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 6955C3 T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2Q2319X 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 B ligand in vitro were increased in Notch2 Q2319X mutants. These effects were suppressed by the ␥-secretase inhibitor LY450139. In conclusion, Notch2 Q2319X mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption.Notch proteins are four single-pass transmembrane receptors that play a critical role in cell fate decisions ( Fig. 1) (1-4). Notch regulates cell renewal and plays a role in skeletal development and homeostasis and in osteoblast and osteoclast differentiation (4 -8). Jagged1 and -2 and DeltaLike1, -3, and -4 are the five classic Notch ligands (4). Notch-ligand interactions result in the proteolytic cleavage and release of the Notch intracellular domain (NICD), 2 which translocates to the nucleus to form a complex with recombination signal-binding protein for immunoglobulin J region (Rbpj) and Mastermind-like to regulate transcription (9 -12). This canonical signaling pathway leads to the transcription of Hairy Enhancer of Split (Hes)1, -5, and -7 and Hes related with YRPW motif (Hey)1, -2, and -L. Skeletal cells express Notch1, Notch2, and low levels of Notch3 transcripts (13-15). Activation of Notch in undifferentiated and differentiated osteoblasts inhibits cell differentiation and function and causes osteopenia (16,17). In contrast, activation of Notch1 in osteocytes causes a pronounced increase in bone mass due to a suppression of bone resorption (18). Results from the conditional inactivation of Notch1 and Notch2 in the developing skeleton confirmed the inhibitory role of Notch in osteoblastogenesis (6,19). Whereas substantial work has characterized the consequences of Notch1 gain-of-function in the skeleton, there is limited knowledge on the function of Notch2 in the postnatal skeleton. This knowledge is particularly important because Notch1 and Notch2 do not have redundant functions, and Notch1 inhibits, wh...

Parathyroid hormone inhibits Notch signaling in osteoblasts and osteocytes

“…To determine the effects of PTH in the context of Notch activation in osteocytes, we tested the consequences of PTH exposure in osteocyte-enriched cells from heterozygous Notch2HCS mice, a model of Notch2 gain-of-function [25]. This was necessary because bone fragments cannot be acutely transfected with Notch expression vectors nor successfully cultured on Notch-specific ligands to achieve activation.…”

“…To achieve Notch2 gain-of-function, mice harboring a point mutation in exon 34 of the Notch2 locus were generated by homologous recombination, as described [25]. A 6955C>T mutation was introduced creating a stop codon leading to the translation of a truncated protein lacking the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain, making it resistant to degradation and resulting in a Notch2 gain-of-function [20].…”

“…In the present study, we tested the effects of PTH on Notch signaling in osteoblasts from wild-type mice and osteocytes from a recently described murine model of Notch2 gain-of-function [25]. These experiments were complemented by in vivo work to determine the impact of PTH on Notch signaling in bone tissue of wild-type mice.…”

NOTCH2 Hajdu-Cheney Mutations Escape SCFFBW7-Dependent Proteolysis to Promote Osteoporosis

“…We previously showed that Notch2 depletion in osteoclast precursors impairs osteoclastogenesis, while ectopic Notch2 ICD expression facilitates osteoclast differentiation (Fukushima et al, 2008). Interestingly, knock-in mice harboring the Notch2 Q2319X HCS mutant exhibited osteopenia due to enhanced osteoclast formation, without significantly affecting osteoblast number and activity (Canalis et al, 2016). This phenotype was alleviated by an antibody against Notch2 negative regulatory region (NRR) (Canalis et al, 2017).…”

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

“…While it is now generally accepted that Notch signaling plays a positive role in the differentiation and function of osteoclasts, variations in both Notch signaling stimulation and osteoclast precursor culture and differentiation led to initially contradictory findings 16,17,18,19 . Closer examination of the differences in methods and use of genetic models have greatly clarified the role of Notch signaling in osteoclastogenesis, but application of standardized Notch stimulation and culture methods could prevent such controversies in future studies of Notch signaling in other cell types 20,21,22,23 .…”