Osteosclerosis Due to Notch Gain of Function Is Solely Rbpj-dependent

doi:10.1002/jbmr.115

J Bone Miner Res. Author manuscript; available in PMC 2011 October 1.

Published in final edited form as:

J Bone Miner Res. 2010 October; 25(10): 2175–2183.

doi: 10.1002/jbmr.115

PMCID: PMC3126919

NIHMSID: NIHMS301974

Osteosclerosis Due to Notch Gain of Function Is Solely Rbpj-dependent

Jianning Tao,¹ Shan Chen,¹ Tao Yang,¹ Brian Dawson,¹ Elda Munivez,¹ Terry Bertin,¹ and Brendan Lee^1,²

¹Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA

²Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA

Jianning Tao: tao/at/bcm.edu; Shan Chen: shanc/at/bcm.edu; Tao Yang: taoy/at/bcm.edu; Brian Dawson: bcd/at/bcm.edu; Elda Munivez: emunivez/at/bcm.edu; Terry Bertin: tbertin/at/bcm.edu

Corresponding Author: Brendan Lee, M.D., Ph.D., One Baylor Plaza, R814, Houston, Texas 77030, USA, Phone: 713-798-8835, Fax: 713-798-5168, Email: blee/at/bcm.edu

The publisher's final edited version of this article is available at J Bone Miner Res

See other articles in PMC that cite the published article.

Abstract

Osteosclerosis is a pathological bone disease characterized by an increase in bone formation over bone resorption. Genetic factors that contribute to the pathogenesis of this disease are poorly understood. Dysregulation or mutation in many components of Notch signaling pathway results in a wide range of human developmental disorders and cancers including bone diseases. Our previous study found that activation of the Notch signaling in osteoblasts promotes cell proliferation and inhibits differentiation, leading to an osteosclerotic phenotype in transgenic mice. In the present study, we report a longer lived mouse model that also develops osteosclerosis and a genetic manipulation that completely rescues the phenotype. Conditionally cre-activated expression of Notch1 intracellular domain (NICD) in vivo exclusively in committed osteoblasts caused massive osteosclerosis with growth retardation and abnormal vertebrae. Importantly, selective deletion of a Notch nuclear effector - Rbpj in osteoblasts completely suppressed the osteosclerotic and growth retardation phenotypes. Furthermore, cellular and molecular analyses of bones from the rescued mice confirmed that NICD-dependent molecular alterations in osteoblasts were completely reversed by removal of the Rbpj pathway. Together, our observations show that the osteosclerosis due to activation of Notch signaling in osteoblasts is canonical in nature since it depends solely on Rbpj signaling. As such, it identifies Rbpj as a specific target for manipulating Notch signaling in a cell autonomous fashion in osteoblasts in bone diseases where Notch may be dysregulated.

Keywords: Osteosclerosis, genetic mouse model, therapeutics, Notch signaling, Osteoblast

Introduction

Physiological bone remodeling is a process specified by a balance between bone formation by osteoblasts and bone resorption by osteoclasts. An imbalance in bone remodeling contributes to several pathological conditions including osteosclerosis, osteopetrosis, and osteoporosis. Those conditions are estimated to affect tens of millions people. Osteosclerosis is a bone disorder characterized by an abnormal thickening and progressive increase in bone mass of the skeleton due to an increased number of osteoblasts⁽¹⁾. In contrast, osteopetrosis results from a primary decrease in osteoclastic function⁽²⁾. Currently, extrinsic causative factors (e.g. fluorosis) that are associated with osteosclerosis have been reported ^(3-6). However, reports of causative genetic factors or signaling pathways that are involved in osteosclerosis have been limited ^(7-10).

Notch signaling is one of several evolutionarily conserved signaling pathways in the development of multi-cellular organisms and its temporal-spatial expression can specify diverse cellular events, including proliferation, differentiation, apoptosis, stem cell maintenance, and binary cell-fate specification⁽¹¹⁾. In mammals and Drosophila, both Notch canonical and non-canonical pathways exist although the non-canonical signaling is less well understood in mammals⁽¹²⁾. The canonical pathway requires the DNA binding protein RBPJ/Su(H) as a nuclear effector for signal transduction and commences with the binding of transmembrane Notch receptors (four in mammals and one in Drosophila) to its ligands. The binding triggers ADAM10- and Presenilin-mediated proteolytic cleavages that then liberate the membrane-bound Notch intracellular domain (NICD), which eventually translocates into the nucleus. In the canonical pathway, NICD forms a complex with the transcription factors RBPJ and MAML to regulate downstream genes, such as HES1 and HEY1, the two classic bHLH targets of Notch. The functional importance of the non-canonical Notch pathway is far less well understood although data primarily generated in the Drosophila and now also in mammalian cell systems are emerging^(11,13). Molecular mechanisms controlling this pathway are still unclear⁽¹²⁾.

From the onset of this decade to date, human genetic studies have indentified mutations in many components of the Notch signaling pathway that cause skeletal defects⁽¹⁴⁾. For example, mutations in the DLL3 gene and subsequently in MESP2 and LFNG genes were identified to cause spondylocostal dysostosis (SCDO), an inherited disorder characterized by abnormal vertebral formation and patterning. Concurrently, studies of the corresponding mutant mouse models have increased our understanding of these birth defects⁽¹⁵⁾. Furthermore, aberrant Notch signaling plays an important role in the pathogenesis of leukemia and several other types of cancer⁽¹⁶⁾. Intensive studies have indentified novel activating mutations in the Notch1 receptor, which are accountable for more than 50% of human T-cell acute lymphoblastic leukemia (T-ALL) samples⁽¹⁷⁾. Importantly, our and other studies suggest that activation of Notch signaling contributes to the pathogenesis of human osteosarcoma^(18-20).

Recently, the in vivo effects of Notch signaling in osteoblast specification, proliferation, and differentiation have been demonstrated, in addition to its regulation of osteoclast activity^(21-24). These studies linked a homeostatic function of Notch to adult bone diseases including osteopenia, osteoporosis, and osteosclerosis. Nevertheless, whether and how both canonical and non-canonical Notch pathways contribute to the Notch function in those pathological conditions in the skeletal system is still unknown. To address the relative contributions of canonical vs. non-canonical Notch signaling in osteosclerosis due to Notch gain of function, we employed genetically engineered mouse models with removal of Rbpj expression. First, we established a bitransgenic mouse model for osteosclerosis through cre-activated expression of the Notch1 intracellular domain (NICD) exclusively in committed osteoblasts. Then we bred the Notch gain of function (GOF) bigenic mice with a floxed allele of Rbpj^f/f to study the effects of conditional Rbpj deletion in osteoblasts. We hypothesized that if NICD mediates the skeletal effects and if the effect is primarily via the canonical pathway, (i.e., Rbpj-dependent), then deletion of Rbpj within osteoblasts should rescue most if not all of the phenotypic alterations. Likewise, we reasoned that if the effect is mediated via both the canonical and non-canonical, then deletion of Rbpj may only partially rescue the phenotypic alterations. Here, we report that osteosclerosis due to Notch gain of function is solely Rbpj-dependent suggesting that Rbpj-independent signaling is dispensable for this model of Notch induced bone pathology in vivo.

Materials and Methods

Animals

Conditional knockout mice Rbpj^flox/flox, Col1a1 2.3kb Cre transgenic mice (TG ^{Col1a1 2.3kb Cre/+}), and Rosa^Notch transgenic mice (TG^{NICD flox/+}) have been described previously ^(25-27). These mice were maintained on a hybrid 129 X C57BL/6 background. PCR genotyping was performed as described. Animals were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All mice were housed in a specific pathogen free facility and under light, temperature and humidity controlled conditions. These studies were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee.

Skeletal Preparation and Histology

Whole-mount skeletal preparations stained with Alcian blue 8GX (Sigma Aldrich) and Alizarin red S (Sigma Aldrich) were prepared as described previously ⁽²¹⁾. Littermates of control, GOF mutant, or GOF:Rbpj^f/f mice were sacrificed at 3 to 8 weeks of age and whole skeleton were fixed in 10% neutral-buffered formalin overnight. We sectioned paraffin-embedded decalcified bones to a 6-μm thickness and stained the section with H&E, toluidine blue, and Goldner's stains using standard protocols. Limb skeletal preparations were photographed with a Nikon 5.0 megapixel digital camera mounted atop of a Nikon SMZ1500 dissecting scope. All microscope and camera settings were identical for the capture of all images. Digital image files were uploaded to an Axiovision software (Carl Zeiss Vision, Munchen-Hallbergmoos, Germany) connected to a Zeiss Axioplan 2 scope and scale equivalency was achieved via transfer of stage micrometer calibration information. These procedures allowed for direct comparison of each image.

Bone micro-computed tomography (microCT) measurements

We analyzed microCT scanning of the trabecular bone of the distal femur by the microCT system (mCT-40, Scanco Medical) using a standard protocol from the MicroCT Core at the Baylor College of Medicine. Briefly, femora from 8-week-old mice were dissected, cleaned of soft tissue and fixed two days in 10% neutral buffered formalin and thereafter stored in 70% ethanol at 4 degrees. Trabecular bone is analyzed at the distal end of the femur starting from the end of the growth plate. 75 slices covering a total length of 1.20 mm were evaluated within the secondary spongiosa at high resolution. A threshold value of 210 was used for the three dimensional evaluation.

RNA extraction and quantitative reverse transcription PCR analysis

RNA extractions, first-strand cDNA syntheses, and real time PCR were carried out as previously described ^(21,28). Briefly, we extracted total RNA from calvaria of 3-week-old mice (n = 4) with TRIzol reagent (Invitrogen). We synthesized cDNAs from extracted RNA with the Superscript III First Strand RT-PCR kit (Invitrogen). We performed real-time quantitative PCR amplifications in a LightCycler (Roche). We used the genes encoding GAPDH and β2-microglobulin as reference genes for the quantity and quality of the cDNAs in real-time PCR assays. The relative amount of each mRNA was determined by the comparative CT method.

Statistical analyses

Results are reported as the mean values ± SD. Statistical significance (P values) was computed by using Student's t-test. A P value of <0.05 was considered statistically significant.

Results

Generation of cre-activated Notch gain of function bitransgenic mice

We previously showed that the constitutively active Notch1 Intracellular Domain (NICD) driven by the 2.3 kb collagen type 1 (Col1a1) promoter was selectively expressed in committed osteoblasts in conventionally generated transgenic mice⁽²¹⁾. These mutant mice displayed a dramatic increase in osteoblast number, proliferation and bone formation resulting in a severe osteosclerotic phenotype, i.e. increased bone formation over bone resorption. Histological analyses of these mice indicated highly disorganized woven bone formation suggesting a maturation defect in committed osteoblastic precursors. Unfortunately, these transgenic founder mice exhibited variable NICD expression and early lethality, which prevented subsequent intercrosses and genetic studies. To generate a longer-lived mouse model for dissecting the genetic modifiers of this osteosclerosis phenotype, we established a bitransgenic line with consistently lower levels of NICD expression and extended longevity. To achieve this, we crossed the Rosa^Notch transgenic mice (TG^{NICD flox/+}) with the Col1a1 2.3kb Cre transgenic line (TG ^{Col1a1 2.3kb Cre/+}) to generate single copy NICD over expression in committed osteoblasts (Fig. 1A). Importantly, the cre-activated bitransgenic gain-of-function (TG ^{NICD flox/+}; TG^{Col1a1 2.3kb Cre/+} or GOF) mice produced a milder osteosclerotic phenotype with long term survival (Fig. 1B-D). The TG^{NICD flox/+} mice have a NICD and a tracer EGFP cDNA with an upstream flox/STOP cassette inserted into the Rosa26 locus⁽²⁹⁾. Expression of tissue specific Cre recombinase activates this gain of function allele in committed osteoblasts in those bitransgenic GOF mice.

FIGURE 1

Cre recombinase-activation of Notch gain of function in bitransgenic mice causes growth retardation and kinky tail phenotypes. (A) Breeding scheme of NICD bitransgenic GOF mice. The transgenic mice (TG^{NICD flox/+}) were generated by Doug Melton using a (more ...)

Phenotypic characterization of NICD bitransgenic mice

The cre-activated bitransgenic GOF mice were indistinguishable from their control littermates mice at birth (wild-type or TG^{NICD flox/+} or TG^{Col1a1 2.3kb Cre/+} genotype, herafter referred to as control mice). They had similar body weight to the control at postnatal day 6 (P6) (Fig. 1B and 1D). However, from 2 weeks of age, the GOF mice showed progressive growth retardation and a unique kinky tail phenotype, which can be used to distinguish between the GOF and control mice (Fig. 1C and 1D). The GOF mice had a significantly smaller body weight compared with the control mice at P24 (p<0.001) (Fig. 1B). Analysis of those mutant mice at 4 weeks of age showed thickened bones consistent with a generalized osteosclerotic phenotype in skulls, rib cages, tail vertebrae, and limb long bones as shown in the skeletal preparation stained with Alcian Blue and Alizarin Red (Fig. 2A-D). The increased thickness of calvarial bone indicates that bones derived from both intramembranous or endochondral ossification were similarly affected. Histologically, these GOF mice recapitulated the phenotype of conventional osteoblast-specific NICD transgenic mice that we previously generated⁽²¹⁾. In those GOF mice, trabecular bone was predominantly composed of immature woven, rather than lamellar bone, while marrow space was enclosed by fibrotic containing cells with morphologic features of early osteoblastic precursors. The cortices of the bones were also composed of woven bone, and this phenotype was typically present in 2 month-old mice (Fig. 4Ae). Goldner's staining of femora from these GOF mice showed significantly altered architecture of trabecular bone (Fig. 4F). A detailed quantitative bone morphometric analysis of 2-months old mice confirmed the significant changes in trabecular bone volume, numbers, thickness, and spaces, which would be consistent with the high bone mass due to increased bone formation (Fig. 4B-E). Notably, the trabecular BV/TV value of GOF mice was increased by >6-fold over control mice (Fig. 4E).

FIGURE 2

Notch gain of function causes generalized osteosclerosis in the bitransgenic mice. Skeletal preparation of 4-week-old GOF mice showed thickened bones of severe osteosclerotic phenotype in skull (A), ribs (B), tail vertebrae (C), and forelimb (D). Skeletal (more ...)

FIGURE 4

The osteosclerotic phenotype in the Notch gain of function (GOF) mice is reversible. (A) Micro-CT reconstruction of distal (a, c) or whole femur (b) from 2-month-old mice shows a shortening in GOF mice compared to control or GOF:Rbpj^f/f. (d-f) is sagittal (more ...)

Consequences of genetic removal of Rbpj pathway in the NICD bitransgenic mice

The GOF mice show severe and more constant osteosclerotic phenotype. Moreover they are more amenable to genetic and therapeutic studies. However, while classical Notch target genes are up-regulated in this model, it is unclear whether the molecular alterations are dependent on canonical or non-canonical Notch signaling within osteoblasts. We hypothesized that the pathological function of Notch might dependend upon contributions from both the canonical and non-canonical Notch pathway, i.e. NICD/Rbpj-dependent signaling in the former and NICD dependent but Rbpj independent in the latter. To probe this hypothesis and to attempt a genetic rescue of the osteosclerotic phenotype, we bred the osteoblast-specific bitransgenic GOF mice onto a floxed allele of Rbpj (Fig. 3A). Initially, our study showed that the osteoblast-specific deletion of Rbpj (TG ^{Col1a1 2.3kb Cre/+}; Rbpj^flox/flox) did not cause any bone phenotype in those conditional knockout mice at two months of age when we applied as an end point for the rescue experiments. This result was consistent with previous loss of function studies of Notch signaling in mice with deletion of Notch1 and Notch2 receptors (TG ^{Col1a1 2.3kb Cre/+}; Notch1^-/fNotch2^f/f, C1NN mice) or deletion of Psen1 and Psen2 (TG ^{Col1a1 2.3kb Cre/+}; Psen1^f/fPsen2^-/-, DKO mice)^(21,22). In this study, the addition of the Rbpj^flox/flox allele in the GOF mice completely and dramatically rescued the growth retardation and osteosclerotic phenotypes of GOF mice (TG ^{NICD flox/+}; TG ^{Col1a1 2.3kb Cre/+}; Rbpj^flox/flox or GOF:Rbpj^f/f or rescued mice) (Fig. 3B). Additionally their body weights and skeletal preparation showed no significant difference from control littermate mice at six weeks of age (Fig. 3C-D). Together, our results indicate that deletion of the Rbpj pathway can block osteosclerosis in GOF mice and the pathological function of Notch depends solely on canonical signaling in committed osteoblasts.

FIGURE 3

Genetic addition of the Rbpj^flox/flox allele rescues growth retardation and kinky tail phenotypes in the bitransgenic mice. (A) Breeding scheme of NICD bitransgenic GOF mice with Rbpj^flox/flox mice. These GOF:Rbpj^f/f mice have an activating NICD allele (more ...)

Bone morphometric and molecular analysis of NICD bitransgenic and rescued osteoblasts

To determine whether the morphological rescue correlated with a complete rescue at the tissue and molecular levels, we first performed bone morphometric analyses by microCT imaging on the femora in 8-week-old control, GOF and GOF:Rbpj^f/f rescued mice. The distribution of trabecular bone and thickness of cortical bone in the rescued femora were comparable to those of control mice (Fig. 4Ad and 4Af), but significantly different from that of GOF mice (Fig. 4Ae). Furthermore, trabecular bone volume and morphometric parameters measuring trabecular number, thickness, and spacing were normalized in the rescued femora compared with GOF mice (Fig. 4B-E). The result from Goldner's staining of femora in the GOF:Rbpj^f/f mice was consistent with that from microCT analysis (Fig. 4F). Thus, these data suggest that the tissue phenotype observed in GOF mice is also reversed by removal of the canonical notch signaling in osteoblasts.

To assess the changes associated with NICD-induced osteosclerosis and the effects of Rbpj deletion, we performed quantitative real time RT-PCR (qRT-PCR) of 4-week-old calvarial total RNA from the control, GOF, and GOF:Rbpj^f/f mice. First, the expression of cell cycle markers cyclin D1 and cyclin A1 were increased in the GOF and then normalized in the Rbpj deleted cavarial osteoblasts. In contrast, p53, another important cell cycle regulator implicated in bone homeostasis, was expressed at a similar level in all of three groups (Fig. 5A). Our analysis also showed increased abundance of early osteoblastic differentiation markers, including osterix (Osx), Runx2, type I collagen α1 (Col1a1), alkaline phosphatase (ALP) and bone sialoprotein (Bsp), and decreased expression of terminal osteoblast differentiation markers, including Osteocalcin (Oc), in the GOF mice. This is similar to the pattern that we observed in the traditional NICD transgenic model⁽²¹⁾. Importantly, all of these were normalized in the rescued mice on an Rbpj mutant background (Fig. 5C). We further analyzed the expression of osteoclastic markers, including Osteoprotegerin (Opg), RANK ligand (RANKL), Tartrate-resistant acid phosphatase (TRAP) and macrophage colony–stimulating factor (M-CSF). The increased expression of both pro- (RANKL and M-CSF) and anti- (Opg) osteoclastic differentiation factors in the GOF were reduced to the normal level in the GOF:Rbpj^f/f mice (Fig. 5B). Notably, we showed that Notch classical target gene Hey1 was significantly increased (about 25 fold) in the GOF, but normalized in the rescued osteoblasts. The nuclear EGFP as a tracer of transgene was significantly expressed in both GOF and GOF:Rbpj^f/f mice although its expression in GOF was the higher because of significantly increased population of immature osteoblasts (Col1a1 positive cells) in calvaria (Fig. 5D). Together, our data show that the morphological and tissue correction of the NICD-dependent osteosclerosis was also completely rescued on a molecular level by the deletion of Rbpj.

FIGURE 5

The molecular signature of osteosclerosis is reversed in the GOF:Rbpj^f/f osteoblasts. Calvarial total RNAs were obtained from the control, GOF, and GOF:Rbpj^f/f mice at three weeks of age (n=4). The transcriptional profile for cell cycle, osteoblast, and (more ...)

Discussion

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 (Fig. 5C). 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 (Fig. 4Ae and F). 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⁾, Osteopoikilosis⁽³⁷⁾, 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 (Fig. 5C) 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:Rbpj^f/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.

Acknowledgments

We thank G. Karsenty (Columbia University) for Col1a1 2.3kb Cre mice, D. Melton (Harvard University) for Rosa^Notch mice, and T. Honjo (Kyoto University) for Rbpj^floxflox mice. This work was supported by US National Institutes of Health grants DE016990 (B.L.) and HD22657 (B.L.) and NIH training grant T32 AI053831 (J.T.) and DK60445 (J.T.).

Footnotes

Conflict of interest: The authors state that they have no conflicts of interest.

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