Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption

Abstract

Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955C→T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2(Q2319X) heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2(Q2319X) cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor κB ligand in vitro were increased in Notch2(Q2319X) mutants. These effects were suppressed by the γ-secretase inhibitor LY450139. In conclusion, Notch2(Q2319X) mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption.

Keywords: Hajdu Cheney syndrome; Notch pathway; Notch protein; animal model; bone; osteoblast; osteoclast.

FIGURE 1.

Domains of Notch2 and engineering of the Hajdu Cheney Notch2^Q2319X mutant allele. A, upper panel shows domains of the Notch2 receptor depicting the following: 1) extracellular domain containing multiple epidermal growth factor (EGF)-like tandem repeats upstream of Lin12-Notch repeats (LNR); 2) heterodimerization domain (HD) which, in association with the LNR, forms the negative regulatory region; 3) transmembrane domain (TMD); and 4) Notch intracellular domain (NICD) consisting of an Rbpjκ-association module (RAM) linked to ankyrin (ANK) repeats and a nuclear localization sequence (NLS), upstream from a PEST domain. Under the Notch2 protein domains, the genomic structure of mutant exon 34 aligned with the corresponding protein structure. Black bars represent exons (E) 30–33, black box exon 34 containing the 6955C→T mutation leading to a Notch2^Q2319X truncated protein, and white box the 3′-untranslated region (UTR). Position of the loxP site remaining following the excision of the neo selection cassette is indicated by the white arrow. Lower panel A shows the region of the Notch2 locus surrounding the 6955C→T substitution; DNA coding for the nuclear localization sequence (green), the PEST domain (blue), and the Notch2 3′-UTR (red). B, identification of four targeted ES clones by long range PCR. Correct targeting of the 5′- and 3′- homology arms into the Notch2 locus was documented by the presence of a 4.4- (left) and 3.2-kb (right) PCR product in each of four clones. The middle lane represents molecular weight markers. Clone 2 was selected for ES cell aggregation. The lower panel in B shows the structure of the linearized targeting vector, consisting of a 4.6-kb 5′-homology arm containing exons 30–33 of Notch2, followed by a phosphoglycerate kinase promoter-driven neo selection cassette flanked by loxP sites ∼400 nucleotides upstream of exon 34. Vertical arrows on the bottom indicate the boundaries of the linearized targeting vector. Black boxes indicate coding sequences, and white box indicates the 3′-UTR. Horizontal arrows indicate binding sites for the primers used for the long range PCR. Primer pairs F1-R1 and F2-R2 were used for amplification of the 5′-homology arm, and primer pairs F3-R3 were used for amplification of the 3′-homology arm. C, genomic DNA from ear samples of F1 pups was used as a template for PCR, and products were sequenced by the Sanger method. The 1:1 signal ratio of C (blue) to T (red) demonstrates the presence of the 6955C→T substitution and heterozygosity of the mutation.

FIGURE 2.

Weight and femoral length of male and female Hajdu Cheney Notch2^Q2319X mutant mice (filled circles) and littermate wild type controls (open circles). Values are means ± S.D. The number of observations is as follows: male control mice n = 5 at 1 month and n = 9 at 3 months; female control mice n = 9 at 1 month and n = 4 at 3 months; male Notch2^Q2319X n = 6 at 1 month and n = 10 at 3 months; and female Notch2^Q2319X n = 8 at 1 month and n = 7 at 3 months of age. *, significantly different between Notch2^Q2319X mutant mice and control, p < 0.05 by unpaired t test.

FIGURE 3.

A, representative microcomputed tomography images of proximal trabecular bone and midshaft of femurs, showing cancellous bone osteopenia and decreased trabecular number and thinner and porous cortical bone in male Notch2^Q2319X mutant mice. Complete data set is shown in Table 2. B, representative static cancellous bone histological sections stained with toluidine blue show decreased number of trabeculae in Hajdu Cheney Notch2^Q2319X mice and calcein and demeclocycline labels show no differences in mineral apposition rate between control and Notch2^Q2319X mice. Complete data set is in Table 3. C, cross-sectional cortical bone stained with hematoxylin and eosin. Arrows in C point to osteoclasts on the endocortical surface. Complete data set is in Table 4. All representative images are from femurs from 1-month-old male Hajdu Cheney Notch2^Q2319X mutant and littermate wild type controls.

FIGURE 4.

Notch^6955C→T (Notch2^Q2319X), Hey1, Hey2 and HeyL, Hes1, Tnfsf11 (Rankl), and Tnfrsf11b (osteoprotegerin) mRNA levels in femoral bones from 1-month-old Hajdu Cheney Notch2^Q2319X mutant mice (black bars) and control littermate mice (white bars). Transcript levels are expressed as copy number corrected for Gapdh. Values are means ± S.D.; n = 8 for control; n = 9 for Notch2^Q2319X for all transcripts. *, significantly different between Notch2^Q2319X mutants and control, p < 0.05 by unpaired t test.

FIGURE 5.

Calvarial osteoblast-enriched cells from Notch2^Q2319X mutant (black bars) and wild type (white bars) littermate controls were isolated and cultured. A, 12× CSL-Luc, Hey1-Luc, and Hey2-Luc reporter constructs were transiently co-transfected with a CMV β-galactosidase construct; data are expressed as luciferase/β-galactosidase activity. Values are means ± S.D.; n = 6 for all data sets. B, total RNA was extracted, and gene expression was measured by qRT-PCR in the presence of specific primers and probes. Data are expressed as Notch2^6955C→T (Notch2^Q2319X), Hey1, Hey2, HeyL, Alpl, Bglap, Tnfsf11 (Rankl), and Tnfrsf11b (osteoprotegerin) copy numbers and corrected for Rpl38. Values are means ± S.D.; number of observations for control, n = 12 at 0 days, n = 10 at 3 days, and n = 8 at 7 days; number of observations for Notch2^Q2319X, n = 12 at 0 and at 3 days and n = 8 at 7 days for all transcripts. For mRNA expression, data were obtained from three experiments and controls normalized to 1. *, significantly different between Notch2^Q2319X mutant and wild type control cells, p < 0.05 by unpaired t test.

FIGURE 6.

Bone marrow mononuclear cells, harvested from femurs of Notch2^Q2319X mutant (black bars) and wild type littermate controls (white bars) were isolated by Ficoll-Hypaque gradient centrifugation and cultured for 6 days in the presence of M-Csf at 30 ng/ml and Rankl at 1–30 ng/ml. A, cultures were assessed for TRAP by enzyme histochemistry. Values are means ± S.D.; n = 6 for all data sets. The lower panel shows representative culture fields of TRAP-stained multinucleated cells. B, cultures were assessed for Notch2^6955C→T (Notch2^Q2319X) and Hes1 mRNA levels expressed as copy number corrected for Rpl38. Values are means ± S.D. Number of observations: n = 4 for control and Notch2^Q2319X at 3 and 6 days. *, significantly different between Notch2^Q2319X mutant and control cells, p < 0.05 by two-way analysis of variance with Holm-Sidak post hoc analysis.

FIGURE 7.

Bone marrow mononuclear cells, harvested from femurs of Notch2^Q2319X mutant (black bars) and wild type littermate controls (white bars) were cultured in the presence of M-Csf at 100 ng/ml for 6 days and switched to 30 ng/ml M-Csf- and 30 ng/ml Rankl-containing medium. Cultures were assessed in A for TRAP by enzyme histochemistry 5–8 days following exposure to Rankl. Values are means ± S.D.; n = 6 for all data sets except for Notch2^Q2319X DMSO at day 5 where n = 5; the right upper panel shows representative culture fields of TRAP-stained multinucleated cells 8 days after the addition of Rankl. B, cultures were assessed for Notch2^6955C→T (Notch2^Q2319X), Hes1, and Acp5 mRNA expression 8 days following exposure to Rankl. Values are means ± S.D.; n = 4 for all data sets. C, cultures were assessed for bone resorption as determined by resorption pit number and area 16 days following exposure to Rankl. Values are means ± S.D.; n = 6 for all data sets in control and in Notch2^Q2319X in DMSO or LY450139 (LY). A representative culture showing resorption pits is presented in the right lower panel. Cultures were conducted in the presence of LY450139 at 1 μm or DMSO vehicle started at the time of Rankl addition and continued throughout the culture period. *, significantly different between Notch2^Q2319X mutant and control cells, p < 0.05. #, significantly different between LY450139 and DMSO, p < 0.05, both two-way analysis of variance with Holm-Sidak post hoc analysis.