JNK Activity Is Essential for Atf4 Expression and Late-Stage Osteoblast Differentiation

• DOI: 10.1359/jbmr.081107
• Publication Date: 2008-11-18T14:32:29Z
• Authors: Tetsuya Matsuguchi; Norika Chiba; Kenjiro Bandow; Kyoko Kakimoto; Akio Masuda; Tomokazu Ohnishi
• Keywords: 0301 basic medicine; Osteoblasts; Sialoglycoproteins; Osteocalcin; Bone Matrix; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Activating Transcription Factor 4; Phosphoric Monoester Hydrolases; Cell Line; Clone Cells; Isoenzymes; Mice, Inbred C57BL; Mice; 03 medical and health sciences; Calcification, Physiologic; Gene Expression Regulation; Sp7 Transcription Factor; Animals; Integrin-Binding Sialoprotein; Mitogen-Activated Protein Kinase 9; Protein Kinase Inhibitors

Abstract Osteoblasts differentiate from mesodermal progenitors and play a pivotal role in bone formation and mineralization. Several transcription factors including runt-related transcription factor 2 (RUNX2), Osterix (OSX), and activating transcription factor4 (ATF4) are known to be crucial for the process, whereas the upstream signal transduction controlling the osteoblast differentiation sequence is largely unknown. Here, we explored the role of c-jun N-terminal kinase (JNK) in osteoblast differentiation using in vitro differentiation models of primary osteoblasts and MC3T3-E1 cells with ascorbic acid/β-glycerophosphate treatment. Terminal osteoblast differentiation, represented by matrix mineralization, was significantly inhibited by the inactivation of JNK with its specific inhibitor and exogenous overexpression of MKP-M (MAP kinase phosphatase isolated from macrophages), which preferentially inactivates JNK. Conversely, enhanced mineral deposition was observed by inducible overexpression of p54JNK2, whereas it was not observed by the overexpression of p46JNK1 or p46JNK2, indicating a distinct enhancing role of p54JNK2 in osteoblast differentiation. Inactivation of JNK significantly inhibited late-stage molecular events of osteoblast differentiation, including gene expression of osteocalcin (Ocn) and bone sialoprotein (Bsp). In contrast, earlier differentiation events including alkaline phosphatase (ALP) activation and osteopontin (Opn) expression were not inhibited by JNK inactivation. Although the expression levels of two transcription factor genes, Runx2 and Osx, were not significantly affected by JNK inactivation, induction of Atf4 mRNA during osteoblast differentiation was significantly inhibited. Taken together, these data indicate that JNK activity is specifically required for the late-stage differentiation events of osteoblasts.