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 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 mutations analogous to those in HCS () 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 () expression. Here, to determine the individual contributions of osteoclasts and osteoblasts to HCS osteopenia, we created a conditional-by-inversion ( ) model in which Cre recombination generates a allele expressing a Notch2 mutant lacking the PEST domain. Germ line inversion phenocopied the mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, mice were bred with mice expressing Cre from the or the promoter, respectively. These crosses created experimental mice harboring a allele in Cre-expressing cells and control littermates expressing a wild-type transcript. inversion in-expressing cells had no skeletal consequences and did not affect the capacity of bone marrow macrophages to form osteoclasts In contrast, 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, activation in osteoblast-enriched cultures from mice induced expression. In conclusion, introduction of the HCS mutation in osteoblasts, but not in osteoclasts, causes osteopenia.

The lateral meningocele syndrome mutation causes marked osteopenia in mice

“…Cultures were carried out until the formation of multinucleated TRAP-positive cells was achieved. TRAP enzyme histochemistry was conducted using a commercial kit (Sigma-Aldrich), in accordance with manufacturer's instructions, as previously reported (50). TRAP-positive cells containing 3 or more nuclei were considered osteoclasts.…”

“…Cells were centrifuged and the sediment suspended in α-minimum essential medium (α-MEM) (Thermo Fisher Scientific) in the presence of 10% FBS and recombinant human M-CSF at 30 ng/ml as described (50). M-CSF cDNA and expression vector were obtained from D. Fremont (Washington University, St. Louis, MO) and M-CSF was purified as previously reported (71).…”

“…To obtain BMMs, the marrow from Notch3 tm1.1Ecn mice and littermate controls was removed by flushing with a 26 gauge needle and erythrocytes were lysed in 150 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA (pH 7.4) as described previously (26,50,70). Cells were centrifuged and the sediment suspended in α-minimum essential medium (α-MEM) (Thermo Fisher Scientific) in the presence of 10% FBS and recombinant human M-CSF at 30 ng/ml as described (50).…”

“…Cells from the last 3 digestions were pooled and seeded at a density of 10,000 cells/cm 2 , as previously described as previously reported (50,68). Osteoblast-enriched cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with non-essential amino acids (both from Thermo Fisher Scientific), 20 mM HEPES, 100 µg/ml ascorbic acid (both from Sigma-Aldrich) and 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA) in a humidified 5% CO2 incubator at 37C.…”

Glucocorticoids inhibit notch target gene expression in osteoblasts

“…NOTCH1 and NOTCH2 regulate bone remodeling and their actions, like those of glucocorticoids, are determined by the degree of osteoblast maturity . Activation of NOTCH1 or NOTCH2 in osteoblasts suppresses cancellous bone formation, whereas activation of NOTCH1 in osteocytes causes osteopetrosis due to the induction of osteoprotegerin and the inhibition of osteoclastogenesis …”