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.