Pro416Arg cherubism mutation in Sh3bp2 knock-in mice affects osteoblasts and alters bone mineral and matrix properties

Abstract

Cherubism is an autosomal dominant disorder in children characterized by unwarranted symmetrical bone resorption of the jaws with fibrous tissue deposition. Mutations causing cherubism have been identified in the adaptor protein SH3BP2. Knock-in mice with a Pro416Arg mutation in Sh3bp2 exhibit a generalized osteoporotic bone phenotype. In this study, we examined the effects of this "cherubism" mutation on spectroscopic indices of "bone quality" and on osteoblast differentiation. Fourier-transform infrared imaging (FTIRI) analysis of femurs from wild-type and Sh3bp2 knock-in mice showed decreased mineral content, decreased mineral crystallinity/crystal size, and increased collagen maturity in homozygous mutants. To assess osteoblast maturation in vivo, knock-in mice were crossed with transgenic mice over-expressing GFP driven by 3.6-kb or 2.3-kb Col1a1 promoter fragments. Reduced numbers of mature osteoblasts were observed in homozygous mice. Neonatal calvarial cultures, which were enriched for osteoblasts by depletion of hematopoietic cells (negative selection for Ter119- and CD45-positive cells) were investigated for osteoblast-specific gene expression and differentiation, which demonstrated that differentiation and mineralization in homozygous osteoblast cultures was impaired. Co-cultures with calvarial osteoblasts and bone marrow macrophages showed that mutant osteoblasts appear to increase osteoclastogenesis resulting in increased bone resorption on bone chips. In summary, the Sh3bp2 mutation in cherubism mice alters bone quality, reduces osteoblast function, and may contribute to excessive bone resorption by osteoclasts. Our data, together with previous osteoclast studies, demonstrate a critical role of Sh3bp2 in bone remodeling and osteoblast differentiation.

Copyright (c) 2010 Elsevier Inc. All rights reserved.

Figures

Fig. 1

Fourier-transform infrared imaging (FTIRI) of wild-type (Sh3bp2+/+) and Sh3bp2 knock-in (Sh3bp2KI/KI) mice showing representative infrared images of cortical bone (A) and trabecular bone (B). Each image represents a 400×400 um area with 6.25 μm spatial resolution. The numerical color scales represent the range of intensity ratio for each parameter and were adjusted to be constant among samples.

Fig. 2

Dynamic histomorphometric analysis of Sh3bp2+/+, Sh3bp2+/KI, and Sh3bp2KI/KI mice. Double labels of calcein (green) and xylenol orange (red) were sequentially injected to 90-day-old mice to assess bone formation activities. Representative images from trabecular bones are shown (A). Mineral deposition rate (B) and bone formation rate (C) were similar between Sh3bp2+/+, Sh3bp2+/KI, and Sh3bp2KI/KI mice. (Student's t-test; n = 5 for Sh3bp2+/+; n = 5 for Sh3bp2+/KI; n = 7 for Sh3bp2KI/KI).

Fig. 3

In vivo monitoring of cherubism Sh3bp2KI/KI osteoblast cultures using Col1a1 promoter-driven GFP. Sh3bp2+/+ and Sh3bp2KI/KI mice were crossed with transgenic mice overexpressing GFP driven by either 3.6-kb or 2.3-kb Col1a1 promoters. Frozen sections (5 μm) of femurs from 10-week-old mice showed increased numbers of 3.6-kb Col1a1 GFP positive cells (3.6GFP) in Sh3bp2KI/KI mice (A), but decreased numbers of 2.3-kb Col1a1 GFP positive cells (2.3GFP) (B). (Student's t-test; n=6 for pOBCol3.6GFPtpz; n=4 for pOBCol2.3GFPemd, *p<0.05). 3.6-kb Col1a1 promoter in osteocytes from femoral cortex of Sh3bp2KI/KI but not of Sh3bp2+/+ mice was active (C). Trab: trabecular bone. Cort: Cortical bone.

Fig. 4

pOBCol3.6GFPtpz and pOBCol2.3GFPemd expression in calvarial cell cultures. Calvarial osteoblasts with GFP driven by a 3.6-kb Col1a1 promoter (pOBCol3.6GFPtpz) from Sh3bp2+/+, Sh3bp2+/KI, and Sh3bp2KI/KI mice showed no difference in GFP activity on day 7 of osteoblast differentiation (A); Calvarial osteoblasts with GFP driven by a 2.3-kb Col1a1 promoter (pOBCol2.3GFPemd) from Sh3bp2KI/KI mice showed reduced GFP activity compared to Sh3bp2+/+ samples on day 18 of osteoblast differentiation (B). BF: bright field; 3.6GFP: GFPtpz filter; 2.3GFP: GFPemd filter.

Fig. 5

Impaired maturation of Sh3bp2KI/KI osteoblasts. Col1a1 (A) and Bsp (B) expression in Sh3bp2KI/KI osteoblasts by quantitative real-time PCR was significantly decreased compared to Sh3bp2+/+ osteoblast cultures. Gene expression for Alp (C), Ocn (D), Runx2 (E) in late/mature-stage cultures only showed a reduced tendency. Three biological repeats with technical triplicates each were performed for each assay. (Two-way ANOVA followed by Bonferroni post-test, n=3, *p<0.05). Alkaline phosphatase (ALP), von Kossa (VK), and alizarin red (AR) (F) staining of calvarial osteoblast cultures indicated reduced osteogenesis in Sh3bp2KI/KI cultures.

Fig. 6

Increased osteoclast-inducing potential of Sh3bp2KI/KI osteoblasts. Sh3bp2+/+ bone marrow macrophages were co-cultured with either Sh3bp2+/+ or Sh3bp2KI/KI osteoblasts for 7 days. Bone chip analysis revealed increased resorption activity in co-cultures containing Sh3bp2KI/KI osteoblasts (Student's t-test, n=15 for resorption pit; n=6 for TRAP staining; *p<0.05) (A&B). The Rankl/Opg ratio in osteoblast cultures was significantly higher (with increase in RANKL and decrease in OPG expression) in Sh3bp2KI/KI cultures compared to Sh3bp2+/+ cultures at day 7 of osteoblastic differentiation, indicating an enhanced ability to generate osteoclasts (C–E). The expression of TNF-α in osteoblasts was significantly reduced in Sh3bp2KI/KI cultures (F). Biological as well as technical triplicates were performed for each assay. (Two-way ANOVA followed by Bonferroni post-test, n=3, *** p<0.001, ** p<0.01)).