Generation of Mig-6 overexpression mice
Mig-6 overexpression animals on a mixed C57Bl/6 and agouti mouse background, with the overexpression cassette in the Rosa26 locus (41) and bred for 10 generations into a C57Bl/6 background. Transcription of Mig-6 is under the control of a ubiquitously expressed chicken beta actin-cytomegalovirus hybrid (CAGGS) promoter, but blocked by a “Stop Cassette” flanked by LoxP sites (LSL) (41). Mig-6 overexpression mice were bred to mice carrying the Cre recombinase gene under the control of the Collagen 2 promoter (42), to induce recombination and removal of the Stop Cassette specifically in cartilage. Throughout the manuscript, animals for homozygote overexpression of Mig-6 from both alleles are termed Mig-6over/over (Mig-6over/overCol2a1-Cre+/-), while control mice are identical but without the Cre gene (noted as “control” in this manuscript for simplicity). Mice were group housed (at least one pair of littermate matched control and overexpression animals), on a standard 12-hour light/dark cycle, without access to running wheels, and with free access to mouse chow and water. Animals were weighed prior to euthanasia by asphyxiation with CO2. All animal experiments were done in accordance with the Animal Use Subcommittee at the University of Western Ontario and conducted in accordance with guidelines from the Canadian Council on Animal Care.
Genotyping
Genotype was determined by polymerase chain reaction (PCR) analysis using DNA processed from biopsy samples of ear tissue from mice surviving to at least 21 days of age. PCR strategy: Primer set P1 and P2 can amplify a 300 bp fragment from the wild-type allele, whereas P1 and P3 can amplify a 450 bp fragment from the targeted ROSA26 locus allele (41) (Supplementary Figure/Table 1).
RNA isolation and quantitative real-time PCR
Total RNA was isolated from post-natal day 0 (P0) mouse cartilage of Mig-6over/over and control littermates using TRIzol® (Invitrogen) as per manufacturer’s instructions and as previously described (43). Complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis kit (Bio-Rad) with 1µg of RNA (Bio-Rad Laboratories), and combined with 300nM of forward and reverse primers (for primer sequences, please see Supplementary Figure 1E) as well as iQ™ SYBR® Green Supermix (Bio-Rad Laboratories) for PCR on a Bio-Rad CFX384 RT-PCR system. Relative gene expression was normalized to the internal control Glyceraldehyde 3-phosphate dehydrogenase (Gapdh), calculated using the ΔΔCT method.
Histopathology of the knee Limbs from Mig-6over/over and control mice were harvested and fixed in 4% paraformaldehyde (Sigma) for 24 hours and decalcified in ethylenediaminetetraacetic acid (5% EDTA in phosphate buffered saline (PBS), pH 7.0. Joints were processed and embedded in paraffin in sagittal or frontal orientation, with serial sections taken at a thickness of 5 μm. Sections were stained with Toluidine Blue (0.04% toluidine blue in 0.2M acetate buffer, pH 4.0, for 10 minutes) for glycosaminoglycan content and general evaluation of articular cartilage. All images were taken with a Leica DFC295 digital camera and a Leica DM1000 microscope.
Thickness of proximal tibia growth plate For early developmental time points such as newborn (P0), sagittal knee sections stained with toluidine blue were used to measure the width of the zones of the epiphyseal growth plate in the proximal tibia. The average thickness of the resting and proliferative zones combined was evaluated by taking three separate measurements at approximately equal intervals across the width of the growth plate. The average hypertrophic zone thickness was also measured using 3 different measurements across the width of the growth plate, starting each measurement at the border of the proliferative and hypertrophic zones and ending at the subchondral bone interface. A third average measurement was then taken of the thickness of the entire growth plate. ImageJ Software (v.1.51) (44) was used for all measures, with the observer blinded to the genotype.
Articular cartilage evaluation Articular cartilage thickness was measured from toluidine blue-stained frontal sections by a blinded observer. Articular cartilage thickness was measured separately for the non-calcified articular cartilage (measured from the superficial tangential zone to the tidemark) and the calcified articular cartilage (measured from the subchondral bone to the tidemark) across three evenly spaced points from all four quadrant of the joint (medial/lateral tibia and femur) in 4 sections spanning at least 500 μm. ImageJ Software (v.1.51) (44) was used to measure the thickness of articular cartilage.
Micro-Computerized Tomography (μCT) Whole body scans were collected in 6 week-, 11 week-, 12 month- and 18 month-old control and Mig-6over/over male and female mice. Mice were euthanized and imaged using General Electric (GE) SpeCZT microCT machine (45) at a resolution of 50μm/voxel or 100μm/voxel. GE Healthcare MicroView software (v2.2) was used to generate 2D maximum intensity projection and 3D isosurface images to evaluate skeletal morphology. MicroView was used to create a line measurement tool in order to calculate the bone lengths, femurs lengths were calculated from the proximal point of the greater trochanter to the base of the lateral femoral condyle. Tibiae lengths were measured from the midpoint medial plateau to the medial malleolus. Humerus lengths were measured from the midpoint of the greater tubercle to the center of the olecranon fossa.
Body composition analysis MicroView software (GE Healthcare Biosciences) was used to analyse the microCT scans at the resolution of 100um/voxel. Briefly, the region of interest (ROI) was used to calculate the mean of air, water and an epoxy-based, cortical bone-mimicking calibrator (SB3; Gammex, Middleton, WI, USA) (1100mg/cm3) (46). A different set of global thresholds was applied to measure adipose, lean and skeletal mass (− 275, − 40 and 280 Hounsfield Units (HU), respectively). Moreover, bone mineral density (BMD) was acquired as the ratio of the average HU (from the value of skeletal region of interest ) in order to calculate HU value of the SB3 calibrator, multiplied by the known density of the SB3 as described (45).
OARSI histopathology scoring
Serial sections through the entire knee joint were scored according to the OARSI histopathology scoring system (47) by two blinded observers on the four quadrants of the knee: lateral femoral condyle (LFC), lateral tibial plateau (LTP), medial femoral condyle (MFC), and medial tibial plateau (MTP). Histologic scoring from 0-6 represent the OA severity, from 0 (healthy cartilage) to 6 (erosion of more than 75% of articular cartilage). Individual scores are averaged first for each observer, then across observers, and OA severity is shown as described for each graph. Scores were compared between male and female Mig-6over/over and control mice at both 12 and 18 months of age. All images were taken with a Leica DFC295 digital camera and a Leica DM1000 microscope.
Immunohistochemistry
Frontal paraffin sections of knees were used to for immunohistochemical analysis, with slides with ‘no primary antibody’ as control. All sections were deparaffinized and rehydrated as previously described (40,48). Subsequently, the sections were incubated in 3% H2O2 in methanol for 15 minutes to inhibit endogenous peroxidase activity. After rising with water, 5% goat or donkey serum in PBS was applied to reduce nonspecific background staining. Sections were incubated overnight at 4°C with primary antibodies against SOX9 (R&D Systems, AF3075), MMP13 (Protein Tech, Chicago, IL, USA, 18165-1-AP), lubricin (Abcam, ab28484) ) (primary concentration antibody for all three 1:100) and phospho-EGFR (phosphoTyr-1173; Cell Signaling Technology) (primary concentration antibody 1:50). After washing, sections were incubated with horseradish peroxidase (HRP)-conjugated donkey anti-goat or goat anti-rabbit secondary antibody (R&D system and Santa Cruz, secondary concentration antibody 1:200), before incubation with diaminobenzidine substrate as a chromogen (Dako, Canada). Finally, sections were counterstained with 0.5% methyl green (Sigma) and mounted. Cell density of articular cartilage chondrocytes from 6 and 11 weeks-old male mice was determined by counting all lacunae with evidence of nuclear staining in the lateral and medial femur/tibia using a centered region of interest measuring 200 μm wide and 70 μm deep from the articular surface by a blinded observer. For newborn (P0) animals the region of interest measured 200 μm wide and 100 μm deep from the proliferative zone.
Statistical analysis
All statistical analyses were performed using GraphPad Prism (v6.0). Differences between two groups were evaluated using Student's t-test, and Two-Way ANOVA was used to compare 4 groups followed by a Bonferroni multiple comparisons test. All n values represent the number of cartilage specific Mig-6 overexpressing mice and control littermates used in each group.