Abstract
Transgenic overexpression of the Notch1 intracellular domain inhibits osteoblast differentiation and causes osteopenia, and inactivation of Notch1 and Notch2 increases bone volume transiently and induces osteoblastic differentiation. However, the biology of Notch is cell-context-dependent, and consequences of Notch activation in cells of the osteoblastic lineage at various stages of differentiation and in osteocytes have not been defined. For this purpose, RosaNotch mice, where a loxP-flanked STOP cassette placed between the Rosa26 promoter and the NICD coding sequence, were crossed with transgenics expressing the Cre recombinase under the control of the Osterix (Osx), Osteocalcin (Oc), Collagen 1a1 (Col2.3), or Dentin matrix protein1 (Dmp1) promoters. At 1 month, Osx-Cre;RosaNotch and Oc-Cre;RosaNotch mice exhibited osteopenia due to impaired bone formation. In contrast, Col2.3-Cre;RosaNotch and Dmp1-Cre;RosaNotch exhibited increased femoral trabecular bone volume due to a decrease in osteoclast number and eroded surface. In the four lines studied, cortical bone was either not present, was porous, or had the appearance of trabecular bone. Oc-Cre;RosaNotch and Col2.3-Cre;RosaNotch mice exhibited early lethality so that their adult phenotype was not established. At 3 months, Osx-Cre;RosaNotch and Dmp1-Cre;RosaNotch mice displayed increased bone volume, and increased osteoblasts although calcein-demeclocycline labels were diffuse and fragmented, indicating abnormal bone formation. In conclusion, Notch effects in the skeleton are cell-context-dependent. When expressed in immature osteoblasts, Notch arrests their differentiation, causing osteopenia, and when expressed in osteocytes, it causes an initial suppression of bone resorption and increased bone volume, a phenotype that evolves as the mice mature.
Osteoblasts are derived from mesenchymal cells, and their number is determined by the rate of replication of precursors, their differentiation toward mature osteoblasts, and the death of mature cells (1–3). Factors that govern these events determine the osteoblastic cell pool and play a substantial role in the formation of new bone (3–5). Osteocytes are terminally differentiated osteoblasts that are embedded in the bone mineralized matrix in distinct lacunae (6, 7). Osteocytes are dendritic cells that communicate through a canalicular network with each other and with osteoblasts and lining cells, which are also derived from osteoblasts. Osteocytes play a fundamental role in mechanotransduction, and osteocyte-ablated mice exhibit bone loss and microstructural deterioration (8). Osteocytes and osteoblasts do not have redundant functions, and the impact of a regulatory signaling pathway may result in different outcomes when it operates in osteoblasts at various stages of cell differentiation or when it is activated in osteocytes.
Notch receptors (Notch 1 to 4) are single-pass transmembrane receptors that play a critical role in cell fate decisions (9–12). Notch regulates cell renewal in multiple organs and cell systems, and is involved in skeletal development and homeostasis, and in osteoblast differentiation (12–15). There are five Notch canonical ligands, which are Jagged1 and 2, and Delta Like1, 3, and 4 (9, 10, 12). Notch–ligand interactions result in the proteolytic cleavage and release of the Notch intracellular domain (NICD), which translocates to the nucleus and interacts with C Promoter Binding factor 1, suppressor of hairless or Lag-1 (CSL), also termed Rbpjκ, and with mastermind-like proteins to regulate transcription (16–19). This is named the Notch canonical signaling pathway, which leads to the induction of hairy enhancer of split (Hes) and Hes related with YRPW motif (Hey) transcription factors (20). Skeletal cells express Notch1 and Notch2 and low levels of Notch3 transcripts, but do not express detectable levels of Notch4 (21–23). Notch1 and Notch2 appear to be responsible for the effects of Notch in the skeleton.
Previously, we have reported that the overexpression of Notch1 NICD in vitro inhibits osteoblast differentiation and that its overexpression in osteoblasts in vivo under the control of a 3.6 kilobase (kb) fragment of the Type Iα1 collagen (Col1a1) promoter causes osteopenia (15). Studies performed by other investigators demonstrated that transgenic expression of Notch1 under the control of a 2.3-kb fragment of the Col1a1 promoter causes the formation of abundant woven bone, possibly because full osteoblastic maturity is not reached (13). In accordance with the observations described, the conditional deletion of Notch1 and Notch2 in the developing skeleton causes a transient increase in bone volume and induces the commitment of mesenchymal precursor cells toward cells of the osteoblastic lineage (13, 14).
Despite former observations indicating an inhibitory effect of Notch on osteoblastic differentiation, it is important to note that the biology of Notch is cell-context-dependent. The consequences of Notch activation on adult skeletal homeostasis in cells of the osteoblastic lineage at early stages of differentiation and in osteocytes have not been defined. It was our hypothesis that Notch would cause cell differentiation-dependent skeletal phenotypes. The intent of the present study was to define the function of Notch in vivo in cells of the osteoblastic lineage at various stages of differentiation and in osteocytes. For this purpose, we used the RosaNotch mouse model, where a STOP cassette, placed between the Rosa26 promoter and the Notch1 NICD coding sequence, is flanked by loxP sites (24, 25). RosaNotch mice were crossed with transgenics expressing the Cre recombinase under the control of the Osterix (Osx), the Osteocalcin (Oc), the 2.3 kb fragment of Col1a1 (Col2.3), or the Dentin matrix protein1 (Dmp1) promoter (26–30). These four models should result in the preferential expression of the NICD by undifferentiated cells of the osteoblastic lineage (Osx), differentiated osteoblasts (Oc), osteocytes (Dmp1), or osteoblasts and osteocytes (Col2.3) (27, 31–35). The skeletal phenotype of these four mouse models was established by microcomputed tomography (μCT) and histomorphometry, and we explored possible mechanisms involved.
Materials and Methods
RosaNotch conditional mice
RosaNotch mice were created by D. A. Melton (Harvard University, Cambridge, Massachusetts) and obtained from Jackson Laboratory (Bar Harbor, Maine) in a 129SvJ/C57BL/6 genetic background (24, 25). In these mice, the Rosa26 locus is targeted with a DNA construct encoding Notch1 NICD, preceded by a STOP cassette flanked by loxP sites, cloned downstream of the Rosa26 promoter. Expression of NICD from the targeted Rosa26 locus occurs following the excision of the STOP cassette by Cre recombination of loxP sequences (36, 37). To study the consequences of the Notch induction in undifferentiated cells of the osteoblastic lineage, we bred homozygous RosaNotch mice to heterozygous Osx-Cre mice in a C57BL/6 genetic background (Jackson Laboratory) to create Osx-Cre+/−;RosaNotch experimental and Osx-Cre−/−;RosaNotch littermate controls (28). Because the expression of Cre is under the control of the tet-off cassette, RosaNotch moms were treated with 625 mg/kg of doxycycline from the time of conception to delivery. To study the induction of Notch in mature osteoblasts, homozygous RosaNotch mice were bred with heterozygous transgenic mice expressing Cre under the control of the OSTEOCALCIN promoter (Oc-Cre), in a C57BL/6 genetic background obtained from T. Clemens (Baltimore, Maryland) (29, 30). To study the induction of Notch in mature osteoblasts and osteocytes, we bred homozygous RosaNotch mice with heterozygous transgenics expressing Cre under the control of the 2.3-kb fragment of the Col1a1 promoter (Col2.3-Cre) in a tropism to friend leukemia virus type B (FVB) background created by the laboratory of G. Karsenty and obtained from Mutated Mouse Regional Resource Center (Davis, California) (26). To study the induction of Notch in osteocytes, we mated homozygous RosaNotch mice with mice expressing Cre under the control of the Dmp1 promoter (Dmp1-Cre) in a C57BL/6 genetic background (27). All mating schemes created Cre+/−;RosaNotch experimental and RosaNotch littermate controls.
Genotyping of Osx-Cre, Oc-Cre, Col2.3-Cre, Dmp1-Cre, and RosaNotch transgenics was carried out by polymerase chain reaction (PCR) in tail DNA extracts (please see Supplemental Table 1A published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org). Deletion of the loxP flanked STOP cassette by the Cre recombinase was documented by PCR in DNA from dissected tibiae devoid of bone marrow of 1-month-old RosaNotch male mice using specific primers (Supplemental Table 1A). The induction of Notch in the skeleton was confirmed by documenting NICD and Notch target gene mRNA expression in calvarial extracts by real time reverse transcription (RT)–PCR. All animal experiments were approved by the Animal Care and Use Committee of Saint Francis Hospital and Medical Center.
Microcomputed tomography (μCT)
Bone microarchitecture of vertebrae and femurs from experimental and control mice was determined using a microcomputed tomographic instrument (μCT 40; Scanco Medical AG, Bassersdorf, Switzerland) calibrated weekly using a phantom provided by the manufacturer (38, 39). The vertebral body of L3 and femurs were scanned in 70% ethanol at high resolution, energy level of 55 kVp, intensity of 145 μA, and integration time of 200 ms. Trabecular bone volume fraction and microarchitecture were evaluated starting approximately 1.0 mm from the cranial side of the vertebral body, or 1.0 mm proximal from the femoral condyles. For L3 and femurs, a total of 100 and 160 consecutive slices, respectively, were acquired at an isotropic voxel size of 6 μm3 and a slice thickness of 6 μm, and chosen for analysis. Contours were manually drawn every 10 slices a few voxels away from the endocortical boundary to define the region of interest for analysis. The remaining slice contours were iterated automatically. Trabecular regions were assessed for total volume, bone volume, bone volume fraction (bone volume/total volume), trabecular thickness, trabecular number, trabecular separation, connectivity density, and structural model index using a Gaussian filter (σ = 0.8, support = 1) and user-defined thresholds (38, 39). For analysis of femoral cortical bone, contours were iterated across 100 slices along the cortical shell of the femoral midshaft, excluding the marrow cavity. Analysis of cortical thickness, bone volume/total volume, porosity, periosteal perimeter, endosteal perimeter, and material density were performed using a Gaussian filter (σ = 0.8. support = 1) and user-defined thresholds. Femoral images acquired with μCT were used to determine femoral length.
Bone histomorphometric analysis
Static and dynamic histomorphometry was carried out on experimental and control mice after they were injected with calcein, 20 mg/kg, and demeclocycline, 50 mg/kg, at an interval of 2 d for 1-month-old animals and 7 d for 3-month-old animals. Col2.3-Cre;RosaNotch female mice were not injected with either calcein or demeclocycline because they appeared frail and early lethality was noted. Mice were sacrificed by CO2 inhalation 2 d after the demeclocycline injection. Longitudinal sections of femurs, 5 μm thick, were cut on a microtome (Microm, Richards-Allan Scientific, Kalamazoo, Michigan) and stained with 0.1% toluidine blue or von Kossa. Static parameters of bone formation and resorption were measured in a defined area between 360 μm and 2160 μm from the growth plate, using an OsteoMeasure morphometry system (Osteometrics, Atlanta, Georgia) (40). For dynamic histomorphometry, mineralizing surface per bone surface and mineral apposition rate were measured on unstained sections under ultraviolet light, using a triple diamidino-2-phenylindole fluorescein texas red set long pass filter, and bone formation rate was calculated. The terminology and units used are those recommended by the Histomorphometry Nomenclature Committee of the American Society of Bone and Mineral Research (40).
Real-time RT-PCR
Total RNA was extracted from calvariae or from femurs following the removal of the bone marrow by centrifugation, and mRNA levels determined by real-time RT-PCR (41, 42). For this purpose, RNA was reverse-transcribed using iScript RT-PCR kit (BioRad, Hercules, California), according to manufacturer's instructions, and amplified in the presence of specific primers (Supplemental Table 1B) and iQ SYBR Green SuperMix (BioRad) at 60°C for 45 cycles. Transcript copy number was estimated by comparison with a standard curve constructed using Notch1 (Thermo Scientific, Rockford, IL), Hey1, Hey2 (both from T. Iso, Los Angeles, CA), or HeyL (from D. Srivastava, Dallas, Texas) (43, 44). Reactions were conducted in a CFX96 RT-PCR detection system (Bio-Rad), and fluorescence was monitored during every PCR cycle at the annealing step (43). Data were pooled from male and female mice and are expressed as copy number corrected for glyceraldehyde-3-phosphate dehydrogenase (Gapdh), which was estimated by comparison to a standard curve constructed with Gapdh cDNA (from R. Wu, Ithaca, New York) (45). Control values were normalized to 1.0.
Serum osteoprotegerin (OPG)
Levels of mouse Opg were measured by enzyme-linked immuno-absorbent assay in serum from 1-month-old male control and experimental mice according to manufacturer's instructions (R and D Systems, Minneapolis, Minnesota).
Statistical analysis
Data are expressed as means ± SEM. Statistical differences were determined by unpaired Student's t test.
Results
General characteristics of RosaNotch mice
To induce the conditional activation of Notch in cells of the osteoblastic lineage at various stages of cell differentiation and in osteocytes, we mated homozygous RosaNotch mice with heterozygous Osx-Cre, Oc-Cre, Col2.3-Cre, or Dmp1-Cre mice, so that approximately half of the pups would express Notch (Cre+;RosaNotch) and approximately half would serve as controls (Cre-;RosaNotch). In preliminary experiments, we documented that at 1 month of age Osx-Cre, Oc-Cre, Col2.3-Cre, and Dmp1-Cre transgenics were not appreciably different from littermate controls not expressing Cre by femoral μCT (not shown). Cre mediated recombination of loxP sites flanking the STOP cassette was documented in extracts from tibiae of 1-month-old mice in the four lines studied (Supplemental Figure 1). NICD, Hey1, Hey2, and HeyL mRNA levels in calvarial extracts from Osx-Cre;RosaNotch, Oc-Cre; RosaNotch, Col2.3-Cre; RosaNotch, and Dmp1-Cre; RosaNotch mice were increased 1.5 to 4 fold (NICD and Hey1), 3- to 20-fold (Hey2) and 9- to 60-fold (HeyL) when compared with control RosaNotch littermates, confirming induction of Notch signaling in the skeleton of the four models studied (Figure 1). Notch activation was verified in femoral bone extracts, where Hey2 mRNA levels were increased (n = 4) 3- to 16-fold in the four lines of Cre+;RosaNotch mice when compared with RosaNotch littermate controls (all Ps < .05). The general appearance of Osx-Cre;RosaNotch or Dmp1-Cre;RosaNotch was normal, although they weighed less than littermate controls, and their femoral length was shorter (Figure 1). In contrast, Oc-Cre;RosaNotch and Col2.3-Cre;RosaNotch appeared frail, were substantially smaller than controls, had short femurs, kinky tails, and kyphosis, tended to drag their hind limbs, and exhibited early lethality; at 1 month of age ∼50% of Oc-Cre;RosaNotch and 33% of Col2.3-Cre;RosaNotch mice died, and Oc-Cre;RosaNotch did not survive beyond 2 months of age. There was no obvious sexual dimorphism in the general or skeletal phenotype of the four RosaNotch lines studied.
Figure 1.
Notch target gene mRNA expression (upper two panels), weight (middle panel), and femoral length (lower panel) in 1-month-old male and female RosaNotch mice (solid bars) and littermate controls (open bars). NICD, Hey1, Hey2, and HeyL mRNA levels in calvarial extracts from both sexes expressed as copy number corrected for Gapdh with controls normalized to 1.0, weight in grams, and femoral length in millimeters for Osterix (Osx)-Cre;RosaNotch, Osteocalcin (Oc)-Cre;RosaNotch, 2.3-kb Collagen (Col2.3)-Cre;RosaNotch, and Dentin matrix protein1 (Dmp1)-Cre;RosaNotch are shown. Values are means ± SEM, n = 3 to 6, except for mRNA levels, n = 2 to 14. *Significantly different from control mice, P < .05. NICD, notch intracellular domain; Hey, Hairy Enhancer of Split, related with YRPW motif.
Femoral microarchitecture and histomorphometry of RosaNotch mice at 1 month of age
μCT revealed that expression of Notch in undifferentiated osteoblastic cells (Osx-Cre;RosaNotch) caused marked femoral trabecular osteopenia (Table 1, Figure 2). Trabecular volume/tissue volume was decreased by 55% due to a decrease in trabecular thickness, and connectivity was reduced by 67%. Structural model index revealed that trabeculae were more rod-like than in control littermates. Cortical bone was modestly affected, and cortical porosity was increased by 2.4-fold in relationship to control littermates. Femoral histomorphometric analysis confirmed the osteopenic phenotype of Osx-Cre;RosaNotch mice, and both trabecular thickness and number were decreased (Table 2, Figure 3). Although osteoblast number was increased, mineralizing surface and bone formation rate were decreased, explaining the phenotype observed and confirming that under the influence of Notch, osteoblasts fail to achieve full maturation or functional capacity so that the surface forming bone was decreased (12). Osteoclast number and eroded surface were not different from controls in Osx-Cre;RosaNotch mice. Accordingly, serum Opg levels were not affected and were (means ± SEM; n = 4 to 6) 529 ± 34 ng/L in control male mice and 583 ± 20 ng/L in Osx-Cre;RosaNotch male littermates. Notch expression in mature osteoblasts (Oc-Cre;RosaNotch) also caused osteopenia. μCT revealed a 55% decrease in trabecular volume/tissue volume due to a decrease in the thickness of trabeculae, which were more rod-like than in control mice (Table 1, Figure 2). Cortical bone was not present. Histomorphometric analysis confirmed that the osteopenia did not reveal a change in osteoblast cell number, and fluorescence microscopy revealed disorganized calcein and demeclocycline labels so that quantitative dynamic histomorphometric analysis could not be performed (Table 2, Figure 3). This labeling pattern suggests abnormal bone formation and dysfunctional osteoblasts. Osteoclast number and eroded surface were modestly reduced in female but not in male Oc-Cre;RosaNotch mice, and serum Opg levels were reduced in Oc-Cre;RosaNotch male mice from (means ± SEM; n = 3 to 4) 562 ± 59 ng/L in control to 458 ± 16 ng/L in Oc-Cre;RosaNotch male littermates (P < .05).
Table 1.
Femoral Microarchitecture Assessed by μCT of 1-month-old Male and Female Conditional RosaNotch Mice and Controls
|
Osx-Cre;RosaNotch |
Oc-Cre;RosaNotch |
Col2.3-Cre;RosaNotch |
Dmp1-Cre;RosaNotch |
|||||
|---|---|---|---|---|---|---|---|---|
| Control | Notch | Control | Notch | Control | Notch | Control | Notch | |
| Males, trabecular bone | ||||||||
| Bone volume/tissue volume (%) | 8.4 ± 1.0 | 3.8 ± 0.4a | 6.1 ± 0.8 | 2.8 ± 0.4a | 14.6 ± 0.3 | 22.3 ± 1.9a | 5.8 ± 0.3 | 18.6 ± 1.6a |
| Trabecular separation (μm) | 239 ± 14 | 286 ± 20 | 261 ± 7 | 277 ± 24 | 169 ± 3 | 128 ± 7a | 257 ± 9 | 89 ± 5a |
| Trabecular number (1/mm) | 4.4 ± 0.3 | 3.7 ± 0.2 | 3.9 ± 0.1 | 3.8 ± 0.2 | 5.8 ± 0.1 | 8.2 ± 0.4a | 3.9 ± 0.1 | 11.6 ± 0.6a |
| Trabecular thickness (μm) | 31.2 ± 0.6 | 25.5 ± 0.1a | 29.5 ± 1.8 | 21.9 ± 1.1a | 34.5 ± 0.5 | 32.0 ± 0.7a | 28.7 ± 0.4 | 26.6 ± 0.7 |
| Connectivity density (1/mm3) | 377 ± 70 | 122 ± 34a | 185 ± 13 | 54 ± 13a | 459 ± 24 | 1612 ± 168a | 202 ± 27 | 2274 ± 156a |
| Structure model index | 2.3 ± 0.1 | 3.0 ± 0.1a | 2.6 ± 0.1 | 3.3 ± 0.1a | 1.8 ± 0.1 | 1.5 ± 0.2 | 2.5 ± 0.1 | 2.3 ± 0.1 |
| Density of material (mg HA/cm3) | 969 ± 11 | 904 ± 19a | 896 ± 6 | 746 ± 24a | 910 ± 10 | 664 ± 34a | 877 ± 8 | 966 ± 40 |
| Males, cortical bone | ||||||||
| Bone volume/tissue volume (%) | 90.7 ± 0.3 | 77.8 ± 0.6a | Cortical bone not present | Cortical bone not present | 87.9 ± 2.0 | 45.3 ± 1.8a | ||
| Porosity (%) | 9.3 ± 0.3 | 22.2 ± 5.6a | 12 ± 2 | 55 ± 2a | ||||
| Cortical thickness (μm) | 106 ± 3 | 127 ± 9a | 99 ± 4 | 422 ± 11a | ||||
| Periosteal perimeter (mm) | 4.3 ± 0.1 | 4.3 ± 0.1 | 4.4 ± 0.1 | 5.1 ± 0.1a | ||||
| Endosteal perimeter (mm) | 3.6 ± 0.1 | 3.5 ± 0.1 | 3.8 ± 0.1 | 2.5 ± 0.1a | ||||
| Density of material (mg HA/cm3) | 1064 ± 7 | 1047 ± 9 | 1072 ± 14 | 941 ± 27a | ||||
| Females, trabecular bone | ||||||||
| Bone volume/tissue volume (%) | 4.9 ± 0.3 | 2.9 ± 0.7a | 6.0 ± 0.7 | 3.6 ± 0.4a | 13.7 ± 0.8 | 22.7 ± 3.6a | 5.5 ± 0.4 | 17.2 ± 2.2a |
| Trabecular separation (μm) | 261 ± 8 | 291 ± 35 | 273 ± 11 | 309 ± 29 | 174 ± 6 | 146 ± 12 | 261 ± 13 | 90 ± 8a |
| Trabecular number (1/mm) | 3.9 ± 0.1 | 3.7 ± 0.5 | 3.7 ± 0.1 | 3.4 ± 0.3 | 5.7 ± 0.2 | 7.5 ± 0.7a | 3.9 ± 0.2 | 11.3 ± 0.9a |
| Trabecular thickness (μm) | 26.7 ± 0.1 | 22.9 ± 0.7a | 30.6 ± 1.2 | 23.5 ± 1.6a | 34.7 ± 0.5 | 33.0 ± 1.5 | 28.5 ± 0.1 | 25.9 ± 0.8a |
| Connectivity density (1/mm3) | 171 ± 15 | 57 ± 36a | 166 ± 22 | 117 ± 45 | 371 ± 23 | 1509 ± 189a | 182 ± 18 | 1887 ± 293a |
| Structure model index | 2.7 ± 0.1 | 3.4 ± 0.1a | 2.5 ± 0.1 | 2.9 ± 0.2b | 2.0 ± 0.1 | 1.2 ± 0.5 | 2.6 ± 0.1 | 2.4 ± 0.2 |
| Density of material (mg HA/cm3) | 912 ± 15 | 865 ± 13a | 888 ± 10 | 724 ± 18a | 911 ± 7 | 646 ± 20a | 917 ± 6 | 885 ± 6a |
| Females, cortical bone | ||||||||
| Bone volume/tissue volume (%) | 90.5 ± 0.5 | 58.0 ± 3.0a | Cortical bone not present | Cortical bone not present | 89.7 ± 0.5 | 34.7 ± 0.5a | ||
| Porosity (%) | 9.5 ± 0.5 | 42.0 ± 3.0a | 10 ± 1 | 65 ± 5a | ||||
| Cortical thickness (μm) | 102 ± 3 | 218 ± 24 | 99 ± 2 | 389 ± 17a | ||||
| Periosteal perimeter (mm) | 4.2 ± 0.1 | 4.6 ± 0.2 | 4.3 ± 0.1 | 4.9 ± 0.1a | ||||
| Endosteal perimeter (mm) | 3.6 ± 0.1 | 3.2 ± 0.1a | 3.6 ± 0.1 | 2.5 ± 0.1a | ||||
| Density of material (mg HA/cm3) | 1065 ± 13 | 993 ± 15a | 1051 ± 6 | 912 ± 15a | ||||
μCT was performed on femurs from Osterix (Osx)-Cre;RosaNotch, Osteocalcin (Oc)-Cre;RosaNotch, 2.3-kb Collagen (Col2.3)-Cre;RosaNotch, and Dentin matrix protein1 (Dmp1)-Cre;RosaNotch and control littermates. Values are means ± sem; n = 3 to 6.
Significantly different from controls by unpaired t test, P < 0.05;
P < 0.053.
Figure 2.
Representative μCT of femurs and L3 vertebrae from 1-month-old male Osterix (Osx)-Cre;RosaNotch, Osteocalcin (Oc)-Cre;RosaNotch, 2.3-kb Collagen (Col2.3)-Cre;RosaNotch, and Dentin matrix protein1 (Dmp1)-Cre; RosaNotch and littermate controls.
Table 2.
Femoral Histomorphometry of 1-month-old Male and Female Conditional RosaNotch Mice and Controls
|
Osx-Cre;RosaNotch |
Oc-Cre;RosaNotch |
Col2.3-Cre;RosaNotch |
Dmp1-Cre;RosaNotch |
|||||
|---|---|---|---|---|---|---|---|---|
| Control | Notch | Control | Notch | Control | Notch | Control | Notch | |
| Males | ||||||||
| Bone volume/tissue volume (%) | 16.1 ± 1.7 | 8.2 ± 1.6a | 9.6 ± 1.5 | 5.3 ± 0.4a | 8.4 ± 0.7 | 33.7 ± 5.7a | 8.0 ± 0.9 | 55.8 ± 2.2a |
| Trabecular separation (μm) | 200 ± 15 | 345 ± 46a | 333 ± 59 | 383 ± 29 | 317 ± 11 | 68 ± 13a | 355 ± 42 | 28 ± 2a |
| Trabecular number (1/mm) | 4.3 ± 0.3 | 2.8 ± 0.3a | 3.2 ± 0.5 | 2.6 ± 0.2 | 2.9 ± 0.1 | 10.3 ± 1.0a | 2.8 ± 0.3 | 15.9 ± 0.6a |
| Trabecular thickness (μm) | 37.3 ± 2.4 | 28.3 ± 3.3a | 30.0 ± 2.1 | 21.0 ± 0.9a | 28.9 ± 2.1 | 32.6 ± 3.7 | 29 ± 1.5 | 35 ± 1.2a |
| Osteoblast surface/bone surface (%) | 21.4 ± 1.4 | 28.4 ± 1.8a | 24.0 ± 1.8 | 21.5 ± 3.7 | 17.5 ± 2.5 | 12.5 ± 5.7 | 26 ± 3 | 28 ± 3 |
| Osteoblasts/bone perimeter (1/mm) | 21.6 ± 1.3 | 29.4 ± 2.5a | 26.0 ± 2.6 | 21.7 ± 4.0 | 18.8 ± 2.3 | 10.8 ± 4.9 | 26.1 ± 2.7 | 26.6 ± 2.8 |
| Osteoclast surface/bone surface (%) | 11.7 ± 0.8 | 11.6 ± 0.4 | 12.6 ± 1.4 | 12.0 ± 0.6 | 11.2 ± 1.0 | 5.7 ± 0.5a | 10 ± 0.6 | 3 ± 0.2a |
| Osteoclasts/bone perimeter (1/mm) | 7.9 ± 0.5 | 8.0 ± 0.3 | 7.9 ± 0.9 | 7.9 ± 0.5 | 7.4 ± 0.8 | 3.7 ± 0.4a | 6.8 ± 0.4 | 2.3 ± 0.2a |
| Eroded surface/bone surface (%) | 22 ± 1 | 21 ± 1 | 22 ± 2 | 24 ± 1 | 18 ± 2 | 11 ± 1a | 17 ± 1 | 7 ± 1a |
| Mineral apposition rate (μm/day) | 2.8 ± 0.2 | 2.2 ± 0.2 | Diffuse and fragmented labels | 3.0 ± 0.3 | 4.7 ± 0.5a | 2.9 ± 0.4 | 2.6 ± 0.43a | |
| Mineralizing surface/bone surface (%) | 7.9 ± 1.0 | 2.4 ± 0.8a | 8.3 ± 1.8 | 2.2 ± 1.1a | 6.4 ± 1.1 | 1.6 ± 0.4a | ||
| Bone formation rate (μm3/μm2/day) | 0.22 ± 0.05 | 0.05 ± 0.02a | 0.26 ± 0.07 | 0.11 ± 0.20 | 0.20 ± 0.05 | 0.04 ± 0.01a | ||
| Females | ||||||||
| Bone volume/tissue volume (%) | 10.9 ± 1.0 | 5.0 ± 1.1a | 7.8 ± 1.0 | 4.7 ± 0.4a | 10.3 ± 1.0 | 21.8 ± 5.5a | 8.1 ± 0.9 | 42.0 ± 2.9a |
| Trabecular separation (μm) | 248 ± 18 | 713 ± 251 | 373 ± 53 | 404 ± 28 | 282 ± 34 | 111 ± 17a | 354 ± 21 | 49 ± 6a |
| Trabecular number (1/mm) | 3.7 ± 0.2 | 1.9 ± 0.5a | 2.7 ± 0.3 | 2.4 ± 0.2 | 3.4 ± 0.3 | 7.7 ± 1.1a | 2.7 ± 0.1 | 12.3 ± 0.7a |
| Trabecular thickness (μm) | 29.8 ± 1.7 | 26.7 ± 1.2 | 29.4 ± 1.1 | 19.4 ± 1.0a | 30.2 ± 1.0 | 27.0 ± 2.6 | 30.3 ± 1.9 | 34.0 ± 0.9 |
| Osteoblast surface/bone surface (%) | 16.9 ± 2.5 | 30.7 ± 3.5a | 18.5 ± 1.7 | 22.1 ± 3.4 | 19.6 ± 1.9 | 10.8 ± 5.7 | 24.6 ± 3.0 | 31.0 ± 2.0 |
| Osteoblasts/bone perimeter (1/mm) | 17.0 ± 2.2 | 33.5 ± 5.2a | 18.8 ± 1.7 | 22.6 ± 3.8 | 18.7 ± 1.5 | 9.7 ± 5.2 | 24.9 ± 3.0 | 27.0 ± 1.7 |
| Osteoclast surface/bone surface (%) | 13.4 ± 0.9 | 13.6 ± 1.5 | 14.5 ± 0.8 | 11.8 ± 0.7a | 11.1 ± 0.4 | 4.8 ± 0.7a | 10.8 ± 0.7 | 5.4 ± 0.5a |
| Osteoclasts/bone perimeter (1/mm) | 8.7 ± 0.5 | 8.1 ± 0.6 | 10.2 ± 0.7 | 8.1 ± 0.4a | 7.6 ± 0.2 | 3.2 ± 0.3a | 7.3 ± 0.4 | 3.6 ± 0.4a |
| Eroded surface/bone surface (%) | 26 ± 1 | 23 ± 1 | 28 ± 2 | 23 ± 1a | 21 ± 1 | 9 ± 1a | 19 ± 1 | 11 ± 1a |
| Mineral apposition rate (μm/day) | 3.4 ± 0.4 | 2.1 ± 0.3a | Diffuse and fragmented labels | Not performed | 2.9 ± 0.3 | 2.9 ± 1.0 | ||
| Mineralizing surface/bone surface (%) | 4.6 ± 1.0 | 1.4 ± 0.5a | 6.7 ± 1.0 | 1.8 ± 0.3a | ||||
| Bone formation rate (μm3/μm2/day) | 0.16 ± 0.05 | 0.03 ± 0.01a | 0.20 ± 0.04 | 0.05 ± 0.02a | ||||
Bone histomorphometry was performed on femurs from 1-month-old male and female Osterix (Osx)-Cre;RosaNotch, Osteocalcin (Oc)-Cre;RosaNotch, 2.3 Collagen (Col2.3)-Cre;RosaNotch, and Dentin matrix protein1 (Dmp1)-Cre;RosaNotch and control littermates. Values are means ± sem; n = 3 to 8.
Significantly different from controls, P < 0.05 by unpaired t test.
Figure 3.
Representative histological sections and calcein/demeclocycline labeling of femoral sections from 1-month-old Osterix (Osx)-Cre;RosaNotch, Osteocalcin (Oc)-Cre;RosaNotch, 2.3-kb Collagen (Col2.3)-Cre;RosaNotch, and Dentin matrix protein1 (Dmp1)-Cre; RosaNotch and littermate controls. Sections from male mice were stained with von Kossa without counterstain (final magnification 40×) or unstained and examined under fluorescence microscopy (final magnification 400×).
In contrast to the osteopenic phenotype observed when Notch was expressed in immature and mature osteoblasts, the phenotype of mice expressing the NICD in osteoblasts and osteocytes (Col2.3-Cre;RosaNotch) or preferentially in osteocytes (Dmp1-Cre;RosaNotch) was distinct. Both RosaNotch models exhibited increased femoral bone volume at 1 month of age. Col2.3-Cre;RosaNotch mice exhibited a 1.5- to 2-fold increase in bone volume/tissue volume due to an increase in trabecular number, and trabecular thickness was modestly decreased (Table 1, Figure 2). Cortical bone was not present. Histomorphometric analysis confirmed the phenotype and revealed a significant decrease in osteoclast number and eroded surface (Table 2, Figure 3). In accordance, serum Opg levels were increased from (means ± SEM; n = 4 to 6) 434 ± 28 ng/L in control mice to 827 ± 113 ng/L in Col2.3-Cre;RosaNotch male littermates (P < .05). Dmp1-Cre;RosaNotch mice exhibited a 3- to 3.2-fold increase in trabecular bone volume/tissue volume due to an increase in trabecular number and had a pronounced increase in connectivity (4- to 10-fold) when compared with control mice. Cortical bone volume was increased by 3-fold, but cortical bone had the appearance of trabecular bone and a compact cortex was not observed, so that the cortical bone was porous (Table 1, Figure 2). Histomorphometric analysis confirmed the increase in trabecular bone volume, and revealed decreased osteoclast number and eroded surface (Table 2, Figure 3). Mineral apposition rate, mineralizing surface, and bone formation rate were decreased, indicating suppressed bone remodeling. The histological appearance and number of osteocytes was not appreciably different in Dmp1-Cre;RosaNotch mice when compared with controls. In accordance with the phenotype observed, serum Opg levels were increased from (means ± SEM; n = 6) 529 ± 17 ng/L in control mice to 698 ± 28 ng/L in Dmp1-Cre;RosaNotch male littermates (P < .05).
Vertebral microarchitecture of RosaNotch mice at 1 month of age
Vertebral microarchitectural analysis confirmed the osteopenic phenotype in 1-month-old Osx-Cre;RosaNotch and Oc-Cre;RosaNotch mice, and the increased trabecular bone volume in Dmp1-Cre;RosaNotch male mice (Table 3, Figure 2). Surprisingly, Col2.3-Cre;RosaNotch mice exhibited vertebral osteopenia so that at femoral sites they phenocopied the osteocytic phenotype of Dmp1-Cre;RosaNotch mice, whereas at vertebral sites they manifested the osteoblastic phenotype of Oc-Cre;RosaNotch mice.
Table 3.
Vertebral Microarchitecture Assessed by μCT of 1-month-old Male and Female Conditional RosaNotch Mice and Controls
|
Osx-Cre;RosaNotch |
Oc-Cre;RosaNotch |
Col2.3-Cre;RosaNotch |
Dmp1-Cre;RosaNotch |
|||||
|---|---|---|---|---|---|---|---|---|
| Control | Notch | Control | Notch | Control | Notch | Control | Notch | |
| Males | ||||||||
| Bone volume/tissue volume (%) | 8.5 ± 0.3 | 7.0 ± 0.4a | 7.8 ± 0.6 | 3.7 ± 0.9a | 13.2 ± 0.1 | 3.9 ± 0.5a | 7.8 ± 0.9 | 14.0 ± 2.5a |
| Trabecular separation (μm) | 212 ± 4 | 200 ± 4 | 197 ± 6 | 269 ± 14 | 221 ± 0.5 | 195 ± 1.1 | 196 ± 4 | 163 ± 26 |
| Trabecular number (1/mm) | 4.7 ± 0.1 | 5.1 ± 0.1 | 5.1 ± 0.2 | 3.9 ± 0.2a | 4.6 ± 0.1 | 5.3 ± 0.3 | 5.1 ± 0.1 | 7.4 ± 1.2 |
| Trabecular thickness (μm) | 27.4 ± 0.5 | 26.7 ± 0.7 | 25.4 ± 0.7 | 26.5 ± 0.2 | 35.2 ± 0.4 | 27.8 ± 0.2a | 24.2 ± 1.1 | 25.3 ± 1.2 |
| Connectivity density (1/mm3) | 322 ± 14 | 259 ± 32 | 335 ± 30 | 129 ± 72 | 245 ± 7 | 163 ± 29a | 384 ± 24 | 1372 ± 317a |
| Structure model index | 2.0 ± 0.1 | 2.7 ± 0.1a | 2.2 ± 0.1 | 3.6 ± 0.2a | 1.4 ± 0.1 | 3.7 ± 0.2a | 2.2 ± 0.1 | 2.5 ± 0.2 |
| Density of material (mg HA/cm3) | 914 ± 15 | 897 ± 20 | 925 ± 11 | 868 ± 34 | 896 ± 8 | 812 ± 40 | 906 ± 15 | 797 ± 9a |
| Females | ||||||||
| Bone volume/tissue volume (%) | 7.9 ± 0.2 | 8.2 ± 1.0 | 6.5 ± 0.9 | 2.9 ± 0.3a | 13.1 ± 0.7 | 4.6 ± 0.7a | 7.0 ± 0.8 | 8.3 ± 1.6 |
| Trabecular separation (μm) | 198 ± 5 | 197 ± 6 | 224 ± 17 | 255 ± 7 | 214 ± 5 | 193 ± 9 | 210 ± 4 | 160 ± 26a |
| Trabecular number (1/mm) | 5.1 ± 0.1 | 5.3 ± 0.2 | 4.6 ± 0.3 | 4.0 ± 0.1 | 4.7 ± 0.1 | 5.3 ± 0.3 | 4.8 ± 0.1 | 6.8 ± 1.1a |
| Trabecular thickness (μm) | 25.3 ± 0.7 | 26.2 ± 0.1 | 25.6 ± 1.0 | 27.2 ± 1.1 | 32.5 ± 0.1 | 26.4 ± 0.1a | 24.5 ± 1.1 | 21.6 ± 0.8 |
| Connectivity density (1/mm3) | 381 ± 40 | 497 ± 107 | 285 ± 47 | 40 ± 9a | 299 ± 16 | 239 ± 72 | 277 ± 21 | 577 ± 272 |
| Structure model index | 2.1 ± 0.1 | 2.9 ± 0.1a | 2.4 ± 0.3 | 3.7 ± 0.1a | 1.3 ± 0.1 | 3.6 ± 0.2a | 2.2 ± 0.1 | 3.1 ± 0.1a |
| Density of material (mg HA/cm3) | 904 ± 10 | 932 ± 20 | 925 ± 17 | 916 ± 22 | 928 ± 14 | 797 ± 26* | 885 ± 10 | 768 ± 22a |
μCT was performed on L3 vertebrae from Osterix (Osx)-Cre;RosaNotch, Osteocalcin (Oc)-Cre;RosaNotch, 2.3-kb Collagen (Col2.3)-Cre;RosaNotch, and Dentin matrix protein1 (Dmp1)-Cre;RosaNotch and control littermates. Values are means ± sem; n = 3 to 6.
Significantly different from controls by unpaired t test, P < 0.05.
Skeletal phenotype of mature RosaNotch mice
Oc-Cre;RosaNotch and 2.3Col-Cre;RosaNotch mice were frail and exhibited early lethality, and their adult skeletal phenotype could not be established. Femoral cancellous bone μCT revealed that Osx-Cre;RosaNotch mice had switched from an osteopenic phenotype at 1 month of age to a high bone mass phenotype at 3 months of age, resembling the phenotype of 1-month-old Col2.3-Cre;RosaNotch and Dmp1-Cre;RosaNotch mice. However, the increased trabecular bone volume was limited to the femur and not observed at vertebral sites (L3) in either male or female Osx-Cre;RosaNotch mice. μCT of cortical bone revealed increased porosity, decreased thickness, and a large endocortical perimeter in Osx-Cre;RosaNotch mature mice, suggesting enhanced bone resorption at cortical sites (Table 4). Femoral histomorphometry confirmed the increased bone volume secondary to an increased trabecular number. The number of osteoblasts was increased, but calcein-demeclocycline labeling revealed diffuse and fragmented labels in male mice, suggesting that the function of osteoblasts was not normal (Table 5). Dynamic histomorphometry in female mice revealed decreased bone formation. Osteoclast number was decreased in female Osx-Cre;RosaNotch mice.
Table 4.
Femoral and Vertebral Microarchitecture Assessed by μCT of 3-month-old Male and Female conditional RosaNotch Mice and Controls
| Femoral |
Vertebral |
|||||||
|---|---|---|---|---|---|---|---|---|
|
Osx-Cre;RosaNotch |
Dmp1-Cre;RosaNotch |
Osx-Cre;RosaNotch |
Dmp1-Cre;RosaNotch |
|||||
| Control | Notch | Control | Notch | Control | Notch | Control | Notch | |
| Males, trabecular bone | ||||||||
| Bone volume/tissue volume (%) | 4.9 ± 0.7 | 12.4 ± 0.3a | 2.4 ± 0.3 | 4.8 ± 0.9a | 5.8 ± 0.6 | 6.3 ± 1.0 | 5.9 ± 0.6 | 16.8 ± 1.8a |
| Trabecular separation (μm) | 240 ± 9 | 154 ± 18a | 313 ± 18 | 262 ± 19 | 254 ± 10 | 247 ± 13 | 251 ± 8 | 159 ± 9a |
| Trabecular number (1/mm) | 4.2 ± 0.2 | 7.1 ± 0.8a | 3.3 ± 0.2 | 4.0 ± 0.3 | 4.1 ± 0.2 | 4.2 ± 0.2 | 4.1 ± 0.1 | 6.9 ± 0.4a |
| Trabecular thickness (μm) | 33 ± 1 | 29 ± 3 | 33.2 ± 0.8 | 22.0 ± 0.9a | 27.6 ± 0.6 | 28.6 ± 1.0 | 27.3 ± 1.1 | 29.0 ± 1.5 |
| Connectivity density (1/mm3) | 141 ± 36 | 921 ± 203a | 31 ± 6 | 322 ± 75a | 246 ± 35 | 296 ± 31 | 226 ± 31 | 1970 ± 277a |
| Structure model index | 3.2 ± 0.1 | 2.9 ± 0.2 | 3.5 ± 0.1 | 2.9 ± 0.1a | 2.4 ± 0.2 | 2.6 ± 0.3 | 2.3 ± 0.1 | 2.3 ± 0.2 |
| Density of material (mg HA/cm3) | 987 ± 21 | 973 ± 17 | 991 ± 16 | 856 ± 17a | 943 ± 27 | 925 ± 34 | 907 ± 16 | 866 ± 36 |
| Males, cortical bone | ||||||||
| Bone volume/tissue volume (%) | 94.3 ± 0.4 | 86.8 ± 1.7a | Cortical bone could not be discerned from trabecular bone | |||||
| Porosity (%) | 5.7 ± 0.4 | 13.2 ± 1.7a | ||||||
| Cortical thickness (μm) | 184 ± 5 | 133 ± 3a | ||||||
| Periosteal perimeter (mm) | 4.70 ± 0.10 | 5.03 ± 0.15 | ||||||
| Endosteal perimeter (mm) | 3.34 ± 0.09 | 3.67 ± 0.07a | ||||||
| Density of material (mg HA/cm3) | 1254 ± 5 | 1217 ± 10a | ||||||
| Females, trabecular bone | ||||||||
| Bone volume/tissue volume (%) | 2.1 ± 0.4 | 10.7 ± 0.1a | 2.3 ± 0.3 | 4.1 ± 0.8a | 6.7 ± 0.9 | 5.2 ± 0.8 | 6.5 ± 0.4 | 14.1 ± 2.7a |
| Trabecular separation (μm) | 362 ± 24 | 162 ± 16a | 319 ± 12 | 226 ± 14a | 261 ± 17 | 277 ± 9 | 252 ± 5 | 162 ± 10a |
| Trabecular number (1/mm) | 2.8 ± 0.2 | 6.6 ± 0.6a | 3.2 ± 0.1 | 4.8 ± 0.4 | 4.1 ± 0.31 | 3.7 ± 0.1 | 4.0 ± 0.1 | 6.7 ± 0.5a |
| Trabecular thickness (μm) | 35 ± 2 | 25 ± 1a | 31.5 ± 0.1 | 21.0 ± 0.1a | 28.9 ± 0.7 | 26.5 ± 0.2 | 25.7 ± 0.8 | 25.4 ± 1.4 |
| Connectivity density (1/mm3) | 21 ± 6 | 758 ± 174a | 28 ± 6 | 97 ± 13 | 292 ± 52 | 289 ± 33 | 287 ± 26 | 1813 ± 450a |
| Structure model index | 3.4 ± 0.1 | 2.9 ± 0.2a | 3.4 ± 0.1 | 3.2 ± 0.1 | 2.3 ± 0.2 | 2.6 ± 0.2 | 1.8 ± 0.1 | 2.4 ± 0.3 |
| Density of material (mg HA/cm3) | 1051 ± 23 | 946 ± 18a | 984 ± 9 | 941 ± 28 | 930 ± 29 | 933 ± 36 | 963 ± 10 | 1060 ± 34a |
| Females, cortical bone | ||||||||
| Bone volume/tissue volume (%) | 94.4 ± 0.2 | 89.2 ± 2.1a | Cortical bone could not be discerned from trabecular bone | |||||
| Porosity (%) | 5.6 ± 0.2 | 10.8 ± 2.1a | ||||||
| Cortical thickness (μm) | 172 ± 5 | 136 ± 6a | ||||||
| Periosteal perimeter (mm) | 4.48 ± 0.02 | 4.64 ± 0.06a | ||||||
| Endosteal perimeter (mm) | 3.26 ± 0.05 | 3.41 ± 0.03a | ||||||
| Density of material (mg HA/cm3) | 1291 ± 8 | 1240 ± 10a | ||||||
μCT was performed on femurs and L3 from Osterix (Osx)-Cre;RosaNotch and Dentin matrix protein1 (Dmp1)-Cre;RosaNotch and control littermates. Values are means ± sem; n = 5 to 6.
Significantly different from controls by unpaired t test, P < 0.05.
Table 5.
Femoral Histomorphometry of 3-month-old Male and Female conditional RosaNotch Mice and Controls
|
Osx-Cre;RosaNotch |
Dmp1-Cre;RosaNotch |
|||
|---|---|---|---|---|
| Control | Notch | Control | Notch | |
| Males | ||||
| Bone volume/tissue volume (%) | 10.5 ± 1.5 | 25.3 ± 2a | 6.3 ± 0.3 | 16.0 ± 3.0a |
| Trabecular separation (μm) | 292 ± 29 | 102 ± 7a | 403 ± 9 | 189 ± 50a |
| Trabecular number (1/mm) | 3.2 ± 0.3 | 7.4 ± 0.3a | 2.3 ± 0.1 | 5.2 ± 0.7a |
| Trabecular thickness (μm) | 32.3 ± 2.2 | 33.9 ± 1.4 | 27.0 ± 0.9 | 29.8 ± 2.1 |
| Osteoblast surface/bone surface (%) | 14.3 ± 1.8 | 43.4 ± 2.3a | 12.5 ± 1.9 | 18.8 ± 4.0 |
| Osteoblasts/bone perimeter (1/mm) | 13.3 ± 1.8 | 29.2 ± 1.9a | 13.3 ± 1.6 | 16.0 ± 3.0 |
| Osteoclast surface/bone surface (%) | 8.1 ± 0.3 | 7.5 ± 0.8 | 8.9 ± 0.5 | 12.4 ± 1.8b |
| Osteoclasts/bone perimeter (1/mm) | 5.1 ± 0.2 | 4.6 ± 0.6 | 6.2 ± 0.4 | 8.5 ± 1.1b |
| Eroded surface/bone surface (%) | 14.0 ± 0.6 | 15.2 ± 1.6 | 15.8 ± 0.8 | 23.9 ± 2.4a |
| Mineral apposition rate (μm/day) | Diffuse and fragmented labels | Diffuse and fragmented labels | ||
| Mineralizing surface/bone surface (%) | ||||
| Bone formation rate (μm3/μm2/d) | ||||
| Females | ||||
| Bone volume/tissue volume (%) | 3.8 ± 0.5 | 25.7 ± 1.3a | 4.2 ± 0.4 | 17.1 ± 4.6a |
| Trabecular separation (μm) | 621 ± 60 | 93 ± 4a | 582 ± 54 | 193 ± 53a |
| Trabecular number (1/mm) | 1.6 ± 0.1 | 8.0 ± 0.3a | 1.7 ± 0.1 | 5.1 ± 1.0a |
| Trabecular thickness (μm) | 23.5 ± 1.4 | 31.9 ± 0.9a | 24.5 ± 0.7 | 32.0 ± 4.2 |
| Osteoblast surface/bone surface (%) | 22.4 ± 1.8 | 45.1 ± 1.4a | 24.4 ± 1.0 | 26.9 ± 3.4 |
| Osteoblasts/bone perimeter (1/mm) | 24.0 ± 1.4 | 36.2 ± 0.9a | 26.1 ± 1.4 | 23.2 ± 2.7 |
| Osteoclast surface/bone surface (%) | 9.0 ± 0.5 | 6.8 ± 0.3a | 14.4 ± 0.8 | 13.1 ± 1.6 |
| Osteoclasts/bone perimeter (1/mm) | 6.0 ± 0.4 | 4.7 ± 0.3a | 9.3 ± 0.6 | 8.8 ± 1.2 |
| Eroded surface/bone surface (%) | 13.3 ± 0.8 | 13.4 ± 0.7 | 21.6 ± 1.6 | 22.7 ± 1.9 |
| Mineral apposition rate (μm/day) | 1.7 ± 0.1 | 1.4 ± 0.1 | Diffuse and fragmented labels | |
| Mineralizing surface/bone surface (%) | 4.8 ± 1.7 | 1.2 ± 0.2a | ||
| Bone formation rate (μm3/μm2/d) | 0.08 ± 0.03 | 0.02 ± 0.01a | ||
Bone histomorphometry was performed on femurs from 3 month old male and female Osterix (Osx)-Cre;RosaNotch and Dentin matrix protein1 (Dmp1)-Cre;RosaNotch and control littermates. Values are means ± sem; n = 4 to 7.
Significantly different from controls, P < 0.05 by unpaired t test;
P < 0.058.
Dmp1-Cre;RosaNotch mice at 3 months of age exhibited a sustained increase in femoral trabecular bone volume and increased connectivity, but cortical bone could not be discerned from trabecular bone and the femur had the general appearance of osteosclerosis (Table 4). Vertebral μCT confirmed the femoral phenotype, and Dmp1-Cre;RosaNotch mice exhibited an increase in vertebral cancellous bone volume due to an increase in trabecular number; connectivity density was increased by ∼9-fold. In contrast to the results obtained at 1 month of age, femoral histomorphometry of Dmp1-Cre;RosaNotch mice revealed increased bone resorption, although this was observed only in male mice (Table 5). Bone formation could not be quantified because calcein-demeclocycline labels were diffuse and fragmented.
Discussion
Our findings demonstrate that Notch expression in cells of the osteoblastic lineage causes a cell-context-dependent skeletal phenotype. The conditional activation of Notch in the skeletal environment was achieved by expressing the Cre recombinase under the control of the Osx, Oc, Col2.3, or Dmp1 promoter, for the removal of a STOP cassette flanked by loxP sites upstream the Notch1 NICD. This resulted in Notch activation prenatally (Col2.3-Cre and Oc-Cre) and throughout the life of the animals studied so that the phenotype observed was in part due to effects on bone development, growth, and modeling (46–48). Osx is expressed at early stages of development in the mouse (embryonic day 13). Therefore, to ensure newborn viability, we prevented prenatal activation of Notch in Osx-Cre;RosaNotch mice by treating pregnant moms with doxycycline, as Cre transcription was under the control of a tet-off cassette (28, 34). Dmp1 is mostly expressed postnatally so that a prenatal phenotype was not expected (49). Activation of Notch in undifferentiated (Osx) or differentiated (Oc) cells of the osteoblastic lineage caused an initial decrease in trabecular bone volume secondary to a decrease in differentiated osteoblastic function. Accordingly, serum levels of Opg were either not changed (Osx-Cre;RosaNotch) or modestly decreased (Oc-Cre;RosaNotch). In contrast, activation of Notch preferentially in osteocytes caused an initial increase in trabecular bone volume due to a decrease in bone resorption. This phenotype correlated with a significant increase in serum Opg levels, which may explain the suppressed bone remodeling observed. It is of interest that Notch activation by the use of a Col2.3-Cre transgenic, known to be active in osteoblasts and osteocytes, caused a phenotype resembling that observed following Notch activation with an Osteocalcin promoter at the spine, and a phenotype resembling that observed following activation with the Dmp1 promoter at femoral sites. A plausible explanation for the results obtained is that vertebrae are composed primarily of trabecular bone, rich in osteoblasts, whereas femurs are composed of trabecular and cortical bone, and cortical bone contains a much greater number of osteocytes than trabecular bone. However, Col2.3-Cre transgenics were in an FVB genetic background, which could have influenced their phenotype since the genetic composition of the other transgenics used was C57BL/6. The induction of Notch in osteocytes did not cause obvious morphological changes of these cells at the microscopic level, and the expression of Dmp1, a gene preferentially expressed by osteocytes, was not suppressed in bone extracts from Dmp1-Cre; RosaNotch mice (E. Canalis, unpublished observations).
Col2.3-Cre;RosaNotch and Dmp1-Cre;RosaNotch exhibited increased bone mass secondary to an inhibition of bone resorption, which correlated with increased serum levels of Opg. Bone formation was not increased in either model. Indeed, bone formation was significantly decreased in Dmp1-Cre;RosaNotch mice, indicating suppressed bone remodeling. Although marked increases in bone volume are often found in response to increased bone formation, they also occur in conditions in which suppressed bone resorption and decreased remodeling are the dominant events, such as the inactivation of the receptor activator of nuclear factor-κB (RANK) and the transgenic overexpression of Opg (50–54). For instance, the transgenic overexpression of Opg causes a 2-fold increase in bone volume secondary to a greater number of bone trabeculae in the context of a 90% suppression of bone formation (53). The greater number of trabeculae probably represents preservation of previously formed bone, which failed to remodel. The phenotype of Dmp1-Cre;RosaNotch mice is analogous to the one reported in Opg transgenics, and may be explained by an increase in serum levels of Opg.
The phenotype of the Col2.3-Cre;RosaNotch mouse model is compatible with the one previously reported in transgenics expressing NICD under the control of the Col2.3 promoter, which was characterized by increased bone mass and osteosclerosis (13). Osteoblast number was increased in Col2.3-NICD transgenics, although osteoblast function appeared to be impaired since immature woven bone was formed. A similar increase in osteoblast number with suppressed bone formation was observed in Osx-Cre;RosaNotch mice, also indicative that the osteoblasts formed were dysfunctional. In agreement with our findings, a reduced number of osteoclasts was noted in Col2.3-NICD transgenics and increased osteoclastogenesis, bone resorption, and osteopenia were reported in Notch loss of function mouse models by the same authors (13). In contrast to the phenotype observed with gain of Notch function under the control of the 2.3-kb fragment of the type I collagen promoter, we reported that transgenics expressing the NICD under the control of a 3.6-kb fragment of the same promoter were osteopenic and exhibited early lethality (15). The phenotype was secondary to a decrease in osteoblast number and presumably bone formation. The differences in the phenotypes observed can be explained by the differential activation of the 2.3-kb and 3.6-kb fragments of the type I collagen promoter resulting in the arrest of osteoblastic cell differentiation at different stages of maturation (55). It is also possible that phenotypic differences in the two models reported are due to the greater and less restricted activity of the 3.6 kb than the 2.3-kb fragment of the type I collagen promoter to direct gene expression in cells of the osteoblastic lineage (48, 56). An inhibitory role of Notch in osteoblastogenesis was also reported following the inactivation of Notch1 and 2 in the limb bud, although the predominant effect in this model was on mesenchymal progenitor cell differentiation and chondrogenesis (14, 57). It is of interest that Notch inactivation during limb development results in a late osteopenic phenotype due to enhanced osteoclastogenesis, confirming the inhibitory effect of Notch on osteoclast differentiation and bone resorption observed in Col2.3-Cre;RosaNotch and Dmp1-Cre;RosaNotch models.
The phenotype of Osx-Cre;RosaNotch and Dmp1-Cre;RosaNotch evolved as the animals matured, and at 3 months of age Osx-Cre;RosaNotch expressed a phenotype resembling that observed with Dmp1-Cre;RosaNotch mice. It is probable that osteoblast precursors, where Notch was expressed following Osx-directed Cre recombination, eventually matured as osteocytes (58–60). Recent work has confirmed a rapid turnover of osteoblasts in vivo, and that a selected number of these cells differentiate into osteocytes (61). Furthermore, osterix-expressing cells are not only a source of osteoblasts but also of osteocytes in vivo, supporting the notion that selected cells expressing Notch under the control of the Osx promoter eventually matured and became embedded in the bone matrix as osteocytes. The phenotype of Dmp1-Cre;RosaNotch mice at 3 months of age revealed continued increase in bone volume although there was a tendency toward increased and not decreased bone remodeling. The studies presented document a unique phenotype characterized by markedly increased bone mass in mouse models of Notch gain of function in osteocytes, which are terminally differentiated osteoblasts embedded in the bone matrix (7). A central function of osteocytes is to sense mechanical signals and transduce these into biological signals that regulate skeletal cell function (7). Recently, we demonstrated that mechanical stimulation of the MLOY4 osteocytic cell line using fluid flow shear stress induces Notch signaling (E. Canalis, unpublished observations), leading to the hypothesis that Notch regulates mechanotransduction in osteocytes in addition to its distinct and previously unrecognized functional role in these cells.
The suppression of bone resorption by Notch is in accordance with the previously reported inhibitory effect of Notch on osteoclastogenesis. Notch inhibits osteoclast function by direct effects on osteoclast precursors and indirectly through its effects on cells of the osteoblastic lineage (14, 21). Inactivation of Notch in osteoclast precursors causes increased osteoclastogenesis, and its inactivation in osteoblasts causes a modest increase in the expression of RANK ligand and a profound decrease in the expression of Opg, and as a consequence increased osteoclastogenesis (14, 21). In accordance with these observations, we report increased serum levels of Opg and have also detected increased expression of Opg mRNA in calvarial extracts from Dmp1-Cre;RosaNotch mice (E. Canalis, unpublished observations). The pronounced inhibition of bone resorption in mice expressing Notch preferentially in osteocytes is in accordance with the recently discovered function of osteocytes in bone remodeling and bone resorption (62–64).
A limitation of the present work is that the Cre recombinase used to activate Notch signaling was under the control of promoters that overlap in their expression pattern in cells of the osteoblastic lineage. Whereas Osteocalcin and Col2.3 are preferentially expressed by osteoblasts, their expression is not restricted to these cells and is also observed in osteocytes (47, 61, 65). Similarly, Dmp1 is preferentially, but not exclusively, expressed by osteocytes (65). As a consequence, preferential but not cell-specific Notch activation was responsible for the phenotypes reported.
In conclusion, activation of Notch in cells of the osteoblastic lineage causes effects that are dependent on the stage of cell maturation. Notch expression in immature and mature osteoblasts impairs osteoblast cell differentiation and causes osteopenia, whereas Notch expression in osteocytes causes an initial suppression of bone resorption and an increase in trabecular bone mass.
Acknowledgments
The authors thank T. Clemens for Osteocalcin-Cre transgenics, T. Iso for Hey1 and Hey2 cDNAs, R. Wu for GAPDH cDNA, Allison Kent and Lauren Kranz for technical assistance, and Mary Yurczak for secretarial assistance.
This work was supported by grant DK045227 (to E.C.) from the National Institute of Diabetes and Digestive and Kidney Diseases.
Disclosure Summary: The authors have nothing to disclose.
Footnotes
- ColIa1
- type Iα1 collagen
- Col2.3
- 2.3 kilobase type 1 collagen fragment
- CSL
- C promoter binding factor 1, Suppressor of Hairless or Lag-1
- Dmp1
- dentix matrix protein 1
- FVB
- friend leukemia virus B type
- Gapdh
- glyceralehyde-3-phosphate dehydrogenase
- Hes
- hairy enhancer of split
- Hey
- Hes related with YRPW motif
- kb
- kilobase
- μCT
- microcomputed tomography
- NICD
- notch intracellular domain
- Oc
- osteocalcin
- Osx
- osterix
- PCR
- polymerase chain reaction
- RANK
- receptor activator of nuclear factor κ B.
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