SOST/Sclerostin Improves Posttraumatic Osteoarthritis and Inhibits MMP2/3 Expression After Injury

Abstract

Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ∼900,000 knee injuries in the United States, which account for ∼12% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post-injury would provide attractive alternatives to surgery. Sclerostin (Sost), a Wnt antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOST^TG ) and knockout (Sost^-/- ) mice. Here we report that SOST^TG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post-injury compared with wild-type (WT) controls and Sost^-/- . In addition, SOST^TG built ∼50% and ∼65% less osteophyte volume than WT and Sost^-/- , respectively. Quantification of metalloproteinase (MMP) activity showed that SOST^TG had ∼2-fold less MMP activation than WT or Sost^-/- , and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra-articular administration of recombinant Sost protein, immediately post-injury, also significantly decreased MMP activity levels relative to PBS-treated controls, and Sost activation in response to injury was TNFα and NF-κB dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Keywords: MMP; OSTEOARTHRITIS; OSTEOPHYTE; SCLEROSTIN; SOST; WNT SIGNALING.

Figure 1

Moderate PTOA phenotype in SOST^TG compared with WT and Sost^‐/‐. Histological staining of Safranin‐O and Fast Green on contralateral (left knee) at 16 weeks post‐injury (A). Cartilage integrity scoring using three distinct regions: femoral surface (I); anterior tibial surface (II); and the posterior tibial surface (III). Lower magnification (×5) of whole joint overview between C57Bl/6 (a, e), Sost^‐/‐ (i, m), and SOST^TG (q, u); higher‐resolution (×20) images are provided for all other images. Injured and contralateral joints were examined and scored in three distinct regions using a modified OARSI scoring method (OA severity: 0∼2, mild; 3∼4, moderate; and 5∼6, severe). Uninjured control (UIC) and contralateral (left knee) were utilized as controls. Scale bar = 0.2 mm. *p < 0.05, ***p < 0.001. Erosion to the growth plate is marked by asterisks (e, m). Black arrows indicated regions of erosion.

Figure 2

Sclerostin upregulates in the articular cartilage post‐injury. Sost immunostaining was conducted on contralateral WT (A) and SOST^TG (E and I) joints at 1 day after injury. Injured WT joints had elevated levels of Sost (B and C), while injured SOST^TG joints had elevated expression of both mouse (F and G) and human Sclerostin (J and K) 1 day after injury. No differences were observed in Sclerostin expression in the osteocytes of injured animals (D, H, and L). Real‐time quantitative PCR (qPCR) analysis of whole‐joint RNA of WTs at 1, 2, 3, and 4 days after injury (M). All images are at ×20 magnification. *p < 0.05, **p < 0.01.

Figure 3

Sost activation in the injured joint is TNFα and NF‐κB dependent. Time line for IA administration of NF‐κB inhibitor (BAY‐11‐7082) and TNFα inhibitor (neutralizing antibody) (A). Immunohistochemical staining of cartilage (Col2a; green), Sost (red), and nucleus (DAPI; blue) between vehicle (DMSO or PBS) and BAY‐11‐7082 (B) or TNFα antibody (C) treated injured joints.

Figure 4

SOST^TG joints are protected from excessive osteophyte formation, while Sost^‐/‐ joints are protected from subchondral bone loss in injured joints. μCT representation of mouse injured joints at 6 and 16 weeks post‐injury. Darker regions in the injured scans depict ectopic bone nodules (A). Osteophyte volume (gray area in A) was quantified at 6, 12, and 16 weeks post‐injury and compared between genotypes (B). Subchondral trabecular bone volume to total volume ratio was quantified and analyzed between injured and uninjured joints at 6, 12, and 16 weeks post‐injury. Scale bar = 1 mm. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

SOST^TG and WT injured joints treated with recombinant Sost (rmSost) protein have reduced levels of activated MMPs post‐injury. MMPSense was administered IV 5 hours post‐injury; rmSost was delivered IA post‐injury, and mean fluorescence intensity was measured 3 days post‐injury as depicted in A. SOST^TG‐ and rmSost‐treated injured joints displayed significantly less fluorescence than Sost^‐/‐ or WT control joints (B). Representative ex vivo images of WT uninjured (C) and injured WT (D), Sost^‐/‐ (E), and SOST^TG (F). Injured joints show reduced fluorescence in SOST^TG (F). Similarly, rmSost‐treated injured joints (H) show less fluorescence than PBS controls (G). **p < 0.01, ***p < 0.001.

Figure 6

Model of Sost action in injured joints.