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. 2013 Dec 31;1(4):336-45.
doi: 10.4248/BR201304004. eCollection 2013 Dec.

Mice Deficient in NF-κB p50 and p52 or RANK Have Defective Growth Plate Formation and Post-natal Dwarfism

Lianping Xing  1 , Di Chen  2 , Brendan F Boyce  1
Affiliations

Mice Deficient in NF-κB p50 and p52 or RANK Have Defective Growth Plate Formation and Post-natal Dwarfism

Lianping Xing et al. Bone Res. .

Abstract

NF-κBp50/p52 double knockout (dKO) and RANK KO mice have no osteoclasts and develop severe osteopetrosis associated with dwarfism. In contrast, Op/Op mice, which form few osteoclasts, and Src KO mice, which have osteoclasts with defective resorptive function, are osteopetrotic, but they are not dwarfed. Here, we compared the morphologic features of long bones from p50/p52 dKO, RANK KO, Op/Op and Src KO mice to attempt to explain the differences in their long bone lengths. We found that growth plates in p50/p52 dKO and RANK KO mice are significantly thicker than those in WT mice due to a 2-3-fold increase in the hypertrophic chondrocyte zone associated with normal a proliferative chondrocyte zone. This growth plate abnormality disappears when animals become older, but their dwarfism persists. Op/Op or Src KO mice have relatively normal growth plate morphology. In-situ hybridization study of long bones from p50/p52 dKO mice showed marked thickening of the growth plate region containing type 10 collagen-expressing chondrocytes. Treatment of micro-mass chondrocyte cultures with RANKL did not affect expression levels of type 2 collagen and Sox9, markers for proliferative chondrocytes, but RANKL reduced the number of type 10 collagen-expressing hypertrophic chondrocytes. Thus, RANK/NF-κB signaling plays a regulatory role in post-natal endochondral ossification that maintains hypertrophic conversion and prevents dwarfism in normal mice.

Keywords: NF-κB; RANK; chondrocytes; dwarfism; growth plate.

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Figures

Figure 1
Figure 1
Growth plate morphology of various osteopetrotic mice. Sections of tibial growth plates from 2-week-old NF-κB p50/p52 dKO, RANK KO, Op/Op and Src KO mice and their littermate control mice stained with Safranin-O-Fast-Green (A), and H&E/alcian blue (B-D). Magnification x4 and x10 in left and right panels, respectively. The proliferative chondrocyte zone is indicated by * and the hypertrophic chondrocyte zone is indicated by #.
Figure 2
Figure 2
Expression of chondrocyte marker genes in tibial growth plates from 2-week-old NF-kB p50/p52 dKO and littermate control mice. (A) In-situ hybridization showing the distribution of type-2 and −10 collagen (Col), and MMP13 mRNA. The proliferative chondrocyte zone is indicated by * and the hypertrophic chondrocyte zone is indicated by #. (B) Safranin O/von Kossa-stained plastic-embedded sections showing a thickened hypertrophic chondrocyte zone and increased volume of mineralized bone matrix in p50/p52 dKO mice.
Figure 3
Figure 3
Histomorphometric analysis of growth plates from RANK KO mice. Histomorphometric analysis was carried out on H&E/Alcian blue-stained sections of tibiae from 1, 2, 3 and 6-week-old RANK KO and their littermate controls. Values are means±D of 3 mice per group. *P<0.05 vs control mice of the same age.
Figure 4
Figure 4
Effects of short-term RANKL treatment on micro-mass cultures from limb buds of WT mice. (A) Cultures were treated with RANKL, BMP2 or RANKL+BMP2 for 24 hrs and the expression levels of Sox9 and type 2 collagen mRNA were determined by qPCR. Fold changes of gene expression levels were calculated using values from PBS-treated cultures as 1. (B) Cells were cultured in chondrocyte-inducing medium and treated with RANKL, BMP2 or RANKL+BMP2 for 8 days. Values are the mean±D of 3 wells. *P<0.05 vs PBS-treated group.
Figure 5
Figure 5
Effects of long-term RANKL treatment on micro-mass chondrocyte cultures. Micro-mass pellets from limb buds of WT mice were cultured in chondrocyte-inducing medium and treated with BMP2±RANKL for 8–24 days, fixed, paraffin embedded, and sectioned. A) and B) H&E/alcian blue-stained sections and type-2 and type-10 collagen (Col) in-situ hybridization (magnification x2 and x20, respectively) at different days of micro-mass cultures. Proliferative chondrocytes are indicated by black arrowheads and hypertrophic chondrocytes are indicated by red arrows in B.
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