Murine Lung Fibroblasts Research Articles

Alcohol induces TGF\u03b21 via downregulation of miR-1946a in murine lung fibroblast

Exaggerated transforming growth factor-beta 1 (TGFβ1) expression worsens fibroproliferation following bleomycin-induced lung injury in alcohol-fed mice. MicroRNA (miR)-1946a is predicted to bind to the TGFβ1 3′ untranslated region (UTR), thereby inhibiting its transcription. We hypothesize that alcohol suppresses miR-1946a and induces TGFβ1. Primary murine lung fibroblasts (PLFs) were cultured ± alcohol, miR-1946a mimic or inhibitor, and TGFβ1 signaling inhibitors. miR-1946a was analyzed after alcohol treatment in vitro and in vivo. TGFβ1 expression and TGFβ1 3′UTR-luciferase activity was quantified. We showed that alcohol suppressed miR-1946a in the alcohol-fed mouse lungs and PLFs. MiR-1946a inhibitor increased TGFβ1 expression in the fibroblast. MiR-1946a mimic treatment suppressed TGFβ1 gene expression and TGFβ1 3′UTR activity. Overexpression of miR1946a inhibited alcohol-induced TGFβ1 gene and protein expression as well as alcohol-induced TGFβ1 and α-smooth muscle actin (SMA) protein expression in PLFs. In conclusion, miR-1946a modulates TGFβ1 expression through direct interaction with TGFβ1 3′UTR. These findings identify a novel mechanism by which alcohol induces TGFβ1 in the lung.

Snail-mediated partial epithelial mesenchymal transition augments the differentiation of local lung myofibroblast

The pathogenesis of pulmonary fibrosis diseases is considered to be related with environmental exposures, but the exact mechanism is unclear and there are no effective treatments. The contribution of epithelial-mesenchymal transition (EMT) to lung fibrosis has been controversial. It was found that partial EMT might play a vital role in renal fibrosis. We also found that partial EMT might be involved in fibrosis diseases. In this study, we used a silicosis animal model of pulmonary fibrosis to observe whether partial EMT existed in pulmonary fibrosis disease and a co-culture system culturing fibroblasts and alveolar epithelial cells stimulated by TGF-β1 to evaluate the probable effects of partial EMT, thus determined the probable role of partial EMT in pulmonary fibrosis diseases. In vivo, the results revealed that partial EMT might exist in silica-induced lung fibrosis model and Snail which is a potent EMT inducer was involved during the process. In vitro, a co-culture system was used to evaluate the effects of EMT in murine alveolar epithelial type II (ATII) cells on the activation of underlying murine lung fibroblasts into myofibroblasts. The results showed that epithelial cells undergoing EMT promoted the differentiation of lung myofibroblast and this epithelial-mesenchymal crosstalk was mainly controlled by Snail. Following Snail silencing the EMT and the activation of NIH-3T3 into myofibroblast were obviously inhibited. It indicated that targeting this novel Snail might be a viable strategy for the treatment of lung fibrosis diseases.

CRel expression regulates distinct transcriptional and functional profiles driving fibroblast matrix production in systemic sclerosis.

NF-κB regulates genes that control inflammation, cell proliferation, differentiation and survival. Dysregulated NF-κB signalling alters normal skin physiology and deletion of cRel limits bleomycin-induced skin fibrosis. This study investigates the role of cRel in modulating fibroblast phenotype in the context of SSc. Fibrosis was assessed histologically in mice challenged with bleomycin to induce lung or skin fibrosis. RNA sequencing and pathway analysis was performed on wild type and Rel-/- murine lung and dermal fibroblasts. Functional assays examined fibroblast proliferation, migration and matrix production. cRel overexpression was investigated in human dermal fibroblasts. cRel immunostaining was performed on lung and skin tissue sections from SSc patients and non-fibrotic controls. cRel expression was elevated in murine lung and skin fibrosis models. Rel-/- mice were protected from developing pulmonary fibrosis. Soluble collagen production was significantly decreased in fibroblasts lacking cRel while proliferation and migration of these cells was significantly increased. cRel regulates genes involved in extracellular structure and matrix organization. Positive cRel staining was observed in fibroblasts in human SSc skin and lung tissue. Overexpression of constitutively active cRel in human dermal fibroblasts increased expression of matrix genes. An NF-κB gene signature was identified in diffuse SSc skin and nuclear cRel expression was elevated in SSc skin fibroblasts. cRel regulates a pro-fibrogenic transcriptional programme in fibroblasts that may contribute to disease pathology. Targeting cRel signalling in fibroblasts of SSc patients could provide a novel therapeutic avenue to limit scar formation in this disease.

The elephant in the lung: Integrating lineage-tracing, molecular markers, and single cell sequencing data to identify distinct fibroblast populations during lung development and regeneration

During lung development, the mesenchyme and epithelium are dependent on each other for instructive morphogenic cues that direct proliferation, cellular differentiation and organogenesis. Specification of epithelial and mesenchymal cell lineages occurs in parallel, forming cellular subtypes that guide the formation of both transitional developmental structures and the permanent architecture of the adult lung. While epithelial cell types and lineages have been relatively well-defined in recent years, the definition of mesenchymal cell types and lineage relationships has been more challenging. Transgenic mouse lines with permanent and inducible lineage tracers have been instrumental in identifying lineage relationships among epithelial progenitor cells and their differentiation into distinct airway and alveolar epithelial cells. Lineage tracing experiments with reporter mice used to identify fibroblast progenitors and their lineage trajectories have been limited by the number of cell specific genes and the developmental timepoint when the lineage trace was activated. In this review, we discuss major developmental mesenchymal lineages, focusing on time of origin, major cell type, and other lineage derivatives, as well as the transgenic tools used to find and define them. We describe lung fibroblasts using function, location, and molecular markers in order to compare and contrast cells with similar functions. The temporal and cell-type specific expression of fourteen “fibroblast lineage” genes were identified in single-cell RNA-sequencing data from LungMAP in the LGEA database. Using these lineage signature genes as guides, we clustered murine lung fibroblast populations from embryonic day 16.5 to postnatal day 28 (E16.5-PN28) and generated heatmaps to illustrate expression of transcription factors, signaling receptors and ligands in a temporal and population specific manner.

Store-operated Ca2+ entry in primary murine lung fibroblasts is independent of classical transient receptor potential (TRPC) channels and contributes to cell migration

Stromal interaction molecules (STIM1, 2) are acting as sensors for Ca2+ in intracellular stores and activate Orai channels at the plasma membrane for store-operated Ca2+ entry (SOCE), while classical transient receptor potential (TRPC) channel mediate receptor-operated Ca2+ entry (ROCE). Several reports, however, indicate a role for TRPC in SOCE in certain cell types. Here, we analyzed Ca2+ influx and cell function in TRPC1/6-deficient (TRPC1/6−/−) and STIM1/2- deficient (STIM1/2ΔpmLF) primary murine lung fibroblasts (pmLF). As expected, SOCE was decreased in STIM1/2- deficient pmLF and ROCE was decreased in TRPC1/6−/− pmLF compared to control cells. By contrast, SOCE was not significantly different in TRPC1/6−/− pmLF and ROCE was similar in STIM1/2-deficient pmLF compared to Wt cells. Most interestingly, cell proliferation, migration and nuclear localization of nuclear factor of activated T-cells (NFATc1 and c3) were decreased after ablation of STIM1/2 proteins in pmLF. In conclusion, TRPC1/6 channels are not involved in SOCE and STIM1/2 deficiency resulted in decreased cell proliferation and migration in pmLF.

Hexokinase 2 couples glycolysis with the profibrotic actions of TGF-β.

Metabolic dysregulation in fibroblasts is implicated in the profibrotic actions of transforming growth factor-β (TGF-β). Here, we present evidence that hexokinase 2 (HK2) is important for mediating the fibroproliferative activity of TGF-β both in vitro and in vivo. Both Smad-dependent and Smad-independent TGF-β signaling induced HK2 accumulation in murine and human lung fibroblasts through induction of the transcription factor c-Myc. Knockdown of HK2 or pharmacological inhibition of HK2 activity with Lonidamine decreased TGF-β-stimulated fibrogenic processes, including profibrotic gene expression, cell migration, colony formation, and activation of the transcription factors YAP and TAZ, with no apparent effect on cellular viability. Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibited an increased abundance of HK2. In a mouse model of bleomycin-induced lung fibrosis, Lonidamine reduced the expression of genes encoding profibrotic markers (collagenΙα1, EDA-fibronectin, α smooth muscle actin, and connective tissue growth factor) and stabilized or improved lung function as assessed by measurement of peripheral blood oxygenation. These findings provide evidence of how metabolic dysregulation through HK2 can be integrated within the context of profibrotic TGF-β signaling.

Amphiregulin modulates murine lung recovery and fibroblast function following exposure to agriculture organic dust.

Exposure to agricultural bioaerosols can lead to chronic inflammatory lung diseases. Amphiregulin (AREG) can promote the lung repair process but can also lead to fibrotic remodeling. The objective of this study was to determine the role of AREG in altering recovery from environmental dust exposure in a murine in vivo model and in vitro using cultured human and murine lung fibroblasts. C57BL/6 mice were intranasally exposed to swine confinement facility dust extract (DE) or saline daily for 1 wk or allowed to recover for 3-7 days while being treated with an AREG-neutralizing antibody or recombinant AREG. Treatment with the anti-AREG antibody prevented resolution of DE exposure-induced airway influx of total cells, neutrophils, and macrophages and increased levels of TNF-α, IL-6, and CXCL1. Neutrophils and activated macrophages (CD11c+CD11bhi) persisted after recovery in lung tissues of anti-AREG-treated mice. In murine and human lung fibroblasts, DE induced the release of AREG and inflammatory cytokines. Fibroblast recellularization of primary human lung mesenchymal matrix scaffolds and wound closure was inhibited by DE and enhanced with recombinant AREG alone. AREG treatment rescued the DE-induced inhibitory fibroblast effects. AREG intranasal treatment for 3 days during recovery phase reduced repetitive DE-induced airway inflammatory cell influx and cytokine release. Collectively, these studies demonstrate that inhibition of AREG reduced, whereas AREG supplementation promoted, the airway inflammatory recovery response following environmental bioaerosol exposure, and AREG enhanced fibroblast function, suggesting that AREG could be targeted in agricultural workers repetitively exposed to organic dust environments to potentially prevent and/or reduce disease.

Sex-specific differences in primary neonatal murine lung fibroblasts exposed to hyperoxia in vitro: implications for bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of the neonate characterized by impaired alveolarization and vascular growth. BPD is more common in premature male infants, but the reasons underlying sexually dimorphic outcomes are not known. It is thought that alterations in fibroblast phenotype in response to environmental stress such as hyperoxia contribute to BPD. Notch signaling creates a profibrotic environment in the lung. However, the role of hyperoxia on differential Notch pathway activation in male vs. female neonatal lung fibroblasts is not known. Primary murine lung fibroblasts from 10-day-old male and female mice were exposed to room air (21% O2, 5% CO2) or hyperoxia (95% O2, 5% CO2), and changes in cell proliferation, viability and expression of fibrosis-related genes and Notch pathway mediators were measured. Upon exposure to hyperoxia, cell proliferation was arrested in male and female fibroblasts, but cell viability was preserved. Increased Notch pathway activation was noted in male fibroblasts along with differential sex-specific modulation of key Notch pathway mediators in response to hyperoxia. α-Smooth muscle actin expression was increased in both male and female fibroblasts upon exposure to hyperoxia. Male and female fibroblasts further demonstrated distinct changes in expression of key fibrosis-related genes upon exposure to hyperoxia. Differential Notch pathway activation and distinct differences in the expression of key fibrosis-related genes might contribute to the sex-specific differences seen in hyperoxia-induced fibrosis and inhibition of lung development in BPD, with more severe implications in male neonates.

The effects of interleukin-33 on airways collagen deposition and matrix metalloproteinase expression in a murine surrogate of asthma.

It has been suggested that interleukin-33 (IL-33) plays an important role in the pathogenesis of asthma through a variety of pathways, but its role in airways fibrosis in asthma has not been fully elucidated. In the present study we evaluated changes in the expression of extracellular matrix proteins (ECMs) as well as matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in an IL-33-induced, antigen-independent murine surrogate of asthma as well as a conventional surrogate employing per-nasal challenge of mice previously sensitized to produce an IgE response to ovalbumin (OVA). In addition, in in vitro experiments we explored the direct effects of IL-33 on the proliferation and function of murine fibroblasts. Per-nasal administration of IL-33 alone was sufficient to induce airways deposition of ECMs, including collagens I, III, V and fibronectin, to a degree comparable with that observed in the OVA-sensitized and challenged mice. These changes were associated with a local imbalance between the expression of extracellular MMPs and TIMPs. Per-nasal challenge of mice with IL-33 also induced elevated airways expression of connective tissue growth factor and fibroblast growth factor receptor 4, two key facilitators of local fibrosis, again to a degree compatible with that observed in OVA-sensitized and challenged mice. Deletion of the ST2 gene, which encodes the IL-33 receptor, abrogated these fibrotic changes in the airways in the OVA surrogate. In vitro, IL-33 significantly increased the proliferation and expression of collagen III by murine lung fibroblasts. These data suggest that direct exposure of murine airways to IL-33 is able to induce local fibrotic changes, at least partially through effects of signalling through the IL-33/ST2 axis on fibroblast function and local expression of MMPs and their inhibitors, and other fibrosis-related proteins.

P3.02-073 Stromal Hedgehog Pathway Activation Suppresses Growth and Metastasis of Lung Adenocarcinoma

Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung adenocarcinoma. Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRASG12Dactivation, p53 loss, and SHH loss compared to wild-type SHH. In human lung adenocarcinoma patients, higher expression of SHH mRNA in lung adenocarcinoma correlated with poor overall and progression free survival. A scan of 35 human lung adenocarcinoma cell lines revealed heterogeneous expression of SHH and IHH with high expression found predominantly in mutant K-Ras lines. Co-culture of high SHH expressing tumor epithelial cells and Shh-Light2 reporter cell lines demonstrated that SHH activated the fibroblast reporter in a paracrine manner, rather than an autocrine effect on cancer cells. Treatment with the SMO inhibitor, KAAD-cyclopamine, also inhibited the growth of tumor epithelial cells in co-culture with NIH-3T3 fibroblast cells but the effect was decreased when co-cultured with lung fibroblasts. Genetic loss of SHH in an autochthonous mouse model, LSL-KrasG12D/+;Trp53fl/fl; Shhfl/fl (KPS) did not affect overall survival compared to LSL-KrasG12D/+; Trp53fl/fl(KP) mice However, early inhibition of the Hh pathway by anti-SHH/IHH antibody, 5E1, on KP mice resulted in significantly worse survival rates with increased metastatic burden compared to IgG treatment. Analysis of KP tumors revealed unexpected high levels of IHH mRNA by in situ hybridization and qPCR that may account for the survival differences seen between genetic ablation and pharmaceutical inhibition of the Hh ligands. The Hh signaling pathway acts upon lung stromal cells in a paracrine fashion and induces distinct transcriptional programs in murine embryonic and lung fibroblasts. Inhibition of paracrine Hh pathway activity in vivo worsened mortality rate due to increase in tumor growth and metastases. Furthermore, mutant Kras lung adenocarcinomas express high levels of IHH that dominates the tumor suppressive effects in our mouse models.

Inhibition of Phosphoglycerate Dehydrogenase Attenuates Bleomycin-induced Pulmonary Fibrosis.

Organ fibrosis, including idiopathic pulmonary fibrosis, is associated with significant morbidity and mortality. Because currently available therapies have limited effect, there is a need to better understand the mechanisms by which organ fibrosis occurs. We have recently reported that transforming growth factor (TGF)-β, a key cytokine that promotes fibrogenesis, induces the expression of the enzymes of the de novo serine and glycine synthesis pathway in human lung fibroblasts, and that phosphoglycerate dehydrogenase (PHGDH; the first and rate-limiting enzyme of the pathway) is required to promote collagen protein synthesis downstream of TGF-β. In this study, we investigated whether inhibition of de novo serine and glycine synthesis attenuates lung fibrosis in vivo. We found that TGF-β induces mRNA and protein expression of PHGDH in murine fibroblasts. Similarly, intratracheal administration of bleomycin resulted in increased expression of PHGDH in mouse lungs, localized to fibrotic regions. Using a newly developed small molecule inhibitor of PHGDH (NCT-503), we tested whether pharmacologic inhibition of PHGDH could inhibit fibrogenesis both in vitro and in vivo. Treatment of murine and human lung fibroblasts with NCT-503 decreased TGF-β-induced collagen protein synthesis. Mice treated with the PHGDH inhibitor beginning 7 days after intratracheal instillation of bleomycin had attenuation of lung fibrosis. These results indicate that the de novo serine and glycine synthesis pathway is necessary for TGF-β-induced collagen synthesis and bleomycin-induced pulmonary fibrosis. PHGDH and other enzymes in the de novo serine and glycine synthesis pathway may be a therapeutic target for treatment of fibrotic diseases, including idiopathic pulmonary fibrosis.

Abstract 2964: Stromal Hedgehog pathway activation suppresses growth and metastases of lung adenocarcinoma

Abstract Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung tumor epithelia. Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRASG12Dactivation, p53 loss,, and SHH loss compared to wild-type SHH. In human lung adenocarcinoma patients, higher expression of SHH mRNA in lung adenocarcinoma correlated with poor overall and progression free survival. A scan of 35 human lung adenocarcinoma cell lines revealed heterogeneous expression of SHH and IHH with high expression found predominantly in mutant K-Ras lines. Co-culture of high SHH expressing tumor epithelial cells and Shh-Light2 reporter cell lines demonstrated that SHH activated the fibroblast reporter in a paracrine manner, rather than an autocrine effect on cancer cells. Treatment with the SMO inhibitor, KAAD-cyclopamine, also inhibited the growth of tumor epithelial cells in co-culture with NIH-3T3 fibroblast cells but the effect was decreased when co-cultured with lung fibroblasts. Genetic loss of SHH in an autochthonous mouse model, LSL-KrasG12D/+;Trp53fl/fl; Shhfl/fl (KPS) did not affect overall survival compared to LSL-KrasG12D/+;Trp53fl/fl (KP) mice However, early inhibition of the Hh pathway by anti-SHH/IHH antibody, 5E1, on KP mice resulted in significantly worse survival rates with increased metastatic burden compared to IgG treatment. Analysis of KP tumors revealed unexpected high levels of IHH mRNA by in situ hybridization that may account for the survival differences seen between genetic ablation and pharmaceutical inhibition of the Hh ligands. In conclusion, the Hh signaling pathway acts upon lung stromal cells in a paracrine fashion. Inhibition of Hh activity in vivo worsened mortality rate due to increase in tumor growth and metastases. Furthermore, mutant Kras lung cancers express high levels of IHH that dominates the tumor suppressive effect of our mouse models. Citation Format: Sahba Kasiri, Baozhi Chen, Alexandra Wilson, Umaru Barrie, Ummay Marriam, Zhiqun Zeng, Luc Girard, James Kim. Stromal Hedgehog pathway activation suppresses growth and metastases of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2964. doi:10.1158/1538-7445.AM2017-2964

XIST/miR-139 axis regulates bleomycin (BLM)-induced extracellular matrix (ECM) and pulmonary fibrosis through β-catenin.

Pulmonary fibrosis (PF), characterized by the destruction of lung tissue architecture and the abnormal deposition of extracellular matrix (ECM) proteins, currently has no satisfactory treatment. In the present study, we investigated the hypothesis that XIST play a promotive role in bleomycin (BLM)-induced ECM and pulmonary fibrosis; XIST exerts its effect through miR-139 regulation. XIST expression was upregulated in lung tissues derived from BLM-induced mouse model of PF, and was positively correlated with β-catenin and ECM protein levels, respectively. LV-sh-XIST-induced XIST knockdown led to decreased PF, reduced β-catenin and ECM protein levels in lung tissues. XIST knockdown suppressed the proliferation of IMR-90 (human fibroblast) and murine lung fibroblasts (MLFCs) and ECM protein expression. Moreover, miR-139 could directly bind to XIST and the 3’UTR of β-catenin; XIST competed with β-catenin for miR-139 binding both in IMR-90 and MLFCs. In MLFCs, miR-139 inversely regulated XIST, and could partially reverse the effect of XIST on β-catenin and ECM proteins. In lung tissues of PF mice, miR-139 expression was downregulated, whereas β-catenin expression was upregulated. In conclusion, XIST exerts positive effects on BLM-induced PF through inhibiting miR-139 to promote human/mouse fibroblast proliferation and ECM proteins.

Lung inflammation after bleomycin treatment in mice: Selection of an accurate normalization strategy for gene expression analysis in an ex-vivo and in-vitro model

Pulmonary fibrosis (PF) is the most common and aggressive interstitial lung disease, characterized by a patchy development of fibrosis leading to progressive destruction of the normal lung architecture which is preceded by an inflammatory process. Gene expression studies are important to understand the development of PF but the accuracy and reproducibility of Real-Time PCR depend on appropriate normalization strategies. This study aimed to analyze the expression variability of eight commonly used reference genes during the initial inflammatory phase of bleomycin-induced PF in a mouse model and to verify whether the selected reference genes could be applied to an in-vitro model of BLM-treated primary murine lung fibroblasts. Wild-type C57BL/6 mice (n=40) were used. Real-Time PCR was carried out on lung tissue of mice either BLM (BLM-tm) or physiological solution-treated (PSS-tm), and in primary lung fibroblasts, isolated from healthy C57BL/6 mice. Histological analysis was performed to confirm the inflammation development. During inflammation, the most stable genes resulted: PPIA, HPRT-1 and SDHA for both models; the normalization strategy was tested analyzing mRNA expression of PTX-3 and TNF-α which resulted up-regulated both in ex-vivo and in-vitro with respect to PSS-tm/fibroblasts. Histological analysis supported the results. This study identified a new set of reference genes expressed both in the in-vitro and ex-vivo models. A higher expression of both markers in BLM-tm with respect to PSS-tm indicated that BLM might lead to increased PTX-3 local production by a co-regulation with TNF-α at lung level.

NADPH Oxidase 4 (Nox4) Suppresses Mitochondrial Biogenesis and Bioenergetics in Lung Fibroblasts via a Nuclear Factor Erythroid-derived 2-like 2 (Nrf2)-dependent Pathway

Mitochondrial bioenergetics are critical for cellular homeostasis and stress responses. The reactive oxygen species-generating enzyme, NADPH oxidase 4 (Nox4), regulates a number of physiological and pathological processes, including cellular differentiation, host defense, and tissue fibrosis. In this study we explored the role of constitutive Nox4 activity in regulating mitochondrial function. An increase in mitochondrial oxygen consumption and reserve capacity was observed in murine and human lung fibroblasts with genetic deficiency (or silencing) of Nox4. Inhibition of Nox4 expression/activity by genetic or pharmacological approaches resulted in stimulation of mitochondrial biogenesis, as evidenced by elevated mitochondrial-to-nuclear DNA ratio and increased expression of the mitochondrial markers transcription factor A (TFAM), citrate synthase, voltage-dependent anion channel (VDAC), and cytochrome c oxidase subunit 4 (COX IV). Induction of mitochondrial biogenesis was dependent on TFAM up-regulation but was independent of the activation of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). The enhancement of mitochondrial bioenergetics as well as the increase in mitochondrial proteins in Nox4-deficient lung fibroblasts is inhibited by silencing of nuclear factor erythroid-derived 2-like 2 (Nrf2), supporting a key role for Nrf2 in control of mitochondrial biogenesis. Together, these results indicate a critical role for both Nox4 and Nrf2 in counter-regulation of mitochondrial biogenesis and metabolism.

Epigenetic Regulation of Caveolin-1 Gene Expression in Lung Fibroblasts.

Fibrotic disorders are associated with tissue accumulation of fibroblasts. We recently showed that caveolin (Cav)-1 gene suppression by a profibrotic cytokine, transforming growth factor (TGF)-β1, contributes to fibroblast proliferation and apoptosis resistance. Cav-1 has been shown to be constitutively suppressed in idiopathic pulmonary fibrosis (IPF), but mechanisms for this suppression are incompletely understood. We hypothesized that epigenetic processes contribute to Cav-1 down-regulation in IPF lung fibroblasts, and after fibrogenic stimuli. Cav-1 expression levels, DNA methylation status, and histone modifications associated with the Cav-1 promoter were examined by PCR, Western blots, pyrosequencing, or chromatin immunoprecipitation assays in IPF lung fibroblasts, normal fibroblasts after TGF-β1 stimulation, or in murine lung fibroblasts after bleomycin injury. Methylation-specific PCR demonstrated methylated and unmethylated Cav-1 DNA copies in all groups. Despite significant changes in Cav-1 expression, no changes in DNA methylation were observed in CpG islands or CpG island shores of the Cav-1 promoter by pyrosequencing of lung fibroblasts from IPF lungs, in response to TGF-β1, or after bleomycin-induced murine lung injury, when compared with respective controls. In contrast, the association of Cav-1 promoter with the active histone modification mark, H3 lysine 4 trimethylation, correlated with Cav-1 down-regulation in activated/fibrotic lung fibroblasts. Our data indicate that Cav-1 gene silencing in lung fibroblasts is actively regulated by epigenetic mechanisms that involve histone modifications, in particular H3 lysine 4 trimethylation, whereas DNA methylation does not appear to be a primary mechanism. These findings support therapeutic strategies that target histone modifications to restore Cav-1 expression in fibroblasts participating in pathogenic tissue remodeling.

Classical transient receptor potential 6 (TRPC6) channels support myofibroblast differentiation and development of experimental pulmonary fibrosis

Pulmonary fibrosis (PF) is a chronic progressive lung disease without effective medical treatment options leading to respiratory failure and death within 3–5years of diagnosis. The pathological process of PF is driven by aberrant wound-healing involving fibroblasts and myofibroblasts differentiated by secreted profibrotic transforming growth factor β (TGF-β1). Classical transient receptor potential 6 (TRPC6), a Na+- and Ca2+-permeable cation channel, is able to promote myofibroblast conversion of primary rat cardiac and human dermal fibroblasts and TRPC6-deficiency impaired wound healing after injury. To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (TRPC6−/−) lungs utilizing a bleomycin-induced PF-model. Fibrotic WT-mice showed a significant higher death rate while bleomycin-treated TRPC6-deficient mice were partly protected from fibrosis as a consequence of a lower production of collagen and an almost normal function of the respiratory system (reduced resistance and elastance compared to fibrotic WT-mice). On a molecular level TGF-β1 induced TRPC6 up-regulation, increased Ca2+ influx and nuclear NFAT localization in WT primary murine lung fibroblasts (PMLFs) resulting in higher stress fiber formation and accelerated contraction rates as compared to treated TRPC6-deficient fibroblasts. Therefore, we conclude that TRPC6 is an important determinant for TGF-β1-induced myofibroblast differentiation during fibrosis and specific channel inhibitors might be beneficial in a future treatment of PF.

The role of microRNA-155/liver X receptor pathway in experimental and idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is progressive and rapidly fatal. Improved understanding of pathogenesis is required to prosper novel therapeutics. Epigenetic changes contribute to IPF; therefore, microRNAs may reveal novel pathogenic pathways.ObjectivesWe sought to determine the regulatory role of microRNA (miR)-155 in the profibrotic function of murine lung macrophages and fibroblasts, IPF lung fibroblasts, and its contribution to experimental pulmonary fibrosis.MethodsBleomycin-induced lung fibrosis in wild-type and miR-155−/− mice was analyzed by histology, collagen, and profibrotic gene expression. Mechanisms were identified by in silico and molecular approaches and validated in mouse lung fibroblasts and macrophages, and in IPF lung fibroblasts, using loss-and-gain of function assays, and in vivo using specific inhibitors.ResultsmiR-155−/− mice developed exacerbated lung fibrosis, increased collagen deposition, collagen 1 and 3 mRNA expression, TGF-β production, and activation of alternatively activated macrophages, contributed by deregulation of the miR-155 target gene the liver X receptor (LXR)α in lung fibroblasts and macrophages. Inhibition of LXRα in experimental lung fibrosis and in IPF lung fibroblasts reduced the exacerbated fibrotic response. Similarly, enforced expression of miR-155 reduced the profibrotic phenotype of IPF and miR-155−/− fibroblasts.ConclusionsWe describe herein a molecular pathway comprising miR-155 and its epigenetic LXRα target that when deregulated enables pathogenic pulmonary fibrosis. Manipulation of the miR-155/LXR pathway may have therapeutic potential for IPF.

Quantitative Analysis of Exosomes From Murine Lung Cancer Cells by Flow Cytometry.

In vivo studies regarding biochemical, molecular biological, and histopathological changes in cancer tissues have been widely performed by the administration of carcinogens in rodents. In these established methods, dissection of the animal following sacrifice must be carried out. Exosomes are cell-derived vesicles that are present in all body fluids and these vesicles have specific roles within cells. Thus, much attention is given to the clinical application of exosomes that can possibly be used for prediction and therapy and as biomarkers related to cancer. To develop a new tool for monitoring in vivo genetic alterations, as a result of carcinogenesis, without the need for frequent euthanasia, we performed quantitative measurement of exosomes in Mlg2908 murine lung fibroblasts and LA-4 and KLN 205 murine lung cancer cells using fluorescence-activated cell sorting. We detected an increase in CD63-specific exosomes in LA-4 lung cancer cells. This result is able to be applied to the classification of cancer-specific proteins and miRNA as diagnostic markers.

Cyclooxygenase-2 Mediated Oxidation of 2-Arachidonoyl-Lysophospholipids Identifies Unknown Lipid Signaling Pathways

Eicosanoid lipids play important roles in cellular signaling as second messengers in inflammation, immune response, vascular tone, and the CNS. Biosynthesis of eicosanoid lipids proceeds via hydrolysis ofesterified arachidonic acid from phospholipids followed by oxidation of the released arachidonic acid by a variety of enzymes including cyclooxygenases (COX). Herein, we demonstrate the remarkable abilityof COX-2, but not COX-1, to directly oxidize 2-arachidonoyl-lysolipids, resulting in the generation of previously unknown classes of eicosanoid-lysolipids, and provide evidence that intracellular lipases can release eicosanoids from their eicosanoid-lysolipid precursors. Importantly, genetic ablation of a phospholipase, iPLA2γ, significantly reduced the amountsof these eicosanoid-lysolipids in murine hepatic tissue and fibroblasts. Furthermore, calcium stimulation of wild-type murine lung fibroblasts produced robust increases in these eicosanoid-lysolipids, which were markedly attenuated in iPLA2γ-/- fibroblasts. Collectively, these results identify an iPLA2γ-initiated pathway generating new classes of lipid metabolites with potential signaling functions resulting from the direct COX-2 catalyzed oxidation of 2-arachidonoyl-lysolipids.