Cell culture medium RPMI 1640 and fetal bovine serum (FBS) were obtained from GIBCO BRL (Lifetechnologies, Rockville, MD, USA). PD98059, AG490, LY294002, phospho-p44/42 MAPK antibody, unphospho-p44/42 MAPK antibody, phospho-Akt antibody, unphospho-Akt antibody, phospho-STAT3 antibody and unphospho-STAT3 antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Human thrombin (T6884), biotin conjugated goat anti-rabbit immunoglobulins, biotin conjugated goat anti-mouse immunoglobulins, extr-Avidin-peroxidase, mouse anti-human β-actin and antibiotics (a mixture of penicillin and streptomycin) were from Sigma (St. Louis, MO, USA). Recombinant hirudin (catalog number 377950) was from Calbiochem (San Diego, CA, USA). PAR-1 agonist peptide (SFLLR-NH2) and its reverse peptide (RLLFS-NH2) were synthesized at the Xian Meiliang Co. (Xian, China). Mouse anti-human PAR-1, mouse anti-human PAR-2, anti-PAR-3 rabbit polyclonal antibody, anti-PAR-4 rabbit polyclonal antibody, goat anti-mouse IgG-cy3, goat anti-mouse IgG-FITC, goat anti-rabbit IgG-FITC, mouse IgG, rabbit IgG and PAR-1 blocking antibody (ATAP2, against amino acids 42–55 of thrombin receptor of human origin) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Sybr green was from Cambrex Bio Science (Baltimore, USA). Most of the other reagents such as salt and buffer components were analytical grade and obtained from Sigma (St. Louis, MO, USA).
Samples of foreskins were obtained from four healthy donors (aged 18, 18, 20, 21 years, respectively) undergoing circumcision in the First Affiliated Hospital, Shantou University Medical College. The study adhered to the tenets of the Declaration of Helsinki, and all subjects signed an informed consent form before undergoing circumcision. HDFs were isolated by sequential enzymatic digestions and cultured in RPMI 1640 medium supplemented with 10% (vol/vol) FBS, 100 units/ml penicillin, and 100 μg/ml of streptomycin at 37°C in an atomosphere of 5% CO2 until confluent islands of cells were observed. Since cells displayed the typical properties of spindle-shaped fibroblasts after the third passage, only passages 3 to 6 HDFs were used for the study. The purity of HDFs was at least 99% as assessed by detecting fibroblast marker using immunostaining with a monoclonal mouse anti-human fibroblast antibody (Dako, Glostrup, Denmark).
Detection of PARs in HDFs by immunofluorescence staining
Confluent fibroblasts were treated with nonenzymatic cell dissociation fluid to obtain single-cell suspensions. After being seeded onto eight-well chamber slides (Lab-Tek Permanox, Nunc, Life Technologies) at a density of 1 × 104 cells/well in 300 μl of RPMI 1640 containing 10% FBS, HDFs were incubated in a humidified atmosphere of air containing 5% CO2 for 48 h at 37°C, and then fixed with 4% paraformaldehyde for 3 min at room temperature. The fixed cells were incubated with mouse anti-human PAR-1, mouse anti-human PAR-2, rabbit anti-human PAR-3, or rabbit anti-human PAR-4 antibody, respectively for 16 h at 4°C. This was followed by addition of the secondary antibodies including goat anti-mouse IgG-cy3 (for PAR-1), goat anti-mouse-FITC (for PAR-2) or goat anti-rabbit IgG-FITC (for PAR-3 and PAR-4) for 60 mins at room temperature. Mouse IgG or rabbit serum (20 ng/ml) was used as negative controls. After three washes with PBS, the chambers were removed, and coverslips were mounted with an antifade glycerol-PBS-based mountant (Dako, Glostrup, Denmark). The cells were visualized by using a confocal microscope (D-ECLIPSE C1, Nikon).
Challenge of HDFs with thrombin and SFLLR-NH2
The cells were plated in 24-well tissue culture plates (5 × 104/well) and serum starved with serum-free medium overnight. After washing, the fresh medium with various concentrations of thrombin (0.1, 1, 5 U/ml, U = NIH unit, 1 U/ml = 6.7 nM), SFLLR-NH2 (1, 10, 100 μM), hirudin (1, 5 U/ml) or thrombin with hirudin was added into each well for 6 h. TGFβ at 10 ng/ml was used as a positive control. The culture supernatant was then collected and stored at -80°C for subsequent analysis.
RT-PCR analysis of expression of PARs and MMP-9 genes in HDFs
After being exposed to various stimuli for 6 h, the total RNA of HDFs was extracted by using an RNeasy Mini Kit (Qiagen, Germany). cDNA was synthesized from 2 μg of RNA by using ProtoScript First Strand cDNA Synthesis Kit (Biolabs, New England, USA) according to the manufacturer's instructions. The cDNA was amplified using forward and reverse specific primers for amplifying human PARs. β-actin was used as an internal control. Primers were designed based on PAR sequences in Genbank using Omiga software and prepared by BioAsia Co.. The primer sequences were summarized in Table . Cycling conditions were as follows: PAR-1, PAR-2, PAR-3, PAR-4, 30 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 1.5 min; β-actin was amplified for 30 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. The reaction for MMP-9 was allowed to proceed for 30 cycles at 94°C for 30 s, 57°C for 45 s, and 72°C for 45 s. The quality of the PCR product was analyzed on a 1.5% agarose gel, visualized with Sybr green and photographed under UV light. The results were expressed as the ratio of mRNAs of PARs or MMP-9 to β-actin after determination of the density of the bands on agarose gel by densitometer.
Primer sequences for PARs and MMP-9
Detection of proMMP-2 and proMMP-9 activities by gelatin zymography
Metalloproteinases generated in culture supernatants were determined using gelatin zymography. Commercial available gelatinases A and B (2 μg; USbiological, New Orleans, LA, USA) were used as standards. Samples or standards were added to platelet-monocyte conditioned medium. The mixture was then placed in 2 × non-reducing SDS sample buffer and electrophoresed in a 10% polyacrylamide gel containing 0.1% gelatin. Following rinsing in 2.5% Triton X-100 for 1 h and in distilled H2O for 2 h at room temperature the gel was subsequently incubated at 37°C for 24 h in the substrate buffer containing 50 mmol/L Tris-base (pH 7.6) and 5 mmol/L CaCl2. After staining with Coomassie Blue and destaining, the presence of gelatinases in the gel was identified as clear bands on a uniform blue and was quantified by densitometry. The density units were integrated optic density, which was area timed optical density. The ScionImage program was used to quantify the band. Identification of each type of gelatinase on the gel is based on the bands produced by the gelatinase standards.
Western blot analysis of signal transduction pathways in HDFs upon thrombin stimulation
To determine the optimal concentrations of the inhibitors of signal transduction pathways in HDFs, 25 and 50 μM of PD98059, 20 and 40 μM of LY294002, and 20 and 40 μM of AG490 were preincubated with HDFs for 30 minutes prior to adding 5 U/ml thrombin. Since 50 μM of PD98059, 40 μM of LY294002 and 40 μM of AG490 almost completely abolished thrombin-induced phosphorylation of ERK1/2, Akt and STAT3 respectively (data not shown), whereas 25 μM of PD98059, 20 M of LY294002 and 20 μM of AG490 inhibited thrombin-induced phosphorylation of ERK1/2, Akt and STAT3 by approximately only 30%, 27% and 40% respectively, the higher concentration of each inhibitor was chosen as the optimal concentration throughout the study.
HDFs, which were planted in 12-well tissue culture plates, 1 × 106 per well, were serum starved overnight and then treated with 50 μM of PD98059, 40 μM of LY294002 or 40 μM of AG490 for 30 min prior to addition of 5 U/ml thrombin. We examined the levels of downstream products of the following pathways: phospho-ERK1/2 in MAPK/ERK pathway, phospho-Akt in the PI3K/Akt pathway, and phospho-STAT3 in the JAK/STAT3 pathway. The levels of these products were determined at 30 min (for phospho-STAT3 only), 2 h and 6 h time points. The levels of proMMP-2 and proMMP-9 in the supernatants were determined by zymography.
The cells were lysed in phosphorylation lysis buffer (0.5%Triton X-100, 150 mM NaCl, 200 μM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 50 mM Hepes, 1.5 mM magnesium chloride, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin) for 60 min at 4°C. After removing cell debris by centrifugation at 10,000 g for 30 min, the supernatants were collected. Equal amount of the cell extracts were loaded on 10% polyacrylamide gel for electrophoresis. Proteins in the gel were then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA). Following blocking the membranes with 5% skim milk in TBST (50 mM Tris, 0.15 M NaCl, 0.1% Tween 20, pH 7.6) for 1 h at room temperature, the primary antibody was added. Biotinylated goat anti-rabbit immunoglobulins or biotinylated goat anti-mouse immunoglobulins were used as secondary antibodies, which was followed by addition of extr-Avidin-peroxidase. The protein bands were detected by a chemiluminescence method according to manufacturer's instructions (supersignal West Pico, Pierce). Radiographs were photographed with a digital scanning system. The same membranes were stripped at 55°C for 30 min in stripped buffer containing 0.7% β-mercaptoethanol, 2% SDS, and 62.5 mM Tris (pH 6.8) prior to being stained with mouse anti-human β-actin monoclonal antibody as described above.
Densitometry analysis of immunoblots was carried out using ScionImage software. The relative levels of phospho-ERK1/2, Akt, and STAT3 were expressed as the ratio to β-actin.
All data were expressed as mean ± SD. Each experiment was repeated four times. Statistical analysis was performed using one-way ANOVA or Students's t test with SPSS (version12.0). P value less than 0.05 was considered as statistically significant.