Surfactant-associated Protein B Research Articles

SFTPB in serum extracellular vesicles as a biomarker of progressive pulmonary fibrosis.

Progressive pulmonary fibrosis (PPF), defined as the worsening of various interstitial lung diseases (ILDs), currently lacks useful biomarkers. To identify novel biomarkers for early detection of patients at risk of PPF, we performed a proteomic analysis of serum extracellular vesicles (EVs). Notably, the identified candidate biomarkers were enriched for lung-derived proteins participating in fibrosis-related pathways. Among them, pulmonary surfactant-associated protein B (SFTPB) in serum EVs could predict ILD progression better than the known biomarkers, serum KL-6 and SP-D, and it was identified as an independent prognostic factor from ILD-gender-age-physiology index. Subsequently, the utility of SFTPB for predicting ILD progression was evaluated further in 2 cohorts using serum EVs and serum, respectively, suggesting that SFTPB in serum EVs but not in serum was helpful. Among SFTPB forms, pro-SFTPB levels were increased in both serum EVs and lungs of patients with PPF compared with those of the control. Consistently, in a mouse model, the levels of pro-SFTPB, primarily originating from alveolar epithelial type 2 cells, were increased similarly in serum EVs and lungs, reflecting pro-fibrotic changes in the lungs, as supported by single-cell RNA sequencing. SFTPB, especially its pro-form, in serum EVs could serve as a biomarker for predicting ILD progression.

HDL proteome remodeling associates with COVID-19 severity

BackgroundBesides the well-accepted role in lipid metabolism, high-density lipoprotein (HDL) also seems to participate in host immune response against infectious diseases.Objective: We used a quantitative proteomic approach to test the hypothesis that alterations in HDL proteome associate with severity of Coronavirus disease 2019 (COVID-19).MethodsBased on clinical criteria, subjects (n=41) diagnosed with COVID-19 were divided into two groups: a group of subjects presenting mild symptoms and a second group displaying severe symptoms and requiring hospitalization. Using a proteomic approach, we quantified the levels of 29 proteins in HDL particles derived from these subjects.ResultsWe showed that the levels of serum amyloid A 1 and 2 (SAA1 and SAA2, respectively), pulmonary surfactant-associated protein B (SFTPB), apolipoprotein F (APOF), and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) were increased by more than 50% in hospitalized patients, independently of sex, HDL-C or triglycerides when comparing with subjects presenting only mild symptoms. Altered HDL proteins were able to classify COVID-19 subjects according to the severity of the disease (error rate 4.9%). Moreover, apolipoprotein M (APOM) in HDL was inversely associated with odds of death due to COVID-19 complications (odds ratio [OR] per 1-SD increase in APOM was 0.27, with 95% confidence interval [CI] of 0.07 to 0.72, P=0.007).ConclusionOur results point to a profound inflammatory remodeling of HDL proteome tracking with severity of COVID-19 infection. They also raise the possibility that HDL particles could play an important role in infectious diseases.

Pulmonary surfactant-associated protein B regulates prostaglandin-endoperoxide synthase-2 and inflammation in chronic obstructive pulmonary disease.

What is the central question of this study? It is reported that polymorphism of the gene for pulmonary surfactant-associated protein B (SFTPB) is associated with chronic obstructive pulmonary disease (COPD): what are the function and mechanism of action of SFTPB in COPD? What is the main finding and its importance? Under stimulation of the risk factors of COPD, SFTPB expression is decreased, which may be involved in the formation of COPD. The progress of COPD induces an inflammatory response and reduces SFTPB expression. Levels of prostaglandin-endoperoxide synthase-2 (PTGS2) and inflammatory responses are changed by SFTPB, which indicates that SFTPB promotes the progression of COPD by PTGS2 and inflammation. Pulmonary surfactant-associated protein B (SFTPB) is a critical protein for lung homeostasis, and polymorphism of its gene is associated with chronic obstructive pulmonary disease (COPD). However, few studies have so far confirmed the functional involvement of SFTPB in COPD. Serum SFTPB and inflammatory cytokine levels were measured in 54 patients with acute exacerbation of COPD and 29 healthy controls. A549 cells were induced using 10% cigarette smoke extract (CSE) and treated with dexamethasone to investigate the effect of inflammation on SFTPB expression, and the effect of SFTPB overexpression and silencing on inflammatory cytokines was measured using real-time PCR and enzyme-linked immunosorbent assay. SFTPB expression was assessed in mouse lung tissues using immunofluorescence. Serum levels of SFTPB were significantly lower in COPD patients than in controls (P=0.009). Conversely, levels of interleukin (IL)-6 and prostaglandin-endoperoxide synthase-2 (PTGS2) were increased in COPD patients (IL-6: P=0.006; PTGS2: P=0.043). After CSE treatment, SFTPB mRNA and protein levels were significantly decreased compared to controls (mRNA: P=0.002; protein: P=0.011), while IL-6, IL-8 and PTGS2 were elevated. Dexamethasone treatment increased SFTPB levels. Following overexpression of SFTPB in A549 cells, mRNA and protein levels of IL-6, IL-8 and PTGS2 were significantly reduced, while gene silencing induced the opposite effect. SFTPB levels were significantly reduced in the lung tissue of a mouse model of COPD compared to controls. Reduced SFTPB levels may induce PTGS2 and inflammatory responses in COPD and SFTPB could be a key protein for evaluation of COPD progression.

Structural and Functional Insight into Recombinant Lung Surfactant Protein B (rSP-B)

Surfactant associated-protein B (SP-B) is the most essential protein component of lung surfactant; its absence is fatal. Lung surfactant associated proteins, especially SP-B, assist in the organization and rearrangement of the lipid monolayers and multilayers that coat the air-water interface of the alveolus, and so help to reduce the work of breathing and prevent lung collapse. Due to SP-B's unique hydrophobic nature however, the overall three dimensional structure of the protein is not yet determined, therefore the molecular basis for its activity is not clearly understood. Recently, our lab has managed to produce the protein in bacteria using recombinant DNA technology. Recombinant SP-B (rSP-B) is shown to retain the expected alpha helical conformation in different membrane mimetic conditions, over a range of temperatures as observed by far UV circular dichroism (CD) spectroscopy. Dynamic Light Scattering (DLS) of rSP-B suspended in detergent micelles indicates two populations, one with a hydrodynamic size of ∼3 nm and the other at ∼100 nm. Nanoparticle Tracking Analysis (NTA) confirmed the ∼100 nm species to be prominent. Preliminary 1D solution NMR experiments have also been carried out.Moreover, rSP-B also shows promising results in in vitro measures of functionality when tested with a Langmuir-Blodgett trough. Lipid films in the presence of rSP-B are able to promote multilayer formation when compressed to a sufficiently high surface pressure, comparable to that of clinical lung surfactants. What is more, films in the presence of rSP-B demonstrate superior compressibility and film recovery when compared to films compressed without rSP-B.

Identification of vitamin D sensitive pathways during lung development.

BackgroundWe have previously shown that vitamin D deficiency has a detrimental impact on lung development. In this study, we aimed to identify the mechanisms linking vitamin D with lung development using a mouse model of dietary manipulation.MethodsFemale offspring were euthanized at different time-points; embryonic day (E)14.5, E17.5 or postnatal day (P)7. Lung tissue was collected for mass spectrometry-based proteomic analysis. Label-free quantitation was used to identify the differentially expressed proteins and ELISA confirmed the expression of selected proteins. Lungs from separate groups of mice were fixed and processed for stereological assessment of lung structure.ResultsNo differences in protein expression between vitamin D deficient and replete mice were detected at E14.5 and E17.5, whereas 66 proteins were differentially expressed in P7 lungs. The expression of pulmonary surfactant-associated protein B (SP-B) and peroxiredoxin 5 (PRDX5) were reduced in P7 lungs of vitamin D deficient mice, while the production of collagen type Ι alpha 1 (COL1A1) was higher in lungs of vitamin D deficient mice. There were no differences in lung volume, parenchymal volume, volume of airspaces or surface area of airspaces between vitamin D deficient and vitamin D replete mice across three time-points.ConclusionsThe difference in protein expression during the early postnatal time-point suggests that vitamin D deficiency may induce alterations of lung structure and function in later life during alveolarization stage through impaired pulmonary surfactant production and anti-oxidative stress ability as well as enhanced collagen synthesis. These data provided a plausible mechanism linking maternal vitamin D deficiency with altered postnatal lung function.

Gene mutation of pulmonary surfactant-associated protein-B and adenosine triphosphate binding cassette transporter A3 with neonatal respiratory distress syndrome

Gene mutation of pulmonary surfactant-associated protein-B and adenosine triphosphate binding cassette transporter A3 with neonatal respiratory distress syndrome

21 CHARACTERIZATION OF THE micro-RNA TRANSCRIPTOME IN LUNG TISSUES OF CLONED CALVES SUFFERING FROM RESPIRATORY DISTRESS SYNDROME

Developmental deficiency leads to low survival rates of newborns, especially in cloned animals. Alveoli collapse leading to respiratory failure is one of the major causes of death in newborn cloned calves. The present study provides an insight into the expression pattern of micro-RNAs (miRNAs) in lung tissues and their role in the respiratory distress syndrome (RDS) in the cloned calves. Short RNA high-throughput sequencing and bioinformatic analysis from small RNA libraries created from collapsed lung tissues from 4 newborn cloned calves with RDS and normal lung tissues from 4 age-matched healthy individuals were implemented. Lung tissues were collected by dissection from newborns that died due to RDS and from healthy individuals on the first day after birth. RNA samples from the lung tissues were processed to generate small RNA libraries that were further used for deep sequencing. Expression profiles of surfactant-associated protein B (SPB), surfactant-associate protein C (SPC), and their key transcription regulator thyroid transcription factor-1 (TTF-1), which are responsible for stabilising alveolar surface, reducing surface tension, and thus preventing alveoli collapse, were verified through real-time RT-PCR, Western blot, and immunohistochemistry (IHC). Differentially expressed (DE) miRNAs were quantified by edgeR (empirical analysis of digital gene expression data in R), and their target genes were predicted by both TargetScan and miRanda software. Only miRNAs with P values <0.05 were considered statistically significant (Fisher exact test). Sequence analysis revealed the presence of 1592 and 1777 miRNAs in the RDS and healthy groups, respectively. A total of 326 miRNAs were DE between the two groups according to our criteria, of which 179 miRNAs were up-regulated and 147 were down-regulated in the RDS group. Gene ontological analysis showed that the DE miRNAs had a primary role in DNA-dependent regulation of transcription, cytoplasm biosynthesis, and nucleotide binding. Eleven miRNAs (bta-miR-186, bta-miR-2284x_R+1, bta-miR-24–3p_R-2, bta-miR-424–3p, bta-miR-592_L-1, bta-miR-660, bta-miR-150_R-1, bta-miR-2478_L-2, bta-miR-450b_R-1, bta-miR-134_L+2R-2 and bta-miR-326_R+1) were DE between the 2 groups and were predicted to target SPB, SPC, and TTF-1, respectively. Among these DE miRNAs, 5 miRNAs (bta-miR-134_L+2R-2, bta-miR-424–3p, bta-miR-660, bta-miR-2478_L-2, bta-miR-450b_R-1) were up-regulated in the RDS group. Western blot and IHC confirmed the down-regulation of SPB, SPC, and TTF-1 at the protein level in RDS group. This increase in abundance of miRNAs targeting key regulatory genes in lung of newborn cloned calves may take part in the dysregulation of alveolus development leading to alveoli collapse and RDS. The assay for target gene verification and analysis of gene transcription profile are under study.Y. Liu and Y. Zhang contributed equally to this work. This project was supported by the National Natural Science Foundation of China (No. 31301977) and the National Nonprofit Institute Research Grant (No. 2011cj-11).

Expression profiling of abundant genes in pulmonary and cardiac muscle tissues of Tibetan Antelope (Pantholops hodgsonii)

The Tibetan Antelope (TA), which has lived at high altitude for millions of years, was selected as the model species of high hypoxia-tolerant adaptation. Here we constructed two cDNA libraries from lung and cardiac muscle tissues, obtained EST sequences from the libraries, and acquired extensive expression data related energy metabolism genes. Comparative analyses of synonymous (Ks) and nonsynonymous (Ka) substitution rates of nucleus-encoded mitochondrial unigenes among different species revealed that many antelope genes have undergone rapid evolution. Surfactant-associated protein A (SP-A) and surfactant-associated protein B (SP-B) genes in the AT lineage experienced accelerated evolution compared to goat and sheep, and these two genes are highly expressed in the lung tissue. This study suggests that many specific genes of lung and cardiac muscle tissues showed unique expression profiles and may undergo fast adaptive evolution in TA. These data provide useful information for studying on molecular adaptation to high-altitude in humans as well as other mammals.

Expression of Pulmonary Surfactant-Associated Protein B in Neonatal Respiratory Distress Syndrome

Summary Background: The aim of this study was to investigate the role of pulmonary surfactant-associated protein B (SP-B) expression in the pathogenesis of neonatal respiratory distress syndrome (RDS) via detecting the protein and mRNA expression of SP-B. Methods: A total of 60 unrelated neonates who died of RDS were chosen as the RDS group and then subgrouped into ≤32 weeks group, 32∼37 weeks group and ≥37 weeks group (n=20). Sixty neonates who died of other diseases were enrolled as controls and subdivided into 3 matched groups based on the gestational age. Western blot assay and RT-PCR were employed. Results: In the RDS group, SP-B protein expression was reduced or deficient in 8 neonates of which 6 had no SP-B protein expression. In the control group, only 1 had reduced SP-B protein expression. The reduced or deficient SP-B protein expression in 9 neonates of both groups was noted in the ≥37 weeks group. In the RDS group, the SP-B mRNA expression was significantly lower than that in the control group. In the ≤37 weeks group, SP-B mRNA expression was comparable between the RDS group and control group. In the 32∼37 weeks group, the SP-B mRNA expression in the RDS group was significantly reduced when compared with the control group. In the ≥37 weeks group, the SP-B mRNA expression in the RDS group was dramatically lower than that in the control group. Conclusions: Alteration of SP-B expression is present at transcriptional and translational levels. Reduction of SP-B mRNA and protein expression is involved in the pathogenesis of RDS.

Gene polymorphism in intron 4 of surfactant protein-B in bronchopulmonary dysplasia infants

Objective To investigate the change of gene polymorphorism of surfactant protein-B (SP-B) intron 4 in infants with bronchopulmonary dysplasia (BPD). Methods Forty-five infants with BPD (BPD group) and ninety-nine infants without lung diseases (control group) who admitted into Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology from July 2008 to July 2011 were selected into this study.Genotyping for fragment length polymorphism of SP-B intron 4 was performed by polymerase chain reaction (PCR),agarose gel electrophoresis,cloning and sequencing methods in both groups.Differences of allele frequencies (invariant allele and variant allele) and genotype frequencies (invariant genotype and variant genotype) between BPD group and control group were analyzed.The differences of gestational age and birth weight between the two groups were compared with Independent-Samples t test.The gender composition and differences of allele or genotype frequencies between the two groups were compared with Chi-square test. Results Invariant allele frequencies in BPD group and control group were 83.3％ (75/90) and 92.0％ (182/198),and variant allele frequencies were 16.7％ (15/90,including eight insertion alleles and seven deletion alleles) and 8.1％ (16/198,including eight insertion alleles and eight deletion alleles).There were significant differences between the two groups (x2 =4.75,P =0.029).In BPD group,there were 32 cases (71.1 ％,32/45) invariant genotypes and 13 cases (28.9 ％,13/45,including seven cases insertions and six cases deletions) variant genotypes; in the control group,there were 85 cases invariant genotypes (85.8％,85/99) and 14 cases (14.1％,14/99,six insertions and eight deletions) variant genotypes.Significant difference was found between the two groups (x2=4.42,P＜0.036). Conclusions Variations of SP-B intron 4 were more in BPD infants,and the variation of SP-B intron 4 might be associated with BPD. Key words: Bronchopulmonary dysplasia; Pulmonary surfactant-associated protein-B; Introns; Polymorphism,genetic

Response to Cummings

We would like to thank Dr Cummings for giving us the opportunity to explain our study results. Results from nine randomized, controlled, trials (RCTs),1, 2, 3, 4, 5, 6, 7, 8, 9 including the one that has been just published (e-pub ahead of print) by Dizdar et al.9 and meta-analyses10, 11, 12 comparing animal-derived surfactants, namely, poractant alfa (PA), beractant (BE), bovactant (BO) and/or calfactant (CA), have consistently shown faster weaning of oxygen and mean airway pressure, less need for redosing, fewer days on oxygen and mechanical ventilation, shorter length of stay (LOS) as well as survival advantage in babies treated with PA. These advantages with PA over BE, CA or BO are likely related to major biological and/or biochemical differences between these animal-derived surfactant preparations. PA contains the highest amount of phospholipids when compared with BE or CA. Higher amount of phospholipids has been shown to downregulate oxidative functions in monocytes and confer better anti-inflammatory properties.13 In addition, bacterial growth in different surfactant preparations is influenced by microbial species and the composition and dose of the surfactant. PA was bactericidal in a dose-dependent fashion and differed from BE and BO, a surfactant preparation similar to CA.14 PA contains the highest amount of plasmalogens (PL) when compared with BE.15 BO contains the lowest amount of PL.15 PL are anti-oxidant phospholipids and presence of higher amounts of PL in the tracheal aspirates from pre-term infants has been shown to be associated with a lower risk for bronchopulmonary dysplasia (BPD).16 Also, the amount of surfactant-associated protein B (SP-B) is highest in PA when compared with BE or CA. SP-B is the most important SP in helping the phospholipids to rapidly adsorb at the air–liquid interphase and in decreasing surface tension. Furthermore, the phospholipid molecular species of PA is much closer to that of human surfactant.17, 18

Surfactant-Associated Protein B Is Critical to Survival in Nickel-Induced Injury in Mice

The etiology of acute lung injury is complex and associated with numerous, chemically diverse precipitating factors. During acute lung injury in mice, one key event is epithelial cell injury that leads to reduced surfactant biosynthesis. We have previously reported that transgenic mice that express transforming growth factor alpha (TGFA) in the lung were protected during nickel-induced lung injury. Here, we find that the mechanism by which TGFA imparts protection includes maintenance of surfactant-associated protein B (SFTPB) transcript levels and epidermal growth factor receptor-dependent signaling in distal pulmonary epithelial cells. This protection is complex and not accompanied by a diminution in inflammatory mediator transcripts or additional stimulation of antioxidant transcripts. In mouse lung epithelial (MLE-15) cells, microarray analysis demonstrated that nickel increased transcripts of genes enriched in MTF1, E2F-1, and AP-2 transcription factor-binding sites and decreased transcripts of genes enriched in AP-1-binding sites. Nickel also increased Jun transcript and DNA-binding activity, but decreased SFTPB transcript. Expression of SFTPB under the control of a doxycycline-sensitive promoter increased survival during nickel-induced injury as compared with control mice. Together, these findings support the idea that maintenance of SFTPB expression is critical to survival during acute lung injury.

Lucinactant: a novel synthetic surfactant for the treatment of respiratory distress syndrome

Lucinactant (Surfaxin®, Discovery Laboratories) is a synthetic surfactant, which contains the novel peptide, sinapultide, a surfactant-associated proteinB mimic. Randomised clinical trials suggest that this compound is a safe and effective treatment for respiratory distress syndrome in preterm infants. It is also being actively investigated for other indications, including meconium aspiration syndrome, treatment of bronchopulmonary dysplasia in neonates, acute respiratory distress syndrome and asthma. A novel aerosol formulation administered with nasal continuous positive airway pressure is also under development for treatment of respiratory insufficiency in neonates. Its non-animal origin may make it an attractive alternative to present animal-derived surfactants by eliminating the risks of infection and immunogenicity related to the latter.

Surfactant-Associated Protein B Kinetics In Vivo in Newborn Infants by Stable Isotopes

Surfactant-associated protein B (SP-B) is critical to the biophysical function of pulmonary surfactant. No information is available on SP-B synthesis and kinetics in humans. We administered a 24-h i.v. infusion of 13C-valine as metabolic precursor of SP-B to six newborn infants (weight 3.5+/-0.5 kg; age 12 d, range 1-43 d). Three of the study infants also received i.v. 2H-palmitate to label surfactant disaturated phosphatidylcholine (DSPC). SP-B and DSPC were isolated from tracheal aspirates, and their respective 13C and 2H enrichments were measured by gas chromatography-mass spectrometry. SP-B kinetics was measured successfully in all six infants. SP-B median (range) fractional synthesis rate was 30% per day (20-78% per day), secretion time was 4.5 h (1-9 h), time to peak was 24 h (12-36 h), and half-life was 21 h (8-35 h). The ascending part of the SP-B kinetic curve was similar to the DSPC curve, suggesting similar secretion pathways. SP-B half-life seemed to be shorter than DSPC half-life. These results agree with existing animal data. We conclude that the measurement of SP-B kinetics is feasible in vivo in humans by stable isotope technology.

Two-dimensional electrophoresis protein profiling and identification in rat bronchoalveolar lavage fluid following allergen and endotoxin challenge.

The protein content of bronchoalveolar lavage fluid (BALF) from actively sensitised Brown Norway (BN) rats challenged with allergen (ovalbumin, OA) and from naïve Brown Norway rats challenged with endotoxin (lipopolysaccharide, LPS) was analyzed and compared to healthy controls treated with vehicle only. BALF proteins were analyzed by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis and identified by peptide mass fingerprinting matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or nanoliquid chromatography-tandem MS (nanoLC-MS/MS) after in-gel trypsin digestion of selected 2-D gel spots. Our study shows that the BALF protein profile is significantly different in animals after allergen (OA) or endotoxin (LPS) challenge as compared to controls, concerning the content of proteins derived from plasma or produced locally in the lung. In both challenges the following proteins presented patterns which differed qualitatively compared to control: T-kininogen I and II, alpha-1-antitrypsin, calgranulin A, fetuin A and B, and haptoglobin. Other proteins were diminished in both challenges, such as Clara cell 10 kDa secretory protein (CC10) and pulmonary surfactant associated protein B (SP-B); c-reactive protein increased in the OA-challenge and decreased in the LPS-challenge, and pulmonary surfactant associated protein A (SP-A) was decreased in the OA-challenge and was not significantly changed in the LPS-challenge. The identified proteins could be important not only for the diagnosis but have also interesting implications for medical treatment of lung inflammatory conditions. Furthermore, even if based on a limited number of animals, our results are of interest for the identification of lung protein markers and a better understanding of the mechanisms involved in the pathogenesis of lung diseases.

Changes in lung-specific molecular expression during differentiation of hamster embryonic M3E3/C3 cell line

To clarify the differentiation mechanisms of bronchiolar epithelial cells, changes at the transcription level of epithelial cell-specific proteins were examined using M3E3/C3, a cell line derived from hamster fetal lung. During a 9-day incubation period with 24 microg/mL of retinol, the cells became attached to each other and formed large bud-like structures which could be detected with periodic acid-Schiff staining. During the incubation period, the mRNA level of surfactant-associated protein-B significantly increased 2.6- and 5.4-fold higher than cells incubated without retinol on days 3 and 9, respectively. The Clara cell-specific secretory protein mRNA level also increased and peaked at 5.1-fold (P < 0.05) on day 5 compared with control cells. In contrast, mRNA for surfactant-associated protein-C, an alveolar type II cell-specific protein, decreased. Moreover, the expression of the gene for hepatocyte nuclear factor 3alpha, a putative transactivating factor for lung-related genes, was up-regulated resulting in consistently higher levels (2.4- to 5.6-fold) compared with controls, while those for transmembrane-type mucin-1 and glyceraldehyde-3-phosphate dehydrogenase were constantly expressed during the incubation. The present study confirms that at the gene transcription level M3E3/C3 cells differentiate into Clara-like cells with mucus granules in the presence of retinol.

Changes in lung-specific molecular expression during differentiation of hamster embryonic M3E3/C3 cell line

To clarify the differentiation mechanisms of bronchiolar epithelial cells, changes at the transcription level of epithelial cell-specific proteins were examined using M3E3/C3, a cell line derived from hamster fetal lung. During a 9-day incubation period with 24 microg/mL of retinol, the cells became attached to each other and formed large bud-like structures which could be detected with periodic acid-Schiff staining. During the incubation period, the mRNA level of surfactant-associated protein-B significantly increased 2.6- and 5.4-fold higher than cells incubated without retinol on days 3 and 9, respectively. The Clara cell-specific secretory protein mRNA level also increased and peaked at 5.1-fold (P < 0.05) on day 5 compared with control cells. In contrast, mRNA for surfactant-associated protein-C, an alveolar type II cell-specific protein, decreased. Moreover, the expression of the gene for hepatocyte nuclear factor 3alpha, a putative transactivating factor for lung-related genes, was up-regulated resulting in consistently higher levels (2.4- to 5.6-fold) compared with controls, while those for transmembrane-type mucin-1 and glyceraldehyde-3-phosphate dehydrogenase were constantly expressed during the incubation. The present study confirms that at the gene transcription level M3E3/C3 cells differentiate into Clara-like cells with mucus granules in the presence of retinol.

Dissociation of surfactant protein B from canine surfactant large aggregates during formation of small surfactant aggregates by in vitro surface area cycling

Pulmonary surfactant isolated by lavage can be separated into large aggregates (LA) and small aggregates (SA). Pulse labeling experiments have shown that the LA subtype is the precursor of the SA subtype. Conversion of LA to SA can be demonstrated in vitro using the technique of surface area cycling. The precise mechanisms of surfactant subtype conversion remain unknown. We have previously reported a decline in surfactant-associated protein B (SP-B) during in vitro subtype conversion of canine surfactant. This led to the hypothesis that SP-B may be degraded by a serine protease ‘convertase’ during cycling. The current studies used a quantitative slot-blot assay to investigate the fates of SP-A and SP-B during in vitro cycling. These studies confirmed some SP-A is present in SA, but SP-B is confirmed to LA. Conversion leads to an apparent loss of SP-B during cycling. However, SP-B can be recovered from the walls of polypropylene and Teflon tubes by washing with chloroform:methanol. Recovered SP-B migrated on non-reducing tricine gels as a single band with an apparent molecular weight of 17 kDa, corresponding to intact SP-B dimer. Reconstitution studies demonstrated that the recovered SP-B retained its surface active properties as determined on a pulsating bubble surfactometer. We conclude in vitro surface area cycling of canine LA results in the dissociation of SP-B from surfactant lipids resulting in an apparent decline in SP-B levels.

Depth profiles of pulmonary surfactant protein B in phosphatidylcholine bilayers, studied by fluorescence and electron spin resonance spectroscopy.

Pulmonary surfactant-associated protein B (SP-B) has been isolated from porcine lungs and reconstituted in bilayers of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) to characterize the extent of insertion of the protein into phospholipid bilayers. The parameters for the interaction of SP-B with DPPC or PC using different reconstitution protocols have been estimated from the changes induced in the fluorescence emission spectrum of the single protein tryptophan. All the different reconstituted SP-B-phospholipid preparations studied had similar Kd values for the binding of the protein to the lipids, on the order of a few micromolar. The depth of penetration of SP-B into phospholipid bilayers has been estimated by the parallax method, which compares the relative efficiencies of quenching of the protein fluorescence by a shallow or a deeper spin-labeled phospholipid probe. SP-B tryptophan was found to be located 10-13 A from the center of bilayers, which is consistent with a superficial location of SP-B in phosphatidylcholine membranes. Parallax experiments, as well as resonance energy transfer from SP-B tryptophan to an acceptor probe located in the center of the bilayer, indicate that there are significant differences in the extent of insertion of the protein, depending on the method of reconstitution. SP-B reconstituted from lipid/protein mixtures in organic solvents is inserted more deeply in PC or DPPC bilayers than the protein reconstituted by addition to preformed phospholipid vesicles. These differences in the extent of insertion lead to qualitative and quantitative differences in the effect of the protein on the mobility of the phospholipid acyl chains, as studied by spin-label electron spin resonance (ESR) spectroscopy, and could represent different functional stages in the surfactant cycle.

Different modes of interaction of pulmonary surfactant protein SP-B in phosphatidylcholine bilayers.

Pulmonary surfactant-associated protein B (SP-B) has been incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) by two different procedures to characterize the dependence of lipid-protein interactions on the method of reconstitution. In method A the protein was dissolved in a small volume of either methanol or 60% (v/v) acetonitrile and injected into an aqueous phase containing phospholipid vesicles. In method B the vesicles were prepared by injection of a mixture of phospholipid and SP-B dissolved in methanol or aqueous acetonitrile. Both methods of reconstitution led to the extensive interaction of SP-B with PC bilayers as demonstrated by co-migration during centrifugation, marked protection against proteolysis, change in the fluorescence emission intensity of SP-B, and protection of SP-B tryptophan fluorescence from quenching by acrylamide. SP-B promoted the rapid adsorption of DPPC on an air/liquid interface irrespective of the method of protein reconstitution. However, the interfacial adsorption activity of SP-B reconstituted by method B remained stable for hours, but that of SP-B prepared by method A decreased with time. Electron microscopy showed that the injection of SP-B into an aqueous phase containing PC or DPPC vesicles (method A) induced a rapid aggregation of vesicles. By contrast, a much longer time was required for detecting vesicle aggregation when the protein was reconstituted by co-injection of SP-B and phospholipids (method B). The presence of 5% (w/w) SP-B in DPPC bilayers prepared by method B broadened the differential scanning calorimetry thermogram and decreased the enthalpy of the transition. In contrast, the injection of SP-B into preformed DPPC vesicles (method A) did not influence the gel-to-liquid phase transition of DPPC bilayers. Taken together, these results indicate that the mode and extent of interaction of SP-B with surfactant phospholipids depends on the conditions of preparation of lipid/protein samples, and that care should be taken in the interpretation of findings from reconstituted systems on the role of these surfactant proteins in the alveolar space.