C-Jun N-terminal Kinase (JNK) Isoform 2 Augments Human Osteoblast Differentiation by Integrating Bone Morphogenetic Protein Signaling and Canonical Notch Signaling

Abstract

The c-Jun N-terminal kinases (JNK) are evolutionary conserved regulators of proliferation, differentiation, and cell death responses. In vivo studies have shown that Jnk1-/- and Jnk2-/- mice display varying degrees of osteopenia due to impaired bone formation. However, JNK1 and JNK2 isoform specific contribution to human osteoblast differentiation is lesser known. Here, we used small interfering RNA-mediated knockdown of JNK1 and JNK2 in bone-marrow derived human mesenchymal stem cells (hMSC) to evaluate their role in BMP-mediated osteoblast differentiation. Histochemical staining for alkaline phosphatase (ALP) expression and extracellular matrix mineralization using Alizarin red S showed that JNK2 activity is essential for hMSC osteoblastic differentiation, whereas JNK1 activity was dispensable. Gene expression analysis revealed a minimal effect on BMP-dependent RUNX2 and SP7 expression after JNK2 knock-down, but their levels were enhanced in JNK1 silenced cells. Since, BMP signaling requires canonical Notch signaling to promote human osteoblastogenesis, we evaluated JNK-isoform specific effects on Notch signaling components. The expression of Notch ligand JAG1 and downstream Notch target genes HES1 and NOTCH3 was reduced in response to BMP stimulation of JNK2 silenced cells, while the effects were modest after JNK1 knock-down. Consequently, the absolute requirement of JAG1 for BMP-mediated hMSC osteoblastogenesis was established as JAG1 silenced hMSC failed to enhance ALP expression and extracellular matrix mineralization. Finally, we generated JNK2-deficient immortalized hMSC using CRISPR-Cas9 genome editing and subjected these cells to BMP2 stimulation. Corresponding to the results with siRNA, JNK2 knock-out cells failed to produce extracellular matrix mineralization in response to BMP2. Collectively, these results establish a regulatory role of JNK2 in human osteoblast differentiation and suggest that it functions by integrating canonical BMP/SMAD signaling and Notch signaling in human cells.