A total of 20 female Sprague-Dawley rats (weight, 180–200 g) were supplied by the Animal Center, Shanghai Jiao Tong University School of Medicine (Shanghai, China). The rats were sacrificed by cervical dislocation prior to surgical removal of normal rat knee cartilage from the tibial platform and the femoral condyle. The experiments were conducted with approval from the Shanghai Jiao Tong University School of Medicine ethics committee. Cartilage pieces were washed with phosphate-buffered saline (PBS) twice and treated with 0.2 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO, USA) in serum-free Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies, Carlsbad, CA, USA) overnight at 37°C. The cells were collected by filtering the disaggregated tissue through a 200-mesh nylon cell strainer, centrifugation at 1,000 × g for 5 min and two washes with PBS. Finally, the cells were re-suspended and cultured in DMEM supplemented with 10% (vol/vol) fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA, USA), plus 1% penicillin and streptomycin (Gibco Life Technologies). The culture medium was replaced every second day. The chondrocytic phenotype of the cultured cells was confirmed by positive immunostaining for type II collagen. Briefly, cells cultured on glass slides were incubated overnight at 4°C with anti-rat type II collagen (1:300 dilution; cat. no. ab34712; Abcam, Cambridge, UK), and immunostaining was performed using immunostaining reagent (Dolichos bifows agglutini Immunohistochemistry kit; Wuhan Boster Biological Technology, Ltd., Wuhan, China), according to the manufacturer's instructions. Positive immunostaining of collagen II was detected using an inverted fluorescence microscope (IX70; Olympus, Tokyo, Japan). Toluidine blue staining (Shanghai Chemical Reagent Co., Ltd., Shanghai, China) of glycosaminoglycans was also conducted. First-passage chondrocytes were used in all experiments.

First-passage chondrocytes were trypsinized and seeded into six-well plates (3×10⁵ cells/well) for 24 h. Chondrocytes were maintained in serum-deficient DMEM for 24 h and subjected to a variety of treatments. Chondrocytes were cultured with or without 10 µg/ml recombinant human TGF-β1 (Perprotech, Rocky Hill, NJ, USA) for 48 h. Type II collagen and aggrecan expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. Chondrocytes were cultured with 10 µg/ml TGF-β1. Following 0, 1, 5, 10, 15, 30, 60 and 180 min of incubation, phosphorylated (p)-Smad3, Smad2/3, p-ERK1/2, and ERK1/2 levels were examined by western blotting. For inhibition studies, chondrocytes were pre-treated in serum-free medium with or without TGF-β receptor I (ALK5) kinase inhibitor SB525334 (1 µM; Selleckchem, Houston, TX, USA), the pharmacological ERK1/2 inhibitor PD98059 (10 µM; Promega Corporation, Madison, WI, USA), and pharmacological Smad3 phosphorylation inhibitor SIS3 (10 µM; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 60 min and then stimulated with or without 10 µg/ml TGF-β1 for 48 h. Expression of type II collagen and aggrecan was evaluated by RT-qPCR and western blotting. To examine the involvement of the ERK1/2 and Smad signaling pathways, chondrocytes were pre-treated in serum-free medium with SB525334, PD98059 or SIS3 for 60 min, followed by stimulation with 10 µg/ml TGF-β1. Following 0, 1, 5, 10, 15, 30, 60 or 180 min of incubation, p-Smad3, Smad2/3, p-ERK1/2, and ERK1/2 levels were examined by western blotting.

Total RNA was extracted from chondrocytes using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer's instructions. For first-strand cDNA synthesis, 2 µg mRNA was reverse-transcribed using the Reverse Transcriptase Moloney murine leukemia virus (M-MLV) cDNA Synthesis kit (Takara, Tokyo, Japan) according to the manufacturer's instructions. Briefly, a 12-µl reaction mixture containing 2 µl oligo d(T)18 primer (50 µM), 2 µg total RNA and RNase-free dH₂O was incubated at 70°C for 10 min. Subsequently, 1 µl deoxynucleotide triphosphate mixture (10 mM each), 4 µl 5X M-MLV buffer, 0.5 µl ribonuclease inhibitor (40 U/µl), 1 µl reverse transcriptase M-MLV (RNase H-free; 200 U/µl) and RNase-free dH₂O were added to a final volume of 20 µl and the mixture was incubated for 60 min at 42°C. Next, the reaction was inactivated by heating at 70°C for 15 min. Subsequently, 2 ml of the 20 ml reverse transcription reaction was subjected to PCR amplification using PCR Master Mix (cat. no. M7502, Promega Corporation) with initial denaturation at 95°C for 10 min, then 35 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec. Products were quantified using a melting curve analysis. Amplification of GAPDH was performed to quantify PCR products and confirm the use of equal amounts of RNA. Relative gene expression was analyzed using the 2^ΔΔCt method (13). Primer sequences (Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China) of rat genes were as follows: Type II collagen forward, 5′-TCCTAAGGGTGCCAATGGTGA-3′, and reverse, 5′-GGACCAACTTTGCCTTGAGGAC-3′; aggrecan forward, 5′-TCCGCTGGTCTGATGGACAC-3′, and reverse, 5′-CCAGATCATCACTACGCAGTCCTC-3′; and GAPDH forward, 5′-CAAGTTCAACGGCACAGTCAAG-3′, and reverse, 5′-ACATACTCAGCACCAGCATCAC-3′.

Whole-cell lysates were prepared using radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA and 2 mM phenylmethanesulfonylfluoride] in the presence of protease and phosphatase inhibitors. For the detection of p-Smad3, Smad2/3, p-ERK1/2, ERK1/2 and aggrecan protein levels, equal amounts of total cellular extracts from rat chondrocytes were then loaded on a 10% SDS-polyacrylamide gel, subjected to electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) by electroblotting. For type II collagen protein levels, equal amounts of total cellular extracts were separated by 8% SDS-polyacrylamide gel electrophoresis. Membranes were then blocked with 5% skimmed milk. Polyclonal anti-rat type II collagen (1:5,000 dilution; cat. no. ab34712; Abcam), polyclonal anti-rat aggrecan (1:200 dilution; cat. no. 251591; Abbiotec, San Diego, CA, USA), monoclonal anti-rat p-Smad3 (1:1,000 dilution; cat. no. ab52903; Abcam), polyclonal anti-rat Smad2/3 (1:500 dilution; cat. no. R-1002–100; Novus, Littleton, CO, USA), monoclonal anti-rat p-ERK1/2 (1:1,000 dilution; cat. no. ab32538; Abcam) and polyclonal anti-rat ERK1/2 antibody (1:1,000; cat. no. 9102; Cell Signaling Technology Inc., Danvers, MA, USA) were incubated with the membranes overnight at 4°C. The membranes were then washed and reacted with secondary horseradish peroxidase-conjugated secondary antibody [donkey anti-rabbit immunoglobulin G for type II collagen, aggrecan, p-Smad3, Smad2/3, p-ERK1/2, and ERK1/2 (1:2,000; cat. no. sc-2305; Santa Cruz Biotechnology, Inc.)] at room temperature for 2 h. Finally, protein bands were visualized by chemiluminescence (EMD Millipore) using the Tanon 4200 SF Imaging Analysis system (Tanon Science and Technology Co., Ltd., Shanghai, China). Western blots were re-probed with a monoclonal anti-GAPDH antibody (1:1,000 dilution; cat. no. 2118; Cell Signaling Technology Inc.) as a control.

All experiments were performed three times and the results are expressed as the mean ± standard deviation. Statistical significance was determined using the Mann-Whitney U test or Kruskal-Wallis analysis of variance test, when appropriate, using SPSS 13.0 statistical software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference between values.

To confirm that TGF-β1 induced upregulation of type II collagen and aggrecan expression in chondrocytes, rat chondrocytes were cultured with or without TGF-β1 for 48 h. RT-qPCR and western blotting were then performed to examine the expression of type II collagen and aggrecan. As shown in Fig. 1A and B, significant upregulation of type II collagen and aggrecan occurred following treatment with TGF-β1. Western blotting also indicated that type II collagen and aggrecan protein levels were increased following TGF-β1 treatment (Fig. 1C). These results confirmed that TGF-β1 stimulated type II collagen and aggrecan expression in rat chondrocytes.

Furthermore, it was evaluated whether the ERK1/2 and Smad2/3 signaling pathways were involved in TGF-β1 stimulation of type II collagen and aggrecan. Rat chondrocytes were cultured in the presence of TGF-β1, and at the indicated time-points, the activation of ERK1/2 and phosphorylation of Smad3 were examined by western blotting. Fig. 1D demonstrates that TGF-β1 induced rapid phosphorylation of ERK1/2 and Smad3. These observations suggested that the ERK1/2 and Smad2/3 signaling pathways may have an important role in TGF-β1-stimulated type II collagen and aggrecan induction in rat chondrocytes.

To further investigate the involvement of the ERK1/2 and Smad2/3 signaling pathways in TGF-β1-induced type II collagen and aggrecan expression, chondrocytes were pre-treated with or without ALK5 kinase inhibitor SB525334 and then stimulated with or without TGF-β1. After 48 h, RT-qPCR and western blotting confirmed that TGF-β1 stimulated type II collagen and aggrecan expression was inhibited by SB525334 (Fig. 2A–C). At the indicated time-points, western blotting demonstrated that phosphorylation of ERK1/2 and Smad3 was blocked by SB525334 (Fig. 2D). These results further validated the role of the ERK1/2 and Smad2/3 signaling pathways in type II collagen and aggrecan induction by TGF-β1.

Next, the the role of ERK1/2 in type II collagen and aggrecan induction by TGF-β1 was examined. Chondrocytes were pre-treated with or without ERK1/2 inhibitor PD98059 followed by culture with or without TGF-β1 for 48 h. RT-qPCR and western blotting revealed that TGF-β1-induced expression of type II collagen and aggrecan was significantly suppressed in the presence of PD98059 (Fig. 3A–C). ERK1/2 signaling pathway activation was then assessed in chondrocytes pre-treated with PD98059 followed by culture with TGF-β1. Activation of the ERK1/2 signaling pathway was inhibited by PD98059 (Fig. 3D). These results suggested that the ERK1/2 signaling pathway may have an important role in type II collagen and aggrecan induction by TGF-β1.

To determine whether the Smad2/3 signaling pathway affected TGF-β1-induced type II collagen and aggrecan, SIS3 was used to suppress Smad3 phosphorylation. First, chondrocytes were cultured with or without TGF-β1 for 48 h following pre-treatment with or without SIS3. RT-qPCR and western blotting indicated that TGF-β1-induced type II collagen and aggrecan expression were significantly inhibited by SIS3 (Fig. 4A–C). Next, chondrocytes were treated with TGF-β1 following pre-treatment with SIS3. At the indicated time-points, western blotting showed that Smad3 phosphorylation was markedly suppressed by SIS3 (Fig. 4C). These results suggested that the Smad2/3 signaling pathway may also have an important role in type II collagen and aggrecan induction by TGF-β1.