Human Surfactant Protein B Research Articles

Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease.

Human surfactant protein-B (SP-B), a hydrophobic protein, is essential for normal lung function. SP-B is expressed and secreted by specific lung cell types, i.e. alveolar type II and Clara cells, of the respiratory epithelium. The SP-B precursor (42 kDa) undergoes post-translational processing to generate an 8 kDa mature SP-B. A single-nucleotide polymorphism (SNP) at nucleotide 1580 (C/T) in exon 4 of SP-B that changes amino acid 131 from threonine to isoleucine (Thr131-->Ile) is associated with several pulmonary diseases. The Thr131-->Ile substitution can eliminate a potential N-linked glycosylation site, Asn129-Gln-Thr131, which is present in the SP-B variant of the C allele (ACT/Thr) but not in that of the T allele (ATT/Ile). To determine whether the C allele SP-B variant is indeed glycosylated at Asn(129)-Gln-Thr131, we first generated stably transfected Chinese hamster ovary cell lines that expressed each version of SP-B, and developed specific SP-B polyclonal anti-peptide antibodies. Using both the stably transfected cell lines and fetal lung explants, we observed that the C allele variant is indeed glycosylated at the Asn129-Gln-Thr131 site, whereas the T allele variant, which served as a control, is not. In addition, we also confirmed that both SP-B variants contain another N-linked glycosylation site, Asn311-Ser-Ser313. Given its association with several pulmonary diseases, this finding provides useful information for future studies in disease systems associated with this SNP. Further, we speculate that, given the fact that this SNP is found frequently in the general population, N-linked glycosylation at residue Asn129 interferes with SP-B processing, secretion and folding under certain disease conditions.

Specific Mode of Interaction Between Components of Model Pulmonary Surfactants Using Computer Simulations

Atomistic molecular dynamics simulations and structural bioinformatics tools enable the identification of the exact mode of interaction between model pulmonary surfactant components. Two nanosecondlong simulations of the N-terminal region of human surfactant protein-B (SP-B 1–25) in dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers of different lipid surface densities reveal the preferential affinity of SP-B 1–25 for anionic phospholipids. In particular, arginine 12 and lysine 24 interact strongly and with high specificity with the phosphate group of the DPPG lipids, stabilizing the position, the orientation, and the secondary structure of the peptide in the monolayer. The peptide lies at an oblique angle to the interfacial plane, ranging between 47° and 62°, increasing with decreasing lipid surface density. In DPPC monolayers the interaction is largely determined by hydrophobic interactions. The non-specific nature of DPPC–SP-B 1–25 interactions allows for significant flexibility in the topology of the peptide in the lipid matrix. Bioinformatics tools are employed to generalize the simulation results to the sequences of SP-B 1–25 in other organisms. The importance of specific residues, and the role of the largely helical and amphiphilic nature of the peptide in the functionality of SP-B 1–25 are established. The synergy of classical mechanics tools with bioinformatics methods greatly enhances the molecular-level interpretation of pulmonary surfactant action and facilitates the development of design rules for synthetic surfactant analogues.

Multilayer Formation upon Compression of Surfactant Monolayers Depends on Protein Concentration as Well as Lipid Composition: AN ATOMIC FORCE MICROSCOPY STUDY

The determinants for the formation of multilayers upon compression of surfactant monolayers were investigated by compressing films, beyond the squeeze-out plateau, to a surface tension of 22 millinewtons/m. Atomic force microscopy was used to visualize the topography of lipid films containing varying amounts of native surfactant protein B (SP-B). These films were compared with films containing synthetic peptides based on the N terminus of human SP-B: monomeric mSP-B-(1-25) or dimeric dSP-B-(1-25). The formation of typical hexagonal network structures as well as the height of protrusions were shown to depend on the concentration of SP-B. Protrusions of bilayer height were formed from physiologically relevant concentrations of 0.2-0.4 mol % (4.5-8.5 wt %) SP-B upwards. Much higher concentrations of SP-B-(1-25) peptides were needed to obtain network structures, and protrusion heights were not equal to those found for films with native SP-B. A striking observation was that while protrusions formed in films of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DPPG) (80/20) had single bilayer thickness, those formed in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (80/20) had various heights of multilayers, whereas those seen in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/DPPG (60/20/20) were mainly of bilayer height. For the first time direct observations by atomic force microscopy show (i) that a certain minimal concentration of SP-B is required for the formation of layered protrusions upon film compression, (ii) that protrusion height depends on whether the phospholipids contain an unsaturated fatty acyl chain, and (iii) that protrusion height also depends on whether the unsaturated acyl chain is present in phosphatidylcholine or in phosphatidylglycerol.

Human surfactant protein B promoter in transgenic mice: temporal, spatial, and stimulus-responsive regulation.

Surfactant protein B (SP-B) is a developmentally and hormonally regulated lung protein that is required for normal surfactant function. We generated transgenic mice carrying the human SP-B promoter (-1,039/+431 bp) linked to chloramphenicol acetyltransferase (CAT). CAT activity was high in lung and immunoreactive protein localized to alveolar type II and bronchiolar epithelial cells. In addition, thyroid, trachea, and intestine demonstrated CAT activity, and each of these tissues also expressed low levels of SP-B mRNA. Developmental expression of CAT activity and SP-B mRNA in fetal lung were similar and both increased during explant culture. SP-B mRNA but not CAT activity decreased during culture of adult lung, and both were reduced by transforming growth factor (TGF)-beta(1). Treatment of adult mice with intratracheal bleomycin caused similar time-dependent decreases in lung SP-B mRNA and CAT activity. These findings indicate that the human SP-B promoter fragment directs tissue- and lung cell-specific transgene expression and contains cis-acting elements involved in regulated expression during development, fetal lung explant culture, and responsiveness to TGF-beta and bleomycin-induced lung injury.

DIMERIC SURFACTANT PROTEIN B PEPTIDE SP-B 1-25 IN NEONATAL AND ACUTE RESPIRATORY DISTRESS SYNDROME

Surfactant protein B (SP-B) is a constituent surfactant protein critical for normal lung function. Monomeric SP-B 1-25 (mSP-B 1-25) , a peptide based on the N-terminal domain of human SP-B, mixed in phospholipids partially restores lung function in surfactant-deficient animals. Because native SP-B is a homodimer, we synthesized and tested dimeric SP-B 1-25 (dSP-B 1-25) . Circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy indicated that the secondary conformation of SP-B 1-25 was not significantly perturbed by dimerization. The effects on lung function were compared to phospholipids and Survanta in models of neonatal respiratory distress syndrome (RDS) and acute RDS (ARDS). Preterm rabbits born at 27 days of gestation received 100 mg surfactant / kg at birth and were ventilated for 1 hour with a tidal volume of 10 mL / kg. Dynamic compliance was monitored every 15 minutes and postmortem pressure-volume curves were measured. Adult rats were lavaged to induce surfactant deficiency, treated with 100 mg surfactant / kg, and ventilated for 2 hours. Lung function was assessed using arterial blood gases and dynamic compliance every 15 minutes and postmortem pressure-volume curves. Lung volumes in both models and oxygenation in the lavaged rats were consistently higher in the dSP-B 1-25 than in the Survanta and mSP-B 1-25 surfactant groups. The data suggest that dSP-B 1-25 is more efficient in restoring lung function in neonatal RDS and ARDS than mSP-B 1-25 surfactant.

Lung surfactant protein B promoter function is dependent on the helical phasing, orientation and combinatorial actions of cis-DNA elements

Surfactant protein B (SP-B), a hydrophobic protein of lung surfactant, is essential for surfactant function, normal respiration and survival. SP-B is expressed in a cell-type specific manner by the alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. Our previous studies showed that the activity of the rabbit SP-B minimal promoter (−236/+39 bp) is dependent on the binding of an array of transcription factors including Sp1, Sp3, thyroid transcription factor 1, hepatocyte nuclear factor 3 and activating transcription factor/cyclic AMP response element binding protein. The SP-B minimal promoter sequence as well as the spacing and orientations of cis-DNA elements are conserved in human, rabbit and mouse SP-B genes. In the present study, we investigated the importance of spacing and orientation of cis-DNA elements on SP-B promoter function in NCI-H441 cells, a human cell line of Clara cell lineage. Further we investigated the effects of transcription factors on SP-B promoter expression by co-transfection experiments. Results showed that disruptions of helical phasing and orientation of cis-DNA elements reduced SP-B promoter activity indicating that proper alignment and orientation of cis-DNA elements are necessary for SP-B promoter function. Co-transfection experiments showed that transcription factors function in a combinatorial rather than in a synergistic manner to enhance SP-B promoter activity.

Membrane activity of (Cys48Ser) lung surfactant protein B increases with dimerisation.

One of the possible functions of lung surfactant protein B (SP-B), an hydrophobic membrane-associated saposin-like protein, is to reduce the alveolar surface tension by promoting insertion of phospholipids into the air/liquid interface of the lung. SP-B is a covalent homodimer; Cys48 of two polypeptides form an intermolecular disulphide bond. In order to test whether dimerisation of SP-B is important for surfactant function, transgenic mice which express (Cys48Ser) human SP-B in a mouse SP-B null background were generated. In previous studies (Cys48Ser)SP-B showed a concentration-dependent in vitro activity, suggesting that it may form non-covalent dimers. Here (Cys48Ser)SP-B isolated from bronchoalveolar lavage of transgenic mice was studied at different concentrations by circular dichroism (CD) spectroscopy, pulsating bubble surfactometry, mass spectrometry and reversed-phase HPLC. The results indicate that (Cys48Ser)SP-B, both in a phospholipid environment and in organic solvents, is largely monomeric and exhibits low activity at concentrations lower than 1 -2 microM, while at higher concentrations it forms non-covalent dimers, which are nearly functionally equivalent to native SP-B in vitro. Furthermore, electrospray mass spectrometry showed that more dimers were found relative to the monomer when the polarity of the solvent was decreased, and when the concentration of SP-B increased. (Cys48Ser)SP-B also eluted earlier than native SP-B in reversed-phase HPLC. Taken together, these results indicate that a polar surface is buried upon dimerisation, thereby promoting formation of interchain ion pairs between Glu51-Arg52' and Glu51'-Arg52.

Protein-Protein Interaction of Retinoic Acid Receptor α and Thyroid Transcription Factor-1 in Respiratory Epithelial Cells

Surfactant protein B (SP-B) is a 79-amino acid peptide critical to postnatal respiratory adaptation and is developmentally regulated. Previous studies demonstrated that retinoic acid receptors (RARs) and thyroid transcription factor 1 (TTF-1) stimulated SP-B gene expression in respiratory epithelial cells. Clustered retinoic acid-responsive element and TTF-1 binding sites were identified in the enhancer region of the SP-B gene and were required for retinoic acid stimulation of the human SP-B (hSP-B) promoter. In addition, RAR and TTF-1 were colocalized in mouse bronchiolar and alveolar type II epithelial cells, the cellular site of SP-B synthesis. In the present studies, RAR and TTF-1 were colocalized in the nucleus of H441 cells. RAR and TTF-1 synergistically stimulated the hSP-B promoter in H441 cells. Direct protein-protein interactions between RAR and TTF-1 were demonstrated by the glutathione S-transferase pull-down assay and the mammalian cell two hybrid assay. Truncation/deletion studies showed that the RAR-TTF-1 interaction was mediated through the RAR DNA binding domain (DBD) and the TTF-1 homeodomain. RAR DBD greatly enhanced TTF-1 homeodomain DNA binding activity to a hSP-B enhancer oligonucleotide, in which retinoic acid-responsive element and TTF-1 DNA binding sites overlap. Chromatin immunoprecipitation assay demonstrated that retinoic acid treatment of H441 cells greatly stimulated both RAR and TTF-1 DNA binding to the hSP-B enhancer region in H441 cells. These findings support a model in which RAR/retinoid X receptor, TTF-1, and coactivators (p160 members and CBP) form an enhanceosome in the enhancer region of the hSP-B gene.

Localization of Misprocessed Pulmonary Surfactant Protein C in Tubular Myelin Enriched Fractions By Cryo Immunogold Labeling

Abstract Pulmonary surfactant is secreted by alveolar type II cells and reduces the surface tension at the air-liquid interface of alveoli. After pulmonary surfactant is secreted into the alveolar space, it transforms into tubular myelin, a highly ordered 3-dimensional lattice-like structure. Pulmonary surfactant protein C (SP-C), one of four pulmonary surfactant associated proteins, is synthesized as a proprotein which is processed to biologically active 35 amino acid mature peptide by proteolytic cleavage of N- and C-terminal peptides from the SP-C propeptide (Weaver, 1998). Processing of SP-C is linked to the expression of pulmonary surfactant protein B (SP-B): In SP-B deficient mice, SP-C is misprocessed and present in the bronchoalveolar lavage (BAL; Vorbroker et. al., 1995a). Although the intracellular localization of SP-C is well established (Vorbroker et. al., 1995b), there is no ultrastructure study available regarding the localization of misprocessed SP-C in the airway. In this study, we used transgenic mice expressing a truncated human SP-B propeptide (hSP-BΔC+/+) bred into the murine granulocyte macrophage colony stimulating factor (GMCSF) and SP-B double knockout background (hSP-BΔC+/+: GMCSF-/-: mSP-B-/-) as a model to localize the misprocessed SP-C by cryoimmunogold labeling.

Intracellular Localization of Processing Events in Human Surfactant Protein B Biosynthesis

Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to alveolar type 2 cell phenotype. Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa SP-B. To examine the intracellular sites of SP-B processing, we carried out immunofluorescence cytochemistry and inhibitor studies on human fetal lung in explant culture and isolated type 2 cells in monolayer culture using polyclonal antibodies to human SP-B(8) (Phe(201)-Met(279)) and specific epitopes within the N- (NFProx, Ser(145)-Leu(160); NFlank Gln(186)-Gln(200)) and C-terminal (CFlank, Gly(284)-Ser(304)) propeptides of pro-SP-B. Fluorescence immunocytochemistry using epitope-specific antisera showed colocalization of pro-SP-B with the endoplasmic reticulum resident protein BiP. The 25-kDa intermediate was partially endo H-sensitive, colocalized with the medial Golgi resident protein MG160, and shifted into the endoplasmic reticulum in the presence of brefeldin A, which interferes with anterograde transport from endoplasmic reticulum to Golgi. The 9-kDa intermediate colocalized in part with MG160 but not with Lamp-1, a transmembrane protein resident in late endosomes and lamellar bodies. Brefeldin A induced a loss of colocalization between MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear tubular array. In pulse-chase studies, brefeldin A blocked all processing of 42-kDa pro-SP-B whereas similar studies using monensin blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We conclude that: 1) the first enzymatic cleavage of pro-SP-B to the 25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2) cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies.

The Role of Homodimers in Surfactant Protein B Function in Vivo

Surfactant protein B (SP-B) is detected in the airways as a sulfhydryl-dependent dimer (M(r) approximately 16,000). To test the hypothesis that formation of homodimers is critical for SP-B function, the cysteine residue reported to be involved in SP-B dimerization was mutated to serine (Cys(248) --> Ser) and the mutated protein was targeted to the distal respiratory epithelium of transgenic mice. Transgenic lines which demonstrated appropriate processing, sorting, and secretion of human SP-B monomer were crossed with SP-B +/- mice to achieve expression of human monomer in the absence of endogenous SP-B dimer (hSP-B(mon), mSP-B-/-). In two of three transgenic lines, hSP-B(mon), mSP-B-/- mice had normal lung structure, complete processing of SP-C proprotein, well formed lamellar bodies, and normal longevity. Pulmonary function studies revealed an altered hysteresis curve for hSP-B(mon), mSP-B-/- mice relative to wild type mice. Large aggregate surfactant fractions from hSP-B(mon), mSP-B-/- mice resulted in higher minimum surface tension in vitro compared with surfactant from wild type mice. Surfactant lipids supplemented with 2% hSP-B monomer resulted in slower adsorption and higher surface tension than surfactant with 2% hSP-B dimer. Taken together, these data indicate a role for SP-B dimer in surface tension reduction in the alveolus.

Surfactant Protein B (SP-B) −/− Mice Are Rescued by Restoration of SP-B Expression in Alveolar Type II Cells but Not Clara Cells

Surfactant protein B (SP-B) mRNA and protein are restricted to alveolar Type II and Clara cells in the respiratory epithelium. In order to investigate the function of SP-B in these distinct cell types, transgenic mice were generated in which SP-B expression was selectively restored in Type II cells or Clara cells of SP-B -/- mice. The 4.8-kilobase murine SP-C promoter was used to generate 3 transgenic lines which expressed human SP-B in Type II cells (mSP-C/hSP-B). Likewise, the 2.3-kilobase murine CCSP promoter was used to generate two transgenic lines which expressed human SP-B in Clara cells (mCCSP/hSP-B). mSP-C/hSP-B and mCCSP/hSP-B transgenic mice were subsequently bred to SP-B +/- mice in order to selectively express SP-B in Type II cells or Clara cells of SP-B -/- mice. Selective restoration of SP-B expression in Type II cells completely rescued the neonatal lethal phenotype in SP-B -/- mice. Expression of SP-B in some, but not all Type II cells of SP-B -/- mice, allowed postnatal survival, but resulted in significantly altered lung architecture and function. Selective restoration of SP-B expression in Clara cells of SP-B -/- mice resulted in respiratory dysfunction and invariable neonatal death, related to the complete absence of mature SP-B peptide in these mice. These results indicate that expression and processing of the SP-B proprotein to the mature peptide in Type II cells is absolutely required for lung function in vivo and that SP-B expression in Clara cells cannot substitute for this function.

Use of Knockout Mice to Study Surfactant Protein Structure and Function

Pulmonary surfactant protein B (SP-B) is a 79 amino acid peptide that is intimately associated with surfactant phospholipids in the alveolar airspace. Mutations of the SP-B gene that result in complete absence of SP-B are invariably fatal in the neonatal period. The pathology associated with SP-B deficiency suggests that SP-B plays a critical role in integrating the synthesis, assembly and metabolism of the surfactant complex. A strategy is described to elucidate the role of SP-B in surfactant homeostasis by characterizing the pathophysiology associated with cell specific expression of SP-B constructs in vivo. Human SP-B constructs, under control of lung cell-specific promoters, were expressed in SP-B knockout mice in order to achieve expression of the human transgene in a null background. The effect of transgene expression on lung structure and function was assessed by biochemical, morphological and physiological analyses of the surfactant system in fetal and postnatal offspring.

Targeting type II and Clara cells for adenovirus-mediated gene transfer using the surfactant protein B promoter.

We examined the ability of the human surfactant protein B (SP-B) promoter to confer cell specificity of transgene expression in an adenoviral vector. Using similar replication-deficient adenoviruses (rAd), we compared lacZ reporter gene expression driven by the human SP-B promoter (rAd.SPBlacZ) with the ubiquitously expressed Rous sarcoma virus promoter (rAd.RSVlacZ). rAd.SPBlacZ expressed lacZ in H-441 and A549 lung epithelial cell lines and not in HeLa cells whereas rAd.RSVlacZ expressed in all three cell lines. In primary human fetal lung fibroblasts, beta-galactosidase activity from rAd.RSVlacZ transduction increased in a dose-dependent manner whereas activity from rAd.SPBlacZ remained low. In mixed cell cultures prepared from human fetal lung explants that contained fibroblasts and type II cells, X-Gal staining localized rAd.SPBlacZ expression to only type II cells whereas rAd.RSVlacZ expressed in both cell types. In 24-wk gestation human fetal tissue explants infected ex vivo, the RSV promoter directed lacZ expression in lung, trachea, heart, liver, and esophagus, whereas with the SP-B promoter lacZ was expressed only in lung, specifically in air space-lining cells. This specificity was maintained in vivo. lacZ expression was undetectable in lung and other tissues after intravenous administration of rAd.SPBlacZ whereas rAd.RSV-lacZ expressed primarily in liver. After intratracheal instillation of rAd.SPBlacZ into mice, X-Gal staining localized expression to type II and Clara cells. In contrast, rAd.RSVlacZ expressed in all pulmonary epithelial cell types. Our results indicate that the SP-B promoter may be useful in targeting type II and Clara cells for gene therapy of conditions such as inherited deficiency of SP-B.

Nuclear localization domain of thyroid transcription factor-1 in respiratory epithelial cells.

Thyroid transcription factor-1 (TITF-1) is a homeodomain containing transcription factor that binds to and selectively activates the expression of genes in thyroid and pulmonary epithelial cells. TITF-1 plays a critical role in gene expression and in organogenesis of lung and thyroid. In the present work, epitope-tagged TITF-1 proteins were used to identify the regions of the TITF-1 polypeptide that mediate nuclear localization and transcriptional activity in human lung adenocarcinoma cells. A series of TITF-1-flag deletion mutants was generated and transfected into H441 cells to determine amino acid sequences involved in translocation to the nucleus. Transfection of the TITF-1-flag mutants demonstrated that a nuclear localization signal (NLS) sequence, located at the N-terminus of the homeodomain, is critical for nuclear targeting. The NLS was essential but not sufficient for translocation of TITF-1 to the nucleus, since deletion of the homeodomain itself also blocked nuclear translocation in the presence of NLS. Deletion of the N-terminal transactivation domain of TITF-1 completely abolished its transcriptional activation on the human surfactant protein-B promoter, and deletion of the C-terminal domain partially reduced its stimulatory activity. Nuclear translocation of TITF-1 depends on both an NLS and the homeodomain of the polypeptide. Both C- and N-terminal regions of TITF-1 are involved in transactivation of surfactant protein B gene expression in pulmonary cells.

Rescue of SP-B Knockout Mice with a Truncated SP-B Proprotein: FUNCTION OF THE C-TERMINAL PROPEPTIDE

The function of the 102-amino acid C-terminal propeptide of surfactant protein B (SP-B) was analyzed by characterizing the phenotype associated with loss of expression of this peptide domain in transgenic mice. A construct encoding the signal peptide, N-terminal propeptide, and mature peptide of human SP-B (hSP-BDeltac) was cloned under the control of the 3.7-kilobase human SP-C promoter and injected into fertilized eggs of the FVB/N mouse strain. Founder mice expressing the hSP-BDeltac transgene were bred with heterozygous SP-B knockout mice (SP-B +/-). Offspring containing the transgene and one allele of mouse SP-B were identified and subsequently crossed to generate a transgenic line that expressed SP-BDeltac in a null background (SP-B(-/-)/hSP-BDeltac(+/+)). Expression of hSP-BDeltac in SP-B(-/-) mice was restricted to type II cells and resulted in a 2-fold increase in mature SP-B relative to wild type littermates. These mice survived without any evidence of respiratory problems and had normal lung function, normal alveolar surfactant phospholipid pool sizes, and typical tubular myelin indicating that the 102-residue C-terminal propeptide of SP-B is not required for normal structure and function of extracellular surfactant. However, proteolytic processing of the SP-C proprotein was perturbed resulting in the accumulation of a processing intermediate, Mr = 11,000, similar to the phenotype detected in SP-B(-/-) mice; furthermore, lamellar bodies in type II cells of SP-B(-/-)/hSP-BDeltac(+/+) mice were much larger than in the wild type animal and saturated phosphatidylcholine content in lung tissue was significantly increased although the incorporation of choline into saturated phosphatidylcholine was normal. Collectively, these results demonstrate a role for the C-terminal propeptide of SP-B in SP-C proprotein processing and the maintenance of lamellar body size. The C-terminal propeptide may be an important determinant of intracellular surfactant pool size.

Surfactant ProteinB (SPB) Processing is Regulated by Glucocorticoids in Human Fetal Type 2 Cells. † 260

SPB gene expression is hormonally regulated by both glucocorticoid and cAMP at the transcriptional level. To characterize the effects of these hormones on SPB protein processing in type 2 cells, we cultured human fetal lung (2123 wk gestation, n = 4) as explants (HFLE) for 1, 3, and 5 d with or without 10 nM Dexamethasone (Dex) or 10 nM Dex/0.1 mM cAMP/0.1 mM IBMX (DCI). After 2h starvation, HFLE were pulsed for 1h with35 Smethionine/cysteinesupplemented media and chased in complete media for 4h. Immunoprecipitation of HFLE homogenates was done using a polyclonal antibody to human SPB. We quantified dpm incorporated into the 42, 2327, and 89 kDa SPB bands, correcting for the numbers of cysteine and methionine in each form. As expected, glucocorticoids ± cAMP increased total dpm incorporated into SPB protein forms over control (3243 ± 593 versus 2236 ± 250 densitometry units, mean±sem, n=24). On days 1, 3, and 5 of culture, control tissue processed 0±0%(mean±sem), 7±5%, and 63±18% of initial incorporated dpm to 8 kDa SPB after 4h, respectively. Hormone treatment markedly enhanced processing to 8 kDa SPB on day 3 (Dex 27±2% and DCI 56±3%) with a less marked effect on day 5 (DCI 81±50%). Processing rates differed considerably between treatments on Day 3. With loss of dpm in 42 and 2327 kD forms (13%/h and 4%/h respectively), there was no accumulation of 8 kD SPB in control samples, consistent with intracellular degradation of these intermediates. After hormone treatment, loss of 42 kD SPB (917%/h) was associated with accumulation of 2327 kD (24%/h) and 8 kD SPB (811%/h). This effect was independent of initial incorporated dpm. We conclude that in addition to transcriptional induction of the SPB gene, hormones have a posttranslational effect on the induction of SPB protein expression. Possible mechanisms include induction of type II cell specific enzymes for further processing of the 2327 kD intermediate and/or increased trafficking of SPB intermediates towards lamellar bodies within the type II cell. Supported by NIH P50HL56401, 5P30HD28815 and the Pennsylvania Thoracic Society.

Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B).

Human SP-B is synthesized by the alveolar Type II epithelial cell as a 381 amino acid preproprotein. The 79 residue mature SP-B peptide is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH2- and COOH-termini, respectively. The purpose of this study was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To this end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-B delta C, in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP-B delta N, in which residues 28-200 were deleted), NH2-terminal propeptide (SP-BN), mature SP-B (SP-BM) and COOH-terminal propeptide (SP-BC), into the mammalian expression vector pcDNA3. The resulting expression constructs were characterized by DNA sequencing and in vitro transcription/translation and subsequently transfected into Chinese hamster ovary cells. 48 h after transfection, cells were labeled with [35S]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiography. Proteins encoded by SP-B, SP-B delta C, SP-BN and SP-BC constructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-B delta N) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rapidly degraded. We conclude that (1) 27 amino acids at the NH2-terminus of SP-B contain a functional signal peptide and (2) the NH2-terminal propeptide of the SP-B precursor is necessary and sufficient for intracellular trafficking of the mature peptide.

Upstream Enhancer Activity in the Human Surfactant Protein B Gene Is Mediated by Thyroid Transcription Factor 1

Surfactant protein B (SP-B) is selectively expressed in bronchiolar and alveolar epithelial cells of the lung. We identified an upstream enhancer located in the 5'-flanking region of the human SP-B gene (-439 to -331 base pair, hSP-B(-439/-331)) by deletion analysis of SP-B-luciferase constructs assessed in transfection assays in vitro. The element cis-activated the expression of an SV40 promoter-luciferase reporter gene in a human pulmonary adenocarcinoma cell line (H441-4). Three distinct binding sites for the nuclear transcription protein, thyroid transcription factor 1 (TTF-1), were identified, and the purified TTF-1 homeodomain was bound to bhe region of hSP-B(-439/-331). Co-transfection of H441-4 cells with the expression vector pCMV-TTF-1 trans-activated the native human SP-B promoter and the SV40 promoter fused with the SP-B enhancer. Mutations of the TTF-1 binding sites in the upstream enhancer blocked TTF-1 binding and transactivation activity. In summary, TTF-1 interacts with distinct proximal (-80 to -110) and distal (-439 to -331) cis-acting elements than regulate lung epithelial cell-specific transcription of the human SP-B gene.

Characterization of the promoter of human pulmonary surfactant protein B gene.

Pulmonary surfactant protein B (SP-B) is required for normal surfactant function and for survival at birth. To further study SP-B gene expression, we sequenced genomic clones and examined promoter activity of SP-B DNA fragments by transient transfection. A plasmid construct containing human SP-B fragment -1039/+431 linked to chloramphenicol acetyltransferase (CAT) reporter gene was readily expressed in H441 cells, which are derived from a human lung adenocarcinoma, but was < 4% as active in Hep G2, HeLa, and Calu 6 cell lines. SP-B promoter activity in H441 cells was orientation dependent and increased by linked Rous sarcoma virus (RSV) enhancer and was stronger than for thymidine kinase (tk) and RSV promoters. Deletional mapping of the 5' flanking region with exonuclease III suggested nonspecific negative (-811/-1039)- and positive (-453/-641)-control regions and a cell-specific enhancer region at -208 to -54. When a fragment from -403 to -35 base pairs (bp) was placed upstream or downstream of tkCAT, in either orientation, expression in H441 cells but not other cell lines was increased 4- to 28-fold relative to tkCAT. Deletional analysis of the 3' terminus indicated a requirement for at least 7 bp 3' of the transcription start site. Promoter activity was strongly inhibited in a dose-dependent fashion by phorbol ester, with responsiveness mapped to bp -208/-54, but was not responsive to glucocorticoid.(ABSTRACT TRUNCATED AT 250 WORDS)