High risk of lung cancer in surfactant-related gene variant carriers.

BACKGROUND: Several rare surfactant-related gene (SRG) variants associated with interstitial lung disease are suspected to be associated with lung cancer, but data are missing. We aimed to study the epidemiology and phenotype of lung cancer in an international cohort of SRG variant carriers. METHODS: We conducted a cross-sectional study of all adults with SRG variants in the OrphaLung network and compared lung cancer risk with telomere-related gene (TRG) variant carriers. RESULTS: We identified 99 SRG adult variant carriers (SFTPA1 (n=18), SFTPA2 (n=31), SFTPC (n=24), ABCA3 (n=14) and NKX2-1 (n=12)), including 20 (20.2%) with lung cancer (SFTPA1 (n=7), SFTPA2 (n=8), SFTPC (n=3), NKX2-1 (n=2) and ABCA3 (n=0)). Among SRG variant carriers, the odds of lung cancer was associated with age (OR 1.04, 95% CI 1.01-1.08), smoking (OR 20.7, 95% CI 6.60-76.2) and SFTPA1/SFTPA2 variants (OR 3.97, 95% CI 1.39-13.2). Adenocarcinoma was the only histological type reported, with programmed death ligand-1 expression ≥1% in tumour cells in three samples. Cancer staging was localised (I/II) in eight (40%) individuals, locally advanced (III) in two (10%) and metastatic (IV) in 10 (50%). We found no somatic variant eligible for targeted therapy. Seven cancers were surgically removed, 10 received systemic therapy, and three received the best supportive care according to their stage and performance status. The median overall survival was 24 months, with stage I/II cancers showing better survival. We identified 233 TRG variant carriers. The comparative risk (subdistribution hazard ratio) for lung cancer in SRG patients versus TRG patients was 18.1 (95% CI 7.1-44.7). CONCLUSIONS: The high risk of lung cancer among SRG variant carriers suggests specific screening and diagnostic and therapeutic challenges. The benefit of regular computed tomography scan follow-up should be evaluated.

The expression of the surfactant proteins SP-A and SP-B during postnatal alveolarization of the rat lung.

OBJECTIVE: Surfactant-specific proteins (SP) are responsible for the functional and structural integrity as well as for the stabilization of the intra-alveolar surfactant. Morphological lung maturation starts in rat lungs after birth. The aim of this study was to investigate whether the expression of the hydrophilic SP-A and the hydrophobic SP-B is associated with characteristic postnatal changes characterizing morphological lung maturation. METHODS: Stereological methods were performed on the light microscope. Using immunohistochemical and molecular biological methods (Western Blot, RT-qPCR), the SP-A and SP-B of adult rat lungs and of those with different postnatal developmental stages (3, 7, 14 and 21 days after birth) were characterized. RESULTS: As signs of alveolarization the total septal surface and volume increased and the septal thickness decreased. The significantly highest relative surface fraction of SP-A labeled alveolar epithelial cells type II (AEII) was found together with the highest relative SP-A gene expression before the alveolarization (3th postnatal day). With the downregulation of SP-A gene expression during and after alveolarization (between postnatal days 7 and 14), the surface fraction of the SP-A labeled AEII also decreased, so they are lowest in adult animals. The surface fraction of SP-B labeled AEII and the SP-B gene expression showed the significantly highest levels in adults, the protein expression increased also significantly at the end of morphological lung maturation. There were no alterations in the SP-B expression before and during alveolarization until postnatal day 14. The protein expression as well as the gene expression of SP-A and SP-B correlated very well with the total surface of alveolar septa independent of the postnatal age. CONCLUSION: The expression of SP-A and SP-B is differentially associated with morphological lung maturation and correlates with increased septation of alveoli as indirect clue for alveolarization.

A dominant negative variant of RAB5B disrupts maturation of surfactant protein B and surfactant protein C.

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.

Immunohistochemical findings in the lungs of COVID-19 subjects: evidence of surfactant dysregulation.

OBJECTIVE: Acute respiratory distress syndrome (ARDS) is characterized by quantitative and qualitative changes in surfactant composition, leading to surfactant dysregulation with alveolar collapse and acute respiratory hypoxic failure. Recently, surfactant has been hypothesized to play a relevant role in COVID-19, representing a strong defender against SARS-CoV-2 infection. The aim of our work was the study of immunohistochemical surfactant expression in the lungs of patients died following SARS-CoV-2 ARDS, in order to shed light on a possible therapeutic surfactant administration. PATIENTS AND METHODS: We investigated four patients who died due to ARDS following SARS-COV-2 infection and four patients submitted to lung biopsy, in the absence of SARS-CoV-2 infection. In all 8 cases, lung specimens were immunostained with anti-surfactant protein A (SP-A) and B (SP-B). RESULTS: In control subjects, reactivity for SP-B was restricted to type II alveolar cells. Immunostaining for SP-A was observed on the surface of alveolar spaces. In the COVID-19 positive lungs, immunoreactivity for SP-B was similar to that observed in control lungs; SP-A was strongly expressed along the alveolar wall. Moreover, dense aggregates of SP-A positive material were observed in the alveolar spaces. CONCLUSIONS: Our immunohistochemical data show the dysregulation of surfactant production in COVID-19 patients, particularly regarding SP-A expression. The increased presence of SP-A in condensed masses inside alveolar spaces could invalidate the therapeutic efficacy of the treatment with exogenous surfactant.

Familial Pulmonary Fibrosis: Genetic Features and Clinical Implications.

Pulmonary fibrosis comprises a wide range of fibrotic lung diseases with unknown pathogenesis and poor prognosis. Familial pulmonary fibrosis (FPF) represents a unique subgroup of patients in which at least one other relative is also affected. Patients with FPF exhibit a wide range of pulmonary fibrosis phenotypes, although idiopathic pulmonary fibrosis is the most common subtype. Despite variable disease manifestations, patients with FPF experience worse survival compared with their counterparts with the sporadic disease form. Therefore, ascertaining a positive family history not only provides prognostic value but should also raise suspicion for the inheritance of an underlying causative genetic variant within kindreds. By focusing on FPF kindreds, rare variants within surfactant metabolism and telomere maintenance genes have been discovered. However, such genetic variation is not solely restricted to FPF, as similar rare variants are found in patients with seemingly sporadic pulmonary fibrosis, further supporting the idea of genetic susceptibility underlying pulmonary fibrosis as a whole. Researchers are beginning to show how the presence of rare variants may inform clinical management, such as informing predisposition risk for yet unaffected relatives as well as informing prognosis and therapeutic strategy for those already affected. Despite these advances, rare variants in surfactant and telomere-related genes only explain the genetic basis in about one-quarter of FPF kindreds. Therefore, research is needed to identify the missing genetic contributors of pulmonary fibrosis, which would not only improve our understanding of disease pathobiology but may offer additional opportunities to improve the health of patients.

A primate-specific RNA-binding protein (RBMXL3) is involved in glucocorticoid regulation of human pulmonary surfactant protein B (SP-B) mRNA stability.

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate levels of surfactant protein B (SP-B). Dexamethasone (DEX) increases human SP-B expression, in part, through increased SP-B mRNA stability. A 30-nt-long hairpin element (RBE) in the 3'-untranslated region of human SP-B mRNA mediates both DEX-induced and intrinsic mRNA stabilities, but the mechanism is unknown. Proteomic analysis of RBE-interacting proteins identified a primate-specific protein, RNA-binding motif X-linked-like-3 (RBMXL3). siRNA directed against RBMXL3 reduces DEX-induced SP-B mRNA expression in human bronchoalveolar cells. Human SP-B mRNA stability, measured by our dual cistronic plasmid assay, is unaffected by DEX in mouse lung epithelial cells lacking RBMXL3, but DEX increases human SP-B mRNA stability when RBMXL3 is expressed and requires the RBE. In the absence of DEX, RBE interacts with cellular proteins, reducing intrinsic SP-B mRNA stability in human and mouse lung epithelial cells. RBMXL3 specifically binds the RBE in vitro, whereas RNA immunoprecipitation and affinity chromatography analyses indicate that binding is enhanced in the presence of DEX. These results describe a model where intrinsic stability of human SP-B mRNA is reduced through binding of cellular mRNA decay factors to RBE, which is then relieved through DEX-enhanced binding of primate-specific RBMXL3.

Recombinant expression of the precursor of rat lung surfactant protein B in Escherichia coli and its antibacterial mechanism.

While the discovery of antibiotics has made a huge contribution to medicine, bacteria that are resistant to many antibiotics pose new challenges to medicine. Antimicrobial peptides (AMPs), a new kind of antibiotics, have attracted people's attention because they are not prone to drug resistance. In this study, glutathione transferase (GST) was used as a fusion partner to recombinantly expressed rat lung surfactant protein B precursor (proSP-B) in E. coli pLySs. Cck-8 evaluated the cytotoxicity of the fusion protein and calculated its 50% inhibitory concentration (IC50). The purified peptides showed broad-spectrum antibacterial activity using filter paper method and MIC, and propidium iodide (PI) was used to explore the antibacterial mechanism against Staphylococcus aureus. In addition, the pEGFP-N2-proSP-B vector was constructed to explore the localization of proSP-B in CCL-149 cells. We found that proSP-B has obvious antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi, and has broad-spectrum antibacterial activity. Besides, proSP-B fusion protein has low toxicity and can change the permeability of Staphylococcus aureus cell membrane to realize its antibacterial. For these reasons, proSP-B can be used as a potential natural antibacterial drug.

A lung-on-chip model of early Mycobacterium tuberculosis infection reveals an essential role for alveolar epithelial cells in controlling bacterial growth.

We establish a murine lung-on-chip infection model and use time-lapse imaging to reveal the dynamics of host-Mycobacterium tuberculosis interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models and to probe the direct role of pulmonary surfactant in early infection. Surfactant deficiency results in rapid and uncontrolled bacterial growth in both macrophages and alveolar epithelial cells. In contrast, under normal surfactant levels, a significant fraction of intracellular bacteria are non-growing. The surfactant-deficient phenotype is rescued by exogenous addition of surfactant replacement formulations, which have no effect on bacterial viability in the absence of host cells. Surfactant partially removes virulence-associated lipids and proteins from the bacterial cell surface. Consistent with this mechanism, the attenuation of bacteria lacking the ESX-1 secretion system is independent of surfactant levels. These findings may partly explain why smokers and elderly persons with compromised surfactant function are at increased risk of developing active tuberculosis.

Tuberculosis is a contagious respiratory disease caused by the bacterium Mycobacterium tuberculosis. Droplets in the air carry these bacteria deep into the lungs, where they cling onto and infect lung cells. Only small droplets, holding one or two bacteria, can reach the right cells, which means that just a couple of bacterial cells can trigger an infection. But people respond differently to the bacteria: some develop active and fatal forms of tuberculosis, while many show no signs of infection. With no effective tuberculosis vaccine for adults, understanding why individuals respond differently to Mycobacterium tuberculosis may help develop treatments. Different responses to Mycobacterium tuberculosis may stem from the earliest stages of infection, but these stages are difficult to study. For one thing, tracking the movements of the few bacterial cells that initiate infection is tricky. For another, studying the molecules, called 'surfactants', that the lungs produce to protect themselves from tuberculosis can prove difficult because these molecules are necessary for the lungs to inflate and deflate normally. Normally, the role of a molecule can be studied by genetically modifying an animal so it does not produce the molecule in question, which provides information as to its potential roles. Unfortunately, due to the role of surfactants in normal breathing, animals lacking them die. Therefore, to reveal the role of some of surfactants in tuberculosis, Thacker et al. used 'lung-on-chip' technology. The 'chip' (a transparent device made of a polymer compatible with biological tissues) is coated with layers of cells and has channels to simulate air and blood flow. To see what effects surfactants have on M. tuberculosis bacteria, Thacker et al. altered the levels of surfactants produced by the cells on the lung-on-chip device. Two types of mouse cells were grown on the chip: lung cells and immune cells. When cells lacked surfactants, bacteria grew rapidly on both lung and immune cells, but when surfactants were present bacteria grew much slower on both cell types, or did not grow at all. Further probing showed that the surfactants pulled out proteins and fats on the surface of M. tuberculosis that help the bacteria to infect their host, highlighting the protective role of surfactants in tuberculosis. These findings lay the foundations for a system to study respiratory infections without using animals. This will allow scientists to study the early stages of Mycobacterium tuberculosis infection, which is crucial for finding ways to manage tuberculosis.

A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency.

Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.

Proteomics of Galápagos Marine Iguanas Links Function of Femoral Gland Proteins to the Immune System.

Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards. Although the lipids and volatile substances secreted by the femoral glands have been investigated in several biochemical studies, the protein composition and functions of secretions remain completely unknown. Applying a proteomic approach, we provide the first attempt to comprehensively characterize the protein composition of femoral gland secretions from the Galápagos marine iguana. Using samples from several organs, the marine iguana proteome was assembled by next-generation sequencing and MS, resulting in 7513 proteins. Of these, 4305 proteins were present in the femoral gland, including keratins, small serum proteins, and fatty acid-binding proteins. Surprisingly, no proteins with discernible roles in partner recognition or inter-species communication could be identified. However, we did find several proteins with direct associations to the innate immune system, including lysozyme C, antileukoproteinase (ALP), pulmonary surfactant protein (SFTPD), and galectin (LGALS1) suggesting that the femoral glands function as an important barrier to infection. Furthermore, we report several novel anti-microbial peptides from the femoral glands that show similar action against Escherichia coli and Bacillus subtilis such as oncocin, a peptide known for its effectiveness against Gram-negative pathogens. This proteomics data set is a valuable resource for future functional protein analysis and demonstrates that femoral gland secretions also perform functions of the innate immune system.

Clinical-pathological and molecular characterization of long-term survivors with advanced non-small cell lung cancer.

Objective: Long-term survivors (LS) of non-small cell lung cancer (NSCLC) without driver alterations, displaying an overall survival (OS) of more than 3 years, comprise around 10% of cases in several series treated with chemotherapy. There are classical prognosis factors for these cases [stage, Eastern Cooperative Oncology Group (ECOG), etc.], but more data are required in the literature. In this multi-center study, we focused on LS of advanced NSCLC with OS above 36 months to perform a clinical-pathological and molecular characterization. Methods: In the first step, we conducted a clinical-pathological characterization of the patients. Afterwards, we carried out a genetic analysis by comparing LS to a sample of short-term survivors (SS) (with an OS less than 9 months). We initially used whole-genome RNA-seq to identify differentiating profiles of LS and SS, and later confirmed these with reverse transcription-polymerase chain reaction (RT-PCR) for the rest of the samples. Results: A total of 94 patients were included, who were mainly men, former smokers, having adenocarcinoma (AC)-type NSCLC with an ECOG of 0-1. We obtained an initial differential transcriptome expression, displaying 5 over- and 33 under-expressed genes involved in different pathways: namely, the secretin receptor, surfactant protein, trefoil factor 1 (TFF1), serpin, Ca-channels, and Toll-like receptor (TLRs) families. Finally, RT-PCR analysis of 40 (20 LS/20 SS) samples confirmed that four genes (surfactant proteins and SFTP) were significantly down-regulated in SS compared to LS by using an analysis of covariance (ANCOVA) model: SFTPA1 (P = 0.023), SFTPA2 (P = 0.027), SFTPB (P = 0.02), and SFTPC (P = 0.047). Conclusions: We present a sequential genetic analysis of a sample of NSCLC LS with no driver alterations, obtaining a differential RNA-seq/RT-PCR profile showing an abnormal expression of SF genes.

Impact of genetic factors on fibrosing interstitial lung diseases. Incidence and clinical presentation in adults.

At least 10% of patients with pulmonary fibrosis, whether idiopathic or secondary, present heritable pulmonary fibrosis suspected on familial aggregation of pulmonary fibrosis, specific syndromes or early age of diagnosis. Approximately 30% of those patients have an identified mutation mostly in telomere related genes (TRG) more rarely in surfactant homeostasis or other genes. TRG mutation may be associated with hematological and hepatic diseases that may worsen after lung transplantation requiring a specific care and adapted immunosuppression. Surfactant genes mutations are usually associated with ground-glass opacities and cysts on CT scan and may improve with steroids, hydroxychloroquine or azithromycin. Moreover relatives should benefit from a genetic analysis associated with a clinical evaluation according to the gene involved. Genetics of pulmonary fibrosis raise specific problems from diagnosis, therapy or genetic counseling varying from one gene to another.

Gestational vitamin D deficiency impairs fetal lung development through suppressing type II pneumocyte differentiation.

Gestational vitamin D deficiency is associated with pulmonary diseases. This study aimed to investigate the effect of gestational vitamin D deficiency on fetal lung development in mice. Absolute and relative weights of fetal lungs were reduced in vitamin D deficient (VDD) group. Incrassate mesenchyme, measured by septal wall thickness, accompanied by lessened saccular space, was shown in VDD group. Numerous immature type II pneumocytes, as determined by PAS staining, were observed in VDD group. Moreover, increased Ki67-positive cells, a marker of cell proliferation, was detected in VDD group. The additional experiments showed that Sftpa, Sftpb, Sftpc and Sftpd, four surfactant genes, were downregulated and pro-surfactant protein B was reduced in VDD group. FoxA1, FoxA2 and TTF-1, three transcription factors that regulate surfactant genes, and VEGF, a key regulator for pulmonary maturation, were downregulated in VDD group. These results suggest that gestational vitamin D deficiency impairs fetal lung development partially through suppressing type II pneumocyte differentiation.

MicroRNA­431 inhibits the expression of surfactant proteins through the BMP4/activin/TGF­ß signaling pathway by targeting SMAD4.

The synthesis and secretion of surfactant proteins (SPs) is an important sign of lung maturation. Furthermore, the morbidity of lung developmental diseases, including respiratory distress syndrome and bronchopulmonary dysplasia which are mainly caused by immature lung development and lack of SPs, is increasing. As is well known, multiple microRNAs (miRs/miRNAs) are able to influence lung development via numerous different signaling pathways. However, few studies examine the association between the miRNAs and lung developmental diseases. A previous study has demonstrated that miR­431 was significantly (F=33.49; P<0.001) downregulated in the lung tissues of Sprague­Dawley rats at 3 time points, embryonic day 19, embryonic day 21 and postnatal day 3. The present study reported that the regulation of miR­431 may influence the expression of SPs. Thus, the further potential mechanisms of miR­431 in negatively regulating lung development were examined in the present study. Stable A549 cell lines overexpressing or knocking down SMAD family member 4 (SMAD4) transfected with miR­431 overexpressed or knocked down, and their control groups were established. Subsequently, the expression of bone morphogenetic protein 4 (BMP4), SMAD4 and SPs (SP­A, SP­B and SP­C) at the RNA and protein levels were validated respectively by reverse transcription quantitative PCR and western blotting. miR­431 exhibited a decreased expression, while BMP4 and SPs exhibited increased expression at the mRNA and protein levels in the SMAD4 knockdown group. Meanwhile, the expression of SPs were reduced in the SMAD4­knockdown group via overexpressing miR­431 and increased in the SMAD4­overexpression group via inhibiting miR­431. The present results indicate that SMAD4 negatively regulates the expression of SPs, and that miR­431 negatively regulates the expression of SPs through inhibiting the BMP4/activin/transforming growth factor­ß signaling pathway by targeting SMAD4.

miR-20a-5p regulates pulmonary surfactant gene expression in alveolar type II cells.

MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR-20a-5p had such regulatory functions on alveolar type II (AT-II) cells. To accomplish this, miR-20a-5p-overexpressed and miR-20a-5p-inhibited adenoviral vectors were constructed and transfected into cultured AT-II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR-20a-5p levels were verified by real-time PCR. The CCK-8 assay was used to compare the proliferation ability of AT-II cells that had over- or underexpressed miR-20a-5p. The expression of surfactant-associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real-time PCR and Western blotting. In AT-II cells, transfection resulted in over- or under-regulation of miR-20a-5p. While overexpression of miR-20a-5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR-20a-5p regulation of PTEN. As a result, when miR-20a-5p was rendered overexpressed, PTEN was down-regulated. By contrast, when miR-20a-5p was underexpressed, PTEN was up-regulated. Neither overexpression nor underexpression of miR-20a-5p altered the cell proliferation. miR-20a-5p plays no role in proliferation of foetal AT-II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.

Expression and potential regulation of miRNA­431 during lung development of Sprague­Dawley rats.

Deficiency of surfactant proteins (SPs) is the main cause of respiratory distress syndrome (RDS) and chronic lung diseases. Our previous study demonstrated that miR­431 was differentially expressed between infants with RDS and infants without RDS using microarray analysis. However, the potential role of miR­431 in the development of lung function is still unknown. In the present study, the morphological characteristics of lung tissues and the expression levels of miR­431 were examined at three time points of rat lung development [gestational days 19 and 21 (E19, and E21) and postnatal day (P3)]. The protein and mRNA levels of SMAD4 and SPs (SP­A, SP­B, SP­C and SP­D) were also validated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis, respectively. The expression levels of miR­431 were gradually decreased over time periods of E19, E21 and P3, as determine using RT­qPCR and fluorescence in situ hybridization. Dual luciferase­reporter assays revealed that SMAD4 is a direct target of miR­431. The mRNA and protein expression levels of SMAD4 and SPs increased gradually in rat lung tissues from E19 to P3. The order of magnitude was as follows: E19, E21 and P3. The present study demonstrated that the expression level of miR­431 decreased in the order of E19, E21 and P3 during rat lung development. The target gene of miR­431, SMAD4, was negatively regulated by miR­431, and its expression levels in the rat lung tissue increased from E19 to the P3. Surfactant synthesis was further increased over the E19 to P3 time period. Further studies are required to determine how miR­431 regulates pulmonary surfactant synthesis by targeting SMAD4.

Organic dust inhibits surfactant protein expression by reducing thyroid transcription factor-1 levels in human lung epithelial cells.

Exposure to organic dust is a risk factor for the development of respiratory diseases. Surfactant proteins (SP) reduce alveolar surface tension and modulate innate immune responses to control lung inflammation. Therefore, changes in SP levels could contribute to the development of organic-dust-induced respiratory diseases. Because information on the effects of organic dust on SP levels is lacking, we studied the effects of dust from a poultry farm on SP expression. We found that dust extract reduced SP-A and SP-B mRNA and protein levels in H441 human lung epithelial cells by inhibiting their promoter activities, but did not have any effect on SP-D protein levels. Dust extract also reduced SP-A and SP-C levels in primary human alveolar epithelial cells. The inhibitory effects were not due to LPS or protease activities present in dust extract or mediated via oxidative stress, but were dependent on a heat-labile factor(s). Thyroid transcription factor-1, a key transcriptional activator of SP expression, was reduced in dust-extract-treated cells, indicating that its down-regulation mediates inhibition of SP levels. Our study implies that down-regulation of SP levels by organic dust could contribute to the development of lung inflammation and respiratory diseases in humans.