Chondrocyte-specific RUNX2 overexpression accelerates cartilage degeneration following traumatic injury

Abstract

Purpose: Osteoarthritis (OA) is a debilitating joint disease causing irreversible loss of cartilage within the synovial joints. Studies show roughly 50% of patients with an ACL or meniscus tear will eventually develop knee OA. Despite the high prevalence of OA, exact molecular mechanisms underlying onset are unclear. RUNX2 is a transcription factor that promotes chondrocyte hypertrophy and expression of factors catabolic to the cartilage extracellular matrix (ECM). It is also known to regulate apoptosis signaling in osteoblasts. Importantly, enhanced RUNX2 expression is seen in early stages of human OA and in murine models of injury-induced OA. Here, we investigate whether chondrocyte-specific RUNX2 overexpression can accelerate OA progression following traumatic joint injury. Methods: At 2 months of age, male and female Acan-Cre+/-ERT2; ROSA-Runx2f/+ (RUNX2 GOF) and littermate Cre-negative control mice were given tamoxifen at a dosage of 100 μg/g body weight daily for 5 days (N = 6 per gender and genotype). At 10 weeks of age, mice were subjected to meniscal-ligamentous injury (MLI) in the right hindlimb, and a sham injury in the contralateral limb. Hindlimbs were harvested for histological analysis 4 and 8 weeks post MLI. Hindlimbs were fixed in 10% neutral buffered formalin for 3 days, decalcified in 14% EDTA for 7 days, and embedded into paraffin. Sagittal sections were cut from the medial knee compartment and stained with Safranin-O/Fast Green. On three sections from distinct levels of each sample, manual histomorphometry and modified OARSI scoring were performed (for OARSI scoring, scores from 4 graders were averaged, p < 0.05, N = 6, 2-way ANOVA). Histomorphometry values represent mean cartilage area ± SEM normalized to contralateral sham values (p < 0.05, n = 4, unpaired t-test). MMP13 immunohistochemistry (IHC) was performed using Abcam MMP13 antibody ab75606, and TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche). Preliminary in vitro studies used primary sternal chondrocytes from ROSA-Runx2f/f mice infected with adenovirus encoding GFP or Cre to induce RUNX2 GOF; cells were treated with 1 ng/ml IL-1β for 24 hours to simulate the inflammatory environment within an injured joint and harvested for qPCR and Western blotting. Results: Histology reveals male RUNX2 GOF mice have enhanced cartilage damage and Saf-O staining loss compared to controls; these findings were confirmed by significantly increased OARSI scores in male RUNX2 GOF mice 2 months post MLI (Figure 1A, 1B). Female RUNX2 GOF mice display a modest increase in damage histologically, but did not exhibit a significant change in OARSI score. RUNX2 GOF contralateral sham joints showed no changes relative to controls for either gender. Preliminary histomorphometry data from both genders of mice reveals significant decreases in total and unmineralized femoral cartilage area in RUNX2 GOF mice 2 months post MLI (Figure 1C). RUNX2 GOF mice also exhibit increased TUNEL-positive staining and MMP13 expression 1 month post MLI in the articular cartilage (Figure 1D). In vitro, preliminary data suggests that RUNX2 GOF chondrocytes induce enhanced expression of pro-apoptotic genes Bax and Bid relative to control cells following IL-1β treatment. Conclusions: Our results suggest chondrocyte-specific RUNX2 overexpression can accelerate OA development after traumatic injury. Upregulation of MMP13, a direct downstream target of RUNX2, implies cartilage ECM catabolism after injury is enhanced with RUNX2 GOF. Both the TUNEL staining and our preliminary in vitro data indicate RUNX2 may be exerting its effects in part via positive regulation of apoptosis. Our data highlight the contribution of genetic variability to the development of this disease and suggest that genetic alterations affecting RUNX2 expression levels may, in part, predetermine the rate of OA onset following traumatic injury.