JNK suppresses pulmonary fibroblast elastogenesis during alveolar development
© Liu et al.; licensee BioMed Central Ltd. 2014
Received: 18 November 2013
Accepted: 7 March 2014
Published: 25 March 2014
The formation of discrete elastin bands at the tips of secondary alveolar septa is important for normal alveolar development, but the mechanisms regulating the lung elastogenic program are incompletely understood. JNK suppress elastin synthesis in the aorta and is important in a host of developmental processes. We sought to determine whether JNK suppresses pulmonary fibroblast elastogenesis during lung development.
Alveolar size, elastin content, and mRNA of elastin-associated genes were quantitated in wild type and JNK-deficient mouse lungs, and expression profiles were validated in primary lung fibroblasts. Tropoelastin protein was quantitated by Western blot. Changes in lung JNK activity throughout development were quantitated, and pJNK was localized by confocal imaging and lineage tracing.
By morphometry, alveolar diameters were increased by 7% and lung elastin content increased 2-fold in JNK-deficient mouse lungs compared to wild type. By Western blot, tropoelastin protein was increased 5-fold in JNK-deficient lungs. Postnatal day 14 (PND14) lung JNK activity was 11-fold higher and pJNK:JNK ratio 6-fold higher compared to PN 8 week lung. Lung tropoelastin, emilin-1, fibrillin-1, fibulin-5, and lysyl oxidase mRNAs inversely correlated with lung JNK activity during alveolar development. Phosphorylated JNK localized to pulmonary lipofibroblasts. PND14 JNK-deficient mouse lungs contained 7-fold more tropoelastin, 2,000-fold more emilin-1, 800-fold more fibrillin-1, and 60-fold more fibulin-5 than PND14 wild type lungs. Primarily lung fibroblasts from wild type and JNK-deficient mice showed similar differences in elastogenic mRNAs.
JNK suppresses fibroblast elastogenesis during the alveolar stage of lung development.
KeywordsLung development Elastin c-terminal Jun kinase Rho kinase
Beginning at approximately 35 weeks gestation and continuing for several years postnatally , large saccular air spaces in the lung are progressively divided into smaller ones by secondary alveolar septa leading to a twenty-fold increase in alveolar surface area . This exponential increase in gas-exchange capacity is essential to meeting the bioenergetic needs of ex utero growth and development. Formation of a low-compliance elastin band at the leading edge of a growing secondary alveolar septal ridge likely plays critical mechano-developmental role in alveolar septation [3–6].
Elastin is necessary for structure and function of both vascular tissues and epithelial-containing organs. It is critical to the hysteretic properties of both arteries  and lung  and to the elasticity of other epithelial organs such as skin and intestine. In infants with impaired alveolar development  and in animal models of the same [10–12], elastin fiber density and organization is disrupted. Regulators of elastogenesis (formation of elastin fibers) such as transforming growth factor-β , fibroblast growth factor-2 , epidermal growth factor , and insulin like growth factor-1  are well described. With regards to signaling in the fibroblast, pro-elastogenic cytokines generally activate SMAD-signaling pathways and anti-elastogenic activate mitogen-activated protein kinases 1%3 (erk 2%1) . The spatial-temporal regulation of pro- and anti-elastogenic signals is likely of critical importance since the formation of discrete elastin bands is critical to both alveolar and vascular development, and elastin experiences virtually no turnover with a half live of 74 years  despite expression of pro-elastogenic cytokines throughout life . In the arterial vasculature, JNK signaling has been shown to suppress elastogenesis , but no such role has been demonstrated in the lung. We hypothesized that activation of JNK suppresses elastogenesis during alveolar lung development contributing to the localization of elastin to alveolar septal tips. To test this hypothesis, we assessed JNK activation during lung development, localized phosphorylated JNK by confocal microscopy, quantitated alveolar development and elastin localization in JNK deficient mice, and quantitated mRNA levels of elastin-associated genes in JNK deficient mouse lungs and primary fibroblasts.
C57BL/6J wild-type, B6.Mapk8 tm1Flv /J (JNK1 −/− ), B6.Twist2 tm1.1(cre)Dor /J(Dermo-1-cre), and B6.Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J(tomato) mice were obtained from Jackson Laboratories. Previously described mice with a floxed JNK1 allele (Mapk8 tm2Flv /J, referred to as JNK1 LoxP/LoxP ) on a B6.Mapk9tm1FlvMapk10tm1Flv/J background (hereafter referred to as JNK2%3 −/− ) were utilized with permission from Roger Davis at the University of Massachusetts . Since JNK3 is expressed only in the heart, testis and brain , single JNK2 or JNK3 knockout animals were not utilized. All animals were housed in a barrier facility with purified air and provided purified water and autoclaved food ad libitum. Animal use was approved by the Cincinnati Children’s Hospital Medical Center Animal Use and Care Committee.
To localize lung JNK activity, we utilized a mesenchymal cell lineage tracing strategy with immunofluorescent staining of pJNK. Dermo1 (a.k.a. twist2) is mesenchymal transcription factor expressed as early as E15.5 . The “tomato” mouse constitutively expresses a membrane-bound td-tomato except in the presence of cre in which recombination excises td-tomato and eGFP is expressed. Crossing Dermo1-cre mice with “tomato” mice lineage traces lung mesenchymal cells with eGFP.
Genotyping primers used
Common forward primer
Lung tissue processing and analysis
Tissue procurement and processing
Mice were sacrificed by intraperitoneal injection of ketamine, xylazine, and acepromazine (100, 6, and 2 mg/kg respectively) and severing of the left renal artery. After exsanguination, the trachea was cannulated with a 22 gauge blunt tip needle and lungs isovolumetrically inflated with 4% paraformaldehyde in PBS at a pressure of 25 cm H2O. Lung inflation was maintained by securing a silk ligature around the trachea, and then the chest was opened. The lungs were removed and then fixed overnight at 4°C. After fixation, the lung lobes were removed from the bronchi and dehydrated by serial passage into 70% ethanol and paraffinized. Five μm sections obtained at random angles through all five lobes.
Lung elastin staining and quantification
Lung sections were stained for elastin using the Hart’s Staining method (PolyScientific) and elastin quantitated by previously published methods . Using a Zeiss Axio Imager A.2 three 20X images were obtained from each lobe yielding fifteen images per mouse. Using MatLab (MatWorks) these images were then separated into four components by cluster analysis after defining a color spectrum and intensity values for dense elastin (arterial walls and elastin bands at septal tips), diffuse elastin (elastin in alveolar walls and bases), non-elastin tissue, and airspace. Pixel quantitation for each component was performed for each image and average values per animal used for statistical analysis. For quantitation, the fraction of each component of total tissue (dense elastin + diffuse elastin + non-elastin tissue) was utilized.
By defining secondary alveolar crests as invaginations into distal lung airspace by tissues associated with an elastin bundle, numbers of secondary alveolar septal crests were counted per 20X field.
Dermo1-cre-tomato mice were sacrificed at PND5 and the lungs immunostained for pJNK using an AF647 secondary antibody. Confocal images were obtained using a Nikon AR1si inverted microscope.
Pulmonary fibroblast isolation
Pulmonary fibroblasts were isolated from PND14 mice by previously described methods which yield >85% fibroblast purity [30, 31]. Briefly, PND14 mice were sacrificed and their lungs inflated by gently instilling dispase (BD Biosciences) via the trachea and then plugging the trachea with 1% low melting point agarose. The lungs were incubated in dispase at 25°C for 45 minutes and lung tissue teased from bronchi and large vessels using sterile forceps. Dispase was neutralized using DMEM with 10% FBS and the fibroblasts were allowed to adhere in a 100 cm2 tissue culture dish for 1 hour at 37°C and the plate rinsed with PBS. The fibroblasts were allowed to grow in DMEM with 10% FBS and 1% Penicillin/Streptomycin and used at passage 3.
In vitro Cre-recombinase experiments
JNK2%3 −/− (which also has floxed JNK1 alleles) pulmonary fibroblasts were infected at 50% confluence with 106 plaque forming units of replication-deficient adenovirus expressing GFP or Cre (Vector BioLabs). Efficacy was assessed by Taqman qPCR for JNK1 mRNA (Applied Biosystems, proprietary primers). RNA and cell culture media was collected at 48 hours.
JNK activity, protein, RNA quantification
JNK activity assays
For in vivo experiments, lung homogenate c-Jun phosphorylation was quantitated to measure JNK activity. To do so, JNK was immunoprecipitated from lung homogenates using an isoform nonspecific JNK antibody (Santa Cruz) conjugated to agarose beads. Precipitated JNK was incubated with GST-labeled c-Jun (Michal Karin, University of California at San Diego)  and P-32 adenosine triphosphate, the kinase solution was separated on a polyacrylamide gel, transferred to a PVDF membrane, and radiolabeled c-Jun quantitated using a Storm 860 phosphorimager.
Lungs from mice of at least two different litters were snap frozen and homogenized in RIPA buffer with protease inhibitor cocktail (Sigma) using a Qiagen TissueLyser II. Protein content was determined and the homogenates were electrophoretically separated and transferred to PVDF membrane. When not frozen, lysates were kept on ice until electrophoresis. Western blot for pJNK and JNK (both isoform non-specific, 1:200 dilution, Santa Cruz) and tropoelastin (1:2000 dilution, Elastin Products Company) was performed and chemiluminisence detected using a General Electric LAS3000.
Lungs from mice aged E15.5 to 8 weeks were snap frozen and then homogenized in RLT buffer using a Qiagen TissueLyser II. For primary lung fibroblasts, passage two cells were seeded at 50% density and collected at 48 hours (with cells achieving 100% density). Tropoelastin, emilin-1, fibrillin-1, lysyl oxidase, fibulin-5, surfactant protein B, CD31, and GAPDH mRNA was quantitated by Taqman PCR (Applied Biosystems proprietary primers).
Soluble elastin quantification
Pulmonary fibroblasts were cultured for 48-hours in DMEM, 10% FBS, 1% penicillin/streptomycin and the media analyzed for elastin content using the Fastin kit (Biocolor, UK) per manufacturer instructions.
Statistical comparisons between groups were performed using the Student two-tail t-test or one-way ANOVA using the Holm-Sidak method. p-values of <0.05 were considered significant.
JNK-deficient mice have enlargement of distal lung airspaces
JNK reduces lung elastin content
JNK activity Is increased during postnatal lung development
JNK activity is localized to pulmonary lipofibroblasts during alveolar development
JNK suppresses pulmonary fibroblast elastogenesis
JNK suppresses mRNAs of elastin-associated genes
For the first time, we have demonstrated a role for JNK in suppressing the elastogenic program during the alveolar stage of lung development. JNK activity increased during late alveolar development, and this increased JNK activity was negatively correlated with elastogenic gene mRNAs. JNK-deficiency increased lung elastin content and impaired alveolar septation. Both JNK-deficient lung and lung fibroblasts had increased mRNA levels of elastin-associated genes. Deletion of both JNK1 and JNK2 increased tropoelastin mRNA to a greater extent than either individually demonstrating that both isoforms independently regulate elastogenesis. Our observations are consistent with previous reports demonstrating JNK regulation of elastogenesis in the aorta  and AP-1 mediated suppression of tropoelastin expression . Phosphorylated c-jun, the readout of the JNK luciferase assay, is a component of the AP-1 transcriptional complex.
We utilized two methods to assess JNK activation during lung development. In addition to comparison of total JNK to phosphorylated JNK, we utilized a pull-down technique developed and validated at the University of California at San Diego in a variety of cell lines with increasing exposures to ultraviolet irradiation—a well described activator of JNK signaling . This technique quantitates JNK phosphorylation of c-Jun. A drawback of this assay is the absence of a loading control which likely accounts for the increased variability in this assay compared to Western blot. Nonetheless, by both Western blot and JNK pull-down, we demonstrated a negative association between lung JNK activity and elastogenic mRNAs.
We have provided strong in vitro and in vivo loss of function data to support a role for JNK in regulating the elastogenic program in the lung. When assessed in isolation, these effects are clear and strong. However, multiple dynamic processes regulate elastogenesis which likely accounts for larger in vitro than in vivo effects. JNK activity was reduced in adult lung compared to developing lung. This discrepancy is likely due to reduced transcriptional activation of the elastogenic program in the adult compared to the juvenile. Conceptually, JNK activity may serve as a brake slowing elastogenesis during alveolarization which turns off when the elastogenic program is no longer activated. The localization of pJNK to pulmonary lipofibroblasts is consistent with their reduced elastogenic and fibrogenic potential ; although, the fact that lipofibroblasts increase in number at an earlier age than JNK activity levels increase  makes a direct relationship between the two unlikely. The mechanism of how JNK suppresses elastogenesis needs to be determined in future studies.
The necessity of elastin band formation for secondary alveolar septation is well-established [4, 16]. Both elastin  and the lysyl oxidase  content are increased in infants with chronic lung disease of prematurity. In mice, elastin haploinsufficiency leads to emphysematous changes , and Mammoto et al. recently demonstrated that reduced matrix stiffness also impairs alveolarization . Our data and these previously published data demonstrate that alveolar growth requires both a low compliance elastin band and high compliance alveolar walls. Therapies aimed at restoring alveolar growth should seek to normalize this relationship.
In conclusion, we report that JNK suppresses pulmonary fibroblast elastogenesis during the alveolar stage of lung development plays a role in alveolar septation.
We would like to thank Patrick Lahni of the Division of Critical Care Medicine at Cincinnati Children’s Hospital Medical Center for his assistance with JNK pull down experiments.
This project was supported with grant support from the National Institutes of Health: K12 HD28827.
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