We have now demonstrated that a pathological role of Notch signaling in a mouse model of osteosclerosis depends on the Rbpj signaling. Gain of Notch function in osteoblasts leads to a proliferation of immature osteoblasts and inhibits their terminal differentiation. The bitransgenic strategy in this study offers certain advantages over our previous conventional transgenic approach. It eliminates potential interline variability of transgene expression resulting from different integration sites and/or variable numbers of transgene copies. Notably, although the bitransgenic GOF mice in this model exhibited milder phenotypes and increased longevity, they recapitulated the defining feature of osteosclerosis from our previous study comparing analyses by bone histomorphometry (BV/TV, trabecular bone number, thickness, and space), skeleton morphology, and molecular signatures of gene expression (21)
. Together, our previous and current studies suggest that Notch gain of function in osteoblasts leads to an osteosclerotic phenotype with an increase in osteoblast number and function with a secondary increase in osteoclast number. The greatly increased bone formation over bone resorption contributes to the high bone mass phenotype in the GOF mice. Accordingly, this model will provide a tool for better understanding the molecular pathogenic mechanism of osteosclerosis and may constitute a platform for developing novel therapeutic strategies.
Human osteosclerosis refers to trabecular bone thickening and an overall increase in bone mass. The potential causes of osteosclerosis include hereditary or sporadic gene mutations intrinsic to the bone cells, or dysregulation of a variety of non-cell autonomous factors that result from endocrine, metabolic, hematologic, infectious, neoplastic disorders, and dietary intake(1)
. Genetically engineered mouse models and the molecular basis of some forms of this disease have been previously reported (7,9,10)
. These models resembled ours in that they showed increased bone formation over the entire skeleton via cell autonomous changes within osteoblasts. Particularly in one example, Baron's group showed that transgenic mice overexpressing the naturally occurring ΔFosB or Δ2ΔFosB splice variants of FosB developed severe osteosclerosis, which was caused, at least in part, through a mechanism of FosB-BMP/Smad1 interaction(7,10)
. Relevant to our model of osteosclerosis, it is possible that the Notch gain of function might affect downstream signaling pathways such as BMP-Smad1 pathway, since interaction between Smad1 and Notch1 NICD or cross-talk between BMP-Smad1 and Notch signaling have been reported in many studies(30)
. However, these potential interactions are probably not the main mechanism for the phenotypes in the GOF mice given that Rbpj deletion completely rescues the effect in spite of the presence of high levels of NICD. Furthermore, similar, although milder, osteosclerotic phenotypes have been described in mice lacking Osteocalcin, or Leptin as well as the Leptin receptor(31,32)
. The former mice may share a mechanism with the GOF mice since expression of Osteocalcin (Oc) was significantly decreased in the GOF calvaria (). On the other hand, all of those reported mouse models revealed an increase in the number of trabeculae as well as in cortical thickness. The latter “hyperostotic” effect was not observed in our GOF mice (). One possible explanation for this is that expression of the transgene in those previous reported mice accelerated differentiation of osteoprogenitors into mature osteoblasts rather than increasing proliferation of immature osteoblasts, which is a characteristic feature of our GOF mice. Thus, the GOF mice may represent a unique model to study the pathogenesis of osteosclerosis in the committed immature osteoblastic compartment.
The etiology of sclerosing bone disorders has been elucidated recently in some hereditary diseases (1)
. Osteopetrosis referred as increased bone mass due to osteoclast failure has been associated with deactivation of genes that encode: carbonic anhydrase II (CA II), an α3 subunit of the vacuolar proton pump (TCIRG1), chloride channel 7 (CLCN7, Albers-Schönberg disease), osteopetrosis associated transmembrane protein 1 (OSMT1), RANKL and RANK(2)
. In contrast, most of the non-osteopetrosis sclerosing bone disorders are due to enhanced osteoblast activity with increased bone formation. Among them, activating mutations in genes encoding transforming growth factor beta1 (TGF-β1) and low density lipoprotein receptor-related protein 5 (LRP5), and loss of function mutations in genes encoding sclerostin (SOST) and LEM domain containing 3 (LEMD3) have been linked to non-osteopetrotic sclerosing bone disorders including Camurati-Engelman dysplasia, van Buchen disease/ sclerosteosis, Worth syndrome/high bone mass syndrome, and Buschke-Ollendorff syndrome/osteopoikilosis, respectively. These genes act primarily in the TGFβ/BMP and Wnt signaling pathways. How they might actually interact with Notch signaling downstream is unclear although cross-talk between these signaling pathways during development and in pathological conditions is well described (30,33,34)
. Thus, it may be interesting to examine the status of Notch signaling in those disorders. Moreover, our studies on Notch pathological function may be applied to the understanding of other sclerosing bone disorders that have unknown etiology and pathogenesis. These disorders include Osteopathia striata, Melorheostosis, Fibrogenesis imperfecta ossium, Osteomesopyknosis, Axial osteomalacia, Dermatofibrosis lenticularis disseminate, Hepatitis C–associated osteosclerosis, Fluorosis, Lymphoma, Myelofibrosis, and Mastocytosis, all of which feature focal or generalized osteosclerosis.
It is well known that in certain bone disorders such as Paget disease, a benign or malignant bone tumor is prone to occur(35)
. Solitary cases of osteosarcoma (OS) have been reported in association with Melorheostosis and Osteopathia striata(36)
, and Osteogenesis imperfecta(38)
. Osteoblastic OS tumor cells are highly proliferative osteoblast cells, which express early differentiation gene makers such as Osterix (OSX) and alkaline phosphotase (ALP), but not the late marker, osteocalcin (OCN)(39)
. The molecular signature of calvarial osteoblasts in the GOF mice () is reminiscent of a proliferative disease of osteoblasts prevailing in OS. Our recent study together with those of others on Notch signaling in human OS samples suggest that aberrant Notch signaling contributes to the pathogenesis of human OS (18-20)
. Notably, those studies on OS were mostly carried out using human OS cell lines and/or primary human OS tumor samples that were diagnosed at a late stage. As such they may already have accumulated complex molecular and cytogenetic alterations. To exclude the confounder of cumulative secondary mutational events, genetically engineered mouse models like the GOF mice might enable us to better understand the possible involvement of Notch in initiation and/or progression of OS.
Manipulation of Notch signaling in the bone may offer a new option for molecular therapeutics of OS and other bone-related diseases. In the case of OS, recent studies in human OS xenografts in nude mice showed that chemical and/or genetic inhibition of Notch signaling decreased tumor growth and metastasis (18-20)
. Interestingly, clinical trials using small-molecule inhibitors of the gamma-secretase complex (GSIs), which were originally developed to treat Alzheimer disease, have proven promising in T-ALL, intestine tumors, stroke, and autoimmune encephalomyelitis(17)
. However, resistance to GSIs has been reported in certain types of T-ALL cell lines as well as in patients though the mechanism of their resistance is unknown. Hence, developing alternative targets for manipulating Notch is needed. Our GOF mice expressed an activated, GSIs (γ-secretase inhibitor)-resistant form of the intracellular domain of Notch 1 (NICD). As such, it may serve as an appropriate preclinical model for therapeutically targeting Notch signaling downstream of protease cleavage. Strategies may include monoclonal antibodies, dominant negative forms of RBPJ and MAML1, synthetic peptides that target NOTCH-RBPJ complex, small molecules for RNAi interference, and enzymatic inhibitors since they have been experimentally employed to inhibit Notch signaling effectively(40-44)
. In contrast to the treatment of proliferative disorders of the osteoblast, transient activation of Notch signaling also has the potential as an anabolic bone agent that may benefit to the patients with osteoporosis. However, this approach is complicated by a concern over the temporal and context dependent nature of Notch signaling on the mesenchymal stem cell during osteoblast differentiation(45)
Finally, our understanding of the role of the canonical Notch signaling in the skeletal biology is still evolving, and the physiological role of the non-canonical Notch signaling in this system remains unclear. A handful of in vitro
cell culture studies suggest that the non-canonical Notch signaling exists and may result from the direct interaction of NICD with either cytoplasmic proteins or nuclear transcription factors, thereby facilitating a cross talk between Notch and other pathways such as NF-kB, small GTPase R-Ras, and Wnt signaling(46-55)
. Mechanistically, our data support a model that originated from Drosophila genetic studies where Notch signaling has both canonical and non-canonical versions in precursor or progenitor cells while it only employs the canonical pathway in differentiating or differentiated cells(34,56)
. Future genetic studies in mammals may further confirm whether this model is applicable to other vertebrate species. Clinically, it will be important to determine whether and when these pathways occur so that therapeutic strategies can be rationally developed for Notch-related diseases. However, in current engineered mice models, the proportional contributions of canonical vs. non-canonical versions of Notch pathway need to be first established. By genetic rescue approaches like that shown here, we can begin to determine whether and how much the non-canonical Notch signaling in fact contributes to a specific cellular context. Only then will mechanistic and translational studies targeted at manipulating Notch signaling in a therapeutic context be more fully and rationally informed.
In summary, we report on a bitransgenic mouse model for osteosclerosis that is generated by cre recombinase-activated expression of the Notch1 intracellular domain (NICD) exclusively in committed osteoblasts. These Notch gain of function or GOF mice developed severe osteosclerosis over the entire skeleton. Genetic deletion of Rbpj specifically in osteoblasts abolished the osteosclerotic phenotypes and growth retardation. Furthermore, cellular and molecular analyses of bones from the GOF:Rbpjf/f mice confirmed that NICD-induced proliferation and differentiation markers in osteoblasts were completely normalized by removal of Rbpj. Thus, activation of the canonical Notch pathway in committed osteoblasts represents one potential pathological mechanism for development of osteosclerosis. Moreover, our findings provide the first genetic evidence in the skeletal system that Notch activation in differentiated cells is solely dependent on its canonical pathway. Hence, selective targeting of Rbpj may be an effective therapeutic approach in bone diseases where there is gain of Notch function such as osteosarcoma.