title: JNK activity is essential for Atf4 expression and late-stage osteoblast differentiation.
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/beta-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 p54(JNK2), whereas it was not observed by the overexpression of p46(JNK1) or p46(JNK2), indicating a distinct enhancing role of p54(JNK2) 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.
authors.affiliation: Department of Oral Biochemistry, Field of Developmental Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. tmatsugu@denta.hal.kagoshima-u.ac.jp
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Matsuguchi
authors.forename: Norika
authors.initials: N
authors.lastname: Chiba
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Bandow
authors.forename: Kyoko
authors.initials: K
authors.lastname: Kakimoto
authors.forename: Akio
authors.initials: A
authors.lastname: Masuda
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Ohnishi
mesh_terms: D051701:Activating Transcription Factor 4 / Q000378:metabolism*; D000818:Animals; D001857:Bone Matrix / Q000187:drug effects / Q000201:enzymology; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D058574:Integrin-Binding Sialoprotein; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D048056:Mitogen-Activated Protein Kinase 9 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism; D000076182:Sp7 Transcription Factor; D013379:Substrate Specificity / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C081452:Atf4 protein, mouse; D050659:Core Binding Factor Alpha 1 Subunit; C546872:Ibsp protein, mouse; D058574:Integrin-Binding Sialoprotein; D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; C493716:Runx2 protein, mouse; D012795:Sialoglycoproteins; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors; D015675:Osteocalcin; D051701:Activating Transcription Factor 4; D048056:Mitogen-Activated Protein Kinase 9; D010744:Phosphoric Monoester Hydrolases