ABSTRACT
ErbB4 is a regulator in lung development and disease. Prenatal infection is an important risk factor for the delay of morphologic lung development, while promoting the maturation of the surfactant system. Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to prevent lung injury. We hypothesized that BMSCs in comparison with hematopoietic control stem cells (HPSCs) minimize the lipopolysaccharide (LPS)-induced lung injury only when functional ErbB4 receptor is present. We injected LPS and/or murine green fluorescent protein-labeled BMSCs or HPSCs into the amniotic cavity of transgenic ErbB4heart mothers at gestational day 17. Fetal lungs were analyzed 24 h later. BMSCs minimized significantly LPS-induced delay in morphological lung maturation consisting of a stereologically measured increase in mesenchyme and septal thickness and a decrease of future airspace and septal surface. This effect was more prominent and significant in the ErbB4heart+/- lungs, suggesting that the presence of functioning ErbB4 signaling is required. BMSC also diminished the LPS induced increase in surfactant protein (Sftp)a mRNA and decrease in Sftpc mRNA is only seen if ErbB4 is present. The reduction of morphological delay of lung development and of levels of immune-modulating Sftp was more pronounced in the presence of the ErbB4 receptor. Thus, ErbB4 may be required for the protective signaling of BMSCs.
Subject(s)
Fetal Development , Lung/embryology , Mesenchymal Stem Cells/cytology , Organogenesis , Receptor, ErbB-4/physiology , Animals , Female , Fetus , Lipopolysaccharides , Lung/pathology , Mesenchymal Stem Cell Transplantation , Mice , Mice, TransgenicABSTRACT
We investigated in a unique setup of animal models and a human lung explant culture biological properties, including zoonotic potential, of a representative 2016 highly pathogenic avian influenza virus (HPAIV) H5N8, clade 2.3.4.4 group B (H5N8B), that spread rapidly in a huge and ongoing outbreak series in Europe and caused high mortality in waterfowl and domestic birds. HPAIV H5N8B showed increased virulence with rapid onset of severe disease and mortality in Pekin ducks due to pronounced neuro- and hepatotropism. Cross-species infection was evaluated in mice, ferrets, and in a human lung explant culture model. While the H5N8B isolate was highly virulent for Balb/c mice, virulence and transmissibility were grossly reduced in ferrets, which was mirrored by marginal replication in human lung cultures infected ex vivo. Our data indicate that the 2016 HPAIV H5N8B is avian-adapted with augmented virulence for waterfowl, but has low zoonotic potential. The here tested combination of animal studies with the inoculation of human explants provides a promising future workflow to evaluate zoonotic potential, mammalian replication competence and avian virulence of HPAIV.
Subject(s)
Ducks/virology , Influenza A Virus, H5N8 Subtype/pathogenicity , Influenza in Birds/transmission , Influenza, Human/transmission , Poultry Diseases/virology , Zoonoses/transmission , Zoonoses/virology , Animals , Disease Outbreaks/veterinary , Ferrets/virology , Humans , Influenza in Birds/virology , Influenza, Human/virology , Lung/pathology , Lung/virology , Mice, Inbred BALB C , Poultry Diseases/transmission , Virulence , Virus ReplicationABSTRACT
Streptococcus pneumoniae (S.pn.) is the most common bacterial pathogen causing community acquired pneumonia. The pore-forming toxin pneumolysin (PLY) is the major virulence factor of S.pn. and supposed to affect alveolar epithelial cells thereby activating the immune system by liberation of danger-associated molecular patterns (DAMP). To test this hypothesis, we established a novel live-cell imaging based assay to analyse mitochondrial function and associated release of mitochondrial DNA (mtDNA) as DAMP in real-time. We first revealed that bacterially released PLY caused significant changes of the cellular ATP homeostasis and led to morphologic alterations of mitochondria in human alveolar epithelial cells in vitro and, by use of spectral live-tissue imaging, in human alveoli. This was accompanied by strong mitochondrial calcium influx and loss of mitochondrial membrane potential resulting in opening of the mitochondrial permeability transition pore and mtDNA release without activation of intrinsic apoptosis. Moreover, our data indicate cellular mtDNA liberation via microvesicles, which may contribute to S.pn. related pro-inflammatory immune activation in the human alveolar compartment.
Subject(s)
Alveolar Epithelial Cells/drug effects , DNA, Mitochondrial/metabolism , Mitochondria/drug effects , Streptolysins/toxicity , Adenosine Triphosphate/metabolism , Alveolar Epithelial Cells/metabolism , Bacterial Proteins/toxicity , Calcium/metabolism , Cell Line, Tumor , Cells, Cultured , Humans , Membrane Potential, Mitochondrial , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition PoreABSTRACT
Lung diseases belong to the major causes of death worldwide. Recent innovative methodological developments now allow more and more for the use of primary human tissue and cells to model such diseases. In this regard, the review covers bronchial air-liquid interface cultures, precision cut lung slices as well as ex vivo cultures of explanted peripheral lung tissue and de-/re-cellularization models. Diseases such as asthma or infections are discussed and an outlook on further areas for development is given. Overall, the progress in ex vivo modeling by using primary human material could make translational research activities more efficient by simultaneously fostering the mechanistic understanding of human lung diseases while reducing animal usage in biomedical research.
Subject(s)
Bronchi/cytology , Lung Diseases/therapy , Translational Research, Biomedical , Epithelial Cells/cytology , Humans , Lung Diseases/physiopathologyABSTRACT
Immune response in the lung has to protect the huge alveolar surface against pathogens while securing the delicate lung structure. Macrophages and alveolar epithelial cells constitute the first line of defense and together orchestrate the initial steps of host defense. In this study, we analysed the influence of macrophages on type II alveolar epithelial cells during Legionella pneumophila-infection by a systems biology approach combining experimental work and mathematical modelling. We found that L. pneumophila-infected THP-1-derived macrophages provoke a pro-inflammatory activation of neighboring lung epithelial cells, but in addition render them hypo-responsive to direct infection with the same pathogen. We generated a kinetic mathematical model of macrophage activation and identified a paracrine mechanism of macrophage-secreted IL-1ß inducing a prolonged IRAK-1 degradation in lung epithelial cells. This intercellular crosstalk may help to avoid an overwhelming inflammatory response by preventing excessive local secretion of pro-inflammatory cytokines and thereby negatively regulating the recruitment of immune cells to the site of infection. This suggests an important but ambivalent immunomodulatory role of macrophages in lung infection.
Subject(s)
Alveolar Epithelial Cells/immunology , Legionella pneumophila/immunology , Legionnaires' Disease/pathology , Macrophages/immunology , A549 Cells , Coculture Techniques , Humans , Models, Theoretical , Systems Biology , THP-1 CellsABSTRACT
The severity and lethality of influenza A virus (IAV) infections is frequently aggravated by secondary bacterial pneumonia. However, the mechanisms in human lung tissue that provoke this increase in fatality are unknown and therapeutic immune modulatory options are lacking.We established a human lung ex vivo co-infection model to investigate innate immune related mechanisms contributing to the susceptibility of secondary pneumococcal pneumonia.We revealed that type I and III interferon (IFN) inhibits Streptococcus pneumoniae-induced interleukin (IL)-1ß release. The lack of IL-1ß resulted in the repression of bacterially induced granulocyte-macrophage colony-stimulating factor (GM-CSF) liberation. Specific inhibition of IFN receptor I and III-associated tyrosine kinase 2 (Tyk2) completely restored the S. pneumoniae-induced IL-1ß-GM-CSF axis, leading to a reduction of bacterial growth. A preceding IAV infection of the human alveolus leads to a type I and III IFN-dependent blockade of the early cytokines IL-1ß and GM-CSF, which are key for orchestrating an adequate innate immune response against bacteria. Their virally induced suppression may result in impaired bacterial clearance and alveolar repair.Pharmacological inhibition of Tyk2 might be a new treatment option to sustain beneficial endogenous GM-CSF levels in IAV-associated secondary bacterial pneumonia.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Influenza, Human/drug therapy , Interferons/pharmacology , Pneumonia, Bacterial/drug therapy , Staphylococcal Infections/drug therapy , TYK2 Kinase/antagonists & inhibitors , Humans , Immunity, Innate/drug effects , Immunologic Factors , Influenza A virus , Influenza, Human/immunology , Interleukin-1beta/metabolism , Lung/drug effects , Pneumonia, Bacterial/immunology , Staphylococcal Infections/immunology , TYK2 Kinase/metabolismABSTRACT
BACKGROUND: Resistance to radiotherapy continues to be a limiting factor in the treatment of cancer including head and neck squamous cell carcinoma (HNSCC). Simultaneous targeting of ß1 integrin and EGFR was shown to have a higher radiosensitizing potential than mono-targeting in the majority of tested HNSCC cancer models. As tumor-initiating cells (TIC) are thought to play a key role for therapy resistance and recurrence and can be enriched in sphere forming conditions, this study investigated the efficacy of ß1 integrin/EGFR targeting without and in combination with X-ray irradiation on the behavior of sphere-forming cells (SFC). METHODS: HNSCC cell lines (UTSCC15, UTSCC5, Cal33, SAS) were injected subcutaneously into nude mice for tumor up-take and plated for primary and secondary sphere formation under non-adhesive conditions which is thought to reflect the enrichment of SFC and their self-renewal capacity, respectively. Treatment was accomplished by inhibitory antibodies for ß1 integrin (AIIB2) and EGFR (Cetuximab) as well as X-ray irradiation (2 - 6 Gy single doses). Further, flow cytometry for TIC marker expression and cell cycling as well as Western blotting for DNA repair protein expression and phosphorylation were employed. RESULTS: We found higher primary and secondary sphere forming capacity of SAS cells relative to other HNSCC cell lines, which was in line with the tumor up-take rates of SAS versus UTSCC15 cells. AIIB2 and Cetuximab administration had minor cytotoxic and no radiosensitizing effects on SFC. Intriguingly, secondary SAS spheres, representing the fraction of surviving SFC upon passaging, showed greatly enhanced radiosensitivity compared to primary spheres. Intriguingly, neither AIIB2 nor Cetuximab significantly altered basal sphere forming capacity and radiosensitivity. While an increased accumulation of G0/G1 phase cells was observable in secondary SAS spheres, DNA double strand break repair indicated no difference on the basis of significantly enhanced ATM and Chk2 dephosphorylation upon irradiation. CONCLUSIONS: In the HNSCC model, sphere-forming conditions select for cells, which are unsusceptible to both anti-ß1 integrin and anti-EGFR inhibitory antibodies. With regard to primary and secondary sphere formation, our data suggest that both of these SFC fractions express distinct survival strategies independent from ß1 integrin and EGFR and that future work is warranted to better understand SFC survival and enrichment before and after treatment to untangle the underlying mechanisms for identifying novel, druggable cancer targets in SFC.
ABSTRACT
BACKGROUND AND PURPOSE: Simultaneous targeting of ß1 integrin receptor and epidermal growth factor receptor (EGFR) showed higher level of radiosensitization in head and neck cancers than monotherapies. As EGFR inhibition is similarly performed in colorectal cancer (CRC), we investigated the radiosensitizing and anti-invasive potential of ß1-integrin/EGFR inhibition in CRC cell lines grown in more physiological three-dimensional (3D) matrix-based cell cultures. MATERIALS AND METHODS: DLD-1 and HT-29 cells were used for 3D-colony formation, invasion and proliferation assays and Western blotting. ß1 integrin, focal adhesion kinase and EGFR were inhibited by AIIB2, TAE226 and Cetuximab, respectively. KRAS and BRAF knockdown were accomplished using small-interfering RNA technology. Single doses of X-rays ranged from 2Gy to 6Gy and 5-fluorouracil (5-FU) concentration was 10µM. RESULTS: Neither ß1-integrin/EGFR inhibition nor KRAS or BRAF depletion nor 5-FU significantly modified CRC cell radiosensitivity. Cetuximab, AIIB2 and Cetuximab/AIIB2 differentially modulated MAPK, JNK and AKT phosphorylation. AIIB2 and TAE226 significantly decreased cell invasion. CONCLUSIONS: Our data show inefficiency of Cetuximab and AIIB2 on top of radiochemotherapy. The functions of KRAS and BRAF in therapy resistance remain unanswered and warrant further preclinical molecular-driven investigations. One promising approach might be ß1 integrin targeting for reducing metastatic CRC cell spread.
Subject(s)
Colorectal Neoplasms/radiotherapy , ErbB Receptors/antagonists & inhibitors , Integrin beta1/metabolism , Radiation-Sensitizing Agents/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cetuximab/pharmacology , Colorectal Neoplasms/pathology , Fluorouracil/pharmacology , Focal Adhesion Kinase 1/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , HT29 Cells , Humans , Neoplasm Invasiveness , RNA, Small Interfering/pharmacology , Radiation Tolerance/drug effectsABSTRACT
BACKGROUND: Signaling from integrins and receptor tyrosine kinases (RTKs) contributes substantially to therapy resistance of malignant tumors. We investigated simultaneous ß1 integrin-epidermal growth factor receptor (EGFR) targeting plus radiotherapy in human head and neck squamous cell carcinomas (HNSCCs). METHODS: Ten HNSCC cell lines were grown in three-dimensional laminin-rich extracellular matrix cell cultures and two of them as tumor xenografts in nude mice (n = 12-16 per group). Targeting of ß1 integrin and EGFR with monoclonal inhibitory antibodies (AIIB2 and cetuximab, respectively) was combined with x-ray irradiation. Clonogenic survival, tumor growth, and tumor control (evaluated by Kaplan-Meier analysis), apoptosis, phosphoproteome (interactome, network betweeness centrality analysis), receptor expression (immunohistochemistry), and downstream signaling (western blotting) were assessed. Various mutants of the integrin signaling mediator focal adhesion kinase (FAK) were employed for mechanistic studies. All statistical tests were two-sided. RESULTS: Compared with ß1 integrin or EGFR single inhibition, combined ß1 integrin-EGFR targeting resulted in enhanced cytotoxicity and radiosensitization in eight out of 10 tested HNSCC cell lines, which responded with an FAK dephosphorylation after ß1 integrin inhibition. In vivo, simultaneous anti-ß1 integrin/anti-EGFR treatment and radiotherapy of UTSCC15 responder xenografts enabled better tumor control compared with anti-EGFR monotherapy and irradiation (hazard ratio [HR] = 6.9, 95% confidence interval [CI] = 1.6 to 30.9, P = .01), in contrast to the SAS nonresponder tumor model (HR = 0.9, 95% CI = 0.4 to 2.3, P = .83). Mechanistically, a protein complex consisting of FAK- and Erk1-mediated prosurvival signals for radiation resistance, which was effectively compromised by ß1 integrin and EGFR blocking. CONCLUSIONS: Concomitant targeting of ß1 integrin and EGFR seems a powerful and promising approach to overcome radioresistance of HNSCCs.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Squamous Cell/therapy , ErbB Receptors/antagonists & inhibitors , Head and Neck Neoplasms/therapy , Integrin beta1/drug effects , Molecular Targeted Therapy/methods , Animals , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis , Blotting, Western , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/radiotherapy , Cell Line, Tumor , Cetuximab , Focal Adhesion Kinase 1/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/radiotherapy , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Mice , Mice, Nude , Mitogen-Activated Protein Kinase 3/metabolism , Odds Ratio , Radiation-Sensitizing Agents/therapeutic use , Signal Transduction , Squamous Cell Carcinoma of Head and Neck , Xenograft Model Antitumor AssaysABSTRACT
TTF-1 is an important transcription factor in lung development and lung disease and is essential for lung cell differentiation, specifically surfactant protein (Sftp) expression. The molecular mechanisms that drive the expression and transcriptional control of TTF-1 are not fully understood. In the fetal lung, ErbB4 functions as a transcriptional co-factor and regulates the timely onset of fetal Sftp expression. We speculate that ErbB4 is an upstream regulator of TTF-1 and regulates Sftpb expression via this pathway in alveolar type II cells. Neuregulin-induced ErbB4 and TTF-1 signaling interactions were studied by co-immunoprecipitation and confocal microscopy. Overexpression of ErbB4 and TTF-1 was analyzed in its effect on cell viability, Sftpb expression, TTF-1 expression, and Sftpb and TTF-1 promoter activity. The effect of ErbB4 deletion and ErbB4 nuclear translocation on TTF-1 expression was studied in primary fetal type II epithelial cells, isolated from transgenic HER4(heart(-/-)) mice. ErbB4 ligand neuregulin induces ErbB4 and TTF-1 co-precipitation and nuclear colocalization. Combined ErbB4 and TTF-1 overexpression inhibits cell viability, while promoting Sftpb expression more than single overexpression of each protein. NRG stimulates TTF-1 expression in ErbB4-overexpressing epithelial cells, while this effect is absent in ErbB4-depleted cells. In primary fetal type II cells, ErbB4 nuclear translocation is critical for its regulation of TTF-1-induced Sftpb upregulation. TTF-1 overexpression did not overcome this important requirement. We conclude that ErbB4 is a critical upstream regulator of TTF-1 in type II epithelial cells and that this interaction is important for Sftpb regulation.
Subject(s)
Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/metabolism , DNA-Binding Proteins/metabolism , ErbB Receptors/metabolism , Fetus/cytology , Alveolar Epithelial Cells/drug effects , Animals , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Survival/drug effects , Cell Survival/genetics , DNA-Binding Proteins/genetics , ErbB Receptors/chemistry , Fetus/drug effects , Fetus/metabolism , Gene Expression Regulation, Developmental/drug effects , Humans , Immunoprecipitation , Mice , Models, Biological , Neuregulins/pharmacology , Promoter Regions, Genetic/genetics , Protein Structure, Tertiary , Protein Transport/drug effects , Pulmonary Surfactant-Associated Protein B/genetics , Pulmonary Surfactant-Associated Protein B/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, ErbB-4 , Transcription FactorsABSTRACT
Estrogen is known for its positive stimulatory effects on surfactant proteins. ErbB4 receptor and its ligand neuregulin (NRG) positively stimulate lung development. ErbB receptors interact with nuclear receptors and ErbB4 co-regulates estrogen receptor (ER)α expression in breast cells. ERß is highly expressed in pneumocytes and its deletion leads to fewer alveoli and reduced elastic recoil. A similar picture was seen in ErbB4-deleted lungs. We hypothesized that estrogen signals its effect on surfactant protein B (Sftpb) expression through interactions of ERß and ErbB4. Estrogen and NRG treatment decreased cell numbers and stimulated Sftpb expression in type II cells. Estrogen and NRG both stimulated phosphorylation of ERß and co-localization of both receptors. Overexpression of ERß increased the cell number and Sftpb expression, which was further augmented by estrogen and NRG. Finally, estrogen and NRG stimulated ERß and ErbB4 binding to the Sftpb promoter. Overexpression of these receptors stimulated Sftpb promoter activation, which was further enhanced by estrogen and NRG. The stimulatory effect of estrogen and NRG was abolished in ErbB4 deletion and reconstituted by re-expression of full-length ErbB4 in fetal ErbB4-deleted type II cells. Estrogen-induced nuclear translocation of ErbB4 required the intact γ-secretase cleavage site but not the nuclear localization sequence of the ErbB4 receptor, suggesting that ERß might function as a nuclear chaperone for ErbB4. These studies demonstrate that estrogen effects on Sftpb expression require an interaction of ERß and ErbB4. We speculate that the stimulatory effects of estrogen on Sftpb are under transcriptional control of ErbB4.
Subject(s)
Epithelial Cells/drug effects , ErbB Receptors/physiology , Estrogens/pharmacology , Lung/drug effects , Pulmonary Surfactant-Associated Protein B/genetics , Animals , Cells, Cultured , Embryo, Mammalian , Epithelial Cells/cytology , Epithelial Cells/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , Estrogen Receptor beta/metabolism , Estrogen Receptor beta/physiology , Female , Gene Expression Regulation, Developmental/drug effects , Lung/cytology , Lung/metabolism , Mice , Mice, Transgenic , Pregnancy , Protein Binding/drug effects , Pulmonary Surfactant-Associated Protein B/metabolism , Receptor, ErbB-4 , Transcriptional Activation/drug effects , Up-Regulation/drug effectsABSTRACT
Pulmonary ErbB4 deletion leads to a delay in fetal lung development, alveolar simplification, and lung function disturbances in adult mice. We generated a model of intrauterine infection in ErbB4 transgenic mice to study the additive effects of antenatal LPS administration and ErbB4 deletion during fetal lung development. Pregnant mice were treated intra-amniotically with an LPS dose of 4 µg at E17 of gestation. Lungs were analyzed 24 h later. A significant influx of inflammatory cells was seen in all LPS-treated lungs. In heterozygote control lungs, LPS treatment resulted in a delay of lung morphogenesis characterized by a significant increase in the fraction of mesenchyme, a decrease in gas exchange area, and disorganization of elastic fibers. Surfactant protein (Sftp)b and Sftpc were upregulated, but mRNA of Sftpb and Sftpc was downregulated compared with non-LPS-treated controls. The mRNA of Sftpa1 and Sftpd was upregulated. In ErbB4-deleted lungs, the LPS effects were more pronounced, resulting in a further delay in morphological development, a more pronounced inflammation in the parenchyma, and a significant higher increase in all Sftp. The effect on Sftpb and Sftpc mRNA was somewhat different, resulting in a significant increase. These results imply a major role of ErbB4 in LPS-induced signaling in structural and functional lung development.
Subject(s)
Alveolar Epithelial Cells/metabolism , ErbB Receptors/deficiency , Fetus/metabolism , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Lung/metabolism , Protein Isoforms/metabolism , Signal Transduction/genetics , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/drug effects , Animals , Cell Movement/drug effects , Elastic Tissue , ErbB Receptors/genetics , Female , Fetus/drug effects , Fetus/embryology , Gene Expression Regulation, Developmental/drug effects , Inflammation/embryology , Inflammation/genetics , Intercellular Signaling Peptides and Proteins , Lipopolysaccharides/adverse effects , Lung/cytology , Lung/drug effects , Lung/embryology , Mice , Mice, Knockout , Peptides/genetics , Peptides/metabolism , Pregnancy , Protein Isoforms/genetics , Pulmonary Surfactant-Associated Protein C , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Receptor, ErbB-4 , Signal Transduction/drug effects , UterusABSTRACT
Sufficient pulmonary surfactant production is required for the fetal-neonatal transition, especially in preterm infants. Neuregulin (NRG) and its transmembrane receptor ErbB4 positively regulate the onset of fetal surfactant synthesis. Details of this signaling process remain to be elucidated. ErbB4 is known to regulate gene expression in the mammary gland, where the receptor associates with the signal transducer and activator of transcription Stat5a to transactivate the ß-casein gene promoter. We hypothesized that in the fetal lung, ErbB4 functions as a transcriptional regulator for surfactant protein B (Sftpb), the most critical surfactant protein gene. Re-expressing full-length ErbB4 in primary fetal ErbB4-depleted Type II epithelial cells led to an increased expression of Sftpb mRNA. This stimulatory effect required the nuclear translocation of ErbB4 and association with Stat5a, with the resultant binding to and activation of the Sftpb promoter. We conclude that ErbB4 directly regulates important aspects of fetal lung maturation that help prepare for the fetal-neonatal transition.
Subject(s)
Alveolar Epithelial Cells/metabolism , ErbB Receptors/metabolism , Lung/metabolism , Neuregulin-1/metabolism , Pulmonary Surfactant-Associated Protein B/metabolism , Active Transport, Cell Nucleus , Animals , Binding Sites , Cells, Cultured , ErbB Receptors/deficiency , ErbB Receptors/genetics , Gene Expression Regulation, Developmental , Genes, Reporter , Gestational Age , Humans , Lung/embryology , Mice , Mice, Knockout , Mice, Transgenic , Neuregulin-1/genetics , Promoter Regions, Genetic , Pulmonary Surfactant-Associated Protein B/genetics , RNA, Messenger/metabolism , Receptor, ErbB-4 , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Time Factors , Transcription, Genetic , TransfectionABSTRACT
Maturation of pulmonary fetal type II cells to initiate adequate surfactant production is crucial for postnatal respiratory function. Little is known about specific mechanisms of signal transduction controlling type II cell maturation. The ErbB4 receptor and its ligand neuregulin (NRG) are critical for lung development. ErbB4 is cleaved at the cell membrane by the γ-secretase enzyme complex whose active component is either presenilin-1 (PSEN-1) or presenilin-2. ErbB4 cleavage releases the 80kDa intracellular domain (4ICD), which associates with chaperone proteins such as YAP (Yes-associated protein) and translocates to the nucleus to regulate gene expression. We hypothesized that PSEN-1 and YAP have a development-specific expression in fetal type II cells and are important for ErbB4 signaling in surfactant production. In primary fetal mouse E16, E17, and E18 type II cells, PSEN-1 and YAP expression increased at E17 and E18 over E16. Subcellular fractionation showed a strong cytosolic and a weaker membrane location of both PSEN-1 and YAP. This was enhanced by NRG stimulation. Co-immunoprecipitations showed ErbB4 associated separately with PSEN-1 and with YAP. Their association, phosphorylation, and co-localization were induced by NRG. Confocal immunofluorescence and nuclear fractionation confirmed these associations in a time-dependent manner after NRG stimulation. Primary ErbB4-deleted E17 type II cells were transfected with a mutant ErbB4 lacking the γ-secretase binding site. When compared to transfection with wild-type ErbB4, the stimulatory effect of NRG on surfactant protein mRNA expression was lost. We conclude that PSEN-1 and YAP have crucial roles in ErbB4 signal transduction during type II cell maturation.
Subject(s)
ErbB Receptors/metabolism , Fetus/metabolism , Lung/cytology , Lung/embryology , Presenilin-1/metabolism , Adaptor Proteins, Signal Transducing/analysis , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Cell Cycle Proteins , Cells, Cultured , ErbB Receptors/analysis , Fetus/embryology , Gene Expression Regulation, Developmental , Intercellular Signaling Peptides and Proteins , Mice , Neuregulins/metabolism , Peptides/genetics , Phosphoproteins/analysis , Phosphoproteins/genetics , Phosphoproteins/metabolism , Presenilin-1/analysis , Presenilin-1/genetics , Pulmonary Surfactant-Associated Protein C , RNA, Messenger/genetics , Receptor, ErbB-4 , YAP-Signaling ProteinsABSTRACT
The ErbB4 receptor has an important function in fetal lung maturation. Deletion of ErbB4 leads to alveolar hypoplasia and hyperreactive airways similar to the changes in bronchopulmonary dysplasia (BPD). BPD is a chronic pulmonary disorder affecting premature infants as a consequence of lung immaturity, lung damage, and abnormal repair. We hypothesized that proper ErbB4 function is needed for the timely progression of fetal lung development. An ErbB4 transgenic cardiac rescue mouse model was used to study the effect of ErbB4 deletion on fetal lung structure, surfactant protein (SP) expression, and synthesis, and inflammation. Morphometric analyses revealed a delayed structural development with a significant decrease in saccular size at E18 and more pronounced changes at E17, keeping these lungs in the canalicular stage. SP-B mRNA expression was significantly down regulated at E17 with a subsequent decrease in SP-B protein expression at E18. SP-D protein expression was significantly decreased at E18. Surfactant phospholipid synthesis was significantly decreased on both days, and secretion was down regulated at E18. We conclude that pulmonary ErbB4 deletion results in a structural and functional delay in fetal lung development, indicating a crucial regulatory role of ErbB4 in the timely progression of fetal lung development.
Subject(s)
ErbB Receptors/metabolism , Fetus/physiology , Animals , Bronchopulmonary Dysplasia/metabolism , CD11b Antigen/metabolism , Cells, Cultured , ErbB Receptors/genetics , Female , Fetus/anatomy & histology , Fibroblasts/cytology , Fibroblasts/physiology , Heart/embryology , Heart/physiology , Humans , Infant, Newborn , Mice , Mice, Transgenic , Pregnancy , Pulmonary Surfactants/chemistry , Pulmonary Surfactants/metabolism , Receptor, ErbB-4ABSTRACT
OBJECTIVE: To assess the potential role for Neuregulin-1 (NRG1) as a systemic endogenous protector in the setting of perinatal inflammatory brain damage. METHODS: We measured NRG1-protein and mRNA levels in human umbilical venous endothelial cells (HUVECs) of different gestational ages at various durations of exposure to lipopolysaccharide (LPS). In parallel, we genotyped the donor individuals for SNP8NRG221533, a disease-related single nucleotide polymorphism in the 5' region upstream of the NRG1 sequence. Intracellular NRG1 localization was visualized by confocal microscopy. Furthermore we analyzed the relationship between SNP8NRG221533 genotype and neurodevelopmental outcome in children born preterm. RESULTS: We observed a positive dose-response-relationship between NRG1-mRNA and intracellular protein levels with both advancing gestational age and duration of LPS exposure in HUVECs. The presence of allele C at the SNP8NRG221533 locus was associated with an increased cellular production of NRG1 in HUVECs, and with a significantly reduced risk for cerebral palsy and developmental delay in children born preterm. INTERPRETATION: In conclusion, our data indicate that gestational age, duration of LPS exposure, and the SNP8NRG221533 genotype affect NRG1 levels. Our results support the hypothesis that NRG1 may qualify as an endogenous protector during fetal development.
Subject(s)
Brain/metabolism , Infant, Premature/metabolism , Leukomalacia, Periventricular/metabolism , Neuregulin-1/metabolism , Umbilical Veins/metabolism , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Enzyme-Linked Immunosorbent Assay , Genotype , Gestational Age , Humans , Infant, Newborn , Leukomalacia, Periventricular/genetics , Lipopolysaccharides , Microscopy, Confocal , Neuregulin-1/genetics , Polymorphism, Single Nucleotide , Reverse Transcriptase Polymerase Chain Reaction , Umbilical Veins/cytologyABSTRACT
ErbB4 is a predominant heterodimer for other ErbB receptors in late fetal lung development where it participates in regulating type II cell surfactant synthesis. To further elucidate the role of ErbB4 in pulmonary alveolar epithelial cell function, the authors hypothesized that ErbB4 participates in maintaining adult lung type II cell homeostasis. The authors used small interfering RNA (siRNA) to down-regulate endogenous, ErbB4 receptors in the adult rat lung epithelial L2 cell line and measured neuregulin 1beta (NRG1beta)-, and fibroblast conditioned medium (FCM)-induced effects on L2 cell surfactant phospholipid synthesis and proliferation. Under control conditions, total and phosphorylated ErbB4 were significantly increased after both NRG1beta and FCM treatment, as were surfactant phospholipids synthesis and cell proliferation. Down-regulation of ErbB4 with siRNA reduced stimulation of NRG1beta- and FCM-induced ErbB4 phosphorylation, decreased endogenous surfactant phospholipid synthesis, and blocked NRG1beta- and FCM-stimulated surfactant phospholipid synthesis. NRG1beta- and FCM-induced cell proliferation was not affected. The authors conclude that ErbB4 participates in maintaining adult lung alveolar epithelial cell surfactant synthesis and proliferation with development-specific functions.
Subject(s)
ErbB Receptors/metabolism , Pulmonary Surfactant-Associated Proteins/biosynthesis , Respiratory Mucosa/metabolism , Animals , Cell Count , Cell Line , Cell Proliferation , Culture Media, Conditioned/pharmacology , Down-Regulation/drug effects , ErbB Receptors/genetics , Female , Gene Expression/drug effects , Gene Transfer, Horizontal , Male , Neuregulin-1/genetics , Neuregulin-1/metabolism , Phospholipids/antagonists & inhibitors , Phospholipids/biosynthesis , Phosphorylation , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Receptor, ErbB-4 , Respiratory Mucosa/cytology , Respiratory Mucosa/drug effectsABSTRACT
Neuregulin is an important growth factor in fetal surfactant synthesis, and downregulation of its receptor, ErbB4, impairs fetal surfactant synthesis. We hypothesized that pulmonary ErbB4 deletion will affect the developing lung leading to an abnormal postnatal lung function. ErbB4-deleted lungs of 11- to 14-wk-old adult HER4heart mice, rescued from their lethal cardiac defects, were studied for the effect on lung function, alveolarization, and the surfactant system. ErbB4 deletion impairs lung function and structure in HER4heart mice resulting in a hyperreactive airway system and alveolar simplification, as seen in preterm infants with bronchopulmonary dysplasia. It also leads to a downregulation of surfactant protein D expression and an underlying chronic inflammation in these lungs. Our findings suggest that this animal model could be used to further study the pathogenesis of bronchopulmonary dysplasia and might help design protective interventions.
Subject(s)
Bronchopulmonary Dysplasia/physiopathology , ErbB Receptors/deficiency , Lung/pathology , Lung/physiopathology , Animals , Disease Models, Animal , ErbB Receptors/genetics , Gene Deletion , Humans , Infant, Newborn , Lung/ultrastructure , Mice , Pulmonary Surfactant-Associated Protein D/biosynthesis , Receptor, ErbB-4ABSTRACT
ErbB receptors are important regulators of fetal organ development, including the fetal lung. They exhibit diversity in signaling potential, acting through homo- and heterodimers to cause different biological responses. We hypothesized that ErbB receptors show cell-specific and stimuli-specific activation, heterodimerization, and cellular localization patterns in fetal lung. We investigated this using immunoblotting, co-immunoprecipitation, and confocal microscopy in primary isolated E19 fetal rat lung fibroblasts and epithelial type II cells, stimulated with epidermal growth factor, transforming growth factor alpha, neuregulin 1beta, or treated with conditioned medium (CM) from the respective other cell type. Fetal type II cells expressed significantly more ErbB1, ErbB2, and ErbB3 protein than fibroblasts. ErbB4 was consistently identified by co-immunoprecipitation of all other ErbB receptors in both cell types independent of the treatments. Downregulation of ErbB4 in fibroblasts initiated cell-cell communication that stimulated surfactant phospholipid synthesis in type II cells. Confocal microscopy in type II cells revealed nuclear localization of all receptors, most prominently for ErbB4. Neuregulin treatment resulted in relocation to the extra-nuclear cytoplasmic region, which was distinct from fibroblast CM treatment which led to nuclear localization of ErbB4 and ErbB2, inducing co-localization of both receptors. We speculate that ErbB4 plays a prominent role in fetal lung mesenchyme-epithelial communication.
Subject(s)
ErbB Receptors/metabolism , Lung/metabolism , Animals , Cell Communication , Culture Media, Conditioned , Dimerization , Down-Regulation , Epithelial Cells/metabolism , Female , Fibroblasts/metabolism , Lung/cytology , Lung/embryology , Microscopy, Confocal , Pregnancy , Rats , Rats, Sprague-Dawley , Receptor, ErbB-4ABSTRACT
Insufficient fetal surfactant production leads to respiratory distress syndrome among preterm infants. Neuregulin signals the onset of fetal surfactant phospholipid synthesis through formation of erbB receptor dimers. We hypothesized that erbB4 downregulation in fetal type II epithelial cells will downregulate not only fetal surfactant phospholipid synthesis, but also affect proliferation and erbB receptor localization. We tested these hypotheses using small interfering RNA (siRNA) directed against the erbB4 gene to silence erbB4 receptor function in cultures of primary day 19 fetal rat lung type II cells. ErbB4 siRNA treatment inhibited erbB4 receptor protein expression, fibroblast-conditioned medium induced erbB4 phosphorylation, and fetal surfactant phospholipid synthesis. Cell proliferation, measured as thymidine incorporation, was also inhibited by erbB4 siRNA treatment. Downregulation of erbB4 receptor protein changed erbB1 localization at baseline and after stimulation, as determined by confocal microscopy and subcellular fractionation. We conclude that erbB4 is an important receptor in the control of fetal lung type II cell maturation.