Embryonic Mouse Lung Research Articles

Rac1 modulates mammalian lung branching morphogenesis in part through canonical Wnt signaling

Lung branching morphogenesis relies on a number of factors, including proper epithelial cell proliferation and differentiation, cell polarity, and migration. Rac1, a small Rho GTPase, orchestrates a number of these cellular processes, including cell proliferation and differentiation, cellular alignment, and polarization. Furthermore, Rac1 modulates both noncanonical and canonical Wnt signaling, important pathways in lung branching morphogenesis. Culture of embryonic mouse lung explants in the presence of the Rac1 inhibitor (NSC23766) resulted in a dose-dependent decrease in branching. Increased cell death and BrdU uptake were notably seen in the mesenchyme, while no direct effect on the epithelium was observed. Moreover, vasculogenesis was impaired following Rac1 inhibition as shown by decreased Vegfa expression and impaired LacZ staining in Flk1-Lacz reporter mice. Rac1 inhibition decreased Fgf10 expression in conjunction with many of its associated factors. Moreover, using the reporter lines TOPGAL and Axin2-LacZ, there was an evident decrease in canonical Wnt signaling in the explants treated with the Rac1 inhibitor. Activation of canonical Wnt pathway using WNT3a or WNT7b only partially rescued the branching inhibition. Moreover, these results were validated on human explants, where Rac1 inhibition resulted in impaired branching and decreased AXIN2 and FGFR2b expression. We therefore conclude that Rac1 regulates lung branching morphogenesis, in part through canonical Wnt signaling. However, the exact mechanisms by which Rac1 interacts with canonical Wnt in human and mouse lung requires further investigation.

Loss of Lysyl Oxidase-like 3 Attenuates Embryonic Lung Development in Mice

Lysyl oxidase-like 3 (LOXL3), a human disease gene candidate, is a member of the lysyl oxidase (LOX) family and is indispensable for mouse palatogenesis and vertebral column development. Our previous study showed that the loss of LOXL3 resulted in a severe cleft palate and spinal deformity. In this study, we investigated a possible role for LOXL3 in mouse embryonic lung development. LOXL3-deficient mice displayed reduced lung volumes and weights, diminished saccular spaces, and deformed and smaller thoracic cavities. Excess elastic fibres were detected in LOXL3-deficient lungs, which might be related to the increased LOXL4 expression. Increased transforming growth factor β1 (TGFβ1) expression might be involved in the up-regulation of LOXL4 in LOXL3-deficient lungs. We concluded that the loss of LOXL3 attenuates mouse embryonic lung development.

Marsupial tammar wallaby delivers milk bioactives to altricial pouch young to support lung development.

Our research is exploiting the marsupial as a model to understand the signals required for lung development. Marsupials have a unique reproductive strategy, the mother gives birth to altricial neonate with an immature lung and the changes in milk composition during lactation in marsupials appears to provide bioactives that can regulate diverse aspects of lung development, including branching morphogenesis, cell proliferation and cell differentiation. These effects are seen with milk collected between 25 and 100 days postpartum. To better understand the temporal effects of milk composition on postnatal lung development we used a cross-fostering technique to restrict the tammar pouch young to milk composition not extending beyond day 25 for 45 days of its early postnatal life. These particular time points were selected as our previous study showed that milk protein collected prior to ~ day 25 had no developmental effect on mouse embryonic lungs in culture. The comparative analysis of the foster group and control young at day 45 postpartum demonstrated that foster pouch young had significantly reduced lung size. The lungs in fostered young were comprised of large intermediate tissue, had a reduced size of airway lumen and a higher percentage of parenchymal tissue. In addition, expression of marker genes for lung development (BMP4, WNT11, AQP-4, HOPX and SPB) were significantly reduced in lungs from fostered young. Further, to identify the potential bioactive expressed by mammary gland that may have developmental effect on pouch young lungs, we performed proteomics analysis on tammar milk through mass-spectrometry and listed the potential bioactives (PDGF, IGFBP5, IGFBPL1 and EGFL6) secreted in milk that may be involved in regulating pouch young lung development. The data suggest that postnatal lung development in the tammar young is most likely regulated by maternal signalling factors supplied through milk.

Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

SummaryThe RAS/MAPK-signalling pathway is frequently deregulated in non-small cell lung cancer (NSCLC), often through KRAS activating mutations1-3. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations4-7. We recently showed that advanced lung tumours from KrasG12D/+;p53-null mice frequently exhibit KrasG12D allelic enrichment (KrasG12D/Kraswild-type>1)7, implying that mutant Kras copy gains are positively selected during progression. Through a comprehensive analysis of mutant Kras homozygous and heterozygous MEFs and lung cancer cells we now show that these genotypes are phenotypically distinct. In particular, KrasG12D/G12D cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the TCA cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous NSCLC cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of KrasG12D copy gain), but not in the corresponding early tumours (KrasG12D heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprised of two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated based on their relative mutant allelic content. We also provide the first in vivo evidence of metabolic rewiring during lung cancer malignant progression.

SINCERA: A Pipeline for Single-Cell RNA-Seq Profiling Analysis.

A major challenge in developmental biology is to understand the genetic and cellular processes/programs driving organ formation and differentiation of the diverse cell types that comprise the embryo. While recent studies using single cell transcriptome analysis illustrate the power to measure and understand cellular heterogeneity in complex biological systems, processing large amounts of RNA-seq data from heterogeneous cell populations creates the need for readily accessible tools for the analysis of single-cell RNA-seq (scRNA-seq) profiles. The present study presents a generally applicable analytic pipeline (SINCERA: a computational pipeline for SINgle CEll RNA-seq profiling Analysis) for processing scRNA-seq data from a whole organ or sorted cells. The pipeline supports the analysis for: 1) the distinction and identification of major cell types; 2) the identification of cell type specific gene signatures; and 3) the determination of driving forces of given cell types. We applied this pipeline to the RNA-seq analysis of single cells isolated from embryonic mouse lung at E16.5. Through the pipeline analysis, we distinguished major cell types of fetal mouse lung, including epithelial, endothelial, smooth muscle, pericyte, and fibroblast-like cell types, and identified cell type specific gene signatures, bioprocesses, and key regulators. SINCERA is implemented in R, licensed under the GNU General Public License v3, and freely available from CCHMC PBGE website, https://research.cchmc.org/pbge/sincera.html.

Localized Smooth Muscle Differentiation Is Essential for Epithelial Bifurcation during Branching Morphogenesis of the Mammalian Lung

The airway epithelium develops into a tree-like structure via branching morphogenesis. Here, we show a critical role for localized differentiation of airway smooth muscle during epithelial bifurcation in the embryonic mouse lung. We found that during terminal bifurcation, changes in the geometry of nascent buds coincided with patterned smooth muscle differentiation. Evaluating spatiotemporal dynamics of α-smooth muscle actin (αSMA) in reporter mice revealed that αSMA-expressing cells appear at the basal surface of the future epithelial cleft prior to bifurcation and then increase in density as they wrap around the bifurcating bud. Disrupting this stereotyped pattern of smooth muscle differentiation prevents terminal bifurcation. Our results reveal stereotyped differentiation of airway smooth muscle adjacent to nascent epithelial buds and suggest that localized smooth muscle wrapping at the cleft site is required for terminal bifurcation during airway branching morphogenesis.

Pathogenesis of iridal coloboma in mice

Our research is exploiting the marsupial as a model to understand the signals required for lung development. Marsupials have a unique reproductive strategy, the mother gives birth to altricial neonate with an immature lung and the changes in milk composition during lactation in marsupials appears to provide bioactives that can regulate diverse aspects of lung development, including branching morphogenesis, cell proliferation and cell differentiation. These effects are seen with milk collected between 25 and 100 days postpartum. To better understand the temporal effects of milk composition on postnatal lung development we used a cross-fostering technique to restrict the tammar pouch young to milk composition not extending beyond day 25 for 45 days of its early postnatal life. These particular time points were selected as our previous study showed that milk protein collected prior to ~ day 25 had no developmental effect on mouse embryonic lungs in culture. The comparative analysis of the foster group and control young at day 45 postpartum demonstrated that foster pouch young had significantly reduced lung size. The lungs in fostered young were comprised of large intermediate tissue, had a reduced size of airway lumen and a higher percentage of parenchymal tissue. In addition, expression of marker genes for lung development (BMP4, WNT11, AQP-4, HOPX and SPB) were significantly reduced in lungs from fostered young. Further, to identify the potential bioactive expressed by mammary gland that may have developmental effect on pouch young lungs, we performed proteomics analysis on tammar milk through mass-spectrometry and listed the potential bioactives (PDGF, IGFBP5, IGFBPL1 and EGFL6) secreted in milk that may be involved in regulating pouch young lung development. The data suggest that postnatal lung development in the tammar young is most likely regulated by maternal signalling factors supplied through milk.

Impact of embryonic Fgf10 expression deficiency on embryonic mouse lung development and hyperoxia lung injury

Introduction: Bronchopulmonary dysplasia (BPD), a chronic lung disease of preterm infants, is characterized by impaired alveolar growth and pathologic vascularization. We investigate the impact of Fgf10 expression deficiency on embryonic lung development and hyperoxia lung injury (BPD mouse model).

Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice.

Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

A breath of fresh air on the mesenchyme: impact of impaired mesenchymal development on the pathogenesis of bronchopulmonary dysplasia.

The early mouse embryonic lung, with its robust and apparently reproducible branching pattern, has always fascinated developmental biologists. They have extensively used this embryonic organ to decipher the role of mammalian orthologs of Drosophila genes in controlling the process of branching morphogenesis. During the early pseudoglandular stage, the embryonic lung is formed mostly of tubes that keep on branching. As the branching takes place, progenitor cells located in niches are also amplified and progressively differentiate along the proximo-distal and dorso-ventral axes of the lung. Such elaborate processes require coordinated interactions between signaling molecules arising from and acting on four functional domains: the epithelium, the endothelium, the mesenchyme, and the mesothelium. These interactions, quite well characterized in a relatively simple lung tubular structure remain elusive in the successive developmental and postnatal phases of lung development. In particular, a better understanding of the process underlying the formation of secondary septa, key structural units characteristic of the alveologenesis phase, is still missing. This structure is critical for the formation of a mature lung as it allows the subdivision of saccules in the early neonatal lung into alveoli, thereby considerably expanding the respiratory surface. Interruption of alveologenesis in preterm neonates underlies the pathogenesis of chronic neonatal lung disease known as bronchopulmonary dysplasia. De novo formation of secondary septae appears also to be the limiting factor for lung regeneration in human patients with emphysema. In this review, we will therefore focus on what is known in terms of interactions between the different lung compartments and discuss the current understanding of mesenchymal cell lineage formation in the lung, focusing on secondary septae formation.

Role of marsupial tammar wallaby milk in lung maturation of pouch young.

BackgroundMarsupials such as the tammar wallaby (M.Eugenii) have a short gestation (29.3 days) and at birth the altricial young resembles a fetus, and the major development occurs postnatally while the young remains in the mother’s pouch. The essential functional factors for the maturation of the neonate are provided by the milk which changes in composition progressively throughout lactation (300 days). Morphologically the lungs of tammar pouch young are immature at birth and the majority of their development occurs during the first 100 days of lactation.ResultsIn this study mouse embryonic lungs (E-12) were cultured in media with tammar skim milk collected at key time points of lactation to identify factors involved in regulating postnatal lung maturation. Remarkably the embryonic lungs showed increased branching morphogenesis and this effect was restricted to milk collected at specific time points between approximately day 40 to 100 lactation. Further analysis to assess lung development showed a significant increase in the expression of marker genes Sp-C, Sp-B, Wnt-7b, BMP4 and Id2 in lung cultures incubated with milk collected at day 60. Similarly, day 60 milk specifically stimulated proliferation and elongation of lung mesenchymal cells that invaded matrigel. In addition, this milk stimulated proliferation of lung epithelium cells on matrigel, and the cells formed 3-dimensional acini with an extended lumen.ConclusionsThis study has clearly demonstrated that tammar wallaby milk collected at specific times in early lactation contains bioactives that may have a significant role in lung maturation of pouch young.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0063-z) contains supplementary material, which is available to authorized users.

Interactions between the otitis media gene, Fbxo11, and p53 in the mouse embryonic lung.

ABSTRACTOtitis media with effusion (OME) is the most common cause of hearing loss in children, and tympanostomy (ear tube insertion) to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of otitis media (OM) are known to have a very substantial genetic component; however, until recently, little was known of the underlying genes involved. The Jeff mouse mutant carries a mutation in the Fbxo11 gene, a member of the F-box family, and develops deafness due to a chronic proliferative OM. We previously reported that Fbxo11 is involved in the regulation of transforming growth factor beta (TGF-β) signalling by regulating the levels of phospho-Smad2 in the epithelial cells of palatal shelves, eyelids and airways of the lungs. It has been proposed that FBXO11 regulates the cell's response to TGF-β through the ubiquitination of CDT2. Additional substrates for FBXO11 have been identified, including p53. Here, we have studied both the genetic and biochemical interactions between FBXO11 and p53 in order to better understand the function of FBXO11 in epithelial development and its potential role in OM. In mice, we show that p53 (also known as Tp53) homozygous mutants and double heterozygous mutants (Jf/+ p53/+) exhibit similar epithelial developmental defects to Fbxo11 homozygotes. FBXO11 and p53 interact in the embryonic lung, and mutation in Fbxo11 prevents the interaction with p53. Both p53 and double mutants show raised levels of pSMAD2, recapitulating that seen in Fbxo11 homozygotes. Overall, our results support the conclusion that FBXO11 regulates the TGF-β pathway in the embryonic lung via cross-talk with p53.

Impact of embryonic Fgf10 expression deficiency on embryonic mouse lung development and repair following hyperoxia injury

s of the 51st Workshop for Pediatric Research 51st Workshop for Pediatric Research Gottingen, Germany

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung.

The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells.

An interplay of geometry and signaling enables robust lung branching morphogenesis.

Early branching events during lung development are stereotyped. Although key regulatory components have been defined, the branching mechanism remains elusive. We have now used a developmental series of 3D geometric datasets of mouse embryonic lungs as well as time-lapse movies of cultured lungs to obtain physiological geometries and displacement fields. We find that only a ligand-receptor-based Turing model in combination with a particular geometry effect that arises from the distinct expression domains of ligands and receptors successfully predicts the embryonic areas of outgrowth and supports robust branch outgrowth. The geometry effect alone does not support bifurcating outgrowth, while the Turing mechanism alone is not robust to noisy initial conditions. The negative feedback between the individual Turing modules formed by fibroblast growth factor 10 (FGF10) and sonic hedgehog (SHH) enlarges the parameter space for which the embryonic growth field is reproduced. We therefore propose that a signaling mechanism based on FGF10 and SHH directs outgrowth of the lung bud via a ligand-receptor-based Turing mechanism and a geometry effect.

Inhibition of β-catenin/p300 interaction proximalizes mouse embryonic lung epithelium.

BackgroundWnt/β-catenin signaling has been suggested to regulate proximal-distal determination of embryonic lung epithelium based upon genetically modified mouse models. The previously identified and characterized small molecule inhibitor IQ1 can pharmacologically decrease the interaction between β-catenin and its transcriptional coactivator p300, thereby enhancing the β-catenin/CBP interaction. Inhibition of the β-catenin/p300 interaction by IQ1 blocks the differentiation of embryonic stem cells and epicardial progenitor cells; however, whether differential coactivator usage by β-catenin plays a role in proximal-distal determination of lung epithelium is unknown.MethodsWe examined the effects of inhibiting the β-catenin/p300 interaction with IQ1 on lung branching morphogenesis in mouse embryos in utero and mouse embryonic lung organ culture ex vivo. The phenotype of IQ1 treated lungs was analyzed by epithelial staining, histology, quantitative PCR and in situ hybridization.ResultsInhibition of the β-catenin/p300 interaction by IQ1 disrupted the distal branching of mouse lung epithelium both in utero and ex vivo. IQ1 proximalized lung epithelium with decreased expression of the genes Bmp4 and Fgf10, hallmarks of distal lung determination, and increased expression of the proximal genes Sox2 and Scgb1a1 (CC10) as shown by quantitative PCR and in situ hybridization. The disruption of branching was reversible ex vivo as branching was reinitiated after removal of IQ1 from the media.ConclusionsThe results demonstrate that the β-catenin/p300 interaction plays a critical role in proximal-distal determination of the epithelium in mouse lung branching morphogenesis and β-catenin/p300 inhibition pharmacologically proximalizes lung epithelium.

Hmga2is required for canonical WNT signaling during lung development

BackgroundThe high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. Here we deciphered the role of the high-mobility-group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2-deficient mice (Hmga2 −/− ).ResultsWe found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2 −/− mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA-binding protein 6 and frizzled homolog 2. Analysis of siRNA-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of Hmga2 as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical WNT signaling in mediating the effect of Hmga2-loss-of-fuction. Finally, we found that HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression.ConclusionsOur results support that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium. Our results are the starting point for future studies investigating the relevance of Hmga2-mediated regulation of WNT signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury.

A wavelet-based method to exploit epigenomic language in the regulatory region

Epigenetic landscapes in the regulatory regions reflect binding condition of transcription factors and their co-factors. Identifying epigenetic condition and its variation is important in understanding condition-specific gene regulation. Computational approaches to explore complex multi-dimensional landscapes are needed. To study epigenomic condition for gene regulation, we developed a method, AWNFR, to classify epigenomic landscapes based on the detected epigenomic landscapes. Assuming mixture of Gaussians for a nucleosome, the proposed method captures the shape of histone modification and identifies potential regulatory regions in the wavelet domain. For accuracy estimation as well as enhanced computational speed, we developed a novel algorithm based on down-sampling operation and footprint in wavelet. We showed the algorithmic advantages of AWNFR using the simulated data. AWNFR identified regulatory regions more effectively and accurately than the previous approaches with the epigenome data in mouse embryonic stem cells and human lung fibroblast cells (IMR90). Based on the detected epigenomic landscapes, AWNFR classified epigenomic status and studied epigenomic codes. We studied co-occurring histone marks and showed that AWNFR captures the epigenomic variation across time. The source code and supplemental document of AWNFR are available at http://wonk.med.upenn.edu/AWNFR.

IL-1 Receptor Regulates microRNA-135b Expression in a Negative Feedback Mechanism during Cigarette Smoke–Induced Inflammation

Although microRNA-135b (miR-135b) is known to be associated with cancer, with recent work showing that it is massively induced in the pulmonary tissues of mice challenged with nanoparticles suggests a critical role for this microRNA in mediating inflammatory response. In this study, we investigated the expression and function of miR-135b in mice exposed to cigarette smoke or nontypeable Haemophilus influenzae (NTHi). Exposure to both cigarette smoke and NTHi elicited robust lung inflammation, but increased miR-135b expression was observed only in the lungs of cigarette smoke-exposed mice. Using IL-1R 1 knockout mice, we show that miR-135b expression is IL-1R1 dependent. A series of in vitro experiments confirmed the role of IL-1R1 in regulating miR-135b expression. In vitro activation of the IL-1R1 pathway in mouse embryonic fibroblast (NIH3T3) and lung epithelial (FE1) cells resulted in increased miR-135b, which was blocked by IL-1R1 antagonists or small interfering RNA-mediated silencing of IL-1R1 expression. Overexpression of mature miR-135b in NIH3T3 cells (pEGP-mmu-mir-135b) resulted in the suppression of endogenous levels of IL-1R1 expression. pEGP-mmu-miR-135b cells transiently transfected with luciferase reporter vector containing the 3'UTR of mouse IL-1R1 showed reduced luciferase activity. Finally, we demonstrate that miR-135b targets IL-1-stimulated activation of Caspase-1, the IL-1R1 downstream activator of IL-1β leading to suppressed synthesis of the active form of IL-1β protein. These results suggest that miR-135b expression during cigarette smoke-induced inflammation is regulated by IL-1R1 in a regulatory feedback mechanism to resolve inflammation.