LacZ staining and immunohistochemistry
β-galactosidase (β-gal) staining was performed in embryos. In brief, specimens were dissected in PBS and prefixed in PBS containing 1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA, and 0.02% Nonidet P-40 at 4°C for 30-90 minutes. Samples were washed three times in PBS containing 0.02% Nonidet P-40 at room temperature for 30 minutes before they were stained in PBS containing 1 mg/ml X-Gal, 5 mM potassium ferricyanide, 2 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40 at 30°C for 6-16 hours. The specimens were subsequently fixed in formaldehyde and processed for paraffin or frozen sections.
Generation of Col2a1-ICAT transgenic mice
TOP-gal mice were obtained from The Jackson Laboratory (Bar Harbor, ME). To generate the Col2a1-ICAT
transgenic mice, DNA fragments encoding ICAT were cloned into the Not
I site of a collagen 2 α1 (Col2a1)-based expression vector PKN185 (Tanaka et al., 2000
; Tsuda et al., 2003
). The resulting vector contains the Flag-ICAT expression unit and includes the 5′ Nde
I site of the Col2a1
promoter (nucleotides 1940-2971, GenBank accession number: M65161), the β-globin intron cassette, Flag-ICAT, SV40 poly (A) and the Col2a1 enhancer (nucleotide 4930–5571, GenBank accession number: M65161). The expression unit of Flag-ICAT was excised by NdeI
III digestion. The ICAT
transgene was then purified and injected into pronuclei of fertilized eggs (C57BL/6J). Positive transgenic founder mice were identified by PCR and confirmed by Southern blot analysis.
Isolation of primary mouse sternal chondrocytes
Primary mouse sternal chondrocytes were isolated as described (Li et al., 2006b
). Briefly, 3-day-old neonatal mice were sacrificed and genotyped using tail tissues obtained at sacrifice. The anterior rib cage and sternum were harvested en-bloc, washed with 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 removed and the remaining sterna and costosternal junctions were further digested in 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 Swinex 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]. Cells were then 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.
Antibodies and reagents
The following antibodies were used in this study: anti-β-catenin monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-cyclin D1 monoclonal antibody (1:1000 dilution, Upstate, Charlottesville, VA), anti-Flag monoclonal antibody (1:200 dilution for immuno-fluorescence assay and 1:1000 dilution for western blotting, Sigma), rabbit anti-Ki67 monoclonal antibody (1:200 dilution for immunostaining, Lab Vision, Fremont, CA), and anti-PECAM-1 antibody (1:100 for immunostaining, Santa Cruz Technology). BIO was purchased from Calbiochem (San Diego, CA), and dissolved in DMSO. BMP2, Wnt3a, noggin and Dkk1 were obtained from R&D Systems (Minneapolis, MN).
Western blot analysis
Cells were lysed on ice for 30 minutes in a buffer containing 50 mM Tris-HCL pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 10 μg/ml asprotinin) and phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4). Proteins were fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and detected using the respective antibodies. Bound primary antibodies were detected with horseradishperoxidase-conjugated secondary antibodies followed by ECL-mediated visualization (Amersham, Piscataway, NJ).
Mouse tissues were dissected in PBS, fixed in 10% buffered formalin, and paraffin embedded. Tissue sections were subject to immune complex staining using an avidin–biotinylated-enzyme complex according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA). Rabbit monoclonal anti-Ki-67 was used in the analysis. Bound primary antibodies were detected with fluorescein-conjugated secondary antibodies (Amersham, Piscataway, NJ). Immunostaining of cultured cells was performed using indirect fluorescent staining technique. Briefly, cells were fixed with 4% paraformaldehyde for 10 minutes and treated with 0.5% Triton X-100 for 15 minutes and 50 mM glycine for 10 minutes followed by blocking with the PBS-Triton buffer containing 3% BSA for 30 minutes. Samples were then incubated with primary antibody for 1 hour and fluorescent conjugated secondary antibody for 45 minutes and mounted with vectashield (Lab Vision, Fremont, CA). The anti-Flag mouse monoclonal antibody was used as primary antibody.
Alkaline phosphatase (ALP) activity assay
Primary sternal chondrocytes isolated from wild-type (WT) or Col2a1-ICAT transgenic mice were plated into 12-well culture plates and grown to 60% confluence. The cells were treated with the growth factors as indicated. 48 hours after incubation, cells were washed twice with PBS, and cell lysates extracted with passive lysis buffer (Promega, Madison, WI). ALP activity in cell lysates was measured using a Sigma ALP assay kit (Sigma, St Louis, MO) and normalized by the protein content.
Total RNA extraction and real-time reverse transcriptase (RT)-PCR analysis
Total cellular and tissue RNA was prepared by Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. One microgram total RNA was used to synthesize cDNA using iScripts cDNA Synthesis Kit (Bio-Rad, Hercules, CA). PCR primers were: VEGF Fw: 5-TTACTGCTGTACCTCCACC-3, Rev: 5′-ACAGGACGGCTTGAAGATG-3′; MMP13 Fw: 5′-TTTGAGAACACGGGGAAGA-3′, Rev: 5-ACTTTGTTGCCAATTCCAGG-3′; Col-X Fw: 5′-ACCCCAAGGACCTAAAGGAA-3′, Rev: 5′-CCCCAGGATACCCTGTTTTT-3′; BMP2 Fw: 5′-GCTTTTCTCGTTTGTGGAGC-3′, Rev: 5′-TGGAAGTGGCCCATTTAGAG-3′; BMP-4 Fw: 5′-GAGGAGGAGGAAGAGCAGAG-3′, Rev: 5′-TGGGATGTTCTCCAGATGTT-3′; ALP Fw: 5′-TGACCTTCTCTCCTCCATCC-3′, Rev: 5′-CTTCCTGGGAGTCTCATCCT-3′.
Transferase UTP nick-end labeling (TUNEL) staining
A TUNEL staining kit (DeadEnd Fluorometric TUNEL System, Promega, Madison, WI) was used to assess cell death by catalytically incorporating fluorescein-12-dUTP at 3′-OH DNA ends using the terminal deoxynucleotidyl transferase and recombinant enzyme (rTDT). After paraffin removal, the tissue sections were placed in equilibration buffer and then in a solution containing the equilibration buffer, nucleotide mix, and rTDT enzyme and incubated at 37°C for 1 hour. The reaction was stopped with 2×saline sodium citrate (SSC). Hoechst dye 33342 was used to stain the nuclei. Fluorescence microscopy (Zeiss, Axiovert 40 CFL, Chester, VA) was used to identify apoptotic cells.
Cells were transfected by Lipofectamine 2000 (Invitrogen) with various combinations of plasmids. 0.5 μg of reporter plasmids (TOP-flash, 12×SBE-luc) and 0.01 μg of internal control SV-40 Renilla plasmid were using in the reporter assays. The luciferase activity was measured using the Promega dual system kit.
Chromatin immunoprecipitation (ChIP) assay
Chromatin IP was performed as described in the manual of the ChIP Assay kit (Upstate, Charlottesville, VA). Briefly, RCJ3.1C518 chondrocytes were cultured in 4×15 cm culture dishes to 70-80% confluence. Chondrocytes were then treated with 1 μM BIO for 4 hours, followed by crosslinking using formaldehyde at 37°C for 10 minutes. The crosslink reaction was stopped using glycine buffer, cells were washed with a protease inhibitor cocktail (Roche Applied Science) and harvested. Chondrocytes were then incubated with lysis buffer on ice for 30 minutes and the cell lysate was sonicated to shear the genomic DNA to 200-bp to 500-bp fragments. After centrifugation, the supernatant was incubated with protein G beads saturated with salmon sperm DNA for 30 minutes to pre-clean the cell lysate. A total of 50 μl anti-β-catenin antibody (Santa Cruz Technology) was added to the cell lysate and incubated for overnight at 4°C. Lysates were then incubated with 100 μl of proteinG beads for 2 hours. Beads were pelleted by centrifugation for 2 minutes at 4000 rpm and washed according to the manufacturer's protocol. The protein-DNA complex was eluted with ChIP elution buffer containing 0.01M NaHCO3 and 0.5% SDS. After centrifugation, 5M NaCl was added to the eluted solution to a final concentration of 200 mM and then incubated at 65°C overnight. After reversing the crosslink, the samples were further digested by proteinase K. The DNA was extracted by phenol-chloroform, precipitated with ethanol and prepared for PCR. Primer sequences were: TRE Fw: 5′-ACTCTAGTTGTCCCTATCCTCA-3′, TRE Rev: 5′-TCTGCGCTTCTCACCGGTAACA-3′; Cont Fw: 5′-AGAGCTTGCCCGAGGAATGT-3′, Cont Rev: 5′-CTCCGATACCTGTGGGAAGA-3′; GAPDH Fw: 5′-AACGACCCCTTCATTGAC-3′, GAPDH Rev: 5′-TCCACGACATACTCAGCAC-3′.
Quantification of vascularity using microCT analysis
The bone vascular network was examined on tissue sections of animals following perfusion of a lead-chromate-based contrast agent using microCT analysis. Microfil MV-122 contrast medium, a radiopaque silicone rubber compound containing lead chromate, was perfused through the heart together with 4% paraformaldehyde. After perfusion, the hind limbs were decalcified using 10% EDTA solution. After complete decalcification, the samples were scanned again to image vascularization.