Downregulation of protease activated receptor expression and cytokine production in P815 cells by RNA interference
© Qiao et al; licensee BioMed Central Ltd. 2009
Received: 31 March 2009
Accepted: 7 September 2009
Published: 7 September 2009
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.
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.
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.
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 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[3–5]; 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, to induce inflammatory cell accumulation in the peritoneum of mice, to elicit histamine release from mast cells6 and to enhance monocyte chemoattractant protein-1 (MCP-1) and IL-8 production in human endothelial cells. Trypsin was found to be able to induce histamine release from human tonsil and skin mast cells and to stimulate IL-8 and IL-6 release from human respiratory epithelial cells.
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 double-stranded fragments known as siRNAs. 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
Reduction of PAR-2 expression in P815 cells by siRNA
Reduction of PAR-4 expression in P815 cells by siRNA
Inhibition of IL-12 induced changes in PAR expression in P815 cells by siRNA
Inhibition of protease-induced cytokine release by siRNA
Tryptase at 1 μg/ml, but not thrombin at 3 U/ml, trypsin at 100 ng/ml, SFLLR-NH2 at 100 μM, SLIGKV-NH2 at 100 μM and GYPGQV-NH2 at 100 μM was able to provoke 2.1 fold increase in IL-6 secretion from P815 cells at 16 h following incubation. 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 (Figure 5B).
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 . 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. 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 siRNAs 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-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. Another study found that the activity of siRNA in mammalian cells is related to structural target accessibility.
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. It has been implicated in the pathogenesis of allergy. 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, 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. 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 and IL-6 production from synovial fibroblasts 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. PAR independent release of IL-6 from P815 cells induced by tryptase was unexpected. However, a study demonstrated that beta-tryptase regulates IL-8 expression in airway smooth muscle cells by a PAR-2-independent mechanism may help to explain our above observation.
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.
The mouse mastocytoma cell line P815 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells culture reagents including Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from HyClone (Logan, UT, USA). siPORT™ NeoFX™ Transfection Agent was from Ambion (Huntingdon, UK). TRIZOL Reagent was from Invitrogen (Carlsbad, CA, USA). ExScriptTM RT reagent kit and SYBR® Premix Ex Taq TM (perfect real time) was from TaKaRa (TaKaRa Biotechnology Co. Ltd. DaLian, China). Primers for mouse PAR-1, PAR-2 and PAR-4 were synthesized by Invitrogen Biotechnology Co. Ltd, (Nanjing, China). Rabbit anti-mouse PAR-1, PAR-2, PAR-4 and rabbit anti-mouse β-actin monoclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FITC-conjugated goat anti-rabbit polyclonal antibody was from BD Pharmingen (San Jose, CA, USA). Paraformaldehyde, bovine serum albumin (BSA, fraction V), trypsin, thrombin and peroxidase conjugated goat anti-rabbit immunoglobulins were from Sigma Inc. (St. Louis, MO, USA). Recombinant human lung β-tryptase was from Promega (Madison, WI, USA). Agonist peptides of PARs, as well as their reverse forms were synthesized in CL Bio-Scientific Inc (XiAn, China). The sequences of the active and reverse peptides were: PAR-1, SFLLR-NH2 and RLLFS-NH2; PAR-2, SLIGKV-NH2 and VKGILS-NH2; PAR-4, GYPGQV-NH2 and VQGPYG-NH2. Mouse IL-4 and IL-6 ELISA kits were from Pierce Biotechnology Inc. (Rockford, IL, USA). Most of other reagents such as salt and buffer components were analytical grade and obtained from Sigma.
Small interfering RNA synthesis
siRNA sequences of PARs
Sense strand (5'-3')
Antisense strand (5'-3')
P815 cell culture
Cells were cultured with ATCC complete growth medium including DMEM with 4 mM L-glutamine, 1.5 mg/ml sodium bicarbonate, 4.5 mg/ml glucose, 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin in 75-cm2 tissue culture flasks (Falcon) at 37°C in a 5% (v/v) CO2, water-saturated atmosphere.
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 × 105 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 being added into each well. The transfected cells were cultured in the normal cell culture conditions for 24, 48 and 72 h before their total RNA being extracted as described above. Control cells were not transfected but processed following a similar procedure.
Quantitative real-time PCR analysis of PAR mRNAs
Primer sequences for real time PCR analysis
Size of product (bp)
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 Tris-buffered saline (TBS) containing 0.05% Tween 20 before rabbit anti-mouse PARs (1:500) being added for 2 h at 37°C. After washing with TBS, the membranes were incubated with peroxidase conjugated goat anti-rabbit immunoglobulins antibody (1:2000) for 1 h at 37°C, and the membranes were developed with DakoCytomation Liquid DAB + Substrate. Not transfected cells were used as negative control.
Flow cytometry analysis
P815 cells were pelleted by centrifugation at 800 g for 5 min, and fixed with 4% Paraformaldehyde (VWR international) for 30 min on ice before being washed twice with 0.5% BSA. 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. Cells were finally resuspended in PBS and analyzed on a FACS Calibur flow cytometer with CellQuest software (BD Biosciences).
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
Agonist peptides and reverse peptides of PARs used in the study
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.
This project was sponsored by the grants from the Li Ka Shing Foundation, Hong Kong, China (No. C0200001); the Major State Basic Research Program of China (973 Program) (No. 2007CB512400); the National Natural Science Foundation of China (No. 30570813, 30772032); the Key Project of the Natural Science Foundation of Jiangsu Province, China (No. BK2007730).
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