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. 2012 Mar;26(3):414-22.
doi: 10.1210/me.2011-1241. Epub 2012 Jan 26.

Accumulation of p100, a precursor of NF-κB2, enhances osteoblastic differentiation in vitro and bone formation in vivo in aly/aly mice

Yoshinori Seo  1 , Hidefumi Fukushima, Toshimasa Maruyama, Kayoko Nakao Kuroishi, Kenji Osawa, Kenichi Nagano, Kazuhiro Aoki, Falk Weih, Takahiro Doi, Min Zhang, Keiichi Ohya, Takenobu Katagiri, Ryuji Hosokawa, Eijiro Jimi
Affiliations

Accumulation of p100, a precursor of NF-κB2, enhances osteoblastic differentiation in vitro and bone formation in vivo in aly/aly mice

Yoshinori Seo et al. Mol Endocrinol. 2012 Mar.

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.

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Figures

Fig. 1.
Fig. 1.
The alternative NF-κB signaling pathway regulates osteoblast differentiation in vitro. POB from calvariae of 1-d-old WT or aly/aly mice were cultured in the presence (closed column) or absence (open column) of β-glycerophosphate (β-GP, 10 mm) and ascorbic acid (AA, 50 μg/ml) for 7 d. A, Cells were stained for ALP activity. B, Cells were fixed with an acetone-ethanol mixture and incubated with a substrate solution. ALP activity was then determined. The data are means ± sd (n = 3), *, P < 0.01. C, von Kossa staining of calvaria cultures in the presence (closed column) or absence (open column) of β-glycerophosphate and ascorbic acid for 14 d. D, The stained area was measured. The data are means ± sd (n = 3), *, P < 0.01.
Fig. 2.
Fig. 2.
BMP2-induced osteoblast differentiation is enhanced in POB from aly/aly mice. A, POB from calvariae of 1-d-old WT (open column) or aly/aly mice (closed column) were treated with BMP2 (0, 50, 100 ng/ml) for 72 h. The cells were fixed with an acetone-ethanol mixture and incubated with a substrate solution. ALP activity was then determined. The data are means ± sd (n = 3), *, P < 0.01. B, POB were treated with (closed column) or without (open column) BMP2 (100 ng/ml) for 72 h. Total RNA was isolated and the expression level of osteocalcin relative to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was measured by quantitative real-time PCR analysis. The data are means ± sd (n = 3), *, P < 0.01. C, POB from calvariae of 1-d-old WT (open column) or aly/aly (closed column) mice were treated with or without BMP2 (100 ng/ml) for 24 h. Total RNA was isolated, and the expression levels of Id1, Osterix, or Runx2 relative to GAPDH were measured by quantitative real-time PCR analysis. The data are means ± sd (n = 3), *, P < 0.01.
Fig. 3.
Fig. 3.
BMP2/Smad signaling is enhanced in POB from aly/aly mice. A, POB from calvariae of 1-d-old WT or aly/aly mice were cultured in the presence of BMP2 (100 ng/ml) for the indicated times. Total cell lysates were immunoblotted with antiphosphorylated Smad1/5/8, Smad1, phosphorylated ERK, ERK, phosphorylated p38, and p38 antibodies. Anti-β-actin was used as a loading control. B, Total RNA was prepared and then the expression levels of R-Smad (Smad1 and Smad5), Co-Smad (Smad4), I-Smad (Smad6 and Smad7), Smurf1, BMP receptors (BMPR1A, BMPR1B, BMPR2, ALK1, and ALK2), and GAPDH mRNA were analyzed using RT-PCR. C, POB were treated with or without BMP2 (100 ng/ml) for 30 min. Total cell lysates were immunoblotted with anti-NF-κB2 antibodies. Anti-β-actin was used as a loading control. D, POB from aly/aly mice were transfected with or without FLAG-tagged NIK for 24 h. The cells were further cultured for 72 h in the presence of BMP2 (100 ng/ml). ALP activity was measured. The data are means ± sd (n = 3), *, P < 0.01. After transfection, cells were treated with or without BMP2 (100 ng/ml) for 1 h. Smad 1/5/8 phosphorylation was determined by immunoblotting.
Fig. 4.
Fig. 4.
BMP2-induced ectopic bone formation is enhanced in aly/aly mice in vivo. BMP2 (4 μg) was implanted sc to induce ectopic bone formation in WT or aly/aly mice. A, After 4 wk, implants were removed and examined by soft x-ray analysis. Bar, 5 mm. B, μCT reconstruction images of ectopic bone in WT or aly/aly mice. Bar, 1 mm. C, BMC and BMD of the ectopic bone were measured by dual-energy x-ray absorptiometry. *, P < 0.05; **, P < 0.01. D, Sections of implants at 4 wk were stained with H&E. Bar, 25 μm.
Fig. 5.
Fig. 5.
Accumulation of p100 enhanced the Smad phosphorylation and ALP activity induced by the constitutively activated BMP receptor. A, ALP activity was induced by overexpression of V5-tagged ALK2(Q207D) with FLAG-tagged Smad1 in the presence or absence of either p100ΔGRR or p52 in POB from calvariae of 1-d-old Nfkb2−/− mice. The data are presented as the mean ± sd (n = 3). *, P < 0.01. B, Phosphorylation of Smad1/5/8 was induced by overexpression of V5-tagged ALK2(Q207D) with FLAG-tagged Smad1 in the presence or absence of either p100ΔGRR or p52 in Nfkb2−/− POB. C, POB from calvariae of 1-d-old WT (open column) or p100−/− mice (closed column) were treated with BMP2 (0, 50, 100 ng/ml) for 72 h. The cells were fixed with an acetone-ethanol mixture and incubated with a substrate solution. ALP activity was then determined. The data are means ± sd (n = 3), *, P < 0.01. D, POB from calvariae of 1-d-old WT or p100−/− mice were cultured in the presence of BMP2 (100 ng/ml) for 1 h. Total cell lysates were immunoblotted with antiphosphorylated Smad1/5/8, anti-Smad1, and anti-NFκB2 antibodies. Anti-β-actin was used as a loading control.
Fig. 6.
Fig. 6.
p52 induced BMP receptor degradation by interacting BMP receptor. A, COS7 cells were cotransfected with V5-tagged ALK2(Q207D) in the presence or absence of either p100ΔGRR or p52. Whole-cell lysates were immunoprecipitated (IP) with an anti-V5 antibody and immunoblotted (IB) with anti-NF-κB2 antibodies. POB from calvariae of 1-d-old NfκB2−/− mice were cotransfected with V5-tagged ALK2(Q207D) in the presence or absence of either p100ΔGRR (panel B) or p52 (panel C). Total cell lysates were immunoblotted with anti-V5 and anti-NFκB2 antibodies. Anti-β-actin was used as a loading control. D, POB from calvariae of 1-d-old NfκB2−/− mice were cotransfected with V5-tagged ALK2(Q207D) in the presence or absence of p52. Cells were treated 24 h after transfection with vehicle or with 50 μm lactacystin for 9 h. Total cell lysates were immunoblotted with anti-V5 and anti-NFκB2 antibodies. Anti-β-actin was used as a loading control.
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