title: Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation.
abstract: Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.
authors.affiliation: National Institute of Radiological Sciences, Chiba, Japan. nifuji-a@tsurumi-u.ac.jp
authors.forename: Akira
authors.initials: A
authors.lastname: Nifuji
authors.forename: Hisashi
authors.initials: H
authors.lastname: Ideno
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Ohyama
authors.forename: Rieko
authors.initials: R
authors.lastname: Takanabe
authors.forename: Ryoko
authors.initials: R
authors.lastname: Araki
authors.forename: Masumi
authors.initials: M
authors.lastname: Abe
authors.forename: Masaki
authors.initials: M
authors.lastname: Noda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shibuya
mesh_terms: D000818:Animals; D001846:Bone Development / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000235:genetics / Q000502:physiology; D004622:Embryo, Mammalian; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D051379:Mice; D008813:Mice, Inbred ICR; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism* / Q000502:physiology*; D047495:PPAR gamma / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D017346:Protein Serine-Threonine Kinases; D055550:Protein Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D014162:Transfection
chemical_list: D047495:PPAR gamma; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C408347:Nlk protein, mouse; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases