Sustained Notch2 signaling in osteoblasts, but not in osteoclasts, is linked to osteopenia in a mouse model of Hajdu-Cheney syndrome

Abstract

Individuals with Hajdu-Cheney syndrome (HCS) present with osteoporosis, and HCS is associated with NOTCH2 mutations causing deletions of the proline-, glutamic acid-, serine-, and threonine-rich (PEST) domain that are predicted to enhance NOTCH2 stability and cause gain-of-function. Previously, we demonstrated that mice harboring Notch2 mutations analogous to those in HCS (Notch2HCS) are severely osteopenic because of enhanced bone resorption. We attributed this phenotype to osteoclastic sensitization to the receptor activator of nuclear factor-κB ligand and increased osteoblastic tumor necrosis factor superfamily member 11 (Tnfsf11) expression. Here, to determine the individual contributions of osteoclasts and osteoblasts to HCS osteopenia, we created a conditional-by-inversion (Notch2^COIN ) model in which Cre recombination generates a Notch2^ΔPEST allele expressing a Notch2 mutant lacking the PEST domain. Germ line Notch2^COIN inversion phenocopied the Notch2HCS mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, Notch2^COIN mice were bred with mice expressing Cre from the Lyz2 or the BGLAP promoter, respectively. These crosses created experimental mice harboring a Notch2^ΔPEST allele in Cre-expressing cells and control littermates expressing a wild-type Notch2 transcript. Notch2^COIN inversion in Lyz2-expressing cells had no skeletal consequences and did not affect the capacity of bone marrow macrophages to form osteoclasts in vitro In contrast, Notch2^COIN inversion in osteoblasts led to generalized osteopenia associated with enhanced bone resorption in the cancellous bone compartment and with suppressed endocortical mineral apposition rate. Accordingly, Notch2 activation in osteoblast-enriched cultures from Notch2^COIN mice induced Tnfsf11 expression. In conclusion, introduction of the HCS mutation in osteoblasts, but not in osteoclasts, causes osteopenia.

Keywords: Hajdu Cheney Syndrome; Notch pathway; Notch2; bone remodeling; conditional by inversion; genetic disease; mouse genetics; osteoblast; osteoclast.

Figure 1.

Engineering of the Notch2^COIN allele. A, genomic structure and size of the Notch2 locus with the position of the 34 exons indicated by vertical black bars for coding sequences or white boxes for untranslated regions (UTR). B, position of the AAG codon (underlined) for lysine 2384 in exon 34. The sequence of the insertion site of the COIN module is in lowercase, and gray and white boxes indicate the coding sequence and the 3′-UTR (rβglpA), respectively. C, structure of exon 34 and of the targeting construct correctly integrated. From 5′ to 3′: lox71 (L71), rabbit β-globin polyadenylation signal, eGFP-coding sequence, internal ribosome entry site (Gtx ires)₅, human influenza hemagglutinin (HA) tag coding sequence, 3′-splice region from the second intron of the rabbit β-globin gene (white curved arrow), and lox66 (L66) that constitute the COIN module and a flippase (FLP) recognition site (FRT)-flanked neo cassette downstream of the human UBp promoter (FRT-hUBp-neo-FRT). Removal of the neo cassette by FLP recombination is indicated (gray dotted lines). D, representation of the silent COIN module in the antisense orientation and of the splicing event (black dotted lines) that excises the COIN module from the nascent transcript, allowing expression of a wild-type Notch2 mRNA and protein. E, generation of the Notch2^ΔPEST allele by Cre recombinase-mediated permanent inversion of the COIN module, and illustration of the splicing event (black dotted lines) that occurs during the maturation of the Notch2^ΔPEST transcript. The latter is translated into a Notch2 mutant lacking the PEST domain. The position of the silent lox72 (L72) sequence and of the wild-type loxP site created by Cre recombination of L71 and L66 is indicated. Images are scaled either in kilobase (kb) or bp.

Figure 2.

Inversion of the Notch2^COIN allele in the germ line causes osteopenia. One-month-old male Notch2^ΔPEST/WT mutants (black bars; Notch2^ΔPEST) were compared with wild-type littermate controls (white bars) of the same sex. A, DNA was extracted from tail, and Notch2^COIN inversion was documented by gel electrophoresis of PCR products obtained with primers specific for the Notch2^ΔPEST allele. Arrows indicate the position of the 250-bp amplicon. B, total RNA was extracted from tibiae, and expression of the Notch2^ΔPEST and Notch2^WT mRNA was measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38 mRNA levels; data for Notch2^WT were normalized to corrected expression in control. Values are means ± S.D.; n = 4 for control, n = 5 for Notch2^ΔPEST, all biological replicates. Two technical replicates were used for each qPCR. *, significantly different between control and Notch2^ΔPEST, p < 0.05 by t test. C, representative μCT images of femoral proximal trabecular bone and midshaft cortical bones of control and Notch2^ΔPEST mice; complete data set in Table 1.

Figure 3.

Inversion of the Notch2^COIN allele in Lyz2-expressing cells has no skeletal consequences. Documentation of Notch2^COIN inversion, analysis of gene expression, and osteoclastogenesis in 1-month-old Lyz2^Cre/WT;Notch2^{ΔPEST/ΔPEST} or Lyz2^Cre/Cre;Notch2^{ΔPEST/ΔPEST} (black bars; Notch2^ΔPEST) and sex-matched Notch2^COIN/COIN or Lyz2^Cre/Cre;Notch2^WT/WT (white bars) controls, respectively. A and D, BMMs from 1-month-old Lyz2^Cre/WT;Notch2^{ΔPEST/ΔPEST} (A) or Lyz2^Cre/Cre;Notch2^{ΔPEST/ΔPEST} (D) mice and respective controls were cultured for 72 h in the presence of M-CSF at 30 ng/ml. DNA was extracted, and Notch2^COIN inversion was demonstrated by gel electrophoresis of PCR products obtained with primers specific for the Notch2^ΔPEST allele. The arrows indicate the position of the 250-bp amplicon. B and E, Notch2^ΔPEST transcript levels were measured by qRT-PCR in total RNA from the parietal bones of Lyz2^Cre/WT;Notch2^{ΔPEST/ΔPEST} (B) or Lyz2^Cre/Cre;Notch2^{ΔPEST/ΔPEST} (E) mice and respective controls. Transcript levels are reported as copy number corrected for Rpl38 mRNA levels. Values are means ± S.D.; n = 4–6 biological replicates. Values are means ± S.D.; n = 4 for both controls, n = 4 for Lyz2^Cre/WT;Notch2^{ΔPEST/ΔPEST}, n = 6 for Lyz2^Cre/Cre;Notch2^{ΔPEST/ΔPEST}, all biological replicates. Two technical replicates were used for each qPCR. C and F, BMMs from 1-month-old Lyz2^Cre/WT;Notch2^{ΔPEST/ΔPEST} (C) or Lyz2^Cre/Cre;Notch2^{ΔPEST/ΔPEST} (F) mice and respective controls were cultured for 72 h in the presence of M-CSF at 30 ng/ml and then with the addition of Rankl at 10 ng/ml until the formation of osteoclasts. Trap activity was assessed by enzyme histochemistry, and data are expressed as number of osteoclasts per well. Values are means ± S.D.; n = 4 for Notch2^COIN/COIN, n = 3 for Lyz2^Cre/Cre;Notch2^WT/WT, n = 5 for Lyz2^Cre/WT;Notch2^{ΔPEST/ΔPEST}, and n = 4 for Lyz2^Cre/Cre;Notch2^{ΔPEST/ΔPEST}, all biological replicates.

Figure 4.

Inversion of the Notch2^COIN allele in osteoblasts leads to Notch2 activation in vivo. Documentation of Notch2^COIN inversion and analysis of gene expression in BGLAP-Cre;Notch2^{ΔPEST/ΔPEST} (black bars; Notch2^ΔPEST) and Notch2^COIN/COIN littermate controls (white bars). A, DNA was extracted from the parietal bones of 1- and 4-month-old male mice, and Notch2^COIN inversion was demonstrated by gel electrophoresis of PCR products obtained with primers specific for the Notch2^ΔPEST allele. The arrows indicate the position of the 250-bp amplicon. B, gene expression was measured by qRT-PCR in total RNA from tibiae of 4-month-old mice. Transcript levels are reported as Notch2^ΔPEST, Hey1, Hey2, and HeyL mRNA copy number corrected for Rpl38 expression. Values are means ± S.D.; n = 11 biological replicates for both groups. Two technical replicates were used for each qPCR. *, significantly different between Notch2^ΔPEST and control, p < 0.05 by t test.

Figure 5.

Notch2 activation in osteoblasts causes osteopenia. One- and 4-month-old male and female BGLAP-Cre;Notch2^{ΔPEST/ΔPEST} (black dots; Notch2^ΔPEST) were compared with sex-matched littermate Notch2^COIN/COIN controls (open circles). A, weight and femoral length. Values are means ± S.D.; in males at 1 month of age n = 7 for control, n = 12 for Notch2^ΔPEST, and at 4 months of age n = 6 for control, n = 6 for Notch2^ΔPEST; in females at 1 month of age n = 7 for control, n = 7 for Notch2^ΔPEST, and at 4 months of age n = 5 for control, n = 6 for Notch2^ΔPEST, all biological replicates. B, representative μCT images of femoral proximal trabecular bone and midshaft. Complete data set in Table 4.

Figure 6.

Notch2 activation in osteoblasts induces Tnfsf11 expression. Calvarial osteoblast-enriched cells from 3- to 5-day-old Notch2^COIN/COIN mice of both sexes were infected with Ad-CMV-Cre (Notch2^ΔPEST; black bars) or Ad-CMV-GFP (control, white bars). A, DNA was extracted, and Notch2^COIN inversion was documented by gel electrophoresis of PCR products obtained with primers specific for the Notch2^ΔPEST allele. The arrow indicates the position of the 250-bp amplicon. B, total RNA was extracted, and gene expression was measured by qRT-PCR in the presence of specific primers. Transcript levels are reported as Notch2^ΔPEST, Hey1, HeyL, and Tnfsf11, corrected for Rpl38 expression. Values are means ± S.D.; n = 4 for all groups, all technical replicates from the same cell preparation. Two technical replicates were used for each qPCR. *, significantly different between Notch2^ΔPEST and control, p < 0.05; +, significantly different from day 0, p < 0.05; two-way analysis of variance with Holm-Šídák post-hoc analysis.