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. 2019 Sep;34(9):1676-1689.
doi: 10.1002/jbmr.3737. Epub 2019 Jun 12.

Chondrocyte-Specific RUNX2 Overexpression Accelerates Post-traumatic Osteoarthritis Progression in Adult Mice

Sarah E Catheline  1   2 , Donna Hoak  1   3 , Martin Chang  1   3 , John P Ketz  1   3 , Matthew J Hilton  4 , Michael J Zuscik  1   3   5   6 , Jennifer H Jonason  1   3
Affiliations

Chondrocyte-Specific RUNX2 Overexpression Accelerates Post-traumatic Osteoarthritis Progression in Adult Mice

Sarah E Catheline et al. J Bone Miner Res. 2019 Sep.

Abstract

RUNX2 is a transcription factor critical for chondrocyte maturation and normal endochondral bone formation. It promotes the expression of factors catabolic to the cartilage extracellular matrix and is upregulated in human osteoarthritic cartilage and in murine articular cartilage following joint injury. To date, in vivo studies of RUNX2 overexpression in cartilage have been limited to forced expression in osteochondroprogenitor cells preventing investigation into the effects of chondrocyte-specific RUNX2 overexpression in postnatal articular cartilage. Here, we used the Rosa26Runx2 allele in combination with the inducible Col2a1CreERT2 transgene or the inducible AcanCreERT2 knock-in allele to achieve chondrocyte-specific RUNX2 overexpression (OE) during embryonic development or in the articular cartilage of adult mice, respectively. RUNX2 OE was induced at embryonic day 13.5 (E13.5) for all developmental studies. Histology and in situ hybridization analyses suggest an early onset of chondrocyte hypertrophy and accelerated terminal maturation in the limbs of the RUNX2 OE embryos compared to control embryos. For all postnatal studies, RUNX2 OE was induced at 2 months of age. Surprisingly, no histopathological signs of cartilage degeneration were observed even 6 months following induction of RUNX2 OE. Using the meniscal/ligamentous injury (MLI), a surgical model of knee joint destabilization and meniscal injury, however, we found that RUNX2 OE accelerates the progression of cartilage degeneration following joint trauma. One month following MLI, the numbers of MMP13-positive and TUNEL-positive chondrocytes were significantly greater in the articular cartilage of the RUNX2 OE joints compared to control joints and 2 months following MLI, histomorphometry and Osteoarthritis Research Society International (OARSI) scoring revealed decreased cartilage area in the RUNX2 OE joints. Collectively, these results suggest that although RUNX2 overexpression alone may not be sufficient to initiate the OA degenerative process, it may predetermine the rate of OA onset and/or progression following traumatic joint injury. © 2019 American Society for Bone and Mineral Research.

Keywords: ANIMAL MODELS; CELL/TISSUE SIGNALING; CHONDROCYTE AND CARTILAGE BIOLOGY; DISEASES AND DISORDERS OF/RELATED TO BONE; GENETIC ANIMAL MODELS; HYPERTROPHY; OSTEOARTHRITIS; POST-TRAUMATIC OSTEOARTHRITIS; RUNX2; TRANSCRIPTION FACTORS.

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Figures

Fig. 1.
Fig. 1.. Chondrocyte-specific RUNX2 overexpression during development leads to chondrodysplasia.
(A) Alcian blue/Alizarin red skeletal staining of E18.5 R26Runx2/+ (Control), Col2a1CreERT2/+; R26Runx2/+ (C2T2; R26Runx2/+), and Col2a1CreERT2/+; R26Runx2/Runx2 (C2T2; R26Runx2/Runx2) embryos. (B) Femur (left), tibia (middle), and humerus (right) lengths from E18.5 Control, C2T2; R26Runx2/+, and C2T2; R26Runx2/Runx2 embryos (n = 3 to 5 for all groups). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test. (C) Alcian blue Hematoxylin/Orange G staining of humerus growth plate sections from E18.5 Control, C2T2; R26Runx2/+, and C2T2; R26Runx2/Runx2 embryos. Top panels are 5X images of the proximal humerus; middle panels are high magnification images (20X) of the proliferating and pre-hypertrophic zones (yellow boxes); bottom panels are high magnification images (20X) of the hypertrophic zone (orange boxes). (D) In situ hybridization for Col10a1 (top panels) and Mmp13 (bottom panels) on E18.5 Control, C2T2; R26Runx2/+, and C2T2; R26Runx2/Runx2 proximal humerus sections. (E) TUNEL staining of proximal humerus sections from E18.5 Control, C2T2; R26Runx2/+, and C2T2; R26Runx2/Runx2 embryos (5X images). (F) Quantification of TUNEL+ cell number as percentage of total cell number within the growth plate of the proximal humeri of E18.5 Control, C2T2; R26Runx2/+, and C2T2; R26Runx2/Runx2 embryos (n = 3 for all groups). *p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test.
Fig. 2.
Fig. 2.. Knee joint injury leads to increased RUNX2 expression in human articular cartilage.
Representative results of RUNX2 immunohistochemistry on a tissue microarray of normal human cartilage or cartilage from patients undergoing arthroscopic surgery 4 weeks following meniscal injury (Post-Injury). Left panels within each group are 10X images; right panels are 20X images of the corresponding boxed regions.
Fig. 3.
Fig. 3.. Chondrocyte-specific RUNX2 overexpression is insufficient to induce phenotypic changes in the articular cartilage.
(A, D) Safranin O/Fast green staining of knee joint sections from male R26Runx2/+ (Control) and AcanCreERT2/+; R26Runx2/+ (RUNX2 OE) mice injected with tamoxifen at 2 months of age daily for 5 consecutive days and harvested 48 hours following the last tamoxifen injection at 9 weeks of age (A) or at 8 months of age (D). Top panels are 5X images of the knee joint; bottom panels are high magnification images (20X) of the boxed regions. (B, E) RUNX2 immunohistochemistry on knee joint sections from Control and RUNX2 OE mice at 9 weeks of age (B) and 8 months of age (E). Left panels are 20X images of the articular cartilage; right panels are high magnification images (40X) of the boxed regions. (C) Quantitative RT-PCR from tibial articular cartilage of Control and RUNX2 GOF mice harvested at 3 months of age following tamoxifen injections at 2 months of age (Control, n = 3, RUNX2 OE, n = 3). (F) Quantification of RUNX2+ cartilage area as percentage of total cartilage area and RUNX2+ cell number as percentage of total cell number from Control and RUNX2 OE mice (Control, n = 3, RUNX2 OE, n = 3). *p < 0.05, Student’s t-test.
Fig. 4.
Fig. 4.. Postnatal RUNX2 overexpression accelerates articular cartilage degeneration following meniscal-ligamentous injury (MLI) in male mice.
(A) Safranin O/Fast green staining of knee joint sections from male R26Runx2/+ (Control) and AcanCreERT2/+; R26Runx2/+ (RUNX2 OE) mice injected with tamoxifen at 2 months of age and subjected to sham or MLI at 2.5 months of age. Joints were harvested 2 months following injury. Top panels are 5X images of the knee joint; bottom panels are high magnification images (20X) of the boxed regions. (B) Modified OARSI scoring of Control and RUNX2 OE slides from the knee joint receiving MLI. (C, D) Quantitative histomorphometric analyses of total tibial cartilage area (C, left panel), total tibial SafO+ area (C, right panel), unmineralized and mineralized tibial cartilage areas (D, left panels), and unmineralized and mineralized tibial SafO+ areas (D, right panels) where MLI cartilage was normalized to corresponding sham cartilage in knee joint sections from Control (n = 6) and RUNX2 OE (n = 7) mice. *p < 0.05, **p < 0.01, Student’s t-test.
Fig. 5.
Fig. 5.. Postnatal RUNX2 overexpression induces MMP13 expression in unmineralized articular chondrocytes prior to accelerating articular cartilage degeneration following MLI.
(A) Safranin O/Fast green staining of knee joint sections from R26Runx2/+ (Control) and AcanCreERT2/+; R26Runx2/+ (RUNX2 OE) mice injected with tamoxifen at 2 months of age and subjected to sham or MLI at 2.5 months of age. Joints were harvested one month following injury. Top panels are 5X images of the knee joint; bottom panels are high magnification images (20X) of the boxed regions. (B) Cartilage degeneration in Control (n = 6) and RUNX2 OE (n = 6) mice was evaluated by modified OARSI scoring (top graph) and quantitative histomorphometric analysis (bottom graph, data normalized to contralateral sham control sample). (C) MMP13 (left panels) and COL10A1 (right panels) immunohistochemistry of knee joint sections from Control or RUNX2 OE mice subjected to sham or MLI at 2.5 months of age and harvested 1 month following injury (20X images). (D) Quantification of MMP13+ cell number as percentage of total cell number from Control and RUNX2 OE mice (MLI samples only, n = 4 for both groups). (E) Quantification of COL10A1+ cartilage area as percentage of total cartilage area from Control and RUNX2 OE mice (MLI samples only, n = 4 for both groups). *p < 0.05, Student’s t-test.
Fig. 6.
Fig. 6.. Postnatal RUNX2 overexpression results in enhanced articular chondrocyte apoptosis following meniscal-ligamentous injury (MLI).
(A) TUNEL staining of knee joint sections from R26Runx2/+ (Control) and AcanCreERT2/+; R26Runx2/+ (RUNX2 OE) mice subjected to MLI at 2.5 months of age and harvested 1 month following injury (10X images). (B) Quantification of TUNEL+ cell number as percentage of total cell number from the tibial (left) or femoral (right) articular cartilage of Control or RUNX2 OE mice (MLI samples only, n = 4 for both groups). *p < 0.05, **p < 0.01, Student’s t-test.
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