Calmodulin-dependent protein kinase kinase 2 as a novel therapeutic target against osteoarthritis

Abstract

Purpose: Osteoarthritis (OA) is a disabling disease that affects more than 250 million people worldwide causing tremendous individual and socioeconomic burden. OA is a complex disease encompassing multiple molecular/biological mechanisms that result in progressive cartilage loss, subchondral bone remodeling, joint tissue inflammation, and osteophytes formation. OA is often diagnosed at a late stage and treatment is confined to management of symptoms due to a lack of clinically available therapies to stop or delay the disease progression. Thus, there is a critical need to identify novel therapeutic targets that could prevent and/or delay multiple facets of OA pathogenesis. Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) plays an important role in bone remodeling and macrophage-mediated inflammation. Hence, we hypothesized that genetic ablation or pharmacological inhibition of CaMKK2 will suppress inflammation and subchondral bone remodeling associated with OA. Methods: Male wild-type (WT) and/or Camkk2-/- mice (both C57BL6; n = 5 per group) underwent unilateral destabilization of medical meniscus (DMM) or sham surgery. WT mice undergoing sham and DMM surgeries were administered STO-609 (CaMKK2 inhibitor; 10 μM/kg body weight) or saline solution tri-weekly by i.p injection for 8 weeks. Damage to the joint tissues was evaluated using microcomputed tomography (microCT) and histology. The impact of CaMKK2 deficiency in cartilage pathophysiology was assessed in vitro using primary WT and Camkk2-/- murine articular chondrocytes (MACs) stimulated by interleukin-1β (IL-1β; 10 ng/ml). Quantitative real-time PCR was used to assess the expression of markers of cartilage matrix and turnover as well as inflammation. Results: Three dimensional microCT analysis of tibial subchondral bone showed a significant increase of tissue volume (TV; by 40%) and bone volume (BV/TV; 18%) in saline-treated DMM mice relative to sham-operated control mice. In addition, the thickness of subchondral bone plate (SPB) was 32% higher in DMM mice compared to sham operated cohorts. These results demonstrated an enhanced subchondral bone sclerosis after DMM surgery. Interestingly, such increases in TV and BV/TV were significantly diminished in CaMKK2 inhibitor-treated WT and Camkk2-/- mice, indicating protection from subchondral bone sclerosis induced by DMM. Moreover, SBP thickness was significantly lower in STO-609 treated WT and Camkk2-/- mice, compared to saline treated WT cohorts. Histological analyses revealed lower OARSI score and prevention of cartilage breakdown following DMM surgery in STO-609 treated WT and Camkk2-/- mice. Moreover, inhibition or loss of CaMKK2 also resulted in significantly reduced macrophage infiltration and inflammation of the synovium following DMM. Further, data obtained from isolated MACs reveal that the genetic ablation of CaMKK2 was able to counteract the effects of IL-1β by affecting the expression levels of inflammatory (iNOS, COX2, TNF-α, and IL6) cytokines and markers of cartilage catabolism (MMP2, MMP-13, and ADAMTS5). In addition, Camkk2-/- MACs expressed significantly higher levels of extracellular matrix proteins such as Col2a1, COMP and glycosaminoglycans (GAGs) in response to IL-1β, compared to WT. Whereas an increase in the activation of ERK was observed, the activity of p38 MAPK was significantly inhibited in theCamkk2-/- MACs. Inhibition of ERK with a specific inhibitor resulted in an increase of inflammatory and catabolism markers in Camkk2-/- MACs, indicating that the protective effects of CaMKK2 loss on OA pathogenesis is potentially through its regulation of MAPK activity. Conclusions: In conclusion, targeting CaMKK2 offers an overall protection of joint tissues in trauma-induced OA, owing to a suppression of inflammation and catabolism through a regulation of the ERK pathway as well as an increase in cartilage matrix production. Our study provides novel information on the role of CaMKK2 in OA pathogenesis and opens new avenues to design innovative therapeutic strategies against OA.