Effects of ventilation with different positive end-expiratory pressures on cytokine expression in the preterm lamb lung.

Ventilator-induced lung injury increases proinflammatory cytokines in the adult lung. We asked if positive end-expiratory pressure (PEEP) affects proinflammatory cytokine mRNA expression in the preterm lung. Preterm lambs at 129 +/- 3 d gestation were treated with 100 mg/kg recombinant human surfactant protein-C surfactant and ventilated for 2 or 7 h with 0, 4, or 7 cm H(2)O of PEEP. Unventilated fetal lambs were used as controls. Within 2 h of ventilation, alveolar total protein and activated neutrophils were increased and expression of mRNAs for the proinflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha) was increased in lung tissue of all ventilated animals relative to unventilated controls. Alveolar protein and neutrophils were higher for 0 and 7 PEEP animals than 4 PEEP animals. IL-1beta, IL-6, and IL-8 mRNAs were significantly elevated in animals ventilated with 0 PEEP compared with 4 PEEP. The percentage fractional area of collapsed alveoli was significantly higher for 0 PEEP compared with 4 and 7 PEEP groups. Mechanical ventilation increased the expression of proinflammatory mediators in surfactant-treated preterm lungs and the use of 4 PEEP minimized this response.

Identification of a decidua-specific enhancer on the human prolactin gene with two critical activator protein 1 (AP-1) binding sites.

Deletion analysis of the human PRL promoter in endometrial stromal cells decidualized in vitro revealed a 536-bp enhancer located between nucleotide (nt) -2,040 to -1,505 in the 5'-flanking region. The 536-bp enhancer fragment ligated into a thymidine kinase (TK) promoter-luciferase reporter plasmid conferred enhancer activity in decidual-type cells but not nondecidual cells. DNase I footprint analysis of decidualized endometrial stromal cells revealed three protected regions, FP1-FP3. Transfection of overlapping 100-bp fragments of the 536-bp enhancer indicated that FP1 and FP3 each conferred enhancer activity. Gel shift assays indicated that both FP1 and FP3 bind activator protein 1 (AP-1), and JunD and Fra-2 are components of the AP-1 complex in decidual fibroblasts. Mutation of the AP-1 binding site in either FP1 or FP3 decreased enhancer activity by approximately 50%, while mutation of both sites almost completely abolished activity. Coexpression of the 536-bp enhancer and A-fos, a dominant negative to AP-1, decreased enhancer activity by approximately 70%. Conversely, coexpression of Fra-2 in combination with JunD or c-Jun and p300 increased enhancer activity 6- to 10-fold. Introduction of JunD and Fra-2 into nondecidual cells is sufficient to confer enhancer activity. JunD and Fra-2 protein expression was markedly increased in secretory phase endometrium and decidua of early pregnancy (high PRL content) compared with proliferative phase endometrium (no PRL). These investigations indicate that the 5'-flanking region of the human PRL gene contains a decidua-specific enhancer between nt -2,040/-1,505 and AP-1 binding sites within this enhancer region are critical for activity.

Tumor necrosis factor-alpha inhibits aquaporin 5 expression in mouse lung epithelial cells.

Aquaporin 5 (AQP5), the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down-regulated during pulmonary inflammation and edema. The mechanisms that underlie this decrease in AQP5 levels are therefore of considerable interest. Here we show that AQP5 expression in cultured lung epithelial cells is decreased 2-fold at the mRNA level and 10-fold at the protein level by the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Treatment of murine lung epithelial cells (MLE-12) with TNF-alpha results in a concentration- and time-dependent decrease in AQP5 mRNA and protein expression. Activation of the p55 TNF-alpha receptor (TNFR1) with an agonist antibody is sufficient to cause decreased AQP5 expression, demonstrating that the TNF-alpha effect is mediated through TNFR1. Inhibition of nuclear factor kappaB (NF-kappaB) translocation to the nucleus blocks the effect of TNF-alpha on AQP5 expression, indicating that activation of NF-kappaB is required, whereas inhibition of extracellular signal-regulated or p38 mitogen-activated protein kinases showed no effect. These data show that TNF-alpha decreases AQP5 mRNA and protein expression and that the molecular pathway for this effect involves TNFR1 and activated NF-kappaB. The ability of inflammatory cytokines to decrease aquaporin expression may help explain the connection between inflammation and edema.

Surfactant protein A recruits neutrophils into the lungs of ventilated preterm lambs.

We tested the effects of surfactant protein A (SP-A) on inflammation and surfactant function in ventilated preterm lungs. Preterm lambs of 131 d gestation were ventilated for 15 min to initiate a mild inflammatory response, and were then treated with 100 mg/ kg recombinant human SP-C surfactant or with the same surfactant supplemented with 3 mg/kg ovine SP-A. Addition of SP-A to the SP-C surfactant did not change lung function. After 6 h of ventilation, cell numbers in the alveolar wash were 4.9 times higher in SP-A + SP-C-surfactant-treated animals. Cellular infiltrates consisted of neutrophils that produced less hydrogen peroxide than did cells from SP-C-surfactant-treated animals. Expression of adhesion molecules CD11b and CD44 was significantly greater after SP-A treatment, whereas the expression of CD14 was unchanged. Messenger RNAs (mRNAs) for the proinflammatory cytokines interleukin (IL)-1beta, IL-6, and IL-8, but not tumor necrosis factor-alpha, were increased in SP-A-treated lungs. Surfactant protein mRNAs and protein leakage into alveolar washes were not altered by SP-A, indicating that SP-A treatment produces no evidence of lung injury. The present study identifies an unanticipated role of SP-A in neutrophil recruitment in the lungs of preterm lambs.

Intra-amniotic endotoxin increases pulmonary surfactant proteins and induces SP-B processing in fetal sheep.

Intra-amniotic (IA) endotoxin induces lung maturation within 6 days in fetal sheep of 125 days gestational age. To determine the early fetal lung response to IA endotoxin, the timing and characteristics of changes in surfactant components were evaluated. Fetal sheep were exposed to 20 mg of Escherichia coli 055:B5 endotoxin by IA injection from 1 to 15 days before preterm delivery at 125 days gestational age. Surfactant protein (SP) A, SP-B, and SP-C mRNAs were maximally induced at 2 days. SP-D mRNA was increased fourfold at 1 day and remained at peak levels for up to 7 days. Bronchoalveolar lavage fluid from control animals contained very little SP-B protein, 75% of which was a partially processed intermediate. The alveolar pool of SP-B was significantly increased between 4 and 7 days in conjunction with conversion to the fully processed active airway peptide. All SPs were significantly elevated in the bronchoalveolar lavage fluid by 7 days. IA endotoxin caused rapid and sustained increases in SP mRNAs that preceded the increase in alveolar saturated phosphatidylcholine processing of SP-B and improved lung compliance in prematurely delivered lambs.

Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs.

The inflammatory and lung maturational effects of intra-amniotic exposure to endotoxin were assessed in fetal lambs. Five hours to 25 days after intra-amniotic injection of endotoxin, preterm lambs were delivered at 119-125 days gestation. Intra-amniotic endotoxin caused an inflammatory cell infiltration in amnion/chorion at 5 h, which persisted for 25 days. At 5-15 h after endotoxin, amnion/chorion cytokine mRNAs increased [12- to 26-fold for interleukin (IL)-1beta, IL-6, and IL-8 mRNA and 3-fold for tumor necrosis factor-alpha mRNA]. At 1-2 days after endotoxin, lung cytokine mRNAs increased 6- to 49-fold. Endotoxin caused modest changes in peripheral white blood cell counts and no significant cytokine mRNA responses in fetal liver, placenta, or jejunum. Lung maturation, as characterized by increased lung volumes and alveolar saturated phosphatidylcholine, occurred at 7 days and persisted for 25 days after endotoxin. We conclude that exposure to a single dose of intra-amniotic endotoxin causes inflammation and increases in cytokine mRNA in amnion/chorion and the fetal lung before lung maturation, consistent with the hypothesis that proinflammatory cytokines signal lung maturation.

Functional genomics of oxidant-induced lung injury.

In summary, acute lung injury is a severe (>40% mortality) respiratory disease associated with numerous precipitating factors. Despite extensive research since its initial description over 30 years ago, questions remain about the basic pathophysiological mechanisms and their relationship to therapeutic strategies. Histopathology reveals surfactant disruption, epithelial perturbation and sepsis, either as initiating factors or as secondary complications, which in turn increase the expression of cytokines that sequester and activate inflammatory cells, most notably, neutrophils. Concomitant release of reactive oxygen and nitrogen species subsequently modulates endothelial function. Together these events orchestrate the principal clinical manifestations of the syndrome, pulmonary edema and atelectasis. To better understand the gene-environmental interactions controlling this complex process, we examined the relative sensitivity of inbred mouse strains to acute lung injury induced by ozone, ultrafine PTFE, or fine particulate NiSO4 (0.2 microm MMAD, 15-150 microg/m3). Measuring survival time, protein and neutrophils in bronchoalveolar lavage, lung wet: dry weight, and histology, we found that these responses varied between inbred mouse strains, and susceptibility is heritable. To assess the molecular progression of NiSO4-induced acute lung injury, temporal relationships of 8734 genes and expressed sequence tags were assessed by cDNA microarray analysis. Clustering of co-regulated genes (displaying similar temporal expression patterns) revealed the altered expression of relatively few genes. Enhanced expression occurred mainly in genes associated with oxidative stress, anti-proteolytic function, and repair of the extracellular matrix. Concomitantly, surfactant proteins and Clara cell secretory protein mRNA expression decreased. Genome wide analysis of 307 mice generated from the backcross of resistant B6xA F1 with susceptible A strain identified significant linkage to a region on chromosome 6 (proposed as Aliq4) and suggestive linkages on chromosomes 1, 8, and 12. Combining of these QTLs with two additional possible modifying loci (chromosome 9 and 16) accounted for the difference in survival time noted in the A and B6 parental strains. Combining these findings with those of the microarray analysis has enabled prioritization of candidate genes. These candidates, in turn, can be directed to the lung epithelium in transgenic mice or abated in inducible and constitutive gene-targeted mice. Initial results are encouraging and suggest that several of these mice vary in their susceptibility to oxidant-induced lung injury. Thus, these combined approaches have led to new insights into functional genomics of lung injury and diseases.

Pathogenomic mechanisms for particulate matter induction of acute lung injury and inflammation in mice.

To begin identifying genes controlling individual susceptibility to particulate matter, responses of inbred mouse strains exposed to nickel sulfate (NiSO4*) were compared with those of mice exposed to ozone (O3) or polytetrafluoroethylene (PTFE). The A strain was sensitive to NiSO4-induced lung injury (quantified by survival time), the C3H/He (C3) strain and several other strains were intermediate in their responses, and the C57BL/6 (B6) strain was resistant. The strains showed a pattern of response similar to the patterns of response to O3 and PTFE. The phenotype of A x B6 offspring (B6AF1) resembled that of the resistant B6 parental strain, with strains exhibiting sensitivity in the order A > C3 > B6 = B6AF1. Pathology was comparable for the A and B6 mice, and exposure to NiSO4 at 15 microg/m3 produced 20% mortality in A mice. Strain sensitivity for the presence of protein or neutrophils in lavage fluid differed from strain sensitivity for survival time, suggesting that they are not causally linked but are controlled by an independent gene or genes. In the B6 strain, exposure to nickel oxide (NiO) by instillation (40 to 1000 nm) or inhalation (50 nm) produced no changes, whereas inhalation of NiSO4 (60 or 250 nm) increased lavage proteins and neutrophils. Complementary DNA (cDNA) microarray analysis with 8,734 sequence-verified clones revealed a temporal pattern of increased oxidative stress, extracellular matrix repair, cell proliferation, and hypoxia, followed by a decrease in surfactant-associated proteins (SPs). Certain expressed sequence tags (ESTs), clustered with known genes, suggest possible coregulation and novel roles in pulmonary injury. Finally, locus number estimation (Wright equation) and a genomewide analysis suggested 5 genes could explain the survival time and identified significant linkage for a quantitative trait locus (QTL) on chromosome 6, Aliq4 (acute lung injury QTL4). Haplotype analysis identified an allelic combination of 5 QTLs that could explain the difference in sensitivity to acute lung injury between parental strains. Positional candidate genes for Aliq4 include aquaporin-1 (Aqp1), SP-B, and transforming growth factor-alpha (TGF-alpha). Transgenic mice expressing TGF-alpha were rescued from NiSO4 injury (that is, they had diminished SP-B loss and increased survival time). These findings suggest that NiSO4-induced acute lung injury is a complex trait controlled by at least 5 genes (all possibly involved in cell proliferation and surfactant function). Future assessment of these susceptibility genes (including evaluations of human synteny and function) could provide valuable insights into individual susceptibility to the adverse effects of particulate matter.

Differential gene expression in the initiation and progression of nickel-induced acute lung injury.

Acute lung injury, an often fatal condition, can result from a wide range of insults leading to a complex series of biologic responses. Despite extensive research, questions remain about the interplay of the factors involved and their role in acute lung injury. We proposed that assessing the temporal and functional relationships of differentially expressed genes after pulmonary insult would reveal novel interactions in the progression of acute lung injury. Specifically, 8,734 sequence-verified murine complementary DNAs were analyzed in mice throughout the initiation and progression of acute lung injury induced by particulate nickel sulfate. This study revealed the expression patterns of genes previously associated with acute lung injury in relationship to one another and also uncovered changes in expression of a number of genes not previously associated with acute lung injury. The overall pattern of gene expression was consistent with oxidative stress, hypoxia, cell proliferation, and extracellular matrix repair, followed by a marked decrease in pulmonary surfactant proteins. Also, expressed sequence tags (ESTs), with nominal homology to known genes, displayed similar expression patterns to those of known genes, suggesting possible roles for these ESTs in the pulmonary response to injury. Thus, this analysis of the progression and response to acute lung injury revealed novel gene expression patterns.

Activator protein-2 regulates human placental lactogen gene expression.

DNase I footprint analysis of the human placental lactogen-A (hPL-A) promoter using nuclear extracts from purified human trophoblast cells and BeWo choriocarcinoma cells revealed five protected regions within the proximal 325 bp. Two of the protected regions, FP4 (-289--267) and FP5 (-167--154), are homologous to regions on the human growth hormone (hGH) promoter that bind transcription factors AP-2 and/or NFI. Competitive gel shift assays and supershift assays indicated that FP4 forms complexes with activator protein-2 (AP-2) and nuclear factor I (NFI), while FP5 forms a complex with AP-2 alone. In transient transfection studies in human trophoblast cells, hPL promoter constructs containing point mutations in the AP-2 binding sites of FP4 and/or FP5 were 60-80% less active than plasmids containing the wild-type promoter. A mutation in the NFI binding site of FP4, however, had little effect on promoter activity in these cells. Overexpression of AP-2 in HepG2 cells co-transfected with the wild-type hPL promoter resulted in a significant increase in promoter activity. Taken together, these findings suggest a critical role for AP-2 in the regulation of hPL gene expression.

IL-4 increases surfactant and regulates metabolism in vivo.

Mice that express interleukin (IL)-4 in Clara cells (CCSP-IL-4) develop chronic airway inflammation and an alveolar proteinosis-like syndrome. To identify the role of IL-4 in surfactant homeostasis, we measured lipid and protein metabolism in the lungs of CCSP-IL-4 mice in vivo. Alveolar saturated phosphatidylcholine (Sat PC) pools were increased 6.5-fold and lung tissue Sat PC pools were increased 4. 8-fold in the IL-4 transgenic mice. Whereas surfactant protein (SP) A was increased proportionately to Sat PC, SP-D was increased approximately 90-fold in the IL-4 mice compared with wild-type mice and was associated with 2.8-fold increase in SP-D mRNA. The incorporation of palmitate and choline into Sat PC was increased about twofold in CCSP-IL-4 mice. Although trace doses of radiolabeled Sat PC were cleared from the air spaces and lungs of CCSP-IL-4 mice more slowly than in wild-type mice, net clearance of Sat PC from the lungs of CCSP-IL-4 mice was sixfold higher in the IL-4 mice than in wild-type mice because of the larger Sat PC pool sizes. Expression of IL-4 in Clara cells increased surfactant lipid synthesis and clearance, establishing a new equilibrium with increased surfactant pools and an alveolar proteinosis associated with a selective increase in SP-D protein, demonstrating a previously unexpected effect of IL-4 in pulmonary surfactant homeostasis.

Functional Genomics of Particle-Induced Lung Injury.

Currently, the biological mechanisms controlling adverse reactions to particulate matter are uncertain, but are likely to include oxidative lung injury, inflammation, infection, and preexisting pulmonary disease (e.g., chronic obstructive pulmonary diseaseJ. Each mechanism can be viewed as a complex trait controlled by interactions of host (genetic) and environmental factors. We propose that genetic factors play a major role in susceptibility to particulate matter because the number of individuals exposed (even in occupational settings) is often large, but relatively few people respond with increases in morbidity and even mortality. Previous clinical studies support this hypothesis, having discovered marked individual variation in diminished lung function following oxidant exposures. Advances in functional genomics have facilitated the examination of this hypothesis and have begun to provide valuable new insights into gene-environmental interactions. For example, genome-wide scans can be completed readily in mice that enable assessment of chromosomal regions with linkage to quantitative traits. Recently, we and others have identified linkage to oxidant-induced inflammation and mortality. Such linkage analysis can narrow and prioritize candidate gene(s) for further investigation, which, in turn, is aided by existing transgenic mouse models. In addition, differential expression (microarray) analysis enables simultaneous assessment of thousands of genes and expressed sequence tags. Combining genome-wide scan with microarray analysis permits a comprehensive assessment of adverse responses to environmental stimuli and will lead to progress in understanding the complex cellular mechanisms and genetic determinants of susceptibility to particulate matter.

Regulation of mouse SP-B gene promoter by AP-1 family members.

The regulatory role of activator protein-1 (AP-1) family members in mouse surfactant protein (SP) B (mSP-B) promoter function was assessed in the mouse lung epithelial cell line MLE-15. Expression of recombinant Jun B and c-Jun inhibited mSP-B promoter activity by 50-75%. Although c-Fos expression did not alter mSP-B transcription, Jun D enhanced mSP-B promoter activity and reversed inhibition of mSP-B by c-Jun or Jun B. A proximal AP-1 binding site (-18 to -10 bp) was identified that overlaps a thyroid transcription factor-1 binding site. Mutation of this proximal AP-1 site blocked both Jun B inhibition and Jun D enhancement and partially blocked c-Jun inhibition of promoter activity. Promoter deletion mutants were used to identify additional sequences mediating the inhibitory effects of c-Jun in the distal region from -397 to -253 bp. The AP-1 element in this distal site (-370 to -364 bp) is part of a composite binding site wherein AP-1, cAMP response element binding protein, thyroid transcription factor-1, and nuclear factor I interact. Point mutation of the distal AP-1 binding site partially blocked c-Jun-mediated inhibition of the SP-B promoter. Both stimulatory (Jun D) and inhibitory (c-Jun/Jun B) effects of AP-1 family members on mSP-B promoter activity are mediated by distinct cis-acting elements in the mSP-B 5'-flanking region.

Keratinocyte growth factor stimulates transduction of the respiratory epithelium by retroviral vectors.

Cell proliferation is required for transduction by standard retrovirus vectors derived from viruses in the murine leukemia virus (MuLV) group. Since proliferation rates are low in the mature pulmonary epithelium, we tested the hypothesis that the efficiency of retrovirus-mediated transduction of respiratory epithelial cells can be enhanced by stimulation of cell proliferation with recombinant human keratinocyte growth factor (rhKGF). A marked increase in proliferation of bronchiolar and alveolar epithelial cells was observed after intratracheal administration of rhKGF (30 mg/kg) to adult FVB/N mice. Two days after rhKGF or saline treatment, 10(7) AP+ FFU of LAPSN, a recombinant amphotropic retrovirus that expresses human placental alkaline phosphatase (AP), was instilled intratracheally into the mice. Transduction efficiency, measured 2 days after infection, was increased approximately 70-fold by rhKGF pretreatment. However, even after KGF treatment the total numbers of AP-expressing cells were few. Transduction efficiency was similar using either LAPSN packaged by amphotropic host range packaging cells or LAPSN pseudotyped with 10A1 MuLV envelope protein (0.091 +/- 0.006 versus 0.094 +/- 0.028 transduction events/mm2, respectively). Amphotropic vectors use Pit-2 for cell entry, while 10A1 MuLV vectors can use Pit-1 or Pit-2 for cell entry. By in situ hybridization the retroviral receptor Pit-2 (Ram-1) mRNA was expressed only in the pulmonary vasculature, and Pit-1 (Glvr-1) mRNA was expressed at low levels throughout the lung. In vitro studies demonstrated that retrovirus was inactivated by pulmonary surfactant. Stimulating proliferation of the respiratory epithelium increased retroviral transduction in vivo, but the paucity of retroviral receptors and inactivation by surfactant are additional barriers to high-level retroviral gene transfer in the lung.

Adenovirus-mediated granulocyte-macrophage colony-stimulating factor improves lung pathology of pulmonary alveolar proteinosis in granulocyte-macrophage colony-stimulating factor-deficient mice.

Mutation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene by homologous recombination causes progressive pulmonary alveolar proteinosis (PAP) in GM-CSF-deficient mice (GM-/-). The present study tested whether adenovirus-mediated expression of GM-CSF alters the progression of PAP in GM-/- mice. Adult mice were pretreated with an anti-T cell receptor (TCR) antibody to block T cell-mediated immune response, followed by intratracheal instillation of deltaE1-E3 replication-deficient adenovirus expressing mouse GM-CSF (Av1mGM). Mice were killed 1, 3, and 5 weeks after treatment to assess lungs for GM-CSF, surfactant protein B (SP-B), alveolar macrophage maturation, and type II cell proliferation. GM-CSF was detected in BAL fluid from GM-/- mice 1 week after Av1mGM treatment, and GM-CSF mRNA was detected by RT-PCR through 5 weeks. Five weeks after Av1mGM treatment, PAP was improved and SP-B decreased as assessed by ELISA and immunostaining. Increased numbers of alveolar macrophages stained with alpha-naphthyl acetate esterase (alpha-NAE) following treatment with Av1mGM. Local expression of GM-CSF with a recombinant adenovirus ameliorated PAP in the GM-/- mice in association with enhanced maturation of alveolar macrophages.

Recombinant adenoviral vector disrupts surfactant homeostasis in mouse lung.

Although replication-deficient adenoviruses efficiently transfer genes into epithelial cells of the lung, host immune responses limit the extent and duration of gene expression. To define further the role of inflammatory responses to first-generation, recombinant, deltaE1, deltaE3 adenovirus in lung pathology and surfactant protein homeostasis, expression of the surfactant proteins SP-A, SP-B, and proSP-C was determined by immunohistochemistry 2, 7, and 14 days following intratracheal administration of 2 x 10(9) pfu of a recombinant adenovirus, Av1Luc1, to BALB/c nu/nu and BALB/c wild-type mice. Two to 7 days after virus administration, an acute inflammatory response was observed in both mouse strains. Respiratory epithelial cells were sloughed, and extracellular accumulation of SP-A and SP-B was detected in the airways. Diminished immunostaining for SP-A and SP-B was noted in type II cells, and SP-A and SP-B mRNA expression was decreased in focal regions of the lungs from both mouse strains. One week after virus administration, immunostaining for proSP-C was markedly increased in cells lining the regenerating alveolar epithelial surfaces. Two weeks after Av1Luc1 treatment of nu/nu mice, immunostaining for SP-A, SP-B, and proSP-C was similar to those patterns observed prior to adenoviral administration. In immunocompetent wild-type mice, however, immunostaining for surfactant proteins was absent in areas associated with chronic lymphocytic infiltration. The recombinant adenoviral vector, Av1Luc1, caused acute inflammatory responses in the respiratory epithelium with disruption of surfactant protein homeostasis in both wild-type and nu/nu mice. Alterations in surfactant homeostasis persisted in wild-type mice. Thus, both acute and thymic-dependent immune responses limit transgene expression and disrupt surfactant protein gene expression and homeostasis. Because surfactant proteins are critical to host defense and to the maintenance of alveolar stability following injury, these findings raise concerns regarding both acute and chronic toxicity of first-generation recombinant adenoviral vectors for gene transfer.

Nuclear factor I family members regulate the transcription of surfactant protein-C.

Transcription of the surfactant protein-C (SP-C) gene is restricted to Type II epithelial cells in the adult lung. We have shown previously that the 0.32-kilobase pair (kb) mouse SP-C promoter is functional in transient transfection assays of the lung epithelial cell-derived cell line, MLE-15, and that thyroid transcription factor 1 (TTF-1) transactivates promoter activity. The 0.32-kb SP-C promoter can be separated into a proximal promoter region (-230 to +18) and an enhancer region (-318 to -230). Three DNase I footprints were mapped in the promoter region (C1 through C3) and two in the enhancer region (C4 and C5). We now show that nuclear factor I (NFI) family members bind to both individual NFI half-sites in footprints C1, C3, and C5, and to a composite site in footprint C4 by competition gel retardation and antibody supershift analyses. Mutational analysis of the 0.32-kb mouse SP-C promoter and transient transfection of MLE-15 cells demonstrated that the NFI binding sites are required for promoter activity in this cell type. Site-specific mutation of the proximal or distal NFI sites drastically reduced transactivation by a co-transfected NFI-A expression vector in HeLa cells. These data indicate that NFI family member(s), binding to sites in both the promoter and enhancer regions, regulate SP-C gene expression in a process independent of TTF-1.

Altered surfactant function and structure in SP-A gene targeted mice.

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.

Transcription of the lung-specific surfactant protein C gene is mediated by thyroid transcription factor 1.

Surfactant protein C (SP-C) is expressed in alveolar Type II epithelial cells of the lung. In order to determine the mechanism(s) that regulate gene transcription, we have analyzed the activation of the murine SP-C promoter in mouse lung epithelial cells (MLE cells) and in HeLa cells after co-transfection with a vector expressing rat thyroid transcription factor-1 (TTF-1). TTF-1 transactivated SP-C-chloramphenicol acetyltransferase constructs containing -13 kilobase pairs to -320 base pairs (bp) of the 5 flanking region of the SP-C gene. Essential cis-acting elements were functionally localized to between -320 and -180 bp from the start of transcription by transfection analysis. Five DNase-protected regions, indicating multiple protein-DNA interactions within the -320 bp TTF-1-responsive region of the SP-C gene, were identified by DNase footprint analysis. A 40-bp segment of SP-C DNA from -197 to -158 linked to a heterologous promoter-chloramphenicol acetyltransferase construct activated expression after co-transfection with CMV-TTF-1 in HeLa and MLE cells. The -197 to -158 segment contained two consensus TTF-1 sites, which were specifically identified as TTF-1 binding sites by gel retardation and antibody supershift with MLE cell nuclear extracts and purified TTF-1 homeodomain protein. Site-specific mutagenesis of either of the TTF-1 binding sites completely blocked activation by TTF-1, indicating both sites are required for TTF stimulation of SP-C transcription.

Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice.

Surfactant protein B (SP-B) is an 8.7-kDa, hydrophobic protein that enhances the spreading and stability of surfactant phospholipids in the alveolus. To further assess the role of SP-B in lung function, the SP-B gene was disrupted by homologous recombination in murine mouse embryonic stem cells. Mice with a single mutated SP-B allele (+/-) were unaffected, whereas homozygous SP-B -/- offspring died of respiratory failure immediately after birth. Lungs of SP-B -/- mice developed normally but remained atelectatic in spite of postnatal respiratory efforts. SP-B protein and mRNA were undetectable and tubular myelin figures were lacking in SP-B -/- mice. Type II cells of SP-B -/- mice contained no fully formed lamellar bodies. While the abundance of SP-A and SP-C mRNAs was not altered, an aberrant form of pro-SP-C, 8.5 kDa, was detected, and fully processed SP-C peptide was markedly decreased in lung homogenates of SP-B -/- mice. Ablation of the SP-B gene disrupts the routing, storage, and function of surfactant phospholipids and proteins, causing respiratory failure at birth.