Dulbecco’s modified Eagle’s medium with 1 g/L glucose (DMEM-LG), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phenylmethanesulfonyl fluoride (PMSF), sodium fluoride, and fetal bovine serum (FBS) was purchased from FUJIFILM Wako Chemical Corp. (Osaka, Japan). Recombinant canine IL-1β and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) were purchased from Kingfisher Biotech, Inc. (Saint Paul, MN) and MedChemExpress (Monmouth Junction, NJ, USA), respectively. Lipofectamine 2000 and TRIzol were purchased from Life Technologies (Carlsbad, CA, USA). CELLBANKER 1 plus medium, Thermal Cycler Dice Real Time System II, TP900 Dice Real Time v4.02B, SYBR Premix Ex Taq II, and PrimeScript RT Master Mix were purchased from TaKaRa Bio, Inc. (Shiga, Japan). Rabbit monoclonal anti-phosphorylated p65 (93H1), anti-total p65 (D14E12), anti-phosphorylated p105 (18E6), anti-total p105 (D4P4D), and mouse monoclonal anti-total IκBα (L35A5) antibodies were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). Anti-β-actin mouse monoclonal antibody (AC74), small interfering RNA (siRNA) for p65 and p105, and scrambled siRNA were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG antibodies, the ECL western blotting Analysis System, and Amersham ImageQuant 800 were purchased from Cytiva Japan (Tokyo, Japan). Mini-PROTEAN TGX gel, polyvinylidene difluoride (PVDF) membranes, and iCycler were obtained from Bio-Rad (Hercules, CA). The complete mini ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor mixture and Block Ace were purchased from Roche (Mannheim, Germany) and KAC Co., Ltd. (Hyogo, Japan), respectively. An enzyme-linked immunosorbent assay (ELISA) kit for canine IL-8 (#CA8000), a freezing vessel (BICELL), and StatMate IV were purchased from R&D Systems, Inc. (Minneapolis, MN), Nihon Freezer Co., Ltd. (Tokyo, Japan), and ATMS (Tokyo, Japan), respectively.

Canine primary cardiac fibroblasts were purchased from Cell Biologics (Chicago, IL, USA). The thawed-out cell suspension was transferred into a centrifuge tube contained DMEM-LG containing 20% FBS. After centrifugation at 300×g for 1 min and removal of the supernatant, the cell pellet was resuspended in DMEM-LG containing 20% FBS and transferred into a 75-cm² culture flask. The cells were maintained in static culture in an incubator at 5% carbon dioxide (CO₂) and 37°C using DMEM-LG supplemented with 20% fetal calf serum, and the medium was changed once a week. When the fibroblasts reached 90–95% confluence, they were harvested using 0.25% trypsin-EDTA. The collected cardiac fibroblasts were suspended using CELLBANKER 1 plus medium at a density of 2 × 10⁶ cells/500 μl, divided into 500 μl each, and placed into a sterilized serum tube. The tubes were then placed into the freezing vessel BICELL and cryopreserved at −80°C. Before being used in the experiments, serum tubes were removed from the BICELL vessel and immersed in a water bath at 37°C. The thawed-out cell suspension was transferred into a centrifuge tube contained DMEM-LG containing 20% FBS. After centrifugation at 300×g for 1 min and removal of the supernatant, the pellet was suspended in DMEM-LG containing 20% FBS and transferred into a 75-cm² culture flask. Static cultures were then performed under the same conditions as those before cryopreservation.

Real-time RT-PCR was performed as previously reported (25–36). Total RNA was extracted from cultured canine cardiac fibroblasts using TRIzol reagent, following the instructions of the manufacturer. Synthesis of first-strand cDNA was performed with 500 ng of total RNA using PrimeScript RT Master Mix. Real-time RT-PCR was performed with 2 μl of first-strand cDNA in 25 μl (total reaction volume), SYBR Premix Ex Taq II, and primers targeting canine IL-8 or hypoxanthine phosphoribosyltransferase 1 (HPRT1), the housekeeping gene (Table 1). Real-time RT-PCRs of “no-template” controls or “no-reverse transcription” controls were performed with 2 μl of RNase- and DNA-free water or 2 μl of each RNA sample, respectively. PCR was conducted using the Thermal Cycler Dice Real-Time System II. The protocol was as follows: one cycle of denaturation at 95°C for 30 s, 40 cycles of denaturation at 95°C for 5 s, and annealing/extension at 60°C for 30 s. The results were analyzed by the second derivative maximum method and the comparative cycle threshold (ΔΔCt) method using real-time RT-PCR analysis software. The amplification of HPRT1 from the same amount of cDNA was used as an endogenous control, whereas cDNA amplification from canine cardiac fibroblasts at time 0 was used as the calibration standard.

Western blotting was performed as described previously (25–27, 29–36). Canine cardiac fibroblasts were lysed in 20 mM HEPES buffer (pH 7.4) containing 1 mM PMSF, 10 mM sodium fluoride, and a complete mini EDTA-free protease inhibitor cocktail. Protein concentrations were determined using the Bradford method (37) and adjusted. After boiling at 98°C for 5 min in the sodium dodecyl sulfate buffer, the extracted protein samples were loaded into separate lanes of 12 or 7.5% Mini-PROTEAN TGX gel and electrophoretically separated. Separated proteins were transferred to PVDF membranes, treated with Block Ace for 50 min at room temperature, and incubated with the following primary antibodies [phosphorylated p-65 (p-p65, 1:1,000), total p65 (t-p65, 1:1,000), phosphorylated p-105 (p-p105, 1:1,000), total p105 (t-p105, 1:1,000), total IκBα (t-IκBα, 1:1,000), and β-actin (1:10,000)] for 120 min at room temperature. After washing, membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG (1:10,000) for 90 min at room temperature. Immunoreactivity was detected using the ECL western blotting Analysis System, and chemiluminescent signals of the membranes were measured using an Amersham ImageQuant 8000.

Canine cardiac fibroblasts were seeded at a density of 3.0 × 10⁵ cells/well in 6-well culture plates. After starvation for 24 h, fibroblasts were treated with IL-1β for 0–24 h and the culture medium was collected. The IL-8 concentration in the culture medium was assayed using an ELISA kit, according to the instructions of the manufacturer.

siRNA transfection was performed as previously described with slight modifications (25–27, 29–36). Canine cardiac fibroblasts were seeded at a density of 1 × 10⁵ cells/35 mm dish or 5 × 10⁵ cells/90 mm dish, and transfected using Opti-MEM containing 3 μl/ml Lipofectamine 2000 and 2 μM p65, p105, or scramble siRNA (Table 2) for 24 h. After transfection, the medium was changed to DMEM-LG containing 20% FBS and the cultures were maintained in an incubator with 5% CO₂ at 37°C for 2 d. The efficiency of siRNA-mediated knockdown was validated by western blotting.

All statistical analyses were performed using EZR (38), a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of the R commander designed to add statistical functions frequently used in biostatistics. Data from all experiments are shown as the mean ± standard error and were analyzed using one-way analysis of variance (ANOVA) with Holm–Bonferroni multiple comparisons test as a post hoc test. Statistical significance was set at p <0.05.

When canine cardiac fibroblasts were incubated with 100 pM IL-1β for 0–48 h, the concentration of IL-8 in the incubation medium increased in a time-dependent manner (Figure 1A). In fibroblasts treated with various concentrations of IL-1β (0–200 pM) for 24 h, IL-1β stimulated IL-8 release in a dose-dependent manner (Figure 1B). We examined the effect of IL-1β on IL-8 mRNA expression in canine cardiac fibroblasts. As shown in Figures 1C, D, IL-1β stimulated IL-8 mRNA expression in a time- and dose-dependent manner. These observations suggest that IL-1β induces IL-8 expression and release in canine cardiac fibroblasts.

IL-1β is known to mediate the mRNA expression of various proteins via the activation of the transcription factor NF-κB. We then examined the involvement of NF-κB in IL-1β-mediated IL-8 expression in canine cardiac fibroblasts using the NF-κB inhibitor TPCA-1. In fibroblasts pretreated with TPCA-1 (10 μM) for 1 h, the effects of IL-1β on IL-8 mRNA expression (Figure 2A) and IL-8 release (Figure 2B) were clearly attenuated. These observations suggest that NF-κB activation is involved in the IL-1β-induced IL-8 mRNA expression in canine cardiac fibroblasts.

Next, we examined the effects of IL-1β on the activation of NF-κB in canine cardiac fibroblasts. When fibroblasts were stimulated with 100 pM IL-1β for 0–300 min, IκBα degradation was observed at 15–30 min (Figures 3A, B). Phosphorylation of p65 and p105 was also observed 15–30 min after stimulation, indicating the activation of NF-κB by IL-1β (Figures 3A, C, D). In fibroblasts pretreated with the NF-κB inhibitor, TPCA-1, for 1 h, IL-1β-mediated phosphorylation of p65 and p105 was clearly attenuated (Figure 4). These observations suggest that IL-1β induces IL-8 expression via the activation of NF-κB.

To confirm the contribution of NF-κB to IL-1β-induced IL-8 expression, we examined the effect of IL-1β on IL-8 mRNA expression in p65 or p105 knockdown fibroblasts using siRNA transfection. In fibroblasts transfected with p65 or p105 siRNA, p65 or p105 protein expression was clearly reduced compared with that in control fibroblasts transfected with scramble RNA (Figures 5A–C). IL-1β-mediated IL-8 mRNA expression was clearly decreased in fibroblasts transfected with p65 siRNA compared to that in the control (Figure 5D). However, p105 siRNA transfection failed to attenuate the IL-1β-mediated IL-8 mRNA expression (Figure 5D). Taken together, it is likely that NF-κB p65 activation contributes to IL-1β-mediated expression of IL-8, but not p105, in canine cardiac fibroblasts.