[Show abstract][Hide abstract] ABSTRACT: Surfactant protein D (SP-D) has important immuno-modulatory properties. The absence of SP-D results in an inducible NO synthase (iNOS, coded by NOS2 gene) related chronic inflammation, development of emphysema-like pathophysiology and alterations of surfactant homeostasis.
In order to test the hypothesis that SP-D deficiency related abnormalities in pulmonary structure and function are a consequence of iNOS induced inflammation, we generated SP-D and iNOS double knockout mice (DiNOS).
Structural data obtained by design-based stereology to quantify the emphysema-like phenotype and disturbances of the intracellular surfactant were correlated to invasive pulmonary function tests and inflammatory markers including activation markers of alveolar macrophages and compared to SP-D (Sftpd(-/-)) and iNOS single knockout mice (NOS2(-/-)) as well as wild type (WT) littermates.
DiNOS mice had reduced inflammatory cells in BAL and BAL-derived alveolar macrophages showed an increased expression of markers of an alternative activation as well as reduced inflammation. As evidenced by increased alveolar numbers and surface area, emphysematous changes were attenuated in DiNOS while disturbances of the surfactant system remained virtually unchanged. Sftpd(-/-) demonstrated alterations of intrinsic mechanical properties of lung parenchyma as shown by reduced stiffness and resistance at its static limits, which could be corrected by additional ablation of NOS2 gene in DiNOS.
iNOS related inflammation in the absence of SP-D is involved in the emphysematous remodeling leading to a loss of alveoli and associated alterations of elastic properties of lung parenchyma while disturbances of surfactant homeostasis are mediated by different mechanisms.
PLoS ONE 01/2014; 9(1):e85722. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the ER folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility and reduced the expression level of wild type ABCA3 in a dose dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that while single motif mutants exhibited a vesicular distribution pattern similar to wild type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability and its disruption results in protein destabilization and proteasomal degradation.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Surfactant protein D (SP-D) is a collection that plays important roles in modulating host defense functions and maintaining phospholipid homeostasis in the lung. The aim of current study was to characterize comparatively the SP-D response in bronchoalveolar lavage (BAL) and serum in three murine models of lung injury, using a validated ELISA technology for estimation of SP-D levels. METHODS: Mice were exposed to lipopolysaccharide, bleomycin, or Pneumocystis carinii (Pc) and sacrificed at different time points. RESULTS: In lipopolysaccharide-challenged mice, the level of SP-D in BAL increased within 6 h, peaked at 51 h (4,518 ng/ml), and returned to base level at 99 h (612 ng/ml). Serum levels of SP-D increased immediately (8.6 ng/ml), peaked at 51 h (16 ng/ml), and returned to base levels at 99 h (3.8 ng/ml). In a subacute bleomycin inflammation model, SP-D levels were 4,625 and 367 ng/ml in BAL and serum, respectively, 8 days after exposure. In a chronic Pc inflammation model, the highest level of SP-D was observed 6 weeks after inoculation, with BAL and serum levels of 1,868 and 335 ng/ml, respectively. CONCLUSIONS: We conclude that serum levels of SP-D increase during lung injury, with a sustained increment during chronic inflammation compared with acute inflammation. A quick upregulation of SP-D in serum in response to acute airway inflammation supports the notion that SP-D translocates from the airways into the vascular system, in favor of being synthesized systemically. The study also confirms the concept of using increased SP-D serum levels as a biomarker of especially chronic airway inflammation.
Beiträge zur Klinik der Tuberkulose 02/2013; · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The integrity of the alveolar epithelium is a key factor in the outcome of acute lung injury. Here, we investigate alveolar epithelial injury and the expression of epithelial-selective markers in Streptococcus pneumoniae-induced acute lung injury. S. pneumoniae was instilled into rat lungs and alveolar type I (RTI(40)/podoplanin, MMC6 antigen) and alveolar type II (MMC4 antigen, surfactant protein D, pro-surfactant protein C, RTII(70)) cell markers were quantified in lavage fluid and lung tissue at 24 and 72 hours. The alveolar epithelium was also examined using electron, confocal, and light microscopy. S. pneumoniae induced an acute inflammatory response as assessed by increased total protein, SP-D, and neutrophils in lavage fluid. Biochemical and morphological studies demonstrated morphologic injury to type II cells but not type I cells. In particular, the expression of RTI(40)/podoplanin was dramatically reduced, on the surface of type I cells, in the absence of morphologic injury. These data demonstrate that type II cell damage can occur in the absence of type I cell injury without affecting the ability of the lung to return to a normal morphology. These data also demonstrate that RTI(40)/podoplanin is not a type I cell phenotypic marker in experimental acute lung injury caused by S. pneumoniae. Given that RTI(40)/podoplanin is an endogenous ligand for the C-type lectin receptor and this receptor plays a role in platelet aggregation and neutrophil activation, we hypothesize that the reduction of RTI(40)/podoplanin on type I cells might be important for the regulation of platelet and/or neutrophil function in experimental acute lung injury.
Experimental Lung Research 06/2012; 38(5):266-76. · 1.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Furthermore, Cryptococcus neoformans has become a primary human pathogen, causing infection in seemingly healthy individuals. Although numerous studies have elucidated the virulence properties of C. neoformans, less is understood regarding lung host immune factors during early stages of fungal infection. Based on our previous studies documenting that pulmonary surfactant protein D (SP-D) protects C. neoformans cells against macrophage-mediated defense mechanisms in vitro (S. Geunes-Boyer et al., Infect. Immun. 77:2783-2794, 2009), we postulated that SP-D would facilitate fungal infection in vivo. To test this hypothesis, we examined the role of SP-D in response to C. neoformans using SP-D⁻/⁻ mice. Here, we demonstrate that mice lacking SP-D were partially protected during C. neoformans infection; they displayed a longer mean time to death and decreased fungal burden at several time points postinfection than wild-type mice. This effect was reversed by the administration of exogenous SP-D. Furthermore, we show that SP-D bound to the surface of the yeast cells and protected the pathogenic microbes against macrophage-mediated defense mechanisms and hydrogen peroxide (H₂O₂)-induced oxidative stress in vitro and in vivo. These findings indicate that C. neoformans is capable of coopting host SP-D to increase host susceptibility to the yeast. This study establishes a new paradigm for the role played by SP-D during host responses to C. neoformans and consequently imparts insight into potential future preventive and/or treatment strategies for cryptococcosis.
Infection and immunity 04/2012; 80(7):2444-53. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epithelial cell dysfunction is now recognized as an important mechanism in the pathogenesis of interstitial lung diseases. Surfactant Protein C (SP-C), an alveolar type II cell specific protein, has contributed to this concept with the observation that heterozygous expression of SFTPC gene mutations are associated with chronic interstitial lung disease. We have shown that transient expression of aggregation prone mutant SP-C isoforms (SP-C BRICHOS) destabilize ER quality control mechanisms resulting in the intracellular accumulation of aggregating propeptide, inhibition of the ubiquitin/proteasome system, and activation of apoptosis. The goal of the present study was to define signaling pathways linking the unfolded protein response (UPR) and subsequent ER stress with intrinsic apoptosis events observed following mutant SP-C expression. In vitro expression of the SP-C BRICHOS mutant, SP-C(Δexon4), was used as a model system. Here we show stimulation of a broad ER stress response in both transfected A549 and HEK293 cells with activation of all 3 canonical sensing pathways, IRE1/XBP-1, ATF6, and PERK/eIF2α. SP-C(Δexon4) expression also resulted in activation of caspase 3, but failed to stimulate expression of the apoptosis mediating transcription factors ATF4/CHOP. However, inhibition of either caspase 4 or c-jun kinase (JNK) each blocked caspase 3 mediated cell death. Taken together, these results suggest that expression of SP-C BRICHOS mutants induce apoptosis through multiple UPR signaling pathways, and provide new therapeutic targets for the amelioration of ER stress induced cytotoxicity observed in fibrotic lung remodeling.
The international journal of biochemistry & cell biology 01/2012; 44(1):101-12. · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Surfactant protein (SP)-D and SP-A have been implicated in immunomodulation in the lung. It has been reported that patients with idiopathic pulmonary fibrosis (IPF) often have elevated serum levels of SP-A and SP-D, although their role in the disease is not known.
The goal of this study was to test the hypothesis that SP-D plays an important role in lung fibrosis using a mouse model of fibrosis induced by bleomycin (BLM).
Triple transgenic inducible SP-D mice (iSP-D mice), in which rat SP-D is expressed in response to doxycycline (Dox) treatment, were administered BLM (100 U/kg) or saline subcutaneously using miniosmotic pumps.
BLM-treated iSP-D mice off Dox (SP-D off) had increased lung fibrosis compared with mice on Dox (SP-D on). SP-D deficiency also increased macrophage-dominant cell infiltration and the expression of profibrotic cytokines (transforming growth factor [TGF]-β1, platelet-derived growth factor-AA). Alveolar macrophages isolated from BLM-treated iSP-D mice off Dox (SP-D off) secreted more TGF-β1. Fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, were increased to a greater extent in the lungs of the BLM-treated iSP-D mice off Dox (SP-D off). Fibrocytes isolated from BLM-treated iSP-D mice off Dox (SP-D off) expressed more of the profibrotic cytokine TGF-β1 and more CXCR4, a chemokine receptor that is important in fibrocyte migration into the lungs. Exogenous SP-D administered intratracheally attenuated BLM-induced lung fibrosis in SP-D(-/-) mice.
These data suggest that alveolar SP-D regulates numbers of macrophages and fibrocytes in the lungs, profibrotic cytokine expression, and fibrotic lung remodeling in response to BLM injury.
American Journal of Respiratory and Critical Care Medicine 12/2011; 185(5):525-36. · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interstitial lung disease in both children and adults has been linked to mutations in the lung-specific surfactant protein C (SFTPC) gene. Among these, the missense mutation [isoleucine to threonine at codon 73 = human surfactant protein C (hSP-C(I73T) )] accounts for ∼30% of all described SFTPC mutations. We reported previously that unlike the BRICHOS misfolding SFTPC mutants, expression of hSP-C(I73T) induces lung remodeling and alveolar lipoproteinosis without a substantial Endoplasmic Reticulum (ER) stress response or ER-mediated intrinsic apoptosis. We show here that, in contrast to its wild-type counterpart that is directly routed to lysosomal-like organelles for processing, SP-C(I73T) is misdirected to the plasma membrane and subsequently internalized to the endocytic pathway via early endosomes, leading to the accumulation of abnormally processed proSP-C isoforms. Functionally, cells expressing hSP-C(I73T) demonstrated both impaired uptake and degradation of surfactant phospholipid, thus providing a molecular mechanism for the observed lipid accumulation in patients expressing hSP-C(I73T) through the disruption of normal phospholipid recycling. Our data provide evidence for a novel cellular mechanism for conformational protein-associated diseases and suggest a paradigm for mistargeted proteins involved in the disruption of the endosomal/lysosomal sorting machinery.
[Show abstract][Hide abstract] ABSTRACT: Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.
Proceedings of the National Academy of Sciences 06/2011; 108(26):10562-7. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: All tissues and organs can be classified according to their ability to repair and regenerate during adult homeostasis and after injury. Some exhibit a high rate of constant cell turnover, while others, such as the lung, exhibit only low-level cell regeneration during normal adult homeostasis but have the ability to rapidly regenerate new cells after injury. Lung regeneration likely involves both activation of progenitor cells as well as cell replacement through proliferation of remaining undamaged cells. The pathways and factors that control this process and its role in disease are only now being explored. In this Review, we will discuss the connection between pathways required for lung development and how the lung responds to injury and disease, with a particular emphasis on recent studies describing the role for the epithelium in repair and regeneration.
The Journal of clinical investigation 06/2011; 121(6):2065-73. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pulmonary phenotype of Hermansky-Pudlak syndrome (HPS) in adults includes foamy alveolar type 2 cells, inflammation, and lung remodeling, but there is no information about ontogeny or early disease mediators.
To establish the ontogeny of HPS lung disease in an animal model, examine disease mediators, and relate them to patients with HPS1.
Mice with mutations in both HPS1/pale ear and HPS2/AP3B1/pearl (EPPE mice) were studied longitudinally. Total lung homogenate, lung tissue sections, and bronchoalveolar lavage (BAL) were examined for phospholipid, collagen, histology, cell counts, chemokines, surfactant protein D (SP-D), and S-nitrosylated SP-D. Isolated alveolar epithelial cells were examined for expression of inflammatory mediators, and chemotaxis assays were used to assess their importance. Pulmonary function test results and BAL from patients with HPS1 and normal volunteers were examined for clinical correlation.
EPPE mice develop increased total lung phospholipid, followed by a macrophage-predominant pulmonary inflammation, and lung remodeling including fibrosis. BAL fluid from EPPE animals exhibited early accumulation of both SP-D and S-nitrosylated SP-D. BAL fluid from patients with HPS1 exhibited similar changes in SP-D that correlated inversely with pulmonary function. Alveolar epithelial cells demonstrated expression of both monocyte chemotactic protein (MCP)-1 and inducible nitric oxide synthase in juvenile EPPE mice. Last, BAL from EPPE mice and patients with HPS1 enhanced migration of RAW267.4 cells, which was attenuated by immunodepletion of SP-D and MCP-1.
Inflammation is initiated from the abnormal alveolar epithelial cells in HPS, and S-nitrosylated SP-D plays a significant role in amplifying pulmonary inflammation.
American Journal of Respiratory and Critical Care Medicine 05/2011; 184(4):449-58. · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.
The Journal of Lipid Research 05/2011; 52(8):1471-82. · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pulmonary surfactant protein D (SP-D) is considered as a candidate biomarker for the functional integrity of the lung and for disease progression, which can be detected in serum. The origin of SP-D in serum and how serum concentrations are related to pulmonary concentrations under inflammatory conditions is still unclear.
In a cross-sectional study comprising non-smokers (n=10), young--(n=10), elderly smokers (n=20), and smokers with COPD (n=20) we simultaneously analysed pulmonary and serum SP-D levels with regard to pulmonary function, exercise, repeatability and its quaternary structure by native gel electrophoresis. Statistical comparisons were conducted by ANOVA and post-hoc testing for multiple comparisons; repeatability was assessed by Bland-Altman analysis.
In COPD, median (IQR) pulmonary SP-D levels were lower (129(68) ng/ml) compared to smokers (young: 299(190), elderly: 296(158) ng/ml; p<0.01) and non-smokers (967(708) ng/ml; p<0.001). The opposite was observed in serum, with higher concentrations in COPD (140(89) ng/ml) as compared to non-smokers (76(47) ng/ml; p<0.01). SP-D levels were reproducible and correlated with the degree of airway obstruction in all smokers. In addition, smoking lead to disruption of the quaternary structure.
Pulmonary and serum SP-D levels are stable markers influenced by smoking and related to airflow obstruction and disease state. Smaller subunits of pulmonary SP-D and the rapid increase of serum SP-D levels in COPD due to exercise support the translocation hypothesis and its use as a COPD biomarker.
no interventional trial.
Respiratory research 03/2011; 12:29. · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Serum levels of surfactant protein D (SP-D) have been suggested as reflecting epithelial damage in acute lung injury, COPD, and idiopathic pulmonary fibrosis (IPF). However, little is known about SP-D levels in the setting of lung transplantation.
We examined plasma SP-D levels in 104 subjects from a prospective, multicenter cohort study of lung allograft recipients. Plasma SP-D was measured by enzyme-linked immunosorbent assay prior to transplant and daily for 3 days after transplant.
Subjects undergoing transplant for IPF had higher baseline SP-D levels (median, 325 ng/mL) compared with subjects with cystic fibrosis, COPD, and pulmonary hypertension (median, 100, 80, and 82 ng/mL, respectively; P = .0001). Among subjects with IPF undergoing bilateral transplant, SP-D levels declined rapidly postoperatively. In contrast, SP-D levels in subjects undergoing single lung transplant for IPF remained significantly higher than those of bilateral allograft recipients. Among subjects undergoing single lung transplant for IPF, the development of primary graft dysfunction (PGD) was associated with a subsequent rise in SP-D levels, whereas SP-D levels in IPF subjects undergoing bilateral transplant declined, even in the presence of grade 3 PGD. Importantly, single lung allograft recipients without PGD had higher postoperative SP-D levels than bilateral allograft recipients with PGD.
Subjects undergoing lung transplant for IPF have significantly higher baseline plasma SP-D levels compared with those with other diagnoses. Plasma SP-D is likely a biomarker of the air-blood barrier integrity in the native IPF lung, but may be less useful as a biomarker of PGD after transplant.
[Show abstract][Hide abstract] ABSTRACT: Acute lung injury (ALI) acts as a complex genetic trait, yet its genetic risk factors remain incompletely understood. Large-scale genotyping has not previously been reported for ALI.
To identify ALI risk variants after major trauma using a large-scale candidate gene approach.
We performed a two-stage genetic association study. We derived findings in an African American cohort (n = 222) using a cardiopulmonary disease-centric 50K single nucleotide polymorphism (SNP) array. Genotype and haplotype distributions were compared between subjects with ALI and without ALI, with adjustment for clinical factors. Top performing SNPs (P < 10(-4)) were tested in a multicenter European American trauma-associated ALI case-control population (n = 600 ALI; n = 2,266 population-based control subjects) for replication. The ALI-associated genomic region was sequenced, analyzed for in silico prediction of function, and plasma was assayed by ELISA and immunoblot.
Five SNPs demonstrated a significant association with ALI after adjustment for covariates in Stage I. Two SNPs in ANGPT2 (rs1868554 and rs2442598) replicated their significant association with ALI in Stage II. rs1868554 was robust to multiple comparison correction: odds ratio 1.22 (1.06-1.40), P = 0.0047. Resequencing identified predicted novel splice sites in linkage disequilibrium with rs1868554, and immunoblots showed higher proportion of variant angiopoietin-2 (ANG2) isoform associated with rs1868554T (0.81 vs. 0.48; P = 0.038).
An ANGPT2 region is associated with both ALI and variation in plasma angiopoietin-2 isoforms. Characterization of the variant isoform and its genetic regulation may yield important insights about ALI pathogenesis and susceptibility.
American Journal of Respiratory and Critical Care Medicine 01/2011; 183(10):1344-53. · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Surfactant protein D (SP-D), a 43-kD collectin, is synthesized and secreted by airway epithelia as a dodecamer formed by assembly of four trimeric subunits. We have previously shown that the quaternary structure of SP-D can be altered during inflammatory lung injury through its modification by S-nitrosylation, which in turn alters its functional behavior producing a proinflammatory response in effector cells.
We hypothesized that alterations in structure and function of SP-D may occur in humans with acute allergic inflammation.
Bronchoalveolar lavage (BAL) fluid was collected from 15 nonsmoking patients with mild intermittent allergic asthma before and 24 hours after segmental provocation with saline, allergen, LPS, and mixtures of allergen and LPS. Structural modifications of SP-D were analyzed by native and sodium dodecyl sulfate gel electrophoresis.
The multimeric structure of native SP-D was found to be disrupted after provocation with allergen or a mixture of allergen and LPS. Interestingly, under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that 7 of 15 patients with asthma developed an abnormal cross-linked SP-D band after segmental challenge with either allergen or a mixture of allergen with LPS but not LPS alone. Importantly, patients with asthma with cross-linked SP-D demonstrated significantly higher levels of BAL eosinophils, nitrogen oxides, IL-4, IL-5, IL-13, and S-nitrosothiol-SP-D compared with patients without cross-linked SP-D.
We conclude that segmental allergen challenge results in changes of SP-D multimeric structure and that these modifications are associated with an altered local inflammatory response in the distal airways.
American Journal of Respiratory and Critical Care Medicine 12/2010; 183(7):856-64. · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.
American Journal of Respiratory Cell and Molecular Biology 12/2010; 45(3):498-509. · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lung is continuously exposed to inhaled pathogens (toxic pollutants, micro-organisms, environmental antigens, allergens) from the external environment. In the broncho-alveolar space, the critical balance between a measured protective response against harmful pathogens and an inappropriate inflammatory response to harmless particles is discerned by the innate pulmonary immune system. Among its many components, the surfactant proteins and specifically the pulmonary collectins (surfactant proteins A [SP-A] and D [SP-D]) appear to provide important contributions to the modulation of host defense and inflammation in the lung. Many studies have shown that multimerization of SP-A and SP-D are important for efficient local host defense including neutralization and opsonization of influenza A virus, binding Pneumocystis murina and inhibition of LPS-induced inflammatory cell responses. These observations strongly imply that oligomerization of collectins is a critical feature of its function. However, during the inflammatory state, despite normal pool sizes, chemical modification of collectins can result in alteration of their structure and function. Both pulmonary collectins can be altered through proteolytic inactivation, nitration, S-nitrosylation, oxidation and/or crosslinking as a consequence of the inflammatory milieu facilitated by cytokines, nitric oxide, proteases, and other chemical mediators released by inflammatory cells. Thus, this review will summarize recent developments in our understanding of the relationship between post-translational assembly of collectins and their modification by inflammation as an important molecular switch for the regulation of local innate host defense.