title: Accumulation of p100, a precursor of NF-κB2, enhances osteoblastic differentiation in vitro and bone formation in vivo in aly/aly mice.
abstract: We previously reported that alymphoplasia (aly/aly) mice, which have a natural loss-of-function mutation in the Nik gene, which encodes a kinase essential for the processing of p100 to p52 in the alternative nuclear factor-κB (NF-κB) pathway, show mild osteopetrosis with an increase in several parameters of bone formation: bone formation rate, mineral apposition rate, and osteoblast number. We therefore investigated the molecular mechanisms triggered by the alternative NF-κB pathway in the regulation of osteoblast differentiation using primary osteoblasts (POB) prepared from aly/aly mice. Alkaline phosphatase (ALP) activity and mineralization induced by the presence of β-glycerophosphate and ascorbic acid were enhanced in POB from aly/aly compared with wild-type (WT) mice. Furthermore, osteoblastic differentiation induced by bone morphogenetic protein 2 (BMP2), as shown by ALP activity, mRNA expression of osteocalcin, Id1, Osterix and Runx2, and Sma- and Mad-related protein (Smad)1/5/8 phosphorylation, was also enhanced in POB from aly/aly mice. The ectopic bone formation in vivo that was induced by BMP2 was enhanced in aly/aly mice compared with controls. Transfection of a mutant form of p100, p100ΔGRR, which cannot be processed to p52, stimulated ALP activity and Smad phosphorylation. In contrast to p100ΔGRR, overexpression of p52 inhibited these events. Both BMP2-induced ALP activity and Smad phosphorylation were reduced in POB from p100-deficient mice, which carry a homozygous deletion of the COOH-terminal ankyrin repeats of p100 but still express functional p52 protein. p52 and p100ΔGRR interacted with a BMP receptor, ALK2, in overexpressed COS7 cells and changed the ALK2 protein levels in opposite directions: p52 reduced ALK2 and p100 increased it. Thus, the alternative the NF-κB pathway via the processing of p52 from p100 negatively regulates osteoblastic differentiation and bone formation by modifying BMP activity.
authors.affiliation: Division of Molecular Signaling and Biochemistry, Department of Bioscience, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Seo
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Fukushima
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Maruyama
authors.forename: Kayoko Nakao
authors.initials: KN
authors.lastname: Kuroishi
authors.forename: Kenji
authors.initials: K
authors.lastname: Osawa
authors.forename: Kenichi
authors.initials: K
authors.lastname: Nagano
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Aoki
authors.forename: Falk
authors.initials: F
authors.lastname: Weih
authors.forename: Takahiro
authors.initials: T
authors.lastname: Doi
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ohya
authors.forename: Takenobu
authors.initials: T
authors.lastname: Katagiri
authors.forename: Ryuji
authors.initials: R
authors.lastname: Hosokawa
authors.forename: Eijiro
authors.initials: E
authors.lastname: Jimi
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000502:physiology; D052004:Bone Morphogenetic Protein Receptors / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D052003:NF-kappa B p52 Subunit / Q000378:metabolism / Q000502:physiology*; D010006:Osteoblasts / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D010012:Osteogenesis; D010766:Phosphorylation; D061251:Primary Cell Culture; D019736:Prostheses and Implants; D011485:Protein Binding; D012886:Skull / Q000166:cytology; D051785:Smad Proteins / Q000378:metabolism
chemical_list: C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D052003:NF-kappa B p52 Subunit; D051785:Smad Proteins; D052004:Bone Morphogenetic Protein Receptors; D000469:Alkaline Phosphatase