstream of miR-210. We demonstrate that the miR-210 downstream target MNT is repressed in hypoxic IPF fibroblasts and that knockdown of miR-210 increases MNT expression. Overexpression of MNT inhibits hypoxia-induced IPF fibroblast proliferation. Together, these data indicate that hypoxia potently stimulates miR-210 expression via HIF-2α, and high miR-210 expression drives fibroblast proliferation by repressing the c-myc inhibitor, MNT. In situ analysis of IPF lung tissue demonstrates miR-210 expression in a similar distribution with HIF-2α and the hypoxic marker carbonic anhydrase-IX in cells within the IPF fibrotic reticulum. Our results raise the possibility that a pathological feed-forward loop exists in the IPF lung, in which hypoxia promotes IPF fibroblast proliferation via stimulation of miR-210 expression, which in turn worsens hypoxia.

Regulation of myofibroblast differentiation by poly(ADP-ribose) polymerase 1.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational protein modification effected by enzymes belonging to the poly(ADP-ribose) polymerase (PARP) superfamily, mainly by PARP-1. The key acceptors of poly(ADP-ribose) include PARP-1 itself, histones, DNA repair proteins, and transcription factors. Because many of these factors are involved in the regulation of myofibroblast differentiation, we examined the role of PARylation on myofibroblast differentiation. Overexpression of PARP-1 with an expression plasmid activated expression of the α-SMA gene (Acta2), a marker of myofibroblast differentiation in lung fibroblasts. Suppression of PARP-1 activity or gene expression with PARP-1 inhibitors or siRNA, respectively, had the opposite effect on these cells. PARP-1-deficient cells also had reduced α-SMA gene expression. DNA pyrosequencing identified hypermethylated regions of the α-SMA gene in PARP-1-deficient cells, relative to wild-type cells. Interestingly, and of potential relevance to human idiopathic pulmonary fibrosis, PARP activity in lung fibroblasts isolated from idiopathic pulmonary fibrosis patients was significantly higher than that in cells isolated from control subjects. Furthermore, PARP-1-deficient mice exhibited reduced pulmonary fibrosis in response to bleomycin-induced lung injury, relative to wild-type controls. These results suggest that PARylation is important for myofibroblast differentiation and the pathogenesis of pulmonary fibrosis.

Small-molecule inhibition of oncogenic eukaryotic protein translation in mesothelioma cells.

Deranged cap-mediated translation is implicated in the genesis, maintenance and progression of many human cancers including mesothelioma. In this study, disrupting the eIF4F complex by antagonizing the eIF4E-mRNA-cap interaction is assessed as a therapy for mesothelioma. Mesothelioma cells were treated with 4Ei-1, a membrane permeable prodrug that when converted to the active drug, 7-benzyl guanosine monophosphate (7Bn-GMP) displaces capped mRNAs from the eIF4F complex. Colony formation was measured in mesothelioma treated with 4Ei-1 alone or combined with pemetrexed. Proliferation was examined in cells treated with 4Ei-1. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation in lysates exposed to 4Ei-1. 4Ei-1 treatment resulted in a dose dependent decrease in colony formation and cell viability. Combination therapy of 4Ei-1 with pemetrexed further reduced colony number. Formation of eIF4F cap-complex decreased in response to 4Ei-1 exposure. 4Ei-1 is a novel prodrug that reduces proliferation, represses colony formation, diminishes association of eIF4F with the mRNA cap, and sensitizes mesothelioma cells to pemetrexed.

Non-ATG-initiated translation directed by microsatellite expansions.

Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.

Telomerase and telomere length in pulmonary fibrosis.

In addition to its expression in stem cells and many cancers, telomerase activity is transiently induced in murine bleomycin (BLM)-induced pulmonary fibrosis with increased levels of telomerase transcriptase (TERT) expression, which is essential for fibrosis. To extend these observations to human chronic fibrotic lung disease, we investigated the expression of telomerase activity in lung fibroblasts from patients with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). The results showed that telomerase activity was induced in more than 66% of IPF lung fibroblast samples, in comparison with less than 29% from control samples, some of which were obtained from lung cancer resections. Less than 4% of the human IPF lung fibroblast samples exhibited shortened telomeres, whereas less than 6% of peripheral blood leukocyte samples from patients with IPF or hypersensitivity pneumonitis demonstrated shortened telomeres. Moreover, shortened telomeres in late-generation telomerase RNA component knockout mice did not exert a significant effect on BLM-induced pulmonary fibrosis. In contrast, TERT knockout mice exhibited deficient fibrosis that was independent of telomere length. Finally, TERT expression was up-regulated by a histone deacetylase inhibitor, while the induction of TERT in lung fibroblasts was associated with the binding of acetylated histone H3K9 to the TERT promoter region. These findings indicate that significant telomerase induction was evident in fibroblasts from fibrotic murine lungs and a majority of IPF lung samples, whereas telomere shortening was not a common finding in the human blood and lung fibroblast samples. Notably, the animal studies indicated that the pathogenesis of pulmonary fibrosis was independent of telomere length.