Both JNK1 and JNK2 Are Indispensable for Sensitized Extracellular Matrix Mineralization in IKKβ-Deficient Osteoblasts.

Qianyun Hao,Zhuangzhuang Liu,Lichen Zhang,Li Zuo,Liaoxun Lu

doi:10.3389/fendo.2020.00013

Abstract

Extracellular matrix mineralization is critical for osteogenesis, and its dysregulation could result in osteoporosis and vascular calcification. IKK/NF-κB activation inhibits differentiation of osteoblasts, and reduces extracellular matrix mineralization, however the underlying mechanisms are poorly understood. In this study, we used CRISPR/Cas9 system to permanently inactivate IKKβ in preosteoblast cells and confirmed that such cells displayed dramatic increase in extracellular matrix mineralization associated with JNK phosphorylation. Such observation was also found in our study using IKKβ-deficient primary murine osteoblasts. Interestingly, we found that in Ikbkb−/− Mapk8−/− or Ikbkb−/− Mapk9−/− double knockout cells, the enhanced mineralization caused by IKKβ deficiency was completely abolished, and deletion of either Mapk8 or Mapk9 was sufficient to dampen c-Jun phosphorylation. In further experiments, we discovered that absence of JNK1 or JNK2 on IKKβ-deficient background resulted in highly conserved transcriptomic alteration in response to osteogenic induction. Therefore, identification of the indispensable roles of JNK1 and JNK2 in activating c-Jun and promoting osteoblast differentiation on IKKβ-deficient background provided novel insights into restoring homeostasis in extracellular matrix mineralization.

Highlights

Bone formation requiring extracellular matrix mineralization is an active, highly regulated process primarily mediated by osteoblasts [1]
Since MC3T3–E1 cells and primary osteoblasts can be induced toward differentiation and matrix mineralization by osteogenic medium, we explored intracellular signal in response to βGP and ascorbic acid (AA) stimulation
We found that Ikbkb targeting by CRISPR/Cas9 resulted in significant increase of JNK phosphorylation as measured by mean fluorescence intensity (MFI) following 30 min of stimulation by β-GP and AA when compared to empty plasmid transfected cells (Figures 3E,F)

Summary

Introduction

Bone formation requiring extracellular matrix mineralization is an active, highly regulated process primarily mediated by osteoblasts [1]. Compelling evidences show that NF-κB regulates bone metabolism for its roles in facilitating osteoclasts differentiation, mechanisms underlying NF-κB in osteoblast differentiation and matrix mineralization are little known [4,5,6,7]. IKKβ is the major catalytic subunit of the IKK complex and is required for the activation of NF-κB by inflammatory mediators in the canonical or classical activation pathway [8]. It is known that inhibition of IKK/NF-κB promotes mineralization in osteoblasts [9], but the underlying mechanisms are poorly understood. There remain contradictory reports on some agents that can induce osteoblast differentiation and mineralization

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Conclusion