Generation of transgenic mice
To generate the Col2a1-ICAT
transgene, DNA fragments encoding ICAT were cloned into the Not
I site of a Colα1(II) (Col2a1
)–based expression vector, PKN185 (8
). The resulting vector contains the FLAG-tagged ICAT
) complementary DNA including the 5′ Nde
I site Col2a1
promoter (nucleotide 1940–2971, GenBank accession no. M65161), β-globin intron cassette, SV40 poly(A), and Col2a1
enhancer (nucleotide 4930–5571, GenBank accession no. M65161). The FLAG-ICAT
transgene was excised by Nde
I and Hind
III digestion. The ICAT
transgene was then purified and injected into pronuclei of fertilized eggs from C57BL/6J mice. Positive transgenic founder mice were identified by Southern blot analysis, and mice were genotyped by polymerase chain reaction (PCR). The F3
generation of 2 separate lines of Col2a1-ICAT
–transgenic mice and their wild-type (WT) littermates were used for phenotype analysis and cellular function studies.
Histology and histomorphometry
Initial radiographic and histologic analyses were performed in 2 lines of the transgenic mice (lines 5 and 7), and similar articular cartilage destruction was found. Analysis of time-dependent articular cartilage destruction was then performed in 1 line of these transgenic mice (line 5). Knee joints from 6-, 9-, 12-, and 15-month-old WT and Col2a1-ICAT–transgenic mice were dissected, fixed in 10% formalin, decalcified, and embedded in paraffin. Serial midsagittal sections (3-µm thick) of knee joints from 6-month-old mice were cut every 10 µm from both the medial and lateral compartments. The sections were stained with Alcian blue/hematoxylin and eosin (H&E). Articular cartilage was outlined on the tibial surface, and an area algorithm in the software package ImagePro 4.5 (Leeds Precision Instruments, Minneapolis, MN) was used to determine the pixel area of outlined articular cartilage from each section. Using this approach, average articular cartilage area was determined. Cells in each section were counted, and the ImagePro counting algorithm was used to determine the average number of chondrocytes per unit cartilage area. Six mice per group were used for histomorphometric measurements.
Tissue sections were deparaffinized by immersing in xylene, then fixed with 4% paraformaldehyde for 15 minutes and treated with 0.5% Triton for 15 minutes followed by fixing with 4% paraformaldehyde for another 5 minutes. Sections were then incubated with a mouse anti-FLAG M2 monoclonal antibody (1:200 dilution; Sigma, St. Louis, MO), rabbit anti–cleaved caspase 3 monoclonal antibody (Asp175, 1:200 dilution; Cell Signaling Technology, Danvers, MA), and rabbit anti–poly(ADP-ribose) polymerase (anti-PARP) polyclonal antibody (1:50 dilution; Abcam, Cambridge, MA) overnight. Secondary incubations were performed with a fluorescence-conjugated secondary antibody for 60 minutes, and sections were mounted with Vectashield (LabVision, Fremont, CA). Slides were visualized under a fluorescence microscope.
Cell isolation and cell culture
Three-day-old neonatal mice were euthanized and genotyped using tail tissues obtained at the time of death. The anterior rib cage and sternum were harvested en bloc, washed with phosphate buffered saline (PBS), and then digested with Pronase (Roche Applied Science, Indianapolis, IN) dissolved in PBS (2 mg/ml) in a 37°C water bath with continuous shaking for 60 minutes. This was followed by incubation in a solution of collagenase D (3 mg/ml dissolved in serum-free Dulbecco’s modified Eagle’s medium [DMEM]; Roche Applied Science) for 90 minutes at 37°C. The soft tissue debris was thoroughly removed. The remaining sterna and costosternal junctions were further digested in a fresh collagenase D solution in petri dishes in a 37°C incubator for 5 hours with intermittent shaking. This step allows remnant fibroblasts to attach to the petri dish while the chondrocytes remain afloat in the medium. The digestion solution was filtered through Swinnex filter (Millipore, Bedford, MA) to remove all residual bone fragments. The solution was centrifuged, and the cells were resuspended in complete medium (DMEM with 10% fetal bovine serum [FBS], 1% penicillin/streptomycin, 100 mM L-glutamine, and 50 µg/ml ascorbic acid, pH 7.1). The cells were counted and plated at the appropriate density. To remove any remaining fibroblasts, 24-hour cultures were treated with 0.05% trypsin for 1 minute to lift the fibroblasts from the culture dish while allowing the chondrocytes to remain attached.
For chicken chondrocytes, 6-week-old chicks were euthanized and the femora and tibiae were carefully separated by removing the surrounding tissue at the knee joint. Articular cartilage was cut off and digested with trypsin (1 mg/ml; Sigma) on a rotator for 30 minutes. This was followed by incubation in a solution of hyaluronidase (1 mg/ml; Sigma) for 60 minutes. The remaining tissues were further digested in collagenase A (1 mg/ml; Roche Applied Science) on the rotator overnight. The digestion solution was filtered through Swinnex filter to remove all residual bone fragments. The solution was centrifuged, and the cells were resuspended in complete medium (DMEM with 5% FBS, 1% penicillin/streptomycin, and 50 µg/ml ascorbic acid, pH 7.1). Cells were counted and plated at the appropriate density.
Porcine articular chondrocytes were isolated from the articular cartilage of elbow and metatarsal joints of freshly slaughtered pigs. Briefly, articular cartilage was shaved from the articular surfaces and minced into 2 × 2–mm chunks. Articular chondrocytes were then released from the native ECM with a 1.5-hour treatment in 0.05% (weight/volume) Pronase (Calbiochem, San Diego, CA) followed by 16-hour digestion in 0.2% (w/v) collagenase (Worthington, Lakewood, NJ). Cells were then pelleted and resuspended in 20 ml of cell culture media containing DMEM, 1% penicillin/streptomycin, 10% FBS, and 50 µg/ml ascorbic acid before counting and plating.
Primary sternal chondrocytes were transfected with the Top-flash reporter construct (0.5 µg/well, 12-well plate) and control SV40 Ranilla luciferase construct (0.01 µg/well) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in the absence or presence of BIO (1 µM), the glycogen synthase kinase 3β (GSK-3β) inhibitor. Cell extracts were harvested, and the luciferase activity was measured 2 days after transfection by the Promega Dual Luciferase system (Promega, Madison, WI).
Western blot analysis
Primary chondrocytes were lysed on ice for 30 minutes in a buffer containing 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P40, and 0.1% sodium dodecyl sulfate (SDS) supplemented with protease inhibitors (10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin) and phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4). Proteins were fractionated by SDS– polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and detected using anti–Bcl-2 and anti–Bcl-xL monoclonal antibodies (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-Bax monoclonal antibody (1:1,000 dilution; Santa Cruz Biotechnology).
A TUNEL staining kit (DeadEnd Fluorometric TUNEL System; Promega) was used to assess programmed cell death by catalytically incorporating fluorescein-12-dUTP at 3′-OH DNA ends using the terminal deoxynucleotidyl transferase and recombinant enzyme. After deparaffinization, the tissue sections were placed in equilibrium buffer and then in a solution containing the equilibrium buffer, nucleotide mix, and terminal deoxynucleotidyl transferase and recombinant enzyme and incubated at 37°C for 1 hour. The reaction was stopped with 2× saline–sodium citrate. Hoechst 33342 was used to stain nuclei. Results were visualized under a fluorescence microscope. Articular cartilage area from the tidemark to the surface and from the anterior to posterior cartilage margins was outlined in each slide. Chondrocyte numbers within the outlined regions were counted on 4 nonconsecutive sections from each joint sample. The apoptotic cell rates were determined by counting the numbers of TUNEL staining–positive cells in the cartilage area divided by the total cell number. Six Col2a1-ICAT–transgenic mice and 6 WT littermates were analyzed.
Caspase activity assay
Caspase 3/7 activity in primary mouse sternal chondrocytes and chicken and porcine articular chondrocytes was measured by adding Apo-ONE Caspase-3/7 Reagent (Apo-ONE homogeneous caspase 3/7 assay; Promega) to each well of a black 96-well tissue culture plate that was either blank or that had cells in culture. After incubation for 2 hours, the fluorescence of each well was measured. Caspase 9 activity was measured by adding Caspase-Glo 9 Reagent (Promega) to each well of a white 96-well tissue culture plate that was either blank or that had cells in culture. After incubation for 2 hours, the luminescence of each well was measured. The caspase 8 activity was determined by adding the Caspase-Glo 8 Reagent (Promega) to the cell culture.