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.     

Structure-function analysis of the rat prolactin promoter: phasing requirements of proximal cell-specific elements

Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less than or equal to 1 min) and requires fairly high salt concentrations (greater than or equal to 300 mM KCl) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (greater than 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lung specific developmental expression of the Xenopus laevis surfactant protein C and B genes

Efforts to characterize the mechanisms underlying early lung development have been confounded by the absence of a model that permits study of lung development prior to the onset of endodermal differentiation. Since Xenopus laevis development occurs in an extrauterine environment, we sought to determine whether the classical molecular markers of lung development and function, surfactant protein genes, are expressed in X. laevis. Surfactant protein C (SP-C) is a specific marker for lung development, expressed early in development and exclusively in the lung. Surfactant protein B (SP-B) expression is essential for life, as its absence results in neonatal death in mice and gene mutations have been associated with neonatal respiratory failure in humans. Here, we report the cloning of the first non-mammalian SP-C and SP-B genes (termed xSP-C and xSP-B) using the Xenopus model. The processed mature translated regions of both xSP-C and xSP-B have high homology with both human and mouse genes. xSP-C and xSP-B are both expressed throughout the lung of the X. laevis swimming tadpoles soon after the initiation of lung development as assessed by RT-PCR and whole mount in situ hybridization. The temporal expression patterns of xSP-C and xSP-B are consistent with the expression patterns in mammalian models of lung development. In both the tadpole and the adult X. laevis, xSP-C and xSP-B are expressed only in lung. Knowledge of the sequence and expression pattern of these two surfactant proteins in Xenopus might allow for use of this organism to study early lung development.     

HCV envelope protein function is dependent on the peptides preceding the glycoproteins

Although significant advances have been made on the studies of HCV glycoproteins (E1 and E2) recently, the role of the peptides preceding each glycoprotein remains unclear. We expressed E1 and E2 using two individual plasmids to form HCV pseudoparticles (HCVpp) in order to characterize the peptides preceding E1 and E2.Our data show that 14 amino acids from the HCV core and 12 amino acids from the E1 C-terminus are required for E1 and E2 function, respectively. The lack of a long enough peptide preceding E1 or E2 will abolish HCVpp infectivity, and the presence of fewer than 14 amino acids ahead of E1 and 12 amino acids ahead of E2 may alter their glycosylation. Furthermore, the peptides preceding E1 and E2 may be interchanged or may be replaced by those from genotype 2a. Our findings may contribute to the future development of new anti-HCV drugs.     

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.     

Disparate effects of two phosphatidylcholine binding proteins, C-reactive protein and surfactant protein A, on pulmonary surfactant structure and function

C-reactive protein (CRP) and surfactant protein A (SP-A) are phosphatidylcholine (PC) binding proteins that function in the innate host defense system. We examined the effects of CRP and SP-A on the surface activity of bovine lipid extract surfactant (BLES), a clinically applied modified natural surfactant. CRP inhibited BLES adsorption to form a surface-active film and the film's ability to lower surface tension (gamma) to low values near 0 mN/m during surface area reduction. The inhibitory effects of CRP were reversed by phosphorylcholine, a water-soluble CRP ligand. SP-A enhanced BLES adsorption and its ability to lower gamma to low values. Small amounts of SP-A blocked the inhibitory effects of CRP. Electron microscopy showed CRP has little effect on the lipid structure of BLES. SP-A altered BLES multilamellar vesicular structure by generating large, loose bilayer structures that were separated by a fuzzy amorphous material, likely SP-A. These studies indicate that although SP-A and CRP both bind PC, there is a difference in the manner in which they interact with surface films.     

Cholesterol modulates the exposure and orientation of pulmonary surfactant protein SP-C in model surfactant membranes

Cholesterol is the major neutral lipid in lung surfactant, accounting for up to 8-10% of surfactant mass, while surfactant protein SP-C (∼ 4.2 kDa) accounts for no more than 1-1.5% of total surfactant weight but plays critical roles in formation and stabilization of pulmonary surfactant films. It has been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films and this interaction could have a potential role on modulating surfactant function. In the present study, we have analyzed the effect of cholesterol on the structure, orientation and dynamic properties of SP-C embedded in physiologically relevant model membranes. The presence of cholesterol does not induce substantial changes in the secondary structure of SP-C, as analyzed by Attenuated Reflection Fourier Transformed Infrared spectroscopy (ATR-FTIR). However, the presence of cholesterol modifies the orientation of the transmembrane helix and the dynamic properties of the protein, as demonstrated by hydrogen/deuterium exchange kinetics. The effect of cholesterol on SP-C reconstituted in zwitterionic, entirely fluid, membranes made of POPC (palmitoyloleoylphospatidylcholine) or in anionic membranes with coexistence of ordered and disordered phases, such as those made of dipalmitoylphosphatidylcholine (DPPC):POPC:Palmitoyloleoylphosphatidylglycerol (POPG) (50:25:15) is dual. Cholesterol decreases the exposure of the protein to the aqueous environment and the tilt of its transmembrane helical segment up to a ratio Cholesterol:SP-C of 4.8 and 2.4 (mol/mol) in the two lipid systems tested, respectively, and it increases the exposure and tilt at higher cholesterol proportions. The results presented here suggest the existence of an interaction between SP-C and cholesterol-enriched phases, with consequences on the behavior of the protein, which could be of relevance for cholesterol-dependent structure-function relationships in pulmonary surfactant membranes and films.     

Computational protein design: structure, function and combinatorial diversity

Computational protein design has blossomed with the development of methods for addressing the complexities involved in specifying the structure, sequence and function of proteins. Recent applications include the design of novel functional membrane and soluble proteins, proteins incorporating non-biological components and protein combinatorial libraries.     

The maximal cytoprotective function of the heat shock protein 27 is dependent on heat shock protein 70

Endogenous heat shock proteins (HSPs) 70 and 25/27 are induced in renal cells by injury from energy depletion. Transfected over-expression of HSPs 70 or 27 (human analogue of HSP25), provide protection against renal cell injury from ATP deprivation. This study examines whether over-expressed HSP27 depends on induction of endogenous HSPs, in particular HSP70, to afford protection against cell injury. LLC-PK1 cells transfected with HSP27 (27OE cells) were injured by ATP depletion for 2 h and recovered for 4 h in the presence of HSF decoy, HSP70 specific siRNA (siRNA-70) and their respective controls. Injury in the presence of HSF decoy, a synthetic oligonucleotide identical to the heat shock element, the nuclear binding site of HSF, decreased HSP70 induction by 80% without affecting the over-expression of transfected HSP27. The HSP70 stress response was completely ablated in the presence of siRNA-70. Protection against injury, provided by over-expression of HSP27, was reduced by treatment with HSF decoy and abolished by treatment with siRNA-70. Immunoprecipitation studies demonstrated association of HSP27 with actin that was not affected by either treatment with HSF decoy or siRNA. Therefore, HSP27 is dependent on HSP70 to provide its maximal cytoprotective effect, but not for its interaction with actin. This study suggests that, while it has specific action on the cytoskeleton, HSP 25/27 must have coordinated activity with other HSP classes, especially HSP70, to provide the full extent of resistance to injury from energy depletion.     

The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization

In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)- Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Delta, pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin cytoskeleton functions and, of these, bni1Delta affected Kar9p localization most severely. Like kar9Delta, bni1Delta mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Delta, the bni1Delta mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Delta bni1Delta double mutants suggested that Kar9p retained some function in bni1Delta mitotic cells. Unlike the polarization mutants, kar9Delta shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Delta. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks.     

A recombinant homotrimer, composed of the alpha helical neck region of human surfactant protein D and C1q B chain globular domain, is an inhibitor of the classical complement pathway

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM. The globular heads of C1q (gC1q domain) are located C-terminal to the six triple-helical stalks present in the molecule, each head being composed of the C-terminal halves of one A, one B, and one C chain. The gC1q modules are also found in a variety of noncomplement proteins, such as type VIII and X collagens, precerebellin, hibernation protein, multimerin, Acrp-30, and saccular collagen. In several of these proteins, the chains containing these gC1q modules appear to form a homotrimeric structure. Here, we report expression of an in-frame fusion of a trimerizing neck region of surfactant protein D with the globular head region of C1q B chain as a fusion to Escherichia coli maltose binding protein. Following cleavage by factor Xa and removal of the maltose binding protein, the neck and globular region, designated ghB(3), formed a soluble, homotrimeric structure and could inhibit C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. The functional properties of ghB(3) indicate that the globular regions of C1q may adopt a modular organization in which each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer. The finding that ghB(3) is an inhibitor of C1q-mediated complement activation opens up the possibility of blocking activation at the first step of the classical complement pathway.     

The S promoter of hepatitis B virus is regulated by positive and negative elements.

The S promoter, one of the major hepatitis B virus (HBV) promoters, directs the synthesis of mRNA for surface antigen. Transient expression studies revealed that this promoter is highly active in the Alexander hepatoma cell line but not in SK-Hep1 and HeLa cells. We found that a distal element of the promoter (-103 to -48) confers this cell-type-specific behavior through a mechanism in which the promoter activity is repressed in HeLa and SK-Hep1 cells but increased in Alexander cells. By using an inhibitor of protein synthesis, we obtained evidence that a labile repressor(s) confers the negative effect in SK-Hep1 cells. We also found an enhancerlike activity associated with a small DNA segment of the S promoter (-27 to + 30). This proximal element was active in HeLa and SK-Hep1 cells only in the absence of the distal negative element. Finally, analysis of S promoter deletion mutants demonstrated that the -27 to -17 region of the S promoter is crucial for its activity.     

The role of charged amphipathic helices in the structure and function of surfactant protein B.

Surfactant protein B (SP-B) is essential for normal lung surfactant function. Theoretical models predict that the disulfide cross-linked, N- and C-terminal domains of SP-B fold as charged amphipathic helices, and suggest that these adjacent helices participate in critical surfactant activities. This hypothesis is tested using a disulfide-linked construct (Mini-B) based on the primary sequences of the N- and C-terminal domains. Consistent with theoretical predictions of the full-length protein, both isotope-enhanced Fourier transform infrared (FTIR) spectroscopy and molecular modeling confirm the presence of charged amphipathic alpha-helices in Mini-B. Similar to that observed with native SP-B, Mini-B in model surfactant lipid mixtures exhibits marked in vitro activity, with spread films showing near-zero minimum surface tensions during cycling using captive bubble surfactometry. In vivo, Mini-B shows oxygenation and dynamic compliance that compare favorably with that of full-length SP-B. Mini-B variants (i.e. reduced disulfides or cationic residues replaced by uncharged residues) or Mini-B fragments (i.e. unlinked N- and C-terminal domains) produced greatly attenuated in vivo and in vitro surfactant properties. Hence, the combination of structure and charge for the amphipathic alpha-helical N- and C-terminal domains are key to SP-B function.     

Stretch-stimulated surfactant synthesis is coordinated by the paracrine actions of PTHrP and leptin

Intrauterine lung development, culminating in physiological pulmonary surfactant production by epithelial type II (TII) cells, is driven by fluid distension through unknown mechanisms. Differentiation of alveolar epithelial and mesenchymal cells is mediated by soluble factors like parathyroid hormone-related protein (PTHrP), a stretch-sensitive TII cell product. PTHrP stimulates pulmonary surfactant production by a paracrine feedback loop mediated by leptin, a soluble product of the mature lipofibroblast (LF). When LFs and TIIs are stretched in coculture, there is a fivefold increase in surfactant phospholipid synthesis that can be "neutralized" by inhibitors of PTHrP or leptin, implicating a paracrine feedback loop in this mechanism. Stretching LFs stimulates PTHrP binding (2.5-fold) and downstream stimulation of triglyceride uptake quantitatively (15-25%) due to upregulation of adipose differentiation-related protein expression. Stretching TII cells increases leptin stimulation of their surfactant phospholipid synthesis threefold, suggesting that retrograde signaling by leptin to TII cells is also stretch sensitive. We conclude that the effect of stretch on alveolar LF and TII differentiation is coordinated by PTHrP, leptin, and their receptors.