Targeted Inhibition of Aggrecanases Prevents Articular Cartilage Degradation and Augments Bone Mass in the STR/Ort Mouse Model of Spontaneous Osteoarthritis

Abstract

Objective. Cartilage destruction in osteoarthritis (OA) is mediated mainly by matrix metalloproteinases (MMPs) and ADAMTS. The therapeutic candidature of targeting aggrecanases has not yet been defined in joints in which spontaneous OA arises from genetic susceptibility, as in the case of the STR/Ort mouse, without a traumatic or loadinduced etiology. In addition, we do not know the long-term effect of aggrecanase inhibition on bone. We undertook this study to assess the potential aggrecanase selectivity of a variant of tissue inhibitor of metalloproteinases 3 (TIMP-3), called [-1A]TIMP-3, on spontaneous OA development and bone formation in STR/Ort mice.Methods. Using the background of STR/Ort mice, which develop spontaneous OA, we generated transgenic mice that overexpress [-1A]TIMP-3, either ubiquitously or conditionally in chondrocytes. [-1A]TIMP-3 has an extra alanine at the N-terminus that selectively inhibits ADAMTS but not MMPs. We analyzed a range of OA-related measures in all mice at age 40 weeks.Results. Mice expressing high levels of [-1A]TIMP-3 were protected against development of OA, while those expressing low levels were not. Interestingly, we also found that high levels of [-1A]TIMP-3 transgene overexpression resulted in increased bone mass, particularly in females. This regulation of bone mass was at least partly direct, as adult mouse primary osteoblasts infected with [-1A]TIMP-3 in vitro showed elevated rates of mineralization.Conclusion. The results provide evidence that [-1A]TIMP-3-mediated inhibition of aggrecanases can protect against cartilage degradation in a naturally occurring mouse model of OA, and they highlight a novel role that aggrecanase inhibition may play in increased bone mass.

Low protein intake during reproduction compromises the recovery of lactation‐induced bone loss in female mouse dams without affecting skeletal muscles

“…To assess gene expression levels, total RNA was isolated from Obs at the end of mineralization (24 d) using TRiZOL reagent (Invitrogen, CA, USA), cleaned‐up using the RNeasy kit (Qiagen, UK) and cDNA was synthesized with the High Capacity cDNA Transcription kit (Applied Biosystems, UK). Expression levels of alkaline phosphatase ( Alp ), collagen type 1 ( Col1a1 ), Runx‐2, and Bglap mRNA were used as osteogenic markers 42 (Table S1). Quantitative polymerase chain reaction (qPCR) was performed on a RotorGene 6000 (Corbett Research) instrument with SYBR (Bioline, UK) and results were analyzed using beta‐actin ( Actb ) as a stable reference gene for osteoblasts 45 .…”

Targeting Dysregulation of Metalloproteinase Activity in Osteoarthritis

“…Addition of an extra alanine to the N-terminal of TIMP-3 (named [-1A]TIMP-3) or Thr 2 Gly mutation interferes with the inhibition of MMPs by TIMP-3, but inhibitory activity for ADAMTS-4 and 5, and ADAM17 is retained [30,123]. This modified activity is driven by conformational changes, in which the active site is tilted and the interaction of Phe 34 [124] and a naturally occurring OA model in the STR/ort mice [125]. The unusual property of the specific inhibitor of aggrecanases and ADAM17 might provide a clue for the generation of a new type of inhibitor for metalloproteinases.…”

Low protein intake compromises the recovery of lactation-induced bone loss in female mouse dams without affecting skeletal muscles

“…For trabecular bone analysis, VOI was selected using mineralised cartilage as a reference point. The tibial VOI analysed was 400 slices starting 20 levels distal to the reference point, while for cortical bone measurements, a VOI (100 slices) was selected 600 slices below the reference point, as previously described (42,43). Trabecular bone was automatically separated from cortical bone using a macro in CTAn.…”

“…The bone shafts were cut into small pieces using a scalpel. and adhering cells were removed by digestion with collagenase type I (Sigma, 1mg/ml in Hank's balanced salt solution, HBSS) for 45min in a shaking water bath at 37˚C, washed in PBS and cultured in alpha-MEM with Glutamax TM (Gibco, UK) and nucleosides, containing 10% heat-inactivated FBS and penicillin (100 IU/ml)/streptomycin (100 μg/ml) (Invitrogen) in a humidified 5% CO2 incubator at 37˚C, as previously described (42). Upon reaching semi-confluence, primary osteoblasts (Obs), grown out of the cleaned bone chips, were harvested using trypsin/EDTA (Gibco, UK) and seeded onto 6-well plates (10 5 cells/well) in osteogenic medium (50μg/mL L-ascorbic acid-2-phosphate and 5mM β-glycerophosphate) (Sigma, UK) for 24 days.…”

“…Mineralisation capacity was assessed by Alizarin Red S (ARS) (Sigma, UK) staining. Bone nodule surface area was calculated using ImageJ (NIH), as previously described (42,44) .…”