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Published ahead of print on September 29, 2005, doi:10.1165/rcmb.2005-0187OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 49-55, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0187OC

RNA Interference Decreases PAR-2 Expression and Function in Human Airway Smooth Muscle Cells

Thomas Trian, Pierre-Olivier Girodet, Olga Ousova, Roger Marthan, J. Manuel Tunon-de-Lara and Patrick Berger

Université Victor Segalen Bordeaux 2, Laboratoire de Physiologie Cellulaire Respiratoire; INSERM, E356, Bordeaux, France

Correspondence and requests for reprints should be addressed to P. Berger, M.D., Ph.D., Laboratoire de Physiologie Cellulaire Respiratoire, INSERM E356, Universite Victor Segalen Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France. E-mail: patrick.berger{at}lpcr.u-bordeaux2.fr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Asthma is characterized by bronchial inflammation and hyperresponsiveness that involves mast cell tryptase and potentially its specific receptor protease activated receptor 2 (PAR-2). Tryptase increases free intracellular calcium concentration ([Ca2+]i), a key step in activation of human airway smooth muscle cells (HASMC). The aim of this study was to analyze the effect of PAR-2 gene silencing on HASMC, in terms of calcium response, since no antagonist is available for this receptor. Five siRNA against PAR-2 were synthesized and transfected in HASMC using lipid agents, and PAR-2 expression was examined using Western blot, fluorescence-activated cell sorter, immunocytochemistry and RT-PCR. [Ca2+]i was measured using microspectrofluorimetry in response to tryptase, the activating peptide SLIGKV, trypsin, or caffeine. Two siRNA significantly inhibited PAR-2 expression in terms of both total and surface protein expression, as well as mRNA levels. Tryptase- and SLIGKV-induced transient increase in [Ca2+]i was significantly inhibited after transfection with the most appropriate siRNA, whereas neither trypsin nor caffeine response was altered. Two control siRNA had no effect in terms of both PAR-2 expression and calcium response. Transfection efficiency was maximal after 24 h and disappeared after 48 h. Gene silencing using siRNA can thus be used in vitro to assess the function of PAR-2 in HASMC.

Key Words: asthma • calcium • mast cell • siRNA • tryptase


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Mast cell infiltration is an important component of inflammation in asthma, a disease characterized by bronchial hyperresponsiveness (BHR) and infiltration of airway mucosa by several cell types, including eosinophils and activated mast cells (1). It has been clearly demonstrated that inflammatory infiltration also concerns the smooth muscle layer and that the number of mast cells infiltrating the bronchial smooth muscle is higher in patients with asthma than in normal subjects and closely related with BHR (2). The mechanism of mast cell infiltration involves the secretion of chemotactic factors such as transforming growth factor (TGF)-{beta}1, stem cell factor (3), and CXCL10 (4) by human airway smooth muscle cells (HASMC). Mast cell–derived products play a major role in this chemotactic activity of HASMC (3).

The neutral serine protease tryptase (EC 3.4.21.59) is the major mast cell product. Tryptase plays an important role in both airway remodeling through its effect on HASMC proliferation (5, 6), and on BHR. Tryptase enhances the contractile response of isolated bronchi to nonspecific agonists (7, 8) and induces BHR in vivo in a model of allergic sheep (9). Since BHR is closely related with HASMC intracellular calcium homeostasis, we analyzed in a previous work the effects of tryptase on calcium response (10). We found that tryptase induces calcium transients in HASMC through the activation of phospholipase C and the mobilization of endoplasmic reticulum store. The subtype 2 of the protease-activated receptor (PAR-2), belonging to the expanding family of G protein–coupled receptors, is thought to be specifically activated by tryptase (11). Activation of PAR is different from that of other seven transmembrane domains G protein–coupled receptors. Proteases cleave PAR within the extracellular N-terminal domain, exposing a new N-terminus that acts as a tethered ligand by binding to extracellular domains of the receptor and thereby activating the cleaved receptor molecule (11). The peptide agonist SLIGKV-NH2 corresponding to the tethered ligand in human PAR-2 activates PAR-2 and mimics the effects of tryptase on HASMC (e.g., calcium rise [10], proliferation [6], and cytokine synthesis [3]). However, there has been no demonstration so far of a direct role of PAR-2 in tryptase-induced effects on HASMC.

Several experimental approaches have been used to analyze the effect of tryptase and the role of PAR-2 in BHR. On the one hand, pharmacologic studies using protease inhibitors in vivo have yielded various findings. The tryptase inhibitor APC366 failed to alter early asthmatic response or BHR in human (12), whereas it blocked BHR, both early and late responses, after antigen challenge in allergic sheep (13). In addition, the tryptase inhibitor MOL 6131 reduced bronchial inflammation (i.e., eosinophilia, peribronchial edema, release of IL-4 and IL-13) but did not alter BHR in asthmatic mice (14). On the other hand, knockout of PAR-2 in mice delayed onset of inflammation (15) and decreased BHR (16), whereas BHR was aggravated in mice overexpressing PAR-2. Direct analysis of PAR-2 function in human bronchial smooth muscle is thus required to better understand mast cell–HASMC interaction in asthma. However, whereas PAR-1 antagonists have been successfully obtained (17), to the best of our knowledge, there is no available PAR-2 antagonist.

RNA interference (RNAi) is an alternative strategy to examine the function of a receptor that has not been used, so far, in HASMC. The aim of the present study was thus to analyze the effect of PAR-2 gene silencing on HASMC using RNAi on calcium signaling. We found that RNAi decreases: (1) PAR-2 expression in terms of protein expression, and mRNA levels; and (2) tryptase and SLIGKV-NH2-induced calcium rise, whereas neither trypsin nor caffeine response was altered.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Small Interfering RNA Synthesis
Candidate small interfering RNAs (siRNAs) directed against PAR-2 mRNA were designed according to the criteria defined by Elbashir and coworkers (18). Five potential siRNA were selected also using the prediction of single strand domains within the secondary mRNA structure (19) and subsequent negative BLAST analyses (Table 1). Two control sequences of siRNA No. 1 were also designed with either two central base-pair inversion (No. 1-inv) or a scrambled sequence (No. 1-scr) (Table 1). Again, BLAST analyses were performed and no significant matching in human transcripts was found. siRNA were prepared by in vitro transcription using the Silencer siRNA construction kit (Ambion, Huntingdon, UK). For this purpose, 14 oligonucleotides primers containing 8 bases complimentary to T7 promoter and 21 to the target gene were designed and purchased from Sigma Genosys (Poole, Dorset, UK) (Table 2A). Some of these siRNA were labeled with Cy3 using Silencer siRNA labeling kit (Ambion).


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  		TABLE 1. siRNA SEQUENCES AND PROPERTIES

 

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  		TABLE 2. PRIMERS PAIRS FOR BOTH siRNA SYNTHESIS AND QUANTITATIVE RT-PCR

 
Cell Cultures
HASMC were derived by primary culture from bronchial tissue, as described previously (10). Patients' characteristics are provided in Table 3. Smooth muscle squares were cultured and maintained in DMEM (Invitrogen, Cergy Pontoise, France) containing 10% (vol/vol) Fetal Calf Serum (FCS; Invitrogen). Cell purity was assessed by immunocytochemistry as described previously (10). Only cells passaged 2 to 4 times with trypsin-ethylene-diamine-tetra acetic acid (EDTA; Invitrogen) were used for this study. Cells were seeded in either 6-, 12-, or 24-well plates or in coverslips at a density of 2 x 105 cells/ml, and grown in 10% FCS DMEM.


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  		TABLE 3. PATIENT CHARACTERISTICS

 
Cell Transfection
The day before transfection, HASMC were plated at 30–35% confluence in culture medium without antibiotics. Transfections were conducted with the cationic lipid Lipofectamine 2000 (Invitrogen). Briefly, 0.5 nmol of siRNA was diluted into free DMEM without serum and antibiotics and mixed in various quantity of lipofectamine. Cells were then transfected at 37°C in 5% CO2 for 6 h and changed with DMEM containing 10% FCS without antibiotics. Control cells were not transfected but processed under similar culture condition.

Immunocytochemistry and Confocal Microscopy
Twenty-four hours after transfection, HASMC were rinsed in PBS and then fixed with cold methanol (VWR international, Strasbourg, France) for 20 min on ice. After drying, the cells were either counterstained with yoyo (Molecular Probes, Eugene, OR) or processed for immunocytochemistry. Cells were treated with 3% bovine serum albumin (BSA; Sigma, Saint Quentin Fallavier, France) and incubated with 1 µg/ml of a mouse monoclonal anti-human PAR-2 antibody (SAM11; Santa Cruz Biotechnology, Santa Cruz, CA) or isotype controls. After rinsing, cells were further incubated with goat anti-mouse IgG-fluorescein isothiocyanate (FITC)-conjugated (Dako, Trappes, France) secondary antibody. After rinsing, the slides were mounted with 10% fluorescent mounting medium (Dako). Confocal differential-interference-contrast images were obtained using Fluoview laser scanning microscope (Nikon, Paris, France) and x60 oil-immersion objective. Z-series sections were recorded in successive z-axis serial sections at 0.5-µm intervals and were composed of optical sections in the x-y optical plane.

Immunoblotting
Whole lysates from transfected HASMC, were collected using 1% Triton X-100 lysis buffer for 15 min in the presence of 2 mM Na orthovanadate, 1 mM EDTA, 50 µg/ml aprotinin, 100 µM Leupeptin, 1 mM 1.4 Dithio-DL-Treitol (DTT) and 1 mM amino-ethyl-benzenesulfonyl fluoride hydrochloride (AEBSF) (all from Sigma). The supernatant was reduced with {beta}-mercaptoethanol, subjected to electrophoresis on a 10% acrylamide reducing gel, and transferred to Immobilon TM-P PVDF membranes (Millipore, Saint-Quentin-en-Yvelines, France). The immunoblots were then developed using 1 µg/ml of mouse monoclonal anti-human PAR-2 antibody (SAM11), 0.8 µg/ml of mouse anti-human {beta}-actin, or irrelevant antibody (all from Santa Cruz Biotechnology). A biotinylated swine secondary antibody (Dako) and a streptavidin–biotinylated horseradish peroxidase complex (Dako) were used for amplification. Immunoblots were revealed by enhanced chemiluminescence (Uptima; Interchim, Montlucon, France). For quantification we used BioCaptMW software (Fischer Bioblock Scientific, Illkieck Groffenstaden, France). The experiments were repeated six times for each experimental condition.

Fluorescence-Activated Cell Sorter Analysis
PAR-2 surface expression was analyzed by fluorescence-activated cell sorter (FACS) on transfected HASMC. Cells were fixed with Para formaldehyde 4% (VWR international) for 15 min in ice and washed twice with 0.5% BSA (Sigma). Mouse IgG anti-human PAR-2 (SAM11; Santa Cruz Biotechnology) or isotype control (Sigma) at the same concentration of 20 µg/ml was added to HASMC for 30 min in ice. After an additional wash, secondary FITC-conjugated goat anti-mouse IgG (Immunotech, Marseille, France) was then added for another 30 min in ice. Cells were analyzed for their fluorescence intensity using FACS Facscalibur (Beckton Dickinson, Pont de Claix, France). Results were presented as mean percentage ± SEM of cells that expressed PAR-2 compared with the relevant isotype control and mean of the median fluorescent intensities ± SEM.

RNA Extraction, Reverse Transcription, and Real-Time Quantitative PCR
The RNA was extracted as described previously using Trizol (Invitrogen) and chloroform (Sigma) (3). Total pure RNA (1 µg) was reverse transcribed into cDNA using AMV reverse transcriptase (Promega, Charbonnieres, France), RNase inhibitor, and oligo d(T) as a primer at 42°C for 60 min followed by heating at 94°C for 3 min. Real-time quantitative PCR was performed on a Rotor-Gene 2000 (Corbett Research, Mortlake, Australia), as described previously (3). Briefly, appropriate primers were designed using the primer analysis software (Oligo 6.6; Molecular Biology Insights, Cascade, CO) and ordered from Sigma-Genosys (Table 2B). The RT-PCR expression of the target gene (PAR-2) was presented as a ratio, normalized to an endogenous reference (glyceraldehyde-3-phosphate deshydrogenase [GAPDH], or tyrosin 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide [YWHAZ]) and relative to a calibrator (control untransfected cells) (20).

Microspectrofluorimetry
Changes in HASMC intracellular calcium concentration ([Ca2+]i), a key step in cell activation (21), were assessed using the Ca2+-sensitive probe indo-1 as described previously (10). Briefly, cells were loaded with indo-1 (Calbiochem, La Jolla, CA) and mounted in a perfusion chamber continuously perfused. HASMC were stimulated with 5 mM Caffeine (Merck, Darmstadt, Germany), 2.10–5 M porcine trypsin (Sigma), 10–4 M SLIGKV-NH2 (Sigma-genosys), or 30 mU/ml human recombinant {beta} II tryptase (kind gift from Dr. A. F. Walls, Southampton, UK). Calcium levels were monitored continuously. Cells were considered as responding when a rise in calcium bigger than 100 nM was obtained. Results were expressed as mean ± SEM peak of [Ca2+]i and percentage of responding cells. Experiments were done at room temperature (22–25°C).

Statistical Analysis
Comparison of the effectiveness of the different siRNA was performed by means of one-way ANOVA, or Kruskall-Wallis when values did not follow a normal distribution. Comparison between siRNA No. 1 transfected cells and control cells was performed by means of paired Student's t test, or Aspin-Welch test when variance were unequal. Finally, {chi}2 test was used to compare the percentage of responding cells. A P value < 0.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
siRNA Decrease PAR-2 Expression in HASMC
Five siRNA directed against PAR-2 were synthesized in vitro (Table 1). To validate the ability of siRNA to be transfected in HASMC, we used Cy3-conjugated siRNA and assessed the number of cells containing Cy3 within the cytosol by confocal microscopy (Figure 1A). Using optimal conditions (i.e., 370 nM siRNA and 10 µg/ml lipofectamine 2,000), 94 ± 3.5% (n = 4) of cells were Cy3-positive 24 h after transfection.



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  		Figure 1. Effect of siRNA on PAR-2 expression assessed by confocal microscopy. Before transfection, siRNA were labeled in red with Cy3. HASMC are transfected for 24 h with siRNA No. 1 (A and B), No. 1-inv (C), or No. 1-scr (D). Cells were stained in green using either counterstaining of nuclei with yoyo (A) or anti–PAR-2 and FITC-conjugated secondary antibodies (B, C, D). Bars indicate 30 µm. Representative confocal images are presented as three axis slices.

 
We then performed Western blot analysis to quantify the effect of the five different siRNA sequences on total PAR-2 protein expression. Compared with control cells, HASMC transfected with siRNA No. 1, 2, and 3 showed a significant decrease in PAR-2 total expression (Figure 2; P = 0.011, 0.005, and 0.004 respectively, paired t tests). Next, to determine whether siRNA also decreased PAR-2 surface expression, we performed FACS analysis using the same primary monoclonal anti–PAR-2 antibody on nonpermeabilized cells (Figures 3A and 3B). PAR-2 surface expression was significantly decreased in HASMC transfected with only siRNA No. 1, 2, and 3 when considering median fluorescent intensity (Figure 3C, P = 0.0001, 0.01, and 0.0004, respectively, paired t tests). However, focusing on the percentage of positive cells, only siRNA No. 1 and 3 revealed a significant effect (76 ± 5.5% and 81 ± 2.0%, respectively, versus 89 ± 3.0% for control cells, P = 0.03 and 0.01, respectively, paired t tests).



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  		Figure 2. Effect of siRNA on PAR-2 protein total expression assessed by Western blot. HASMC were untransfected (C) or transfected for 24 h with siRNA No. 1, 2, 3, 4, or 5. (A) Representative blot stained with anti–PAR-2 or anti–{beta}-actin antibody. (B) Results from six different primary HASMC cultures are expressed in mean density ratio of PAR-2 normalized to {beta}-actin, as mean ± SEM. *P < 0.05 using paired t test.

 


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  		Figure 3. Effect of siRNA on PAR-2 protein surface expression assessed by flow cytometry analysis. (A and B) representative flow cytometry data from one experiment showing control cells (A) and cells transfected with siRNA No. 1 (B), stained with isotype control antibody (gray lines) or anti–PAR-2 antibody (black lines). (C) HASMC were untransfected (C) or transfected for 24 h with siRNA No. 1, 2, 3, 4, or 5 (n = 5). Results are expressed as median fluorescent intensity (MFI) from isotype control (mean ± SEM). *P < 0.05 using paired t test.

 
Since RNAi is known to degrade mRNA, we then quantified the amount of PAR-2 transcript using real-time quantitative RT-PCR and normalized results to the expression of two house-keeping genes: GAPDH and YWHAZ. In HASMC transfected with siRNA No. 1, 3, and more surprisingly, with 4 and 5, PAR-2 mRNA levels were significantly decreased using paired t tests (Figure 4A). Collectively, these results indicate that, both siRNA No. 1 and 3 significantly decrease HASMC PAR-2 in terms of total protein, surface protein, and mRNA expression. We thus decided to focus our attention on siRNA No. 1 because it is more potent in terms of decrease in mRNA level (Figure 4A).



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  		Figure 4. Effect of siRNA on PAR-2 mRNA levels assessed by real-time quantitative RT-PCR. Results are expressed as a ratio normalized to an endogenous reference (housekeeping gene) and relative to a calibrator (control untransfected cells) (mean ± SEM). (A) HASMC were untransfected (C) or transfected for 24 h with various siRNA No. 1, 2, 3, 4, or 5 (n = 6). The housekeeping gene is either GAPDH (black bars) or YWHAZ (gray bars). (B) Time course of PAR-2 mRNA at 6, 24, and 36 h after transfection (n = 3). HASMC were untransfected (white bars) or transfected with siRNA No. 1-inv (light gray bars), No. 1-scr (dark gray bars), or No. 1 (black bars). *P < 0.05 using paired t test. Error bars not shown when smaller than size of symbol.

 
Two control sequences of siRNA No. 1 named siRNA No. 1-inv or 1-scr were synthesized in vitro by inverting two central bases or scrambling bases, respectively (Table 1). Confocal microscopy was used to assess PAR-2 expression on HASMC transfected with Cy3-conjugated siRNA. Whereas siRNA No. 1 decreased PAR-2 staining (Figure 1B), neither siRNA No. 1-inv nor No. 1-scr displayed such an effect (Figures 1C and 1D), although the presence of siRNA inside the cells was confirmed by the red fluorescence of Cy3 (Figures 1C and 1D). Similarly, both siRNA No. 1-inv and No. 1-scr failed to alter PAR-2 in terms of mRNA (Figure 4B) or protein levels. Using Western blot, mean density ratios of PAR-2 normalized to {beta}-actin were similar for control siRNA No. 1-inv and No. 1-scr, and for nontransfected cells (0.92 ± 0.15, 0.90 ± 0.05, and 0.95 ± 0.12, respectively, P = NS, paired t tests). Using FACS, median fluorescent intensities were also similar for control siRNA No. 1-inv and No. 1-scr, and for nontransfected cells (32.7 ± 4.46, 32.9 ± 7.58, and 35.3 ± 4.46, respectively, P = NS, paired t tests). The effect of siRNA No. 1 was, however, transient, since it was abolished 36 h after transfection (Figure 4B).

siRNA Decrease PAR-2 Function in HASMC
Calcium signaling in HASMC 24 h after transfection with siRNA No. 1, No. 1-inv, or No. 1-scr was examined using microspectrofluorimetry. Basal calcium concentrations were consistent in control (untransfected) cells (149 ± 16 nM) and cells transfected with siRNA No. 1, No. 1-inv, or No. 1-scr (134 ± 17 nM, 132 ± 17 nM, 135 ± 12 nM, respectively, P = 0.86, Kruskall Wallis). Caffeine was then used to evaluate the calcium response to an agent bypassing membrane receptor, that is, an agonist of the ryanodine receptor present on the endoplasmic reticulum. Caffeine response was not altered by the transfection of HASMC with siRNA No. 1 (ANOVA, Figure 5). We then evaluated the effect of an agonist of membrane receptor not restricted to PAR-2, namely, trypsin. Trypsin-induced calcium rise was not altered by siRNA No. 1 transfection in terms of both peak amplitude (ANOVA) and percentage of responding cells ({chi}2 test) (Figures 5, 6A, and 6B). Collectively the results indicate that transfection of HASMC with siRNA does not alter PAR-2–independent calcium responses.



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  		Figure 5. Effect of siRNA on HASMC calcium response assessed by microspectrofluorimetry. Cells were untransfected (white bars) or transfected with siRNA No. 1-inv (light gray bars), No. 1-scr (dark gray bars), or No. 1 (black bars). Intracellular calcium concentration ([Ca2+]i) was monitored in response to 5 mM caffeine, 2 · 10–5 M trypsin, 10–4 M SLIGKV-NH2, or 30 mU/ml human lung recombinant tryptase. Results from four different primary HASMC cultures are expressed as mean ± SEM of calcium rise in all cells (A), responding cells only (B), or as a percentage of responding cells (C). *P < 0.05 using ANOVA (A and B) or {chi}2 test (C).

 


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  		Figure 6. Effect of siRNA on HASMC calcium response assessed by microspectrofluorimetry. Representative intracellular calcium responses are presented following stimulation by 2 · 10–5 M trypsin (A, B), 10–4 M SLIGKV-NH2 (C, D), or 30 mU/ml human lung recombinant tryptase (E, F). Cells were transfected for 24 h with siRNA No. 1-inv (A, C, E) or No. 1 (B, D, F). Responses are presented as black lines for responding cells and as gray lines for nonresponding cells.

 
Since siRNA inhibits PAR-2 expression in HASMC, we verified whether it could also alter PAR-2–induced calcium response. Specific activation of PAR-2 by the synthetic peptide SLIGKV-NH2 induced a calcium rise in control cells and in cells transfected with the control sequences No. 1-inv (Figure 6C) or No. 1-scr. In contrast, in HASMC transfected with siRNA No. 1, stimulation with SLIGKV-NH2 revealed two cell populations: responding and nonresponding cells (Figure 6D). The mean calcium response induced by SLIGKV-NH2 was significantly reduced in siRNA No. 1-transfected cells (Figure 5A, ANOVA). However, the mean calcium response remained unchanged by siRNA No. 1 when responding cells were only considered (Figure 5B, ANOVA). Therefore, siRNA No. 1 significantly reduced the percentage of responding cells to SLIGKV-NH2 (Figure 5C, {chi}2 test). Finally, the effect of tryptase was examined because it is supposed to specifically activate PAR-2. siRNA No. 1 also reduced tryptase-induced calcium response as a consequence of a significant decrease in the percentage of responding cells (ANOVA, {chi}2 test) (Figures 5, 6E, and 6F). As for PAR-2 expression, the effect of siRNA on calcium responses was transient, since 48 h after transfection with siRNA No. 1, calcium response induced by SLIGKV-NH2 or tryptase were similar to those of control cells (data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study, we have demonstrated that an appropriately designed siRNA induces PAR-2 gene silencing in HASMC. We found that siRNA selectively decreases both PAR-2 expression and function in terms of calcium signaling without altering alternative transduction signaling pathways. Finally, we directly demonstrate that tryptase-mediated calcium response in HASMC is altered by PAR-2 RNA interference.

RNAi is a highly conserved gene silencing mechanism that uses double-stranded RNA as a signal to trigger the degradation of homologous mRNA. Elbashir and coworkers initially demonstrated that siRNA specifically suppresses the expression of various genes in different mammalian cell lines, such as HeLa cells (18). This new tool for studying gene function in mammalian cells has then been used in primary somatic cells including T lymphocytes, endothelial cells, hepatic stellate cells, and many others (22–24). In the present study, we have applied, for the first time, RNAi mechanism for gene silencing in HASMC to examine the function of a receptor for which conventional antagonists are lacking.

The design of siRNA is a critical step for using RNAi mechanism. Initially, Elbashir and colleagues defined different parameters including 21 nucleotide siRNA with overhanging 3' ends, a percentage of GC between 30 and 50% and a target localized in the open reading frame (25). Since the activity of siRNA in mammalian cells is related to structural target accessibility (26), we have focused our attention on parts of PAR-2 mRNA with the highest probability to remain in a single-strand manner, according to Zucker's mRNA secondary structure prediction software (19). We thus have designed five siRNA against PAR-2 mRNA using all these criteria. siRNA No. 4 and 5 have shown limited, if any, effect on PAR-2 expression. siRNA No. 1 and 3 have decreased protein and mRNA expression, whereas siRNA No. 2 has decreased protein expression but not mRNA levels. This latter type of response has been described as micro-RNA (27). However, no experimental data have confirmed this hypothesis in our present work. We therefore focus our attention on siRNA No. 1, since it was the most potent in terms of mRNA levels and surface protein expression. The selection of appropriate controls for RNAi mechanism is also a matter of debate. On the one hand, control siRNA should be scrambled because direct silencing of nontargeted genes has been demonstrated using siRNA containing as few as eleven contiguous nucleotides homology (28). On the other hand, control siRNA should be built with two inverted central base pairs, since scrambled sequences are too unrelated to the original transcript (29). In this study, we compared cells transfected with siRNA No. 1 with cells transfected with the two control siRNAs (inv and scr), as well as with untransfected cells. Regarding mRNA level or calcium response, cells transfected with the control siRNA No. 1-inv are indistinguishable from untransfected cells. The mechanisms of RNAi involves (1) 21- to 23-nt siRNAs homologous in sequence to the target gene, (2) activation of the RNA inducing silencing complex (RISC), and (3) target mRNA degradation (18). Increasing the size of siRNA to more than 29-nt, results in protein kinase R activation, nonspecific mRNA degradation and apoptosis (25, 30). In addition, because the activation of RISC is saturable, the concentration of siRNA should be kept as low as possible (25). In our study, we used low amount of 21-nt siRNA to avoid these nonspecific effects.

As a consequence of its effect on PAR-2 expression, RNAi impaired PAR-2–mediated functional effects. Regarding the most selective PAR-2 agonist, the activating peptide SLIGKV-NH2, RNAi decreased the mean [Ca2+]i response by ~ 50% (Figure 5A) in agreement with the amplitude of the decrease in PAR-2 expression (Figure 3C). However, this mean decrease was due to the coexistence of two cell populations after siRNA transfection: one population of fully responding cells and one population of nonresponding cells (i.e., RNAi decreased the percentage of responding cells). It can therefore be concluded that, in our hand, RNAi was an all-or-none phenomenon. This result derived from functional data is supported by the analysis of the distribution of PAR-2 expression on HASMC (Figures 3A and 3B) showing the existence of both populations. We have previously shown that HASMC PAR-2–mediated calcium response results from receptor activation of pertussis toxin–insensitive G protein, phopholipase C, and inositol 3-phosphate (IP3) formation, leading to a calcium release from intracellular stores (10). RNAi effect of SLIGKV-NH2–induced calcium response was undoubtedly related to PAR-2 extinction, since IP3 formation via activation of alternative PAR by means of trypsin (an activator of PAR-2 but also PAR-1 and PAR-4 [31]) remained unaltered by siRNA. Finally, the fact that RNAi effect on tryptase-induced response was identical to that on SLIGKV-NH2 provides a molecular confirming proof that tryptase-mediated effects involve PAR-2 activation.

As already demonstrated, the time course of siRNA-mediated gene silencing was limited to the first few hours (32). Both PAR-2 protein/mRNA expression and PAR-2–induced calcium response were restored 36–48 h after transfection. Whereas such a transient effect does not interfere when examining short-term PAR-2–mediated effects such as HASMC contraction, further technical developments are required to analyze long-term effects implicating PAR-2, such as HASMC proliferation (6) or secretion (3). In this connection, such developments are also required to evaluate the PAR-2 as a possible therapeutic target in BHR.


   Acknowledgments
 
The authors thank the staff of "Service de Chirurgie Thoracique et Service d'Anatomo-pathologie, Hôpital Haut Lévêque, CHU de Bordeaux" for the supply of human lung tissue. The authors also thank Dr. Christian Cazenave (INSERM U386, Bordeaux) for expert help in RNA structure analysis and Vincent Pitard (CNRS UMR 5164, Bordeaux), for cytometry facilities.


   Footnotes
 
This study was funded by the "Fondation de l'avenir pour la recherche medicale appliquée, Paris, France, ET4–373, 2004."

Originally Published in Press as DOI: 10.1165/rcmb.2005-0187OC on September 29, 2005

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 May 17, 2005

Accepted in final form August 2, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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  4. Brightling CE, Ammit AJ, Kaur D, Black JL, Wardlaw AJ, Hughes JM, Bradding P. The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle. Am J Respir Crit Care Med 2005;171:1103–1108.[Abstract/Free Full Text]
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