Cell culture and reagents
NHLFs (Lonza, Basel, Switzerland) were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS) and 1% penicillin/streptavidin (Life Technologies, Paisley, UK) in a humidified 5% CO2 atmosphere at 37°C. Prior to treatment, cells were serum starved overnight. Cells were stimulated with collagen I from rat tail (25 μg/mL) or vehicle (acetic acid, 0.1 M).
RNA isolation and real-time quantitative reverse transcription (qRT)-PCR
RNA from NHLFs was extracted using RNeasy Plus Kit (Qiagen, Crawley, UK) according to manufacturer's instructions. For reverse transcription, 100 ng of total RNA was added to 50 μL of reaction buffer containing RT-PCR Buffer, MgCl2 (5.5 mM), desoxyribonucleoside triphosphate mixture (500 μM), random hexamers (2.5 μM), RNase inhibitors (0.4 U/μL) and MultiScribe Reverse Transcriptase (1.25 U/μL) (all reagents from Applied Biosystems), and incubated for 10 minutes at 25°C followed by 30 minutes at 48°C and 5 minutes at 95°C. Real-time PCR was performed using TaqMan system 7900HT (Life Technologies, Paisley, UK). TaqMan probes and primers used for the reaction is as follows, DDR1: 5'-GCGTCTGTCTGCGGGTAGAG-3', (forward) 5'-ACGGCCTCAGATAAATACATTGTCT-3' (reverse), 6-FAM-AGGGATGGACTCCTGTC-MGB (TaqMan probe) (99 bp), DDR2: 5'-TGTTCCTGCTGCTGCCTATCTT-3' (forward), 5'-AGGATAGCGGCATATAGCTGGAT-3' (reverse), 6-FAM-AGTTCTGCAAAAGCTCAGGT-MGB (TaqMan Probe) (68 bp), MMP-10: 5'-TCACAGAGCTCGCCCAGTT-3' (forward), 5'-CGTAGAGAGACTGAATGCCATTCA-3' (reverse), 6-FAM-CCTTTCGCAAGATGAT-MGB (TaqMan Probe) (63 bp), MMP-2: 5'-CGTCTGTCCCAGGATGACATC-3' (forward), 5'-TGTCAGGAGAGGCCCCATAG-3' (reverse), 6-FAM-AGGGCATTCAGGAGC-MGB (TaqMan Probe) (58 bp). For real-time qRT-PCR 1 μL cDNA was added to a total volume of 20 μL of reaction buffer containing TaqMan Fast Universal master mix (Life Technologies, Paisley, UK), TaqMan probes (0.3 μM) and forward and reverse primers (0.9 μM). PCR was performed by denaturation at 95°C for 20 s, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The expression changes (fold increase) were calculated relative to unstimulated control cells using the crossing points of the log linear portion of the amplification curve after normalizing with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) endogenous control (VIC/MGB) (Life Technologies, Paisley, UK).
Small interference RNA (siRNA) and transfection
Negative control 2# siRNA, human DDR2-specific siRNA, human DDR1-specific siRNA, human MMP-10-specific siRNA, and human MMP-2-specific siRNA were synthesized by Applied Biosystems. The sequence of DDR2 siRNA is as follows: 5'-GCACUGUCAGUUACACCAATT-3' (sense) and 5'-UUGGUGUAACUGACAGUGCGT-3' (antisense). The sequence of DDR1 siRNA is as follows: 5'-GGCUAUGCAGGUCCACUGUTT-3' (sense) and 5'-ACAGUGGACCUGCAUAGCCTG-3' (antisense). The sequence of MMP-10 siRNA is as follows: 5'-GAGAAUAUCUGUUCUUUAATT-3' (sense) and 5'-UUAAAGAACAGAUAUUCUCCC-3' (antisense). The sequence of MMP-2 siRNA is as follows: 5'-GGAAAAGAUUGAUGCGGUATT-3' (sense) and 5'-UACCGCAUCAAUCUUUUCCGG-3' (antisense). JAK2-specific and ERK1/2-specific siRNAs were obtained from Cell Signaling Technologies. For siRNA delivery, NHLFs were reverse transfected with 10 nM siRNA using lipofectamine RNAiMAX (Life Technologies, Paisley, UK) according to manufacturer's instructions. At 48 h after transfection, cells were serum starved for 24 h prior to treatment with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M). Transfection of NHLFs with specific siRNAs suppressed DDR2 by 91.7% (P = 0.002), DDR1 by 85.7% (P = 0.008), JAK2 by 93.5% (P = 0.003), ERK1/2 by 90% (P = 0.004), MMP-10 by 88.2% (P = 0.006), and MMP-2 by 90.7% (P = 0.004), versus control.
Western blotting and immunoprecipitation
NHLFs were lysed in radioimmune precipitation assay buffer containing 50 mM Tris (pH 8), 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate acid, 0.1% SDS, proteinase inhibitors (Roche Diagnostics, Burgess Hill, UK) and protease inhibitors (Roche Diagnostics, Burgess Hill, UK). Cell debris was removed by centrifugation and supernatants were denatured at 94°C in 4 × NuPAGE sample buffer (Life Technologies, Paisley, UK) containing 5% 2-mercaptoethanol. Protein samples (20 μg) were subjected to electrophoresis on a 4-10% Bis-Tris (MOPS) NuPAGE gel (Life Technologies, Paisley, UK) and blotted onto a nitrocellulose membrane (Life Technologies, Paisley, UK). Membranes were blocked for 1 h in Tris-buffered saline (TBS), 0.1% (v/v) Tween-20 and 5% (w/v) non-fat dried milk. Immunodetection was carried out using anti-phospho-ERK1/2 (T185+Y187) (Life Technologies, Paisley, UK), anti-ERK1/2 (Cell Signaling Technologies, Danvers, MA, USA), anti-phospho-JAK2 (Y1007/Y1008) (Abcam, Cambridge, UK), anti-JAK2 (Santa Cruz Heidelberg, Germany,), anti-DDR1, anti-DDR2 (both from R&D Systems, Abingdon, UK), and anti-GAPDH (Santa Cruz, Heidelberg, Germany). The bands were visualized using the ECL system (GE Healthcare Life Sciences, Little Chalfont, UK).
Enzyme-linked immunosorbent assay (ELISA) analysis
The concentration of pro-MMP-2 and mature MMP-2, and pro-MMP-10 in NHLFs culture supernatants was determined by MMP-2, and MMP-10 Quantikine ELISA (R&D Systems), respectively, according to manufacturer's instructions.
Cell migration assay
NHLFs were seeded into polycarbonate inserts with an 8.0 μm pore size in a 24-well plate (Corning, Corning, NY, USA) at 75,000 cells/well. Where indicated, inserts were coated with collagen I (10 μg/cm2), collagen IV (10 μg/cm2) (both from Sigma-Aldrich, Gillingham, UK) or human plasma-derived fibronectin (5 μg/cm2) (R&D Systems, Abingdon, UK). Cells were serum starved overnight, and where indicated collagen I (25 μg/mL) was added to top and bottom chamber for 16 h. Bottom chamber was treated with cell dissociation buffer (Life Technologies, Paisley, UK) and acetomethoxycalcein (calcein AM; Trevigen, AMS Biotechnology, Abingdon, UK) for 1 h at 37°C. The suspension of detached cells was transferred to a 96-well plate and fluorescence was measured at 485 nm excitation and 520 nm emission.
Cell proliferation assay
Cell proliferation was determined using the DELFIA proliferation assay kit (PerkinElmer, Cambridge, UK) according to manufacturer's instructions. Briefly, NHLFs were seeded into a 96-well black walled plate at 3,000 cells/well and grown in humidified 5% CO2 atmosphere at 37°C. Cells were serum starved overnight, and incubated with collagen I (25 μg/mL) or vehicle (acetic acid, 0.1 M) for 16 h. NHLFs were incubated with 5-bromo-2'-deoxyuridine (BrdU) for an additional 16 h, fixed and incubated with anti-BdrU labeled with europium for 90 minutes. Time-resolved fluorescence was measured at 340 nm excitation and 615 nm emission.
Data were expressed as the mean ± SD of six or more independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Tukey test. Significance was set as P < 0.05.