Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival.

Q-S Zhang,H Ichijo,T A Freeman,G J Eaton,C Diallo,M J Steinbeck,A Matsuzawa

doi:10.1038/cddis.2014.480

Abstract

Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species 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 tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 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. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy.

Highlights

Apoptosis signal-regulating kinase 1 (ASK1) is constitutively produced by all cells and exists in the cytoplasm as an inactive complex bound to inhibitory proteins thioredoxin, glutaredoxin or 14-3-3.9–12 In the presence of oxidative stress, disulfide bond formation occurs, accompanied by a conformational change of the inhibitory proteins and their dissociation from ASK1.9 This dissociation allows the self-dimerization, autophosphorylation and activation of ASK1.4,13 Activated ASK1 selectively phosphorylates c-Jun
We found that inhibition of ASK1 activation accelerated diaphyseal bone formation, increased the amount of trabecular bone and enhanced bone volume in an ectopic endochondral ossification model
We show that the absence (ASK1 KO mouse) or inhibition (ASK1 inhibitor, NQDI-1) of ASK1 enhances hypertrophic chondrocyte survival resulting in increased endochondral bone formation

Summary

Introduction

ASK1 is constitutively produced by all cells and exists in the cytoplasm as an inactive complex bound to inhibitory proteins thioredoxin, glutaredoxin or 14-3-3.9–12 In the presence of oxidative stress, disulfide bond formation occurs, accompanied by a conformational change of the inhibitory proteins and their dissociation from ASK1.9 This dissociation allows the self-dimerization, autophosphorylation and activation of ASK1.4,13 Activated ASK1 selectively phosphorylates c-Jun. Oxidative stress is known to have a role in bone formation, growth and remodeling of adult bone.[17,18,19,20] oxidative stress drives the process of hypertrophy and death of terminally differentiated chondrocytes in the growth plate, which is necessary for long bones to lengthen and form properly. Chondrocyte death is thought to be initiated through caspase-3 (CASP3), and a report by Hatai, et al 21 implicates ASK1 as an upstream activator of mitochondria-dependent. JNK and p38 MAP kinase pathways are activated by ASK1 and both have important roles in chondrogenesis, bone formation and turnover

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