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Published ahead of print on March 6, 2008, doi:10.1165/rcmb.2007-0293OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 227-234, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0293OC

Leukotriene D4 Up-Regulates Furin Expression through CysLT1 Receptor Signaling

Charles Thompson1, Stéphanie McMahon1, Ynuk Bossé1, Claire M. Dubois1, Jana Stankova1 and Marek Rola-Pleszczynski1

1 Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada

Correspondence and requests for reprints should be addressed to Marek Rola-Pleszczynski, MD, Immunology Division, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 North 12th Avenue, Sherbrooke, PQ, J1H 5N4 Canada. E-mail: marek.rola-pleszczynski{at}usherbrooke.ca


   Abstract
Top
 Abstract
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Leukotriene (LT)D4 is suggested to play a role in airway remodeling, which is characterized by fibrogenesis and airway smooth muscle cell hyperplasia. In this study, we investigated the effects of LTD4 on the expression of furin, a proprotein convertase involved in the maturation/activation of several substrates implicated in the remodeling processes. HEK293 cells stably transfected with the CysLT1 receptor were used to study the transcriptional regulation of furin by LTD4. Stimulation of the cells with LTD4 resulted in a time- and concentration-dependent induction of furin mRNA and protein expression. The study of furin gene (fur) promoters P1, P1A, and P1B revealed a selective transactivation of the P1 promoter by LTD4. Mutations in the activator protein (AP)-1–binding element of the P1 promoter resulted in the partial loss of transactivation by LTD4. Binding of AP-1 transcription factor to fur P1 promoter after stimulation with LTD4 was demonstrated by electrophoretic mobility shift assay, and supershift assays indicated the formation of c-Jun/c-Fos complexes. LTD4 induced the maturation of the furin substrates membrane-type 1 matrix metalloproteinase and transforming growth factor-β1, which was inhibited by the furin inhibitor {alpha}1-PDX. Finally, LTD4 induced furin gene expression in monocytic THP-1 cells, which was abrogated using a selective CysLT1 receptor antagonist and inhibitors of the mitogen-activated protein kinases MEK-1, p38, and JunK. Our data show for the first time that LTD4, via the CysLT1 receptor, can transcriptionally activate furin production with consequent maturation of furin substrates relevant to airway remodeling. These findings suggest that CysLT1 is involved in remodeling processes through modulation of furin transcription.

Key Words: cysteinyl-leukotriene • convertase • furin • activator protein-1 • membrane-type 1 matrix metalloproteinase

Cysteinyl-leukotrienes (cysLT) are potent lipid mediators implicated in the pathogenesis of allergy and asthma. They are mainly released by eosinophils, mast cells, and macrophages in the airways (1). Leukotriene (LT)C4, LTD4, and LTE4 act on two G protein–coupled receptors, CysLT1 and CysLT2 (2–6). High levels of expression of CysLT1 have been demonstrated in the lung, spleen, and peripheral blood leukocytes (2, 3). Several studies suggest that LTD4 may play a role as a protagonist of airway remodeling, which is characterized by fibrogenesis and airway smooth muscle cell proliferation. We and others have previously reported that cysLTs may contribute to human bronchial smooth muscle cell proliferation (7, 8). Recently, Lex and coworkers established an association between cysLTs in the exhaled breath condensate and reticular basement membrane thickening in human asthma (9). Moreover, Henderson and colleagues demonstrated that CysLT1 antagonists could reduce airway remodeling in a murine asthma model (10). In addition, increased levels of cysLTs have been found in pathologies that include fibrotic processes, such as severe asthma (11, 12) and cystic fibrosis (13).

Airway remodeling involves many fibrogenic molecules known to increase extracellular matrix (ECM) production and turnover, as well as airway smooth muscle cell proliferation. Several of these proteins are first synthesized as larger inactive propeptides that need maturation by endoproteolytic cleavage to become active. This is the case of transforming growth factor β1 (TGF-β1), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and metalloproteinases (MMP), including membrane-type 1 matrix metalloproteinase (MT1-MMP). The proprotein convertase (PC) furin is one of the proteases responsible for this type of specific cleavage. Furin is a member of seven closely related mammalian subtilisin/kexin-like serine proteases. This group of proprotein convertases includes PC1/PC3, PC2, PC4, PC5/PC6, PC7, PACE4, and furin (14). Within this family, selected members exhibit a tissue-specific distribution, such as PC1, PC2, and PC4, whereas furin, PACE-4, PC5/PC6, and PC7 are expressed in a broad range of tissues (15, 16). The wide distribution and regulation of PCs is in accordance with their involvement in several physiological and pathological processes.

The mechanisms of activation of the fur gene in a context of inflammation have not been fully investigated. Furin gene expression is regulated by three distinct promoters, namely P1, P1A, and P1B. As with housekeeping genes, the P1A and P1B promoters contain several Sp1-binding sites in their sequence. Conversely, the P1 promoter bears inducible gene features, with TATA and CAAT elements in its proximal region. Previous reports demonstrated transactivation of the P1 promoter by C/EBP-β, Smads, and HIF-1 (17–20). Proximal fur P1 promoter analysis also revealed an activator protein (AP)-1–binding site in position –179. We recently demontrated that LTD4, through the CysLT1 receptor, could activate the AP-1 signaling pathway, in the context of IL-8 expression (21).

Little is known about the mechanisms by which cysLTs orchestrate remodeling and fibrosis in asthma. From the proposed relationship between cysLTs and airway remodeling as well as the established relationship between furin and pro-fibrogenic substrate maturation/activation, we hypothesized that LTD4 could modulate the expression and function of furin. In the present report, we demonstrated that LTD4 upregulates furin expression at both the mRNA and protein levels. Up-regulation of furin expression by cysLTs resulted in the maturation of the furin substrates MT1-MMP and TGF-β.


   MATERIALS AND METHODS
Top
 Abstract
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Antibodies and Reagents
Specific antibodies against human MT1-MMP hinge region were purchased from Research Diagnostics Inc., and c-Jun (sc-1694X), c-Fos (sc-52X), and JunD (sc-74X) were from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal mouse IgG1 antibody directed against human furin was purchased from Alexis Biochemicals (San Diego, CA). LTD4 was obtained from Cayman Chemical (Ann Arbor, MI). Anti-actin, Aprotinin, 4-(2 aminoethyl)benzenesulfonyl fluoride (AEBSF), leupeptin, NaF, soybean trypsin inhibitor, and Na3VO4 were from Sigma-Aldrich (Oakville, ON, Canada). Hygromycin B (Wisent, St-Bruno, PQ, Canada), and FuGENE6 were purchased from Roche (Laval, PQ, Canada). Montelukast (MK-476) was obtained from Merck Frosst (Pointe-Claire, PQ, Canada). MEK-1 inhibitor PD98059 was from InvivoGen (Hornby, ON, Canada); p38 inhibitor SB203580 was from Sigma-Aldrich; and SP600125 (c-Jun kinase [JNK] inhibitor II) was from Calbiochem (San Diego, CA).

Plasmids
The human fur promoter luciferase constructs pGL2-P1, pGL2-P1-SacI, pGL2-P1-NheI, pGL2-P1-KpnI, pGL2-P1A, and pGL2-P1B were generously provided by Dr Torik A. Y. Ayoubi (University of Leuven and Flanders Interuniversity, Belgium). The pcDNA3.1–MT1-MMP construct was generously provided by Dr R. Béliveau (Hôpital Ste-Justine-Université du Québec à Montréal, Montréal, PQ, Canada) and pcDNA3–{alpha}1-PDX was prepared as previously described (22).

Cells
Human monocytic THP-1 cells (ATCC, Rockville, MD) and MonoMac-1 cells (German Collection of Microorganisms and Cell Cultures [DSMZ], Braunschweig, Germany) were cultured in RPMI-1640 medium (Life Technologies, Burlington, ON, Canada) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich). HEK293 stably expressing CysLT1 were grown in Dulbecco's modified Eagle's medium (DMEM) with glucose (Life Technologies), supplemented with 10% fetal bovine serum (Sigma-Aldrich), gentamicin sulfate (40 µg/ml), and 100 µg/ml hygromycin B. These cells are referred to as 293LT1. Transient transfections were performed with FuGENE 6 according to the manufacturer's protocol, and experiments were performed 48 hours after transfection.

Northern Blot Analysis
Total cellular RNA was extracted using TriPure according to the manufacturer's instruction (Roche Diagnostics); 15 µg of total RNA were separated by electrophoresis on 1% agarose and transferred onto a Hybond N+ membrane (Amersham Pharmacia Biotech, Baie d'Urfé, PQ, Canada) for Northern blot analysis. Furin riboprobe synthesis and hybridization were done as previously described (23). 18S RNA expression was used as an internal control.

Western Blot Analysis
293LT1 cells were incubated in 6-well plates in medium without serum for 24 hours, stimulated with LTD4 (10 nm) for the indicated times, and lysed. Western blot analysis was performed as previously described (21).

Electrophoretic Mobility Shift Assays
Electrophoretic mobility shift assays were performed as previously described (21).

Luciferase Assays
293LT1 cells were seeded in 12-well tissue culture plates 24 hours before transient transfection using 1.5 µl of FuGENE 6 transfection reagent and 0.5 µg of plasmid DNA (fur promoter-luciferase constructs or promoter-less pGL2 plasmid) per well according to the manufacturer's instructions. The day after transfection, cells were serum-starved overnight before stimulation with LTD4 (10nM) or equivalent vehicle (ethanol: EtOH) concentration for 6 hours. Cell lysates were assayed for luciferase activity, which was normalized for basal activity of promoter-less pGL2.

Site-Directed Mutagenesis
The AP-1 sequence located at position –179 within the P1-Kpn1 promoter was mutated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Distinct mutations were generated by replacing 5'-AGTCAG-3' by 5'-ACTTGG-3'. Mutations were verified by direct sequencing.

Real-Time PCR
In selected experiments, THP-1 and MonoMac-1 cells were pretreated for 30 minutes with different inhibitors or vehicle, then stimulated with 10 nM LTD4 for 20 hours. Total RNA was purified by using Trizol reagent (Invitrogen, Burlington, ON, Canada) according to the manuacturer's instructions and 1.0 µg of RNA was converted to cDNA with oligo dT and reverse transcriptase (M-MLV) from Promega (Madison, WI) in a volume of 20 µl. Furin and RPL-PO (Ribosomal protein large PO, as a housekeeping gene) expression were measured using real-time PCR performed on a Rotor-Gene 3000 (Corbett Research, Kirkland, PQ, Canada). The following oligonucleotide primer sets were used. Human furin: forward, 5-CTACACAGGGCACGGCATTG-3 and reverse, 5-CCACACCTACACCACAGACAC-3; human RPL-PO: forward, 5-GATTACACCTTCCCACTTGC-3 and reverse, 5-CCAAATCCCATATCCTCGTCCG-3. Furin and RPL-PO DNA were cloned in pGEMT plasmid (Promega) using the Promega protocol. Both plasmids were amplified in bacteria, extracted with the Qiagen midiprep plasmid extraction kit (Qiagen, Mississauga, ON, Canada) and diluted to perform standard curve amplification. Each sample for the real-time PCR consisted of: 1 µl cDNA, 2.5 mM MgCl2, 100 µM dNTP, 1 µM of primers, 2.5 µl of 10x PCR buffer, 0.5 units of Taq polymerase (New England Biolabs, Pickering, ON, Canada), and 0.8 µl of SYBR Green (1/1,000; stock dilution Molecular Probes, Eugene, OR) in a reaction volume of 25 µl. The cycling program consisted of an initial denaturation at 95°C for 5 minutes, 40 cycles of amplification conditions as follows: 95°C (15 s), 66°C (45 s), 72°C (30 s), and a final extension at 72°C for 6 minutes. Comparison of the expression of each gene between its control and stimulated states was determined with the delta-delta ({Delta}{Delta})Ct, according to the following formula: ({Delta}{Delta}Ct = [(Ct G.O.I.Ctl – Ct HK.G.Ctl) – (Ct G.O.I.STIM. – Ct HK.G.STIM.)]).

Results were then transformed into fold variation measurements: fold increase = 2{Delta}{Delta}Ct.

TGF-β Bioassay
The presence of active TGF-β in cell cultures or in conditioned media was determined by stimulation of a TGF-β–responsive luciferase reporter p(SBE)4-Luc containing four copies of the Smad-binding element found in the human PAI-1 promoter sequence. Values represent luciferase reporter gene activity as previously described (19).

Statistical Analyses
Where mentioned, statistical significance was determined using the Student's t test for paired data (one-tailed) or two-way ANOVA, as appropriate, using PRISM4 software (GraphPad Software Inc, San Diego CA). Differences were considered significant at P <= 0.05.


   RESULTS
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
LTD4 Regulates Endogenous Furin Synthesis in 293LT1 Cells
Cell populations that express CysLT1 usually also express CysLT2, the second member of the cysLT receptor family, responsive to the same agonists. Hence, Transfected cell lines stably expressing only the CysLT1 receptor, the 293LT1 cells, as previously described (21), were used to dissect selective receptor-mediated mechanisms. Time-course and concentration–response experiments were conducted to investigate the effect of LTD4 on furin expression in 293LT1 cells. Kinetics of furin mRNA expression after LTD4 stimulation are shown in Figure 1A. Results indicate an up-regulation of furin mRNA expression starting at 6 hours and maintained through 24 hours of LTD4 stimulation. Furin mRNA expression was dependent on the concentration of LTD4, as illustrated in Figure 1B, with a maximal effect at 10nM of LTD4 for 8 hours. Figure 1C illustrates the significant accumulation of furin protein in response to LTD4 after 16 hours of stimulation. These results indicate that furin can be induced by cysLTs at both the mRNA and the protein level.


Figure 1
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  		Figure 1. Leukotriene (LT)D4 induces furin expression at both mRNA and protein levels. (A) 293LT1 cells were stimulated with LTD4 (10 nM) or ethanol (EtOH) for indicated times or (B) incubated in presence of LTD4 or EtOH for 8 hours at indicated concentrations. Furin mRNA expression is shown by Northern blot analysis using a specific human furin riboprobe. Densitometry ratios of furin mRNA to 18S RNA were normalized with a value of 1 for unstimulated cells and are indicated below the blots. (C) Western blot analysis of furin expression by 293LT1 cells stimulated with LTD4 (10 nM) or EtOH for 16 hours. The densitometry ratio of furin to β-actin was normalized with a value of 1 for unstimulated cells and is indicated below the blot. Data are representative of three independent experiments.

 
Analysis of Fur Promoter Activity in Response to LTD4
Furin transcription is driven by three alternative promoters, P1, P1A, and P1B. Since we observed that LTD4 increased furin mRNA expression, we investigated whether LTD4 could induce furin transcription and if so, which promoter was involved. In brief, we measured the capacity of LTD4 to activate the transcription of a luciferase reporter gene driven by each promoter. As shown in Figure 2A, LTD4 strongly increased P1 promoter activity (up to 7-fold); in contrast, it had no effect on P1A and P1B promoters. To define the P1 promoter region involved in this response, 5' deletion constructs were tested. Results in Figure 2B indicate that P1 promoter activity in response to LTD4 stimulation gradually decreased after truncation of the distal promoter region. These results suggest the presence of active transcription sites dispersed throughout the P1 promoter. Transactivation activity of P1-Kpn1 deletion mutant was significantly reduced by39% as compared with the full-length promoter. The shorter P1-Kpn1 region, however, still demonstrated significant promoter activity (4-fold) in response to LTD4, which indicates that the proximal region of P1 is responsible for the greater part of the transcriptional activity induced by LTD4. We therefore decided to focus on the proximal region of the P1 promoter in subsequent experiments.


Figure 2
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  		Figure 2. Analysis of fur promoter activity in response to LTD4. (A) 293LT1 cells were transiently transfected with fur P1, P1A, or P1B (0.5 µg/well). Cells were incubated for 6 hours with LTD4 (10 nM) or EtOH before measurement of luciferase activity. (B) Schematic representation of fur promoter fragments shortened in 5' according to endogenous Sac1, Nhe1, or Kpn1 restriction sites. Base positions are numbered relative to the TATA box. 293LT1 cells were transiently transfected with fur P1, P1-Sac1, P1-Nhe1, or P1-Kpn1 constructs (0.5 µg/well). Cells were incubated for 6 hours with LTD4 (10 nM) or EtOH before measurement of luciferase activity. Data are expressed as fold increase relative to respective EtOH control (*P < 0.05; n = 3).

 
Analysis of the AP-1–Binding Site within the P1-Kpn1 Promoter Region
Nucleotide sequence of the proximal fragment (P1-Kpn1) revealed the presence of a potential AP-1–binding site (5'-AGTCAG-3') at position –179. Thus, we assessed whether AP-1 DNA binding to fur P1 promoter was induced by LTD4. As illustrated in Figure 3A, exposure of 293LT1 cells to LTD4 resulted in time-dependent induction of AP-1 DNA binding activity, which could be observed within 60 minutes of stimulation. To study the nature of the complex induced by LTD4, supershift experiments were performed using specific antibodies directed against c-Jun, JunD, or c-Fos. Results in Figure 3B indicate that the AP-1–binding complex induced by LTD4 was composed of c-Jun and c-Fos. To determine the implication of the AP-1 site in the context of LTD4 stimulation, we used a fur P1-Kpn1 promoter construct containing a site-specific mutation. As shown in Figure 4, the AP-1 site mutation significantly reduced the P1 promoter activity induced by LTD4.


Figure 3
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  		Figure 3. LTD4 up-regulates activator protein (AP)-1 DNA binding to fur P1 promoter. (A) 293LT1 cells were cultured in presence of LTD4 (10 nM) or EtOH for indicated times before nuclear extract preparation. The nuclear extracts were analyzed by electrophoretic mobility shift assay (EMSA) using fur P1 promoter AP-1 oligonucleotide radiolabeled probe. (B) Cells were stimulated with LTD4 (10 nM) or EtOH for 2 hours before EMSA analysis. The specificity of complex formation was tested by the inclusion of unlabeled competitors (cold probe, lane 7) and by including specific anti–c-Jun (lane 3), anti–c-Fos (lane 4), or anti-JunD (lane 5) antibodies, or an isotype-matched control antibody (lane 6). SS: Supershift.

 

Figure 4
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  		Figure 4. Implication of AP-1–binding site in fur P1-Kpn1 promoter transactivation by LTD4. 293LT1 cells were transiently transfected with 0.5 µg/well of pGL2 (promoterless vector), P1-Kpn1–WT, or P1-Kpn1–{Delta}AP-1 promoter constructs. Cells were incubated for 6 hours with EtOH or LTD4 (10 nM) before measurement of luciferase activity. Data are expressed as fold increase relative to EtOH control. *P < 0.05 relative to EtOH control cells, n = 3.

 
Impact of Increased Furin Expression on Substrate Maturation
We investigated whether furin expression induced by LTD4 correlated with an increase in maturaton/activation of the furin substrates, MT1-MMP and TGF-β. These substrates contain specific furin cleavage sites, and are involved in remodeling processes. We overexpressed MT1-MMP in 293LT1 cells to analyze the generation of a digestion product in response to LTD4 stimulation. As illustrated in Figure 5A, a high-molecular-weight band (66 kD) corresponding to the unprocessed MT1-MMP precursor is seen in all culture conditions. LTD4 induced conversion of the MT1-MMP precursor into the 60-kD mature form, which was totally abrogated by overexpression of {alpha}1-PDX, a specific furin inhibitor (24). We also studied the impact of furin up-regulation on TGF-β–like activity. After binding to their cognate receptors, TGF-β family members activate the Smad signaling pathway. Hence, to investigate bioactive TGF-β–like production, 293LT1 cells were transfected with the TGF-β–inducible reporter (SBE)4-luc, a reporter gene that specifically responds to Smad protein–induced transactivation (25). As shown in Figure 5B, LTD4 induced a strong activation of the TGF-β–inducible reporter, which was partially prevented by co-tranfection with {alpha}1-PDX.


Figure 5
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  		Figure 5. Biological impact of LTD4-induced furin gene expression. (A) Western blot analysis of furin processing of MT1-MMP. 293LT1 cells were transiently transfected with MT1-MMP and an empty vector or the furin inhibitor {alpha}1-PDX and cultured in presence of LTD4 (10 nM) or EtOH for 24 hours. Total proteins were separated by electrophoresis and analyzed by Western blot using indicated antibodies. Total actin protein expression served as internal control. This experiment is representative of three. (B) 293LT1 cells were transiently transfected with the TGF-β–inducible reporter (SBE)4-Luc and empty vector or the furin inhibitor {alpha}1-PDX and cultured in presence of EtOH or LTD4 (10 nM) for 24 hours before measurement of luciferase activity. Data are expressed as fold increase relative to respective EtOH control (*P < 0.05, using paired Student's t test; n = 3).

 
LTD4 Induces Fur Gene Expression in Human Monocytic THP-1 Cells
To study the effect of LTD4 on furin expression through the endogenous CysLT1 receptor, we used human monocytic THP-1 cells as a model. The CysLT1 receptor is expressed in mononuclear phagocytes and, as we previously reported, it is up-regulated by IL-4 and IL-13 (26). Based on this, THP-1 cells were cultured in the presence of IL-4 (10 ng/ml) for 24 hours before stimulation with LTD4 or vehicle (EtOH) for 8 hours. As shown in Figures 6A and 6B, LTD4 induced expression of furin mRNA in THP-1 cells, which was prevented by pretreatment with the selective CysLT1 antagonist MK-476 (montelukast), indicating that the effect induced by LTD4 was mediated through the CysLT1 receptor.


Figure 6
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  		Figure 6. Induction of furin gene expression by LTD4 in monocytic THP-1 and MonoMac-1 cells. THP-1 cells were cultured for 24 hours in the presence of IL-4 (10 ng/ml) before addition of LTD4. (A) Northern blot analysis of Furin mRNA expression in THP-1 cells preincubated in the absence or presence of the CysLT1 antagonist MK-476 (1 µM) and then cultured for 8 hours in the presence of (10 nM) LTD4 or vehicle (EtOH). The 18S RNA expression was used as an internal control. Results are representative of three separate experiments. (B) Densitometry ratios of Furin/18S RNA of results presented in A (*P < 0.05). THP-1 (C) and MonoMac-1 (D) cells were pretreated for 30 minutes with JNK inhibitor II (10 µM), PD98059 (10 µM), SB203580 (10 µM), or their control vehicle before an 18-hour incubation with LTD4 (50 nM) or its vehicle (EtOH). Alternatively, cells were pretreated with the LTD4 antagonist montelukast (MK476, 10 µM). Furin mRNA expression was measured using quantitative real-time PCR. Results represent fold induction of furine mRNA expression for each treatment when compared with their respective control (n = 4; *P < 0.05).

 
Moreover, since MEK and JNK are upstream activators of the AP-1 transcriptional system, we tested whether inhibition of these kinase would prevent the effect of LTD4 on furin expression. When THP-1 and MonoMac-1 cells were pretreated with inhibitors of JNK, MEK-1, or p38 MAPK, the induction of furin mRNA expression was abrogated (Figures 6C and 6D).


   DISCUSSION
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Airway remodeling is a feature of chronic severe asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), and idiopathic pulmonary fibrosis (27, 28). In chronic asthma, the phenomenon is characterized by airway smooth muscle cell hyperplasia, extracellular matrix deposition, angiogenesis, and microvascular remodeling under the influence of fibrogenic molecules (28). Among these, TGF-β (29, 30), PDGF (31, 32), IGF (33), and MT1-MMP (34, 35) all contain specific furin cleavage sites. The correlation between the presence, and regulation of expression, of the convertase furin and airway pathology, however, has not been demonstrated as yet.

CysLTs are well characterized as major players in pulmonary inflammation. LTD4, through the CysLT1 receptor, is responsible for bronchoconstriction and mucus hypersecretion (36–38). Some evidence also supports the implication of cysLTs in pulmonary remodeling processes, but the mechanisms remain unclear. In this work, we propose a mechanism by which cysLTs could orchestrate airway remodeling, namely through the regulation of furin expression. We demonstrated that LTD4, via the CysLT1 receptor, can induce the expression of furin, a well-characterized proprotein convertase that plays a key role in proteolytic processing/activation of proproteins. Interestingly, a recent study demonstrated that platelet-activating factor (PAF), another inflammatory lipid mediator, can induce furin expression in corneal myofibroblasts (39).

Fur gene transcription is under the control of three alternatively spliced promoters. Regulation of fur gene expression is still poorly understood, especially in the context of inflammation. The P1A and P1B promoters resemble housekeeping genes with multiple Sp1-binding sites. On the other hand, the P1 promoter is inducible in response to different transcription factors (17, 18, 20, 40). In this respect, using a reporter gene assay, our studies demonstrated that LTD4-induced furin expression was transcriptionally regulated, and that, as expected, only the P1 promoter could be induced by LTD4. From this, deletion studies of the P1 promoter revealed that the transactivation gradually decreased in response to LTD4 as the promoter sequence was progressively truncated in 5'. The proximal P1-Kpn1 promoter, however, was still highly inducible through CysLT1 activation, providing evidence that the proximal fragment could drive fur gene activation after LTD4 stimulation. Analysis of the proximal P1-Kpn1 promoter region revealed the presence of an AP-1–binding site at position –179.

We recently reported in 293LT1 cells that LTD4 could induce AP-1–signaling pathways, leading to IL-8 transactivation. AP-1–responsive elements are part of many gene promoters, including IL-8 (41), COX-2 (42), and c-Jun (43). AP-1 is a dimeric transcription factor consisting of the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) family members (44). The AP-1 complex can also be regulated post-translationally by phosphorylation, which can influence the stability of its members, its transactivating potential, and its DNA-binding capacity (44). Our results demonstrated that LTD4 induced a time-dependent induction of AP-1 binding to the proximal P1 promoter. Supershift assays indicated that the AP-1 complex, which bound to the fur gene, was composed of c-Jun and c-Fos. The role of AP-1 in the induction of fur gene expression by LTD4 was demonstrated by a reduction of the P1-Kpn1 promoter transactivation when the the AP-1–binding site was mutated. Moreover, we also found LTD4 to induce phosphorylation of c-Jun in a time-dependent manner (data not shown). This is the first demonstration of the transactivation of the fur gene via the AP-1–binding site, and it suggests that other activators of AP-1 could also induce fur gene transcription. Moreover, since mutation of the AP-1–binding site did not completely abrogate CysLT1-dependent furin gene transcription, our data suggest that additional transcription factors may be involved. Indeed, the proximal P1 fur promoter also contains potential GATA and Sp1 cis-elements.

The induction of furin expression by cysLTs may be involved in the increased bioavailability of fibrogenic/angiogenic mediators such as MT1-MMP and TGF-β. These mediators are well-characterized furin substrates that have been shown to play a role in several aspects of remodeling. MT1-MMP, through proteolytic events, regulates various cellular functions, including ECM turnover, promotion of cell migration, and invasion. MT1-MMP acts either through direct degradation of ECM components or indirectly by activating pro-MMP2 (45). In addition, these MMPs are involved in the construction of the vascular tubular network, in part, through the release or the activation of growth factors (46, 47). TGF-β1, in turn, creates a favorable environment for airway remodeling and fibrosis by repressing immune surveillance, inducing the production of angiogenic factors, such as vascular endothelial growth factor, and by increasing the production of ECM (48, 49). It is well known that TGF-β is up-regulated in patients with asthma (50, 51), and we have recently shown that LTD4 could enhance TGF-β production in human lung epithelial cells (52). In addition to TGF-β and MT1-MMP, a multiplicity of other established furin substrates involved in cell growth and survival (insulin-like growth factor receptor-1 and platelet-derived growth factor), cell invasion (E-cadherin and integrins) (53, 54), and angiogenesis (vascular endothelial growth factor-c) (55) support the contention that the regulation of furin activity within the area of pulmonary inflammation could profoundly impact the course of airway remodeling (Figure 7). Futhermore, ADAM33, a disintegrin and metalloprotease 33, a new member of the ADAM family, has been identified as an asthma susceptibility gene via genetic linkage analysis (56). Interestingly, some reports suggest a role for ADAM33 in airway remodeling (57). Indeed, ADAM33 needs to be activated by cleavage within a putative furin cleavage site (58), reinforcing the impact of furin up-regulation in airway remodeling.


Figure 7
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  		Figure 7. Proposed impact of cysLT-induced furin expression on airway remodeling. In this illustration, we propose a series of sequential events, in a context of airway remodeling, from inflammation and cysLT-induced up-regulation of furin to substrate processing by furin and bioactivation of several fibrogenic/angiogenic factors leading to airway remodeling. Relevance to other pathological processes is not excluded.

 
In summary, we propose a new mechanism for cysLT involvement in airway remodeling. The current study suggests that LTD4 not only acts as an acute inflammatory mediator, but can also promote the production of furin, a protease that activates a number of fibrogenic and angiogenic factors implicated in lung remodeling (Figure 7). Selective CysLT1 antagonists could thus be considered for more chronic aspects of asthma, including asthmatic inflammation and airway remodeling.


   Acknowledgments
 
The authors thank Dr. Torik A. Y. Ayoubi (University of Leuven and Flanders Interuniversity, Belgium) for the fur promoter constructs, and Dr. R. Béliveau (Hôpital Ste-Justine, Montreal, PQ, Canada) for the pcDNA3.1-MT1-MMP construct.


   Footnotes
 
This work was supported by grants (C.M.D., J.S., and M.R.-P.) and studentships (C.T., S.M., and Y.B.) from the Canadian Institutes of Health Research, and by a Canada Research Chair in Inflammation (M.R.-P.).

Originally Published in Press as DOI: 10.1165/rcmb.2007-0293OC on March 6, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 31, 2007

Accepted in final form February 21, 2008


   References
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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