Surfactant protein-C Promoter Research Articles

Bone Marrow-Derived VSELs Engraft as Lung Epithelial Progenitor Cells after Bleomycin-Induced Lung Injury.

Background: Alveolar type 2 (AT2) cells and bronchioalveolar stem cells (BASC) perform critical regenerative functions in response to lung damage. Published data show that nonhematopoietic, bone marrow-derived “very small embryonic-like stem cells” (VSELs) can differentiate in vivo into surfactant protein C (SPC)-producing AT2 cells in the lung. Here, we test directly whether VSEL-derived BASC and AT2 cells function to produce differentiated progeny. Methods: using a reporter mouse in which the H2B-GFP fusion protein is driven from the murine SPC promoter, we tested whether bone marrow-derived VSELs or non-VSEL/nonhematopoietic stem cells (non-VSEL/non-HSCs) can differentiate into AT2 and BASC cells that function as progenitor cells. Immediately following bleomycin administration, WT recipient mice underwent intravenous administration of VSELs or non-VSEL/non-HSCs from SPC H2B-GFP mice. GFP+ AT2 and BASC were isolated and tested for progenitor activity using in vitro organoid assays. Results: after 21 days in vivo, we observed differentiation of VSELs but not non-VSEL/non-HSCs into phenotypic AT2 and BASC consistent with previous data in irradiated recipients. Subsequent in vitro organoid assays revealed that VSEL-derived AT2 and BASC maintained physiological potential for differentiation and self-renewal. Conclusion: these findings prove that VSELs produce functional BASC and AT2 cells, and this may open new avenues using VSELs to develop effective cell therapy approaches for patients with lung injury.

Surfactant protein C dampens inflammation by decreasing JAK/STAT activation during lung repair.

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications.

Targeted Type 2 Alveolar Cell Depletion. A Dynamic Functional Model for Lung Injury Repair.

Type 2 alveolar epithelial cells (AEC2) are regarded as the progenitor population of the alveolus responsible for injury repair and homeostatic maintenance. Depletion of this population is hypothesized to underlie various lung pathologies. Current models of lung injury rely on either uncontrolled, nonspecific destruction of alveolar epithelia or on targeted, nontitratable levels of fixed AEC2 ablation. We hypothesized that discrete levels of AEC2 ablation would trigger stereotypical and informative patterns of repair. To this end, we created a transgenic mouse model in which the surfactant protein-C promoter drives expression of a mutant SR39TK herpes simplex virus-1 thymidine kinase specifically in AEC2. Because of the sensitivity of SR39TK, low doses of ganciclovir can be administered to these animals to induce dose-dependent AEC2 depletion ranging from mild (50%) to lethal (82%) levels. We demonstrate that specific levels of AEC2 depletion cause altered expression patterns of apoptosis and repair proteins in surviving AEC2 as well as distinct changes in distal lung morphology, pulmonary function, collagen deposition, and expression of remodeling proteins in whole lung that persist for up to 60 days. We believe SPCTK mice demonstrate the utility of cell-specific expression of the SR39TK transgene for exerting fine control of target cell depletion. Our data demonstrate, for the first time, that specific levels of type 2 alveolar epithelial cell depletion produce characteristic injury repair outcomes. Most importantly, use of these mice will contribute to a better understanding of the role of AEC2 in the initiation of, and response to, lung injury.

Transgenically-expressed secretoglobin 3A2 accelerates resolution of bleomycin-induced pulmonary fibrosis in mice.

BackgroundSecretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, is predominantly expressed in airway epithelial cells. While SCGB3A2 is known to have anti-inflammatory, growth factor, and anti-fibrotic activities, whether SCGB3A2 has any other roles, particularly in lung homeostasis and disease has not been demonstrated in vivo. The aim of this study was to address these questions in mice.MethodsA transgenic mouse line that expresses SCGB3A2 in the lung using the human surfactant protein-C promoter was established. Detailed histological, immunohistochemical, physiological, and molecular characterization of the Scgb3a2-transgenic mouse lungs were carried out. Scgb3a2-transgenic and wild-type mice were subjected to bleomycin-induced pulmonary fibrosis model, and their lungs and bronchoalveolar lavage fluids were collected at various time points during 9 weeks post-bleomycin treatment for further analysis.ResultsAdult Scgb3a2-transgenic mouse lungs expressed approximately five-fold higher levels of SCGB3A2 protein in comparison to wild-type mice as determined by western blotting of lung tissues. Immunohistochemistry showed that expression was localized to alveolar type II cells in addition to airway epithelial cells, thus accurately reflecting the site of surfactant protein-C expression. Scgb3a2-transgenic mice showed normal lung development and histology, and no overt gross phenotypes. However, when subjected to a bleomycin-induced pulmonary fibrosis model, they initially exhibited exacerbated fibrosis at 3 weeks post-bleomycin administration that was more rapidly resolved by 6 weeks as compared with wild-type mice, as determined by lung histology, Masson Trichrome staining and hydroxyproline content, inflammatory cell numbers, expression of collagen genes, and proinflammatory cytokine levels. The decrease of fibrosis coincided with the increased expression of SCGB3A2 in Scgb3a2-transgenic lungs.ConclusionsThese results demonstrate that SCGB3A2 is an anti-fibrotic agent, and suggest a possible therapeutic use of recombinant SCGB3A2 in the treatment of pulmonary fibrosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12890-015-0065-4) contains supplementary material, which is available to authorized users.

Identification and Functional Characterization of a Novel Mutation in theNKX2-1Gene: Comparison with the Data in the Literature

NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain-thyroid-lung syndrome (BTLS). The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters. The mutation is a deletion of a cytosine at position 834 (ref. sequence NM_003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy-terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. We describe a novel NKX2-1 mutation and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype-phenotype correlations in the NKX2-1 deficiency syndrome.

ERBB2-induced inflammation in lung carcinogenesis

ERBB2/HER2/NEU, a member of the epidermal growth factor receptor family, is overexpressed in more than 25 % of non-small cell lung cancer and is considered to be a significant and independent prognostic factor in lung cancer. Here we generated a lung specific HER2 overexpressing transgenic mouse model. In this model, HER2 was driven by the human surfactant protein-C promoter to investigate the role of the HER2 oncogene in pulmonary carcinogenesis and progression. Notably, significant pathological changes, including lymphocyte infiltration and mesenchymal cells hyperplasia, were found in the lung tissue of transgenic mice aged from 4 to 12 months. The occurrence and severity of those lesions increased as the mice aged. Some inflammatory factors, such as tumor necrosis factor, interleukin 1 and interleukin 6, were upregulated in lung tissue of transgenic mice compared with that of wild-type mice, implying that long-term HER2 overexpression could induce serious lung inflammation and some precancerous lesions. This model would be useful for studying the mechanism of HER2 involvement in lung carcinogenesis and for understanding the relationship between carcinogenesis and inflammation.

Targeted Deletion of Jun/AP-1 in Alveolar Epithelial Cells Causes Progressive Emphysema and Worsens Cigarette Smoke–Induced Lung Inflammation

Chronic obstructive pulmonary disease appears to occur slowly and progressively over many years, with both genetic factors and environmental modifiers contributing to its pathogenesis. Although the c-Jun/activator protein 1 transcriptional factor regulates cell proliferation, apoptosis, and inflammatory responses, its role in lung pathogenesis is largely unknown. In this study, we report decreased expression levels of c-Jun mRNA and protein in the lung tissues of patients with advanced chronic obstructive pulmonary disease, and the genetic deletion of c-Jun specifically in alveolar epithelial cells causes progressive emphysema with lung inflammation and alveolar air space enlargement, which are cardinal features of emphysema. Although mice lacking c-Jun specifically in lung alveolar epithelial cells appear normal at the age of 6 weeks, when exposed to long-term cigarette smoke, c-Jun-mutant mice display more lung inflammation with perivascular and peribronchiolar infiltrates compared with controls. These results demonstrate that the c-Jun/activator protein 1 pathway is critical for maintaining lung alveolar cell homeostasis and that loss of its expression can contribute to lung inflammation and progressive emphysema.

The SP-C promoter facilitates alveolar type II epithelial cell-specific plasmid nuclear import and gene expression

Although nonviral gene therapy has great potential for use in the lung, the relative lack of cell-specific targeting has limited its applications. We have developed a new approach for cell-specific targeting based on selective nuclear import of plasmids in non-dividing cells. Using a microinjection and in situ hybridization approach, we tested several potential DNA sequences for the ability to mediate plasmid nuclear import in alveolar type II epithelial (ATII) cells. Of these, only a sequence within the human surfactant protein C (SP-C) promoter was able to mediate nuclear localization of plasmid DNA specifically in ATII cells but not in other cell types. We have mapped the minimal import sequence to the proximal 318 nucleotides of the promoter, and demonstrate that binding sites for NFI, TTF-1, and GATA-6 and the proteins themselves are required for import activity. Using intratracheal delivery of DNA followed by electroporation, we demonstrate that the SP-C promoter sequence will enhance gene expression specifically in ATII cells in mouse lung. This represents a novel activity for the SP-C promoter and thus ATII cell-specific nuclear import of DNA may prove to be a safe and effective method for targeted and enhanced gene expression in ATII cells.

Epithelial Ablation of Bcl-XL Increases Sensitivity to Oxygen without Disrupting Lung Development

Recent studies indicate that the antiapoptotic Bcl-X(L), one of five isoforms expressed by the Bcl-X gene, protects a variety of cell lines exposed to hyperoxia. However, its role in lung development and protection against oxidative stress in vivo is not known. Here, we show Bcl-X(L) is the predominant isoform expressed in the lung, and the only isoform detected in respiratory epithelium. Because loss of Bcl-X(L) is embryonically lethal, Bcl-X(L) was ablated throughout the respiratory epithelium by mating mice with a floxed exon II of the Bcl-X gene with mice expressing Cre under control of the surfactant protein-C promoter. Interestingly, the loss of Bcl-X(L) in respiratory epithelium was perinatally lethal in approximately 50% of the expected offspring. However, some adult mice lacking the gene were obtained. The epithelial-specific ablation of Bcl-X(L) did not disrupt pulmonary function, the expression of epithelial cell-specific markers, or lung development. However, it shifted the lung toward a proapoptotic state, defined by a reduction in antiapoptotic Mcl-1, an increase in proapoptotic Bak, and increased sensitivity of the respiratory epithelium to hyperoxia. Intriguingly, increased 8-oxoguanine lesions seen during hyperoxia were also evident as lungs transitioned to room air at birth, a time when perinatal lethality in some mice lacking Bcl-X(L) was observed. These findings reveal that the epithelial-specific expression of Bcl-X(L) is not required for proper lung development, but functions to protect respiratory epithelial cells against oxygen-induced toxicity, such as during hyperoxia and the lung's first exposure to ambient air.

Chemopreventive Effects of Gefitinib on Nonsmoking-Related Lung Tumorigenesis in Activating Epidermal Growth Factor Receptor Transgenic Mice

Twenty-five percent of all lung cancer cases are not attributable to smoking. Epidermal growth factor receptor (EGFR) mutations, which are involved in approximately 50% of nonsmoker lung cancer, are positively correlated with responsiveness to gefitinib, and inversely correlated with smoking history. Activating EGFR mutations play a critical role in the carcinogenesis of nonsmoking-related lung cancer. To investigate the chemopreventive effects of gefitinib on nonsmoking-related lung cancer, we generated transgenic mice expressing EGFR L858R in type II pneumocytes constitutively using the surfactant protein-C promoter. The transgenic mice invariably developed atypical adenomatous hyperplasia at age 4 weeks and multifocal adenocarcinoma of varying sizes at age 7 weeks. Notably, the expression levels of phosphorylated and total ErbB2, ErbB3, and thyroid transcription factor-1 were elevated in the transgenic mice compared with wild-type controls at age 3 weeks. Administration of gefitinib to 3-week-old transgenic mice for 1 week before carcinogenesis reduced the amount of phosphorylated EGFR in the lungs of the mice to the baseline level. Gefitinib (5 mg/kg/d; n = 5, 5, and 15) or vehicle (n = 5, 5, and 15) was administered to transgenic mice from age 3 to 8, 13, and 18 weeks, respectively. The numbers of lung tumors in the control and gefitinib-treated groups were 1.75, 5.8, 10.2, and 0 (P < 0.05), respectively. No fatal toxic events occurred in either group, and gefitinib inhibited tumorigenesis completely in this mouse model. These results suggest the utility of molecular targeted chemoprevention against nonsmoking-related lung cancer.

Generation and characterization of JSRV envelope transgenic mice in FVB background

Jaagsiekte sheep retrovirus (JSRV) that causes contagious ovine pulmonary adenocarcinoma (OPA) in sheep carries an oncogenic Envelope gene (Env), which is capable of transforming target cells in vitro and in vivo. We cloned full-length JSRV Env cDNA into an expression vector, SPC/SV40, where the transgene was driven by lung-specific surfactant protein C (SPC) promoter, to obtain SPC-JSRV Env construct. SPC-JSRV Env was microinjected into immunocompetent FVB/N mice embryos to generate Env transgenic mice. We obtained two lines of transgenic mice, both of which were capable of developing spontaneous lung tumors from 1 month onwards and the tumor incidence rate was about 56% at the age of 7 months in Env Transgenic line 1 and about 71% at the age of 6 months in Env Transgenic line 2. We were able to correlate higher tumor incidence rate and tumorigenicity in Env Transgenic line 2 to higher level of expression of Env transgene compared to Env Transgenic line 1. Immunohistochemical analysis showed that the tumor was primarily composed of type II pneumocytes where SPC promoter is known to be active similar to natural infection of JSRV in sheep. Analysis of cellular mitogenic signal transduction pathways revealed significant induction of p44/42 ERK pathway in the transgenic mice lungs with tumors compared to the lungs from non-transgenic FVB/N mice. Tumors in our transgenic mice pose similarities to human lung adenocarcinoma and therefore our mice could serve as a model system for evaluating the mechanisms of lung tumorigenesis in vivo.

Modulation of PLAGL2 transactivation activity by Ubc9 co-activation not SUMOylation

Pleomorphic adenoma gene like-2 (PLAGL2), a developmentally regulated and stress inducible zinc finger protein can be post-translationally modified by small ubiquitin-like modifier peptide (SUMO-1); and SUMOylation attenuates PLAGL2 activity on the interactive promoter. Since PLAGL2 was a transactivator of the surfactant protein-C (SP-C) promoter, we hypothesized that SUMOylation down-regulated PLAGL2-activated SP-C promoter activity. Unexpectedly, the SUMO-conjugating enzyme Ubc9 enhanced, rather than reduced, PLAGL2 activated promoter activity but did not affect TTF-1 activation of the promoter. Ubc9 mutant (Ubc9-C93S) defective in SUMO-conjugating activity also enhanced PLAGL2-driven promoter activity suggesting that the stimulatory effect of Ubc9 on SP-C promoter activation was independent of its enzymatic function. PLAGL2 mutants without the K250 and/or K269 SUMOylation sites did not further improve PLAGL2 programmed transcription nor did they abolish Ubc9 enhanced promoter activity supporting the SUMOylation-independent mechanism. Chromatin immunoprecipitation (ChIP) assay demonstrated the association of PLAGL2 and Ubc9 with the SP-C promoter in vivo. Taken together, our data suggests that Ubc9 can function as a co-factor of PLAGL2, uncoupling from its enzymatic activity, to mediate PLAGL2 interactive SP-C promoter activity.

Foxp2 Inhibits Nkx2.1-Mediated Transcription of SP-C via Interactions with the Nkx2.1 Homeodomain

The transcription factor (TF) Foxp2 has been shown to partially repress surfactant protein C (SP-C) transcription, presumably through interaction of an independent repressor domain with a conserved Foxp2 consensus site in the SP-C promoter. We explored the role of interactions between Foxp2 and the homeodomain TF Nkx2.1 that may contribute to the marked reduction in SP-C expression accompanying phenotypic transition of alveolar epithelial type II (AT2) to type I (AT1) cells. Foxp2 dose-dependently inhibited Nkx2.1-mediated activation of SP-C in MLE-15 cells. While electrophoretic mobility shift assays and chromatin immunoprecipitations revealed an interaction between Foxp2 and the conserved consensus motif in the SP-C promoter, Nkx2.1-mediated activation of the 318-bp proximal SP-C promoter (which lacks a Foxp2 consensus) was attenuated by increasing amounts of Foxp2. Co-immunoprecipitation and mammalian two-hybrid assays confirmed a physical interaction between Nkx2.1 and Foxp2 mediated through the Nkx2.1 homeodomain. Formation of an Nkx2.1 complex with an SP-C oligonucleotide was inhibited dose-dependently by recombinant Foxp2. These findings demonstrate that direct interaction between Foxp2 and Nkx2.1 inhibits Nkx2.1 DNA-binding and transcriptional activity and suggest a mechanism for down-regulation of SP-C (and probably other AT2 cell genes) during transition of AT2 cells to an AT1 cell phenotype.

Erm/Thyroid Transcription Factor 1 Interactions Modulate Surfactant Protein C Transcription

Expression of surfactant protein C (SP-C), which is restricted to alveolar type II epithelial cells of the adult lung, is critically dependent on thyroid transcription factor 1 (TTF-1). In the present study we have demonstrated that Erm, a member of the Ets family of transcription factors, is expressed in the distal lung epithelium during development and is also restricted to alveolar type II cells in the adult. Erm was up-regulated by fibroblast growth factors (FGFs) in culture, and blocking FGF signaling inhibited Erm expression both in vivo and in vitro. The SP-C minimal promoter was found to contain two potential Ets binding sites, and electrophoretic mobility shift assays showed that two 20-bp wild-type oligonucleotides containing the 5'-GGA(A/T)-3' Ets consensus binding motif were shifted by nuclear extracts from MLE15 cells. Co-transfection assays showed that Erm by itself had little effect on SP-C promoter activity but that Erm significantly enhanced TTF-1-mediated SP-C transcription. Mutation of one of the Ets binding sites reduced SP-C transcription to background levels, whereas mutation of the other site resulted in increased SP-C transcription. Protein-protein interactions between Erm and TTF-1 were demonstrated by mammalian two-hybrid assays and by co-immunoprecipitation assays. Mapping studies showed that the Ets domain of Erm and the combined N terminus and homeodomain of TTF-1 were critical for this interaction. Treatment of primary cultures of adult alveolar type II cells with siRNA targeting Erm diminished expression of both Erm and SP-C but had no effect on beta-actin or GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Taken together, these results demonstrate that Erm is involved in SP-C regulation, which results from an interaction with TTF-1.

572. Alveolar Epithelial Cell-Specific Plasmid Nuclear Import

A major concern regarding naked DNA delivery, both in vivo and in vitro, is the relatively low level of gene expression observed following transfection of non-dividing cells. This low level of gene expression is due in part to the relative inaccessibility of plasmid to the nucleus where the cellular transcriptional machinery resides. Many groups have had success customizing plasmid vectors to facilitate DNA nuclear transport. To enhance nuclear import of plasmids, a specific DNA nuclear targeting sequence (DTS) can be included within the plasmid. These sequences bind a subset of newly synthesized transcription factors in the cytoplasm and the plasmid is transported into the nucleus via nuclear localization signals within the DNA-bound transcription factor proteins. Our lab has shown that by using specific DTSs, which bind cell-specific transcription factors, nuclear import of plasmid DNA and subsequent gene expression can be restricted to specific cell types. The experiments presented here describe our identification of a new cell-specific DTS that functions in a class of cells in the lung epithelium. The lung alveolar epithelium contains two morphologically and functionally distinct cell types known as type I (ATI) and type II (ATII) cells. Because several lung diseases specifically affect ATII cells, this cell type is an attractive target for gene delivery. To identify an ATII-specific DTS, a set of ATII-specific promoter sequences were cloned into plasmids lacking any other DTS and these plasmids were microinjected into the cytoplasm of A549 and MLE-12 cells, both ATII-like cell lines. The localization of plasmid DNA was determined eight hours post-injection using in situ hybridization. Of all the sequences tested, only a fragment of the human surfactant protein C (SP-C) promoter (-318 to +18), when included on a plasmid, mediated nuclear import in A549 and MLE-12 cells. When plasmids containing the SP-C promoter were cytoplasmically microinjected into non-ATII cell types (HeLa, TC7, pulmonary smooth muscle cells, and bronchial epithelial cells), no nuclear localization was observed. Thus, the human SP-C promoter functions as an ATII-specific DNA nuclear targeting sequence. Restriction of gene expression to desired cell types represents one of the most important challenges to gene therapy today, and the use of specific nuclear import sequences represents a novel strategy for cell-specific expression of transgenes from plasmids.

90-Day Repeated Inhalation Exposure of Surfactant Protein-C/Tumor Necrosis Factor-α(SP-C/TNF-α) Transgenic Mice to Air Pollutants

Tumor necrosis factor (TNF)-alpha, a cytokine present in inflammed lungs, is known to mediate some of the adverse effects of ozone and inhaled particles. The authors evaluated transgenic mice with constitutive pulmonary expression of TNF-alpha under transcriptional regulation of the surfactant protein-C promoter as an animal model of biological susceptibility to air pollutants. To simulate a repeated, episodic exposure to air pollutants, wild-type and TNF mice inhaled air or a mixture of ozone (0.4 ppm) and urban particles (EHC-93, 4.8 mg/m3) for 4 h, once per week, for 12 consecutive weeks and were sacrificed 20 h after last exposure. TNF mice exhibited chronic lung inflammation with septal thickening, alveolar enlargement, and elevated protein and cellularity in bronchoalveolar lavage fluid (genotype main effect, p < .001). Repeated exposure to pollutants did not result in measurable inflammatory changes in wild-type mice and did not exacerbate the inflammation in TNF mice. The pollutants decreased recovery of alveolar macrophages in tavage fluid of both wild-type and TNF mice (exposure main effect, p < .001). Exacerbation of the rate of protein nitration reactions specifically in the lungs of TNF mice was revealed by the high ratio of 3-nitrotyrosine to L-DOPA after exposure to the air pollutants (Genotype x Exposure factor interaction, p = .014). Serum creatine kinase-MM isoform increased in TNF mice exposed to pollutants (Genotype X Exposure factor interaction, p = .043). The marked pollutant-related nitration in the lungs of the TNF mice reveals basic differences in free radical generation and scavenging in the inflamed lungs in response to pollutants. Furthermore, elevation of circulating creatine kinase-MM isoform specifically in TNF mice exposed to pollutants suggests systemic adverse impacts from lung inflammatory mediators, possibly on muscles and the cardiovascular system.

The murine SP-C promoter directs type II cell-specific expression in transgenic mice

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

Increased Phosphatidylcholine Production but Disrupted Glycogen Metabolism in Fetal Type II Cells of Mice That Overexpress CTP:Phosphocholine Cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in the de novo synthesis of phosphatidylcholine (PtdCho). Alveolar type II cells synthesize large quantities of disaturated PtdCho, the surface-active agent of pulmonary surfactant, particularly at late gestation when the lung prepares itself for postnatal air breathing. To clarify the role of CCTalpha in lung surfactant maturation, we overexpressed CCTalpha(1-367) using the surfactant protein-C promoter. Lungs of transgenic mice were analyzed at day 18 of gestation (term = 19 days). Overexpression of CCTalpha(1-367) increased the synthesis and content of PtdCho in fetal type II cells isolated from the transgenic mice. Also, PtdCho content of fetal lung fluid was increased. No changes in surfactant protein content were detected. Interestingly, fetal type II cells of transgenic mice contained more glycogen than control cells. Incorporation studies with [U-(14)C]glucose demonstrated that overexpression of CCTalpha(1-367) in fetal type II cells increased glycogen synthesis without affecting glycogen breakdown. To determine which domain contributes to this glycogen phenotype, two additional transgenes were created overexpressing either CCTalpha(1-239) or CCTalpha(239-367). Glycogen synthesis and content were increased in fetal type II cells expressing CCTalpha(239-367) but not CCTalpha(1-239)(.) We conclude that overexpression of CCTalpha increases surfactant PtdCho synthesis without affecting surfactant protein levels but that it disrupts glycogen metabolism in differentiating type II cells via its regulatory domain.

TAZ Interacts with TTF-1 and Regulates Expression of Surfactant Protein-C

Thyroid transcription factor-1 (TTF-1/Nkx-2.1) is required for formation of the lung and differentiation of peripheral respiratory epithelial cells. TTF-1 activates transcription of target genes, including the surfactant proteins critical for lung function. A recently identified protein TAZ (transcriptional co-activator with PDZ-binding motif) contains a WW domain and a COOH-terminal PDZ-binding motif that are proposed to mediate its interactions with various transcriptional proteins. To determine the role of TAZ in the regulation of gene expression in the lung, the sites of TAZ expression and the role of TAZ in the regulation of respiratory epithelial gene expression were assessed. TAZ mRNA was detected in immortalized mouse lung epithelial cells, primary isolates of mouse alveolar type II epithelial cells, and epithelial cells of fetal lung. Sites of TAZ mRNA and protein overlapped with those of TTF-1 and surfactant protein C (SP-C) in the respiratory epithelial cells of the mouse lung. In the presence of TTF-1, TAZ synergistically activated the expression of mouse SP-C-luciferase reporter constructs. Mammalian two-hybrid assays and pull-down experiments demonstrated that the TAZ directly interacted with TTF-1. Further, deletion analysis demonstrated that TAZ binds to the NH(2)-terminal domain of TTF-1. TAZ binds to TTF-1, increasing the transcriptional activity of TTF-1 on the SP-C promoter. Developmental and cell-selective regulation of TAZ provides a mechanism by which the activity of TTF-1 on target genes is modulated.

HumanSP-Cgene sequences that confer lung epithelium-specific expression in transgenic mice

We used transgenic mice to identify cis-active regions of the human pulmonary surfactant protein C (SP-C) gene that impart tissue- and cell-specific expression in vivo in the lung. Approximately 3.7 kb of genomic SP-C DNA upstream of the transcription start site was sufficient to direct chloramphenicol acetyltransferase (CAT) reporter gene expression specifically in bronchiolar and alveolar epithelial cells of the lung. To further define cis-active regulatory elements that mediate cell-specific expression, we tested deletions of the parental 3.7-kb human SP-C sequence in transgenic mice. Tissue CAT assays of mice generated with truncations or overlapping internal deletions of the 3.7-kb construct functionally map alveolar cell-specific regulatory elements to within -215 bp of the SP-C promoter. Analysis of SP-C promoter deletions demonstrate that sequences between -3.7 kb and -1.9 kb contain enhancer sequences that stimulate SP-C transgene expression. In situ hybridization studies demonstrate that deletion of the -1,910- to -215-bp region abolishes the ectopic bronchiolar expression seen with the original 3.7-kb SP-C promoter construct. Comparison of sequences from -215 to +1 bp identified consensus binding sites for the homeodomain transcription factor thyroid transcription factor-1 (TTF-1). Cotransfection assays of the human 3.7-kb SP-C or -1,910- to -215-bp SP-C deletion construct with a TTF-1 expression plasmid demonstrates that TTF-1 transactivates the human SP-C gene. These results suggest that the TTF-1 cis-active sites are important in directing cell-specific expression of the SP-C gene in vivo.