Background
Chronic pulmonary obstructive disease (COPD) is predicted to become the fourth leading cause of death worldwide by 2030 [
1],[
2]. Due to the aging population and increasing number of smokers, the burden of medical and social resources for COPD is estimated to be US$47 trillion by 2030 [
3]. Although there are many mediators (i.e., inflammatory cells, lipids, reactivate oxygen species, nitric oxide, peptides, chemokines, cytokines, growth factors, and proteases) and cellular pathways (e.g., inflammation, apoptosis, senescence and repair) involved in the pathogenesis of COPD, increasing evidence indicates that proteases provide vital contributions to all mediators and cellular pathways [
4],[
5]. However, to date, the detailed pathogenic mechanisms of protease-mediated COPD are not fully understood [
3],[
6].
In developed countries, the major factor for the pathogenesis and progression of COPD is cigarette smoke (CS). Exposure to CS results in chronic inflammation, elevated oxidative stress, and protease-anti-protease imbalance within the respiratory system [
7]. The protease-anti-protease imbalance is triggered by the infiltration of inflammatory cells like neutrophils, macrophages, and CD8
+ T lymphocytes [
7]-[
11]. Proteolytic enzymes of neutrophils and macrophages, neutrophil elastase (NE), and matrix metalloproteinase (MMP)-12, degrade their respective inhibitors. Thus, the interaction promotes protease-anti-protease imbalance and destroys the pulmonary parenchyma with alveolar space dilatation, i.e. emphysema, which is a major component of COPD [
12].
Neutrophil elastase is a secreted serine protease that degrades extracellular matrix like elastin, which contributes to the recoil capacity of alveoli [
13]. Other than proteolytic activity, NE up-regulates elafin, interleukin-8, MUC4, and MUC5AC, and promotes the secretion of mucin in LE cells [
14]-[
18]. Excessive NE also results in LE cell apoptosis through protease-activated receptor (PAR)-1, which is abrogated by treatment with retinoic acid [
19],[
20].
Apoptosis of LE cells results in the loss of lung parenchyma and is a potential pathogenic mechanism for emphysema and COPD [
21]. Placenta growth factor (PlGF) induces apoptosis of type II alveolar epithelial cells (AEC II) such that PlGF transgenic mice develop a phenotype of pulmonary emphysema [
22]. PlGF is a member of the vascular endothelial growth factor family that promotes angiogenesis [
23],[
24]. PlGF expression is abundant in the placenta, heart, lungs, thyroid, brain, and skeleton muscle during fetal development, but declines in adulthood [
25]. Higher levels of PlGF have been shown in serum and broncho-alveolar lavage (BAL) fluid of COPD patients and the PlGF levels is inversely proportional to lung function deterioration [
26]. Porcine pancreatic elastase (PPE), a recombinant porcine elastase for the animal model of emphysema, has also been shown to increase PlGF expression in LE cells and promote LE cells apoptosis [
27]. However, the role of NE in human COPD has not been established.
Under the hypothesis that NE, like PPE, up-regulates PlGF expression and leads to LE cell apoptosis and pulmonary emphysema. This study demonstrates that the NE-promoted PlGF expression and secretion in LE cells and lungs. Early growth response gene (Egr)-1 is a transcriptional factor responsible for the up-regulation of PlGF by NE in LE cells. PlGF induces apoptosis through the c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)δ signaling pathways. Ablation of PlGF protects mice from NE-induced pulmonary apoptosis and emphysema. Thus, NE-induced PlGF and the downstream JNK/PKCδ signaling pathways contribute to the pathogenesis of pulmonary emphysema and COPD. Both PlGF and its downstream signaling pathways may be potential therapeutic targets for COPD.
Materials and methods
Reagents
Rabbit antibodies for phosphor-P38 MAPK (p-P38 MAPK), P38 MAPK, MTF-1, p-JNK and p-PKCδ were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies for PlGF, JNK, PKC, and Egr-1, mouse and human PlGF siRNA, mouse and human PKCδ siRNA, and the corresponding scramble siRNA were purchased from Santa Cruz (Santa Cruz, CA, USA), while NE was purchased from Abcam (Cambridge, MA, USA). Trizol reagent, SuperScript III Reverse Transcriptase and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA, USA). Mouse antibody for beta-actin and rabbit antibody for HIF-1alpha were purchased from Genetex (Irvine, CA, USA). Human and mouse recombinant PlGF protein and an enzyme-linked immuno-sorbent assay (ELISA) kit were obtained from R and D Systems (Minneapolis, MN, USA).
A dual-luciferase reporter assay system was obtained from Promega (Madison, WI, USA). Hematoxylin and Eosin, Chromatin immuno-precipitation (ChIP) Assay Kit, and EZ-Zyme Chromatin Prep Kit were purchased from Merck-Millipore (Boston, MA, USA). An in situ cell Death Detection Kit and X-tremeGENE HP DNA Transfection Reagent were purchased from Roche (Mannheim, Germany). The FITC Annexin V apoptosis detection Kit I was obtained from BD Biosciences (San Jose, CA, USA). The JNK inhibitor, SP600125, was obtained from Enzo Life Science (Plymouth Meeting, PA, USA). A SuperSensitive Polymer-HRP IHC Detection System was purchased from Biogenex (Fremont, CA, USA).
Animals
This study conformed to the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85–23, revised 1996). All of the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Center, College of Medicine and Public Health of National Taiwan University. Eight-week-old male C57BL/6 wild type (WT) mice were purchased from the Laboratory Animal Center, College of Medicine and College of Public Health, National Taiwan University. The PlGF knockout (KO) mice in B6 background were provided by Dr. Po-Nien Tsao (National Taiwan University, Taiwan).
Cell culture
Human bronchial epithelial cells, BEAS-2B (ATCC number CRL-9609), were cultured in F12 nutrient mixture (Carlsbad, CA, USA) with 0.5 ng/ml recombinant epidermal growth factor, 500 ng/ml hydrocortisone, 0.005 mg/ml insulin, 0.035 mg/ml bovine pituitary extract, 500 nM ethanolamine, 500 nM phosphoethanolamine, 0.01 mg/ml transferrin, 6.5 ng/ml 3, 3′, 5-triiodothyronine, 500 ng/ml epinephrine, 0.1 ng/ml retinoic acid, 10% FCS 100 unit/ml penicillin, and 100 μg/ml streptomycin in a humidified 95% air-5% CO2 incubator at 37°C. Mouse primary type II alveolar epithelial cells (AEC II) and culture medium were purchased from chi scientific (Maynard, MA, USA). Primary normal human bronchial epithelial (NHBE) cells were kindly provided by Dr. Reen Wu at University of California, Davis.
Plasmids
Human genomic DNA was extracted from BEAS-2B by a Quick-gDNA MiniPrep kit (Zymo Research, CA, USA). The 2.0 kb human PlGF promoter region was amplified from human genomic DNA using polymerase chain reaction (PCR) performed with Hi Fi
Taq DNA polymerase (Geneaid, Taipei, Taiwan) as follows: 2 minutes at 94°C, then 15 sec at 94°C, 30 sec at 59°C, and 2 min and 30 sec at 72°C for 35 cycles. The primers for 2.0 kb human PlGF promoter region were 5′-GC
G GTAC CCA AAC TCA TAC ACA ATA GAC-3′ (forward primer; italic,
KpnI site) and 5′-TT
A AGCT TCC GTA GGT AAG GCT GTG GCT-3′ (reverse primer; italic,
HindIII site). The amplified DNA fragments were cloned into pGL3 vector (Promega, WI, USA) and the sequences were confirmed by DNA sequence analysis. The pGL3 with mouse PlGF promoter was as previously described [
27].
Enzyme-linked immuno-sorbent assay (ELISA)
Cellular medium from BEAS-2B and AEC II, and BAL fluid from mice were analyzed by a PlGF ELISA kit (R & D, MN, USA) according to the manufacturer’s instructions.
Luciferase reporter assay
The BEAS-2B and AEC II were co-transfected with the pGL3-PlGF promoter and pRenilla for 24 h via Lipofectamine 2000 and X-tremeGENE HP DNA Transfection Reagent, and then collected and analyzed on a dual-luciferase reporter assay system (Promega, WI, USA) using a lumicounter Packard BL10000 according to the manufacturer’s instructions.
Protein extraction and immuno-blot analysis
The BEAS-2B and AEC II were lysed using RIPA lysis buffer (Genestar, Taipei, Taiwan), containing 1% NP-40, 0.1% SDS, 150 mM sodium chloride, 0.5% sodium deoxycholate, and 50 mM Tris with a protease inhibitor cocktail (Bionovas, Toronto, Canada) and PhosSTOP (Roche, Basilea, Switzerland). The cell lysates were centrifuged at 12,000 rpm for 5 min and the resulting supernatant was collected.
The extracted protein was quantified by protein assay. Equal amounts of protein were separated using 10% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, MA, USA). After blocking with 5% skimmed milk, the membranes were incubated with various primary antibodies and then incubated with the corresponding secondary antibodies. The protein bands were detected using an Immobilon Western Chemi-luminescent HRP Substrate (Millipore, MA, USA) and quantified by the ImageQuant 5.2 software (Healthcare Bio-Sciences, PA, USA).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
The BEAS-2B and AEC II, and OCT-embedded lung tissue from the mice were analyzed for the apoptosis level using an in situ cell Death Detection Kit (Roche, Basilea, Switzerland) according to the manufacturer’s instructions. Fluorescence-positive cells were photographed by a Leica DM 4000B microscope (Leica, Solms, Germany).
Flow cytometry analysis
The BEAS-2B and AEC II were analyzed on a FITC Annexin V apoptosis detection Kit I (Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The FITC-positive cells were analyzed using a FACS Calibur flow cytometer (Becton Drive, NJ, USA).
Immuno-histochemistry (IHC) assay
Paraffin was removed from paraffin-embedded tissue sections by xylene, dehydrated by ethanol, and re-hydrated by PBS. After treatment with 3% H2O2, the sections were applied to a SuperSensitive Polymer-HRP IHC Detection System (Biogenex, CA, USA) and incubated with PlGF, p-JNK, and p-PKCδ antibodies as primary antibodies. The stained-sections were photographed using a Leica DM 4000B microscope (Leica, Solms, Germany).
Hematoxylin and eosin (H and E) staining
Paraffin was removed from paraffin-embedded tissue sections by xylene, dehydrated by ethanol, and re-hydrated by PBS. Sections stained with H and E were photographed by a Leica DM 4,000 B microscope (Leica, Solms, German).
NE-induced emphysema
The dose of NE was four-fold higher than that of porcine pancreatic elastase according to previous report [
28] and the methodology of intra-tracheal instilling NE was performed as previously described [
29]. Briefly, eight-week-old mice were intra-tracheally given saline (CON), 400 mU/ml NE (NE), 400 mU/ml NE with 50 mg/kg JNK inhibitor SP600125 (NE SP), 3 mg/kg scramble siRNA (NE Si-Sc), 3 mg/kg mouse PKCδ siRNA (NE Si-PK) and 3 mg/kg PlGF siRNA (NE Si-Pl) weekly for one month. The dose of siRNA instillation was according to a previous study [
27],[
30]. Each experimental group had five mice and the processing of lung tissues and BAL fluid were performed as previously described [
27],[
29].
Reverse-transcriptional (RT)-PCR assay
Total RNA of BEAS-2B and AEC II were extracted by Trizol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Total RNA (5 μg) was used in the RT reactions using a SuperScript III Reverse Transcriptase kit (Invitrogen, CA, USA) according to the manufacturer’s instructions to synthesize the cDNA. The human PlGF and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA fragments were amplified from the cDNA by PCR, performed with Dream
Taq DNA polymerase (Fermentas, MA, USA) as follows: 5 min at 95°C, then 30 sec at 98°C, 30 sec at 59°C, and 1 min at 72°C for 35 cycles. The primers for 164 bp human PlGF cDNA fragment were 5′-GGC GAT GAG AAT CTG CAC TGT-3′ and 5′-GAA GAT GAA GCC GGA AAG GTG-3′. The primers for 530 bp human GAPDH cDNA fragment were 5′-GGG CGC CTG GTC ACC AGG GCT G-3′ and 5′-GGG GCC ATC CAC AGT CTT CTG-3′. The primer sets for mouse PlGF and GAPDH was as previously described [
27].
Chromatin immuno-precipitation (ChIP)
Genomic DNA fragment from BEAS-2B were prepared by the EZ-Zyme Chromatin Prep Kit (Millipore, MA, USA) and analyzed using the Chromatin immuno-precipitation (ChIP) Assay Kit (Millipore, MA, USA) to evaluate the associated levels of Egr-1 and PlGF promoter regions. The antibody of Egr-1 was used for immuno-precipitation and the primer set (5′-CAC TTT CCA AGA ATG CCT ATG TCC ATT C-3′ and 5′-TTA AGC TTC CGT AGG TAA GGC TGT GGC T-3′) were used to amplify the human PlGF promoter fragment according to the manufacturer’s instructions.
Statistical analysis
The results were presented as mean ± SEM from five independent experiments and animals. The Mann–Whitney test was used to compare two independent groups. Kruskal-Wallis with Bonferroni post hoc analysis was used for multiple testing. Statistical analyses were performed using the SPSS version 8.0 (SPSS Inc., IL, USA). Statistical significance was set at p < 0.05.
Discussion
There are several conserved trans-elements within the human and mouse PlGF promoter regions, including MRE and HRE [
31],[
32]. Treatment with PlGF does not affect the expressions of MTF-1 and HIF-1α, which are the binding proteins for MRE and HRE. A conserved Egr-1 response element (CCCCGCCCC) [
36] is observed near the transcriptional start site in both mouse and human PlGF promoter. Egr-1 is a rapid response transcription factor for UV and cigarette smoke stimuli that up-regulates several genes, including PTEN, microtubule-associated protein-1 light chain 3, and PAR-1 in LE cells [
36]-[
39]. The Egr-1-upregulated down-stream genes mediate various cellular functions like cell growth, proliferation, differentiation, and apoptosis [
39]. Egr-1 also has an impact on the pathogenesis of acute lung injury [
40]. A previous study has demonstrated that NE inhibitors decrease ventilator-induced Egr-1 expression [
41]. In the present study, NE promotes the transient expression of Egr-1, which is involved in NE-induced PlGF expression.
The present study demonstrates that NE-induced PlGF promotes LE cell apoptosis, which corroborate the results of a previous study [
22]. However, unlike previously established mechanisms of NE-induced LE cell apoptosis [
19],[
20], this study is the first to show that NE-induces LE cell apoptosis through PlGF and PlGF-mediated downstream JNK and PKCδ signaling pathways. The results of NHBE cells further indicate that NE-promoted endogenous PlGF contributes to LE cell apoptosis. Furthermore, NE up-regulates PlGF in endothelial cells and in LE cells. The PlGF-induced LE cell apoptosis may work through both autocrine and paracrine mechanism. In addition, it is interesting to know that the up-regulation of PlGF is identified in an ovalbumin-induced asthma mice model wherein PlGF promotes neutrophilic chemotaxis [
42]. Therefore, the positive feedback loop between NE and PlGF in the pathogenesis of COPD warrants further investigation.
Because of frequently ignored early symptoms and irreversible pulmonary damage, COPD remains a major cause of death worldwide [
2]. As a chronic disease with insidious pathogenesis, COPD is difficult to diagnose early. Useful diagnostic markers will help in the early diagnosis, early treatment, and reduction of mortality and morbidity. A previous report indicates that the NE-digested product, Aα-Val360, may be a marker for COPD [
43]. However, endogenous elastin fragments can disturb the utility of Aα-Val360 for predicting COPD.
The present study demonstrates that PlGF, which physiologically appears only in the embryonic stage, may be a suitable candidate as a diagnostic marker of early COPD. Based on the IHC results and BAL data in a previous study [
26], COPD patients secrete and express more PlGF compared to non-COPD controls. Other than COPD, the up-regulation of PlGF is also associated with higher risk of several human diseases, including age-related macular degradation, sickle cell disease, and most kinds of tumors [
24]. As PlGF expression is barely detectable in healthy adults, further investigation regarding the association between PlGF and COPD may therefore support PlGF as a candidate marker for early COPD.
A previous study indicates that mouse PlGF activates p38 MAPK and JNK signaling pathway in mouse alveolar epithelial cells, and that MLE-15 and human PlGF activates the p38 MAPK and JNK signaling pathway in BEAS-2B. In the present study, PlGF promotes only JNK and PKCδ in AEC II cell. The difference in cell systems may explain why PlGF acts through different down-stream signaling pathways. However, the JNK, p38 MAPK, and PKCδ signaling pathways should all be considered as potential therapeutic targets aside from PlGF for COPD therapy [
44]-[
46].
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Competing interests
The authors declare that they have no competing interests.