Downregulation of protease activated receptor expression and cytokine production in P815 cells by RNA interference

Background Protease-activated receptors (PAR) are seven transmembrane G-coupled receptors comprising four genes (PAR-1 ~ PAR-4). Mast cell has been identified to be able to express PARs and release an array of cytokines upon activation. Recently, it was reported that interleukin (IL)-12 could regulate the expression of PARs in mast cells, and tryptase could induce IL-4 and IL-6 release from mast cells. In order to further investigate the issues, RNA interference (RNAi) technique was employed and small interfering RNAs (siRNA) of PARs were transfected in P815 cells. Results The results showed that siRNAs for PAR-1, PAR-2 and PAR-4 significantly downregulated expression of PAR-1, PAR-2 and PAR-4 mRNAs and proteins in P815 cells at 24, 48 and 72 h following transfection. siRNA PAR-1.2 and siRNA PAR-4.2 significantly reduced IL-12 induced upregulation of PAR-1 and PAR-4 expression, respectively when P815 cells were transfected with them for 48 h. siRNA PAR-2.3 blocked IL-12 induced downregulation of PAR-2 expression on both mRNA and protein levels. It was also observed that siRNA PAR-2.3 and siRNA PAR-1.2 reduced trypsin induced IL-4 release by approximately 92.6% and 65.3%, and SLIGKV-NH2 induced IL-4 release by 82.1% and 60.1%, respectively. Similarly, siRNA PAR-2.3 eliminated tryptase-induced IL-4 release by 75.3%, and siRNA PAR-1.2 diminished SFLLR-NH2 induced IL-4 release by 79.3%. However, siRNA PAR-1.2, siRNA PAR-2.3 and siRNA PAR-4.3 at 10 nM did not show any effect on tryptase-induced IL-6 release from P815 cells. Conclusion In conclusion, siRNAs of PARs can modulate PAR expression and PAR related cytokine production in mast cells, confirming that PARs are likely to play a role in allergic reactions.


Background
As the primary effector cell, mast cell is actively involved in the pathogenesis of both acute and chronic allergic diseases [1,2]. Upon activation, mast cell can release not only its preformed but also newly generated mediators to fulfill its biological functions. Besides histamine, heparin and proteases, mast cells can synthesize and secrete a variety of cytokines [2] such as IL-4, IL-5 and IL-6. These cytokines have been well documented for their ability to regulate cell behavior including growth, secretion and migration in physiological and pathological conditions. In recent years, PARs have been indentified as receptors for serine proteinases. PARs are a subfamily of seven transmembrane G-protein-coupled receptors. Among them, PAR-1, PAR-3 and PAR-4 serve as a receptor of thrombin [3][4][5]; PAR-1, PAR-2 and PAR-4 are receptors of trypsin and PAR-2 is a receptor of tryptase. These serine proteinases have been discovered to play a crucial role in allergic inflammation. Tryptase was reported to be able to stimulate microvascular leakage in the skin of guinea pigs [6], to induce inflammatory cell accumulation in the peritoneum of mice [7], to elicit histamine release from mast cells 6 and to enhance monocyte chemoattractant protein-1 (MCP-1) and IL-8 production in human endothelial cells [8]. Trypsin was found to be able to induce histamine release from human tonsil and skin mast cells [9] and to stimulate IL-8 and IL-6 release from human respiratory epithelial cells [10].
RNAi is a process in which double-stranded RNA (dsRNA) induces the posttranscriptional degradation of homologous transcripts. It has been observed in variety of organisms including plants, fungi, insects, protozans and mammals [11,12]. RNAi can be initiated by exposing cells to dsRNA either via transfection or endogenous expression. dsRNAs are processed into 21-to 23-nt doublestranded fragments known as siRNAs [12]. With unprecedented speed, RNAi has advanced from its basic discovery in lower organisms to becoming a powerful genetic tool in mammals. The aim of this study is to investigate the effect of PAR-1, PAR-2 and PAR-4 gene silencing on the expression of PARs and cytokine production in P815 cells by using RNAi.

Reduction of PAR-1 expression in P815 cells by siRNA
Three target sequences in the coding region of PAR-1 mRNA were selected as siRNA sequences, namely siRNA PAR-1.1, siRNA PAR-1.2 and siRNA PAR-1.3. Among the 3 siRNA PAR-1s, siRNA PAR-1.2 appeared most effective in inhibition of PAR-1 mRNA expression in P815 cells (data not shown). Approximately up to 80.5% reduction of PAR-1 mRNA expression was observed when P815 cells were transfected with 10 nM of siRNA PAR-1.2 for 48 h. At 24 or 72 h following transfection, however siRNA PAR-1.2 showed less degree of inhibition of PAR-1 mRNA expression ( Figure 1A). Flow cytometry analysis revealed that PAR-1 protein expression was eliminated by up to 60.1% when P815 cells were transfected with 10 nM of siRNA PAR-1.2 for 48 h ( Figure 1B). Because siRNA PAR-1.2 showed greater inhibitory effect on PAR-1 expression at 48 h than at 24 or 72 h following incubation, the 48 h incubation period was chosen as the fixed time point for the following Western blot and immunofluorescent analysis. Approximately up to 46.3% and 42.6% inhibition of PAR-1 expression was observed by Western blot ( Figure  1C) and immunofluorescent analysis ( Figure 1D), respec-tively. siRNA PAR-1.1 and siRNA PAR-1.3 also showed significant inhibition of PAR-1 protein expression in P815 cells (data not shown).

Reduction of PAR-2 expression in P815 cells by siRNA
Of the three siRNA PAR-2s, siRNA PAR-2.1 and PAR-2.3, but not PAR-2.2 downregulated PAR-2 mRNA expression by up to 69.3% and 82.1% at 48 h following transfection ( Figure 2A). Therefore siRNA PAR-2.3 was employed as a representative throughout the study. siRNA PAR-2.3 also suppressed PAR-2 protein expression during flow cytometry, Western blot and immunofluorescence staining analysis by up to 56.8% ( Figure 2B), 60.2% ( Figure 2C) and 59.3% ( Figure 2D), respectively following 48 h incubation period. At 24 and 72 h following incubation, siRNA PAR-2.3 showed significant downregulation of PAR-2 expression on both mRNA and protein levels.  Figure 3C) and immunofluorescence staining analysis ( Figure 3D) in P815 cells at 48 h following transfection.

Inhibition of IL-12 induced changes in PAR expression in P815 cells by siRNA
IL-12 at the concentration of 10 ng/ml induced approximately 2.4 and 3.0 fold upregulation of expression of PAR-1 ( Figure 4A) and PAR-4 mRNAs ( Figure 4C) in P815 cells, respectively at 6 h following incubation. In contrast, 10 ng/ml of IL-12 downregulated PAR-2 mRNA expression in P815 cells by approximately up to 40.1% ( Figure  4B). It was observed that IL-12 induced upregulation of PAR-4 ( Figure 4E), but downregulation of PAR-2 protein expression ( Figure 4D). siRNA PAR-1.2 at 10 nM and siRNA PAR-4.2 at 10 nM significantly reduced IL-12 induced upregulation of PAR-1 ( Figure 4A) and PAR-4 expression ( Figure 4C, Fig. 4E), respectively when P815 cells were transfected with them for 48 h. siRNA PAR-2.3 at 10 nM was able to block IL-12 induced downregulation of PAR-2 expression on both mRNA ( Figure 4B) and protein ( Figure 4D) levels in P815 cells.

Discussion
Delivery of synthetic siRNA or vector-based siRNA expression systems to target cells can reverse the expression and function of the gene under consideration. In recent years, siRNA technique has been widely used as one of the most powerful tools for investigation of protein functions in mammalian cells [12]. Although P815 cell is a mouse mastocytoma cell line, rather than primary mast cells, it has been shown to express PAR-1, PAR-2, PAR-3 and PAR-4 genes and proteins [13]. To confirm these findings, we examined the effect of siRNAs on PAR expression in the present study. Our data showed, for the first time, that siR-NAs of PAR-1, PAR-2 and PAR-4 are able to reduce PAR-1, PAR-2 and PAR-4 expression to a great extent, indicating these small RNAs disrupt the normal PAR generation process of P815 cells. As for most dsRNAs, siRNAs of PAR-Effect of siRNA PARs on IL-12 (10 ng/ml) induced expression of PARs in P815 cells 1, PAR-2 and PAR-4 fail to completely block PAR production in P815 cells. Incomplete siRNA-induced gene suppression may result from the presence of a fraction of mRNA in a protected compartment such as spliceosomes, other nuclear locations or non-transfected cells [14]. Another study found that the activity of siRNA in mammalian cells is related to structural target accessibility [15].
PAR-1, PAR-2 and PAR-4 gene silencing can block IL-12 induced alteration of expression of PAR mRNAs and proteins in P815 cells, confirming that IL-12 is an effective modulator of PAR expression in mast cells. To our knowledge, this is the first work demonstrates the effects of PAR siRNAs on cytokine induced alteration of PAR expression. IL-12 regulates Th1 cell differentiation, while suppressing the expansion of Th2 cell clones [16]. It has been implicated in the pathogenesis of allergy [17]. Since mast cell has long been recognized as the primary effector cell of allergy and upregulation of PAR-2 expression was found in the airways of asthma [18], we anticipate that IL-12 is likely to be involved in the pathogenesis of asthma through its ability of regulation of PAR expression on mast cells.
We have previously showed that tryptase and trypsin can induce IL-4 release from P815 cells via a PAR-2 dependent mechanism [13]. Using siRNA technique in the present study, it is confirmed that trypsin provoked IL-4 release is mainly through PAR-2 and partially via PAR-1 related mechanisms, whereas tryptase elicited IL-4 release is dependent on activation of PAR-2. In contrast, tryptase induced IL-6 release is independent on the activation of PARs as siRNAs of PAR-1, PAR-2 and PAR-4 did not show any influence on the event. Since tryptase is a unique secretory product of mast cells and IL-4 is a classic Th2 cytokine which is actively involved in the pathogenesis of allergic reactions, induction of IL-4 release from mast cells by tryptase may suggest a self-amplification mechanism of allergic reactions. Inhibition of PAR dependent cytokine release by siRNAs of PARs has been reported before. Thus, thrombin induced IL-8 and VEGF release from prostate cancer cells [19] and IL-6 production from synovial fibroblasts [20] was blocked by siRNA of PAR-1, trypsin induced IL-8 production from human gastric epithelial cells (MKN45 cells) was inhibited by siRNA of PAR-2 [21]. PAR independent release of IL-6 from P815 cells induced by tryptase was unexpected. However, a study demonstrated Effect of siRNA PARs on IL-4 (A) and IL-6 (B) release from P815 cells that beta-tryptase regulates IL-8 expression in airway smooth muscle cells by a PAR-2-independent mechanism [22] may help to explain our above observation.

Conclusion
In conclusion, siRNAs of PAR-1, PAR-2, PAR-4 not only block their corresponding PAR expression, inhibit IL-12 induced alteration of expression of PARs, but also reduce tryptase and trypsin provoked IL-4 release from P815 cells, confirming that mast cell PARs are likely to be involved in the pathogenesis of allergic reactions. However, a further study using primary human mast cells would be better for evaluation the role of mast cell PARs in allergic reactions.

Small interfering RNA synthesis
The potential siRNAs of PAR-1, PAR-2 and PAR-4 were designed by using the prediction of single strand domains in the secondary mRNA structure and subsequent negative BLAST analyses, 3 sequences for each PAR (Table 1). A scrambled sequence was used as negative control, and βactin siRNA was employed as positive control. No significant matching in mouse transcripts was found in BLAST analyses. siRNA were prepared by in vitro transcription using the siPORT™ NeoFX™ Transfection Agent, a lipidbased agent for reverse transfection.

Cell transfection
The cell transfection was performed according to the manufacturer's instruction. Briefly, P815 cells were collected 1 h before transfection. After washing, cells were resuspended in normal growth medium to a concentration of 1 × 10 5 cells/ml at 37°C. For preparation of the transfection solution, 3 μl of siPORT NeoFX Transfection agent was added into OPTI-MEM I medium to a total volume of 50 μl, and the solution was incubated for 10 min at room temperature. After dilution of small RNA in OPTI-MEM I medium to final concentrations of 3, 10, 30 and 100 nM, the RNA preparations were mixed with transfection solution for 10 min at room temperature. The RNA transfection mixture was then dispensed into a 12-well culture plate, 100 μl per well before 900 μl of cell suspension  [23].

Western blot analysis
Cells were lyzed by using Triton X-100 lysis buffer for 15 min in the presence of 2 mM sodium orthovanadate, 1 mM EDTA, 50 g/ml aprotinin, 100 M leupeptin, 1 mM Dithio-DL-Treitol (DTT) and 1 mM amino-ethyl-benzenesul-fonylfuoride hydrochloride (AEBSF). After centrifugation at 1500 g for 5 min, the supernatant was mixed with β-mercaptoethanol for electrophoresis on a 12% acrylamide reducing gel. The proteins on the gel were then transferred to Immobilon PVDF membranes. The membranes were blocked overnight at 4°C with 1% BSA in

Immunofluorescence cell staining
After being fixed in 4% paraformaldehyde for 30 min, P815 cells were incubated with 1% BSA for 10 min. After washing, the cells were resuspended in PBS and incubated with FITC-conjugated rabbit anti-mouse PAR-1, PAR-2, PAR-4 polyclonal antibody or isotype control (at a final concentration 4 μg/ml) at 37°C for 2 h. Images were obtained on a Nikon EZ-C1 confocal laser scanning microscope (Japan).

Mast cell challenge
P815 cells at a density of 1 × 10 5 cells/ml were transfected with 10 nM of siRNAs of PAR-1, PAR-2 or PAR-4 for 48 h at 37°C before PAR expression being examined. The transfected cells were then exposed to IL-12 (10 ng/ml), trypsin (100 ng/ml, 1 μg/ml = 42 nM), tryptase (1 μg/ml, 1 μg/ml = 7.4 nM), thrombin (3 U/ml, 1 U/ml = 6.7 nM), the agonist peptides of PARs (Table 3), as well as the reverse peptides (100 μM), respectively. At 6 or 16 h following challenge, the culture plates were centrifuged at 800 g for 5 min at 4°C and culture supernatants (1 ml per well) were collected and stored at -80°C until use. The cell pel- let was resuspended in PBS and analyzed on a FACS Calibur flow cytometer and confocal laser scanning microscope. Concentrations of IL-4 and IL-6 in the supernatants were quantified using commercial ELISA kits according to the manufacturer's instructions.

Statistical analyses
Data are expressed as mean ± SEM for the indicated number of independently performed duplicated experiments. Statistical significance between means was analyzed by one-way ANOVA or the Student's t test utilizing the SPSS 13.0 version. P < 0.05 was taken as statistically significant.