Jagged1 expression by osteoblast-lineage cells regulates trabecular bone mass and periosteal expansion in mice

Abstract

Loss-of-function mutations in the Notch ligand, Jagged1 (Jag1), result in multi-system developmental pathologies associated with Alagille syndrome (ALGS). ALGS patients present with skeletal manifestations including hemi-vertebrae, reduced bone mass, increased fracture incidence and poor bone healing. However, it is not known whether the increased fracture risk is due to altered bone homeostasis (primary) or nutritional malabsorption due to chronic liver disease (secondary). To determine the significance of Jag1 loss in bone, we characterized the skeletal phenotype of two Jag1-floxed conditional knockout mouse models: Prx1-Cre;Jag1f/f to target osteoprogenitor cells and their progeny, and Col2.3-Cre;Jag1f/f to target mid-stage osteoblasts and their progeny. Knockout phenotypes were compared to wild-type (WT) controls using quantitative microcomputed tomography, gene expression profiling and mechanical testing. Expression of Jag1 and the Notch target genes Hes1 and Hey1 was downregulated in all Jag1 knockout mice. Osteoblast differentiation genes were downregulated in whole bone of both groups, but unchanged in Prx1-Cre;Jag1f/f cortical bone. Both knockout lines exhibited changes in femoral trabecular morphology including decreased bone volume fraction and increased trabecular spacing, with males presenting a more severe trabecular osteopenic phenotype. Prx1-Cre;Jag1f/f mice showed an increase in marrow mesenchymal progenitor cell number and, counterintuitively, developed increased cortical thickness resulting from periosteal expansion, translating to greater mechanical stiffness and strength. Similar alterations in femoral morphology were observed in mice with canonical Notch signaling disrupted using Prx1-Cre-regulatable dominant-negative mastermind like-protein (dnMAML). Taken together, we report that 1) Jag1 negatively regulates the marrow osteochondral progenitor pool, 2) Jag1 is required for normal trabecular bone formation and 3) Notch signaling through homotypic Jag1 signaling in osteochondral progenitors, but not mature osteoblasts, inhibits periosteal expansion. Therefore, Jag1 signaling within the osteoblast lineage regulates bone metabolism in a compartment-dependent manner. Moreover, loss of Jag1 function in osteoblast lineage cells may contribute to the skeletal phenotype associated with ALGS.

The developmental biology of genetic Notch disorders

“…The systemic deletion of Jag1 or Notch2 did not reveal any somite-related phenotype that would suggest their involvement in the early events of bone formation (Hamada et al, 1999;Xue et al, 1999). However, Jag1 and Notch2 in the skeletogenic mesenchyme negatively regulate the differentiation of mesenchymal progenitors into osteoblasts, both in vitro and in adolescent mice, and their ablation leads to progressive bone loss in adult mice (Hilton et al, 2008;Nobta et al, 2005;Youngstrom et al, 2016). Importantly, Jag1 deletion in mesenchymal progenitors causes expansion of the cortical bone, while diminishing trabecular bone mass, suggesting opposing effects of Jag1 signaling on cortical versus trabecular osteoblasts (Youngstrom et al, 2016).…”

“…However, Jag1 and Notch2 in the skeletogenic mesenchyme negatively regulate the differentiation of mesenchymal progenitors into osteoblasts, both in vitro and in adolescent mice, and their ablation leads to progressive bone loss in adult mice (Hilton et al, 2008;Nobta et al, 2005;Youngstrom et al, 2016). Importantly, Jag1 deletion in mesenchymal progenitors causes expansion of the cortical bone, while diminishing trabecular bone mass, suggesting opposing effects of Jag1 signaling on cortical versus trabecular osteoblasts (Youngstrom et al, 2016). This imbalance leads to spine defects and the formation of butterfly vertebrae, a characteristic feature of Alagille syndrome (Emerick et al, 1999;Youngstrom et al, 2016).…”

“…Importantly, Jag1 deletion in mesenchymal progenitors causes expansion of the cortical bone, while diminishing trabecular bone mass, suggesting opposing effects of Jag1 signaling on cortical versus trabecular osteoblasts (Youngstrom et al, 2016). This imbalance leads to spine defects and the formation of butterfly vertebrae, a characteristic feature of Alagille syndrome (Emerick et al, 1999;Youngstrom et al, 2016). Furthermore, both clinical and genome-wide association studies indicate a positive correlation between mutations in JAG1 and decreased bone mineral density and osteoporotic fractures (Bales et al, 2010;Kung et al, 2010).…”

Identification of novel variants associated with osteoporosis, type 2 diabetes and potentially pleiotropic loci using pleiotropic cFDR method

“…JAG1 participated in normal trabecular bone formation and inhibited periosteal expansion through affecting osteoprogenitor cells and their progeny [69]. Recently, Zfp423 was found to act as a hinge regulated coordinating regulation of adipocyte and osteo­blast differentiation [70].…”

Notch activation promotes osteoblast mineralization by inhibition of apoptosis

“…Particularly, JAG1‐mediated Notch signaling has been shown to play an important role in the regulation of skeletal metabolism (Lawal et al, ). JAG1 transgenic mice showed an increased formation of cortical and cancellous bone, reduced bone loss and improved bone mechanical strength by increasing the proliferation of osteoblast progenitors, stimulating osteoblast differentiation and inhibiting osteoclast proliferation (Youngstrom et al, ). On the other hand, knocking down JAG1 exerts a catabolic effect on bone mass by inhibiting osteoblast differentiation (Liu et al, ).…”