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 (Notch2COIN ) model in which Cre recombination generates a Notch2ΔPEST allele expressing a Notch2 mutant lacking the PEST domain. Germ line Notch2COIN inversion phenocopied the Notch2HCS mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, Notch2COIN mice were bred with mice expressing Cre from the Lyz2 or the BGLAP promoter, respectively. These crosses created experimental mice harboring a Notch2ΔPEST allele in Cre-expressing cells and control littermates expressing a wild-type Notch2 transcript. Notch2COIN 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, Notch2COIN 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 Notch2COIN mice induced Tnfsf11 expression. In conclusion, introduction of the HCS mutation in osteoblasts, but not in osteoclasts, causes osteopenia.
Highlights
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
Exon 34 was split into two exons at a position corresponding to lysine 2384, which is upstream of the PEST domain and downstream of the domains required for the transcriptional activation of Notch2 (NCBI protein database NP035058; Fig. 1B)
The individual contributions of the osteoclast and osteoblast lineages to the bone loss observed in Notch2HCS mutant mice were explored by the conditional introduction of the HCS genetic defect in selected cell lineages
Summary
The condition is associated with mutations in exon 34 of NOTCH2 that create a premature stop codon immediately upstream of the sequences coding for the proline- (P), glutamic acid- (E), serine- (S), and (T) threonine-rich (PEST) domain [22,23,24,25,26]. A conditional by inversion (COIN) approach was utilized to create a conditional mouse model of HCS (Notch2COIN) [28, 29] This system was designed to introduce a premature STOP codon in exon 34 of Notch following Cremediated recombination leading to the translation of a truncated Notch protein, mimicking the genetic defect associated with HCS. Mutant and control mice were examined for skeletal phenotypic changes by microcomputed tomography (CT) and bone histomorphometry, and the potential mechanisms of Notch action were explored
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