Skeletal development is a complex process which requires the tight regulation of gene activation and suppression in response to local signaling pathways. Among these pathways, Notch signaling is implicated in governing cell fate determination, proliferation, differentiation and apoptosis of skeletal cells-osteoblasts, osteoclasts, osteocytes and chondrocytes. Moreover, human genetic mutations in Notch components emphasize the critical roles of Notch signaling in skeletal development and homeostasis. In this review, we focus on the physiological roles of Notch signaling in skeletogenesis, postnatal bone and cartilage homeostasis and fracture repair. We also discuss the pathological gain- and loss-of-function of Notch signaling in bone and cartilage, resulting in osteosarcoma and age-related degenerative diseases, such as osteoporosis and osteoarthritis. Understanding the physiological and pathological function of Notch signaling in skeletal tissues using animal models and human genetics will provide new insights into disease pathogenesis and offer novel approaches for the treatment of bone/cartilage diseases.

Osteogenesis Imperfecta (OI) is a genetic bone dysplasia characterized by bone deformities and fractures caused by low bone mass and impaired bone quality. OI is a genetically heterogeneous disorder that most commonly arises from dominant mutations in genes encoding type‐I collagen (COL1A1 and COL1A2). In addition, OI is recessively inherited with the majority of cases resulting from mutations in prolyl‐3‐hydroxylation complex members, which includes Cartilage Associated Protein (CRTAP). OI patients are at an increased risk of fracture throughout their lifetimes. However, non‐union or delayed healing has been reported in 24% of fractures and 52% of osteotomies. Additionally, re‐fractures typically go unreported making the frequency of re‐fractures in OI patients unknown. Thus, there is an unmet need to better understand the mechanisms by which OI affects fracture healing. Using an open tibial fracture model, our study demonstrates delayed healing in both Col1a2^G610c/+ and Crtap^–/– OI mouse models (dominant and recessive OI, respectively) that is associated with reduced callus size and predicted strength. Callus cartilage distribution and chondrocyte maturation were altered in OI suggesting accelerated cartilage differentiation. Importantly, we determined that healed fractured tibia in female OI mice are biomechanically weaker when compared to the contralateral unfractured bone suggesting that abnormal OI fracture healing OI may prime future re‐fracture at the same location. We have previously shown up‐regulated TGF‐β signaling in OI and we confirm this in the context of fracture healing. Interestingly, treatment of Crtap^–/– mice with the anti‐TGF‐β antibody, 1D11 resulted in further reduced callus size and predicted strength, highlighting the importance of investigating dose response in treatment strategies. These data provide valuable insight into the effect of the extracellular matrix (ECM) on fracture healing, a poorly understood mechanism, and support the need for prevention of primary fractures to decrease incidence of refracture and deformity in OI patients.

Spondylocarpotarsal syndrome (SCT) is a rare musculoskeletal disorder characterized by short stature and vertebral, carpal, and tarsal fusions resulting from biallelic nonsense mutations in the gene encoding filamin B (FLNB). Utilizing a FLNB knockout mouse, we showed that the vertebral fusions in SCT evolved from intervertebral disc (IVD) degeneration and ossification of the annulus fibrosus (AF), eventually leading to full trabecular bone formation. This resulted from alterations in the TGFβ/BMP signaling pathway that included increased canonical TGFβ and noncanonical BMP signaling. In this study, the role of FLNB in the TGFβ/BMP pathway was elucidated using in vitro, in vivo, and ex vivo treatment methodologies. The data demonstrated that FLNB interacts with inhibitory Smads 6 and 7 (i-Smads) to regulate TGFβ/BMP signaling and that loss of FLNB produces increased TGFβ receptor activity and decreased Smad 1 ubiquitination. Through the use of small molecule inhibitors in an ex vivo spine model, TGFβ/BMP signaling was modulated to design a targeted treatment for SCT and disc degeneration. Inhibition of canonical and noncanonical TGFβ/BMP pathway activity restored Flnb^−/− IVD morphology. These most effective improvements resulted from specific inhibition of TGFβ and p38 signaling activation. FLNB acts as a bridge for TGFβ/BMP signaling crosstalk through i-Smads and is key for the critical balance in TGFβ/BMP signaling that maintains the IVD. These findings further our understanding of IVD biology and reveal new molecular targets for disc degeneration as well as congenital vertebral fusion disorders.

Osteogenesis imperfecta (OI) is a genetically heterogenous disorder characterized by brittle bone and recurrent fractures. The majority of OI is clinically categorized into mild, moderate, severe, and perinatal lethal forms. A clinical study on the use of intermittent parathyroid hormone (PTH) as an anabolic therapy for OI found PTH was effective in mild, but not in individuals with moderate/severe OI. However, this sub-group analysis was relatively small and considered preliminary by the authors. Since then, research on PTH therapy in OI has focused on mild OI and no studies of PTH use in moderate/severe OI patients or animal models have been reported. In this work, we used the Aga2/+ mouse, an established autosomal dominant model of moderate/severe OI, to investigate whether PTH treatment has an effect on bone. In vitro, Aga2/+ osteoblasts were sensitive to PTH and exhibited similar transcriptional changes compared to WT. In vivo, PTH treatment improved lumbar trabecular bone, increased cortical bone area fraction in the mid-femur, and, in females, led to improved biomechanical properties. These results demonstrate the Aga2/+ model responded to PTH and supports further investigation into whether PTH is effective in adult individuals with moderate/severe OI who have limited treatment options.

Osteogenesis imperfecta, a genetically heterogeneous disorder, is characterized by brittle bones with recurrent fractures as well as short stature. A recent study investigating cellular signaling in growth plate chondrocytes identified an increased expression of FGF receptors and elevated FGF signaling. We inhibited FGF receptor activation with the small molecule infigratinib in growing Aga2/+ mice and then characterized linear growth, cartilage growth plate histology, and bone morphometrics. Infigratinib treatment resulted in increased female femur length, increased growth plate lengths in both sexes, specifically in the proliferative zone, and reduced phospho-ERK1/2 as well as SOX9 expression in lower hypertrophic chondrocytes. Infigratinib had no impact on cortical bone but reduced male trabecular bone parameters in Aga2/+ mice. Three point bending biomechanical tests showed that Aga2/+-treated males had increased bone strength for some parameters, but reduced bone strength in treated WT males, correlating with the negative impact on trabecular bone parameters. WT and Aga2-treated male mice also showed increased serum testosterone levels. Together, these data establish FGFR as a therapeutic target for short stature in osteogenesis imperfecta and identify potential negative effects of pan-FGFR inhibition, particularly in the male skeleton.

Osteogenesis imperfecta (OI) is a genetically heterogenous disorder characterized by brittle bone and recurrent fractures. The majority of OI is clinically categorized into mild, moderate, severe, and perinatal lethal forms. A clinical study on the use of intermittent parathyroid hormone (PTH) as an anabolic therapy for OI found PTH was effective in mild, but not in individuals with moderate/severe OI. However, this sub-group analysis was relatively small and considered preliminary by the authors. Since then, research on PTH therapy in OI has focused on mild OI and no studies of PTH use in moderate/severe OI patients or animal models have been reported. In this work, we used the Aga2/+ mouse, an established autosomal dominant model of moderate/severe OI, to investigate whether PTH treatment has an effect on bone. In vitro, Aga2/+ osteoblasts were sensitive to PTH and exhibited similar transcriptional changes compared to WT. In vivo, PTH treatment improved lumbar trabecular bone, increased cortical bone area fraction in the mid-femur, and, in females, led to improved biomechanical properties. These results demonstrate the Aga2/+ model responded to PTH and supports further investigation into whether PTH is effective in adult individuals with moderate/severe OI who have limited treatment options.