Apoptosis Signal-Regulating Kinase 1 Modulates Endochondral Bone Formation and Osteoarthritis Progression
Abstract
Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK) kinase kinase that is activated by reactive oxygen species (ROS, chemically volatile molecules containing oxygen) and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including: cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissue development has not been investigated. As ROS, the main activator of ASK1, are increased in aging, injury, and inflammation, this work serves to show that ROS-induced ASK1 activation in chondrocytes results in chondrocyte maturation and death and cartilage degeneration. We studied the activation of ASK1 in endochondral bone formation using wildtype (WT) and ASK1 knockout (KO) mice. We examined long bone length, growth plate morphology, and bone thickness. Using mouse embryonic fibroblasts (MEF) derived from these mice and chondrogenically differentiated, western blot analysis allowed the study of protein expression and signaling levels. Finally, using a subdermal model of ectopic ossification, we studied endochondral bone formation outside of the context of embryonic development, and applied therapeutic inhibition of ASK1 in WT animals using NQDI-1, a small molecule inhibitor of ASK1. To test our hypothesis that increased inflammation and ROS present in OA cartilage leads to ASK1 signaling, we used human OA tissue from total knee arthroplasty (TKA) for immunohistochemical analysis. We also used WT and ASK1 KO MEFs to examine the underlying mechanism of ASK1 MAPK signaling in chondrocyte hypertrophy and maturation in response to stress. Growth plates from WT and KO mice were useful as a model for chondrocyte hypertrophy and death that occurs during OA progression. We also exploited the use of two surgeries to induce either severe or moderate OA in mice: partial meniscectomy and cartilage injury, respectively. Finally, we examined aged mouse limbs to study natural cartilage degradation over time between WT and KO mice. We observed accelerated long bone mineralization in KO mice, concurrent with a longer hypertrophic zone in the growth plate and an increase in trabecular formation. Under normal culture conditions, KO MEFs mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared to WT. However, when cultured with stress activators, H2O 2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways, and a reduction in markers of terminal differentiation. Importantly, ectopic ossification in either KO or NQDI-1 treated animals recapitulated the observed increased endochondral bone formation. In OA articular cartilage, we observed increased expression and signaling of ASK1 in the deep zone of human TKA tissue. Western analysis demonstrated that WT MEFs had an increased expression of ASK1 signaling and chondrogenic differentiation markers in response to stress, but each were abrogated in KO MEFs or after ASK1 inhibition. Both histological and µCT parameters showed a protection against OA induction in KO mice, and immunohistochemistry revealed a decrease in articular chondrocyte maturation. Our data leads to the conclusion that ASK1 serves as a negative regulator of endochondral bone formation by sensitizing the hypertrophic chondrocytes to apoptosis in response to stress. It demonstrates that inhibition of ASK1 may have clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy. We propose that the ASK1 MAP kinase signaling cascade is an important regulator of chondrocyte differentiation and is increased during OA, leading to both catabolic and apoptotic pathways in articular chondrocytes and accelerating OA progression. Further inhibitors of this pathway would be useful for the prevention of chondrocyte maturation in the treatment of OA.
Subject Area
Cellular biology|Histology|Developmental biology
Recommended Citation
Eaton, Gregory J., "Apoptosis Signal-Regulating Kinase 1 Modulates Endochondral Bone Formation and Osteoarthritis Progression" (2015). ProQuest ETD Collection - Thomas Jefferson University. AAI3705071.
https://jdc.jefferson.edu/dissertations/AAI3705071
