Osteocytes are terminally differentiated cells of the osteoblastic lineage that are embedded within the mineralized bone matrix and communicate with each other and with cells at the bone surface via a complex canalicular network through which they extend long dendritic processes.1–3 Osteocytes secrete sclerostin, the protein product of the Sost gene, which is a potent inhibitor of osteoblastic bone formation.4Sost deficiency leads to marked high bone mass phenotypes as observed in the rare human SOST loss-of-function hereditary disorders van Buchem (VB) disease5, 6 and sclerosteosis.7–9 Correspondingly, Sost knockout (KO) mouse models display strikingly elevated bone mass and strength because of increased bone formation in the entire skeleton that is progressive throughout life.10–12
Consistent with the key role of Sost as an inhibitor of osteoblastic bone formation, its expression is regulated by a complex interplay of local and systemic factors and physiological stimuli.12–21Sost expression is controlled by a distant downstream enhancer that is required for Sost expression in adult bone and lies within the 52-kb noncoding DNA region deleted in VB disease patients.19 The activity of this enhancer is suppressed by parathyroid hormone (PTH) signaling and involves binding of myocyte enhancer factor 2 (MEF2) transcription factors to a conserved MEF2 response element.18, 22 Endogenous Sost expression is reduced after siRNA-mediated knockdown of single or multiple Mef2 family members in vitro,18 implying these transcription factors in bone biology.
The mammalian MEF2 family of evolutionary conserved MADS (MCM1, Agamous, Deficiens, SRF) box containing transcription factors is composed of four members named Mef2a, Mef2b, Mef2c, and Mef2d.23, 24 The N-terminal MADS box is flanked by a MEF2 domain, both of which together mediate DNA binding, dimerization, and interaction with transcriptional cofactors. The C-terminal portion consists of a transactivation domain being less conserved between individual MEF2 family members. All MEF2 members are widely expressed and function as transcriptional regulators of cell growth and differentiation.23, 24 Constitutive Mef2c-deficient mice die during embryogenesis at about embryonic day E9.5 because of severe malformations of the heart and severe vascular abnormalities.25 Recently, Mef2c and Mef2d were shown to be required for chondrocyte hypertrophy during endochondral bone formation,26 but a role in osteocytes, representing 90% to 95% of all cell types in bone,1–3 has not been described so far.
Quantitative gene expression analyses in femoral cortical bone containing mostly osteocytes and comparatively few other bone surface-adhering cell types revealed that Mef2c is most strongly expressed, followed by lower levels of Mef2a and Mef2d, whereas Mef2b is not detectable.18 Moreover, MEF2C was recently identified as one of 20 bone mineral density (BMD) loci in a meta-analysis of five genomewide association studies of human femoral neck and lumbar spine BMD.27 Thus, Mef2c might play a role in control of bone mass, putatively in part through regulation of Sost expression in osteocytes.
To test this hypothesis and to analyze the role of Mef2c in osteocytes in vivo, we generated Mef2c conditional KO (cKO) mice in which Mef2c was ablated in osteocytes by virtue of the Cre-loxP technology using 9.6-kb Dmp1-Cre transgenic mice.28 We characterized the bone phenotype of 9.6-kb Dmp1-Cre-directed Mef2c-deficient and control littermates during skeletal growth and aging and compared their bone phenotype to heterozygous Sost KO mice at the tissue and gene expression level. Here, we present a function for Mef2c in control of adult bone mass that has not been described before.
Materials and Methods
Mef2cloxP/loxP mice with a floxed exon 4 encoding part of the MADS box were previously reported29 and were maintained on a mixed 129/Sv;C57BL/6 genetic background. The 9.6-kb Dmp1-Cre transgenic mice were described before28 and were maintained on a mixed CD1;C57BL/6 background. Mef2cloxP/loxP mice were bred with 9.6-kb Dmp1-Cre mice and heterozygous Mef2c cKO offspring (Mef2c+/loxP;Dmp1-Cre) was backcrossed to Mef2cloxP/loxP mice to generate homozygous Mef2c cKO mice (Mef2cloxP/loxP;Dmp1-Cre). Heterozygous and homozygous floxed Mef2c littermates lacking 9.6-kb Dmp1-Cre were pooled and used as controls. Sost KO mice have been previously reported and were maintained on a mixed 129/Sv;C57BL/6 genetic background.10 All mice were kept in cages under standard laboratory conditions with constant temperature of 22°C and a 12-hour/12-hour light/dark cycle. Mice were fed on a standard rodent diet (3302, Provimi Kliba SA, Penthalaz, Switzerland) with food and water ad libitum. Protocols, handling, and care of the mice conformed to the Swiss federal law for animal protection under control of the Basel-Stadt Cantonal Veterinary Office, Switzerland.
Bone cell fractionation
Osteoblast- and osteocyte-enriched cell fractions were isolated by sequential enzymatic digestion of mouse femora using a modified version of the protocol by Gu and colleagues.30 Adhering soft tissue was removed, epiphyses were cut off, and bone marrow was flushed with PBS. Femora from two animals per group were pooled, and the four diaphyses were incubated at 37°C for 30 minutes, shaking in 0.2% collagenase solution (0.2% collagenase type IV in sterile isolation buffer containing 25 mM HEPES, pH 7.4, 70 mM NaCl, 10 mM NaHCO3, 60 mM sorbitol, 30 mM KCl, 3 mM K2HPO4, 1 mM CaCl2, 0.1% bovine serum albumin [BSA], and 0.5% glucose) followed by rinsing with PBS. Supernatants were collected and centrifuged at 800g for 8 minutes and cell pellets were resuspended in TRIzol (Invitrogen, Carlsbad, CA, USA) and frozen at −80°C (fraction 1). The remainders of the diaphyses were digested in PBS with 5 mM EDTA and 0.1% BSA at 37°C for 30 minutes, rinsed once with PBS, and the supernatants were collected and stored as described above (fraction 2). The diaphyses were then incubated twice in 0.2% collagenase solution at 37°C for 60 minutes, rinsed with PBS, and the supernatants were collected and stored as before to yield fractions 3 and 4. Fraction 5 was obtained after digestion of the diaphyses in PBS with 5 mM EDTA and 0.1% BSA at 37°C for another 45 minutes, and cells were collected as described above. The remaining femoral diaphyses were homogenized using a precooled BioPulverizer with spring-loaded hammer (Biospec Products Inc., Bartlesville, OK, USA). The crushed bone powder was transferred into TRIzol and stored at −80°C (fraction 6). Total RNA was extracted from cell fractions 1 to 6 according to the manufacturer's recommendations followed by RNA cleanup (RNeasy MinElute cleanup kit, Qiagen, Valencia, CA, USA).
RNA extraction and quantitative real-time PCR (qPCR) expression analyses
Total RNA isolated from cortical bone of femoral diaphyses by sequential enzymatic digestion or as previously described17 was reverse transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's protocol. Gene expression analyses were performed using an ABI Prism 7900HT sequence detection system (Applied Biosystems), TaqMan Universal PCR Master Mix, and the following mouse TaqMan probes according to the manufacturer's instructions (Applied Biosystems): 18S (4310893E) for normalization, Axin2 (Mm01265783_m1), Lef1 (Mm00550265_m1), MCSF/Csf1 (Mm00432688_m1), Mef2a (Mm01318991_m1), Mef2c (Mm01340839_m1), Mef2d (Mm01218042_g1), Opg/Tnfrsf11b (Mm00435452_m1), RANKL/Tnfsf11 (Mm00441908_m1), Sfrp2 (Mm01213947_m1), Sfrp3 (Mm00441378_m1), and Sost (Mm00470479_m1).
Animals were placed laterally under inhalation narcosis (isoflurane, 2.5%) and the left leg was stretched and fixed. Cross-sectional cortical thickness and cancellous bone mineral density (BMD) were evaluated in vivo in the distal femur metaphysis at 1-month intervals or ex vivo at 3.5 and 4 months of age in the distal femur metaphysis and midshaft region using an adapted Stratec-Norland XCT-2000 fitted with an Oxford (Oxford, UK) 50-mm X-ray tube (GTA6505M/LA) and a collimator of 0.5 mm diameter. The following setup was chosen for the measurements: voxel size: 0.07 mm × 0.07 mm × 0.4 mm; scan speed: 5 mm/s 1 block, contour mode 1, peel mode 2; cortical and inner thresholds: 350 mg/cm3.
Micro-computed tomography (µCT) analyses
Generation of three-dimensional (3D) bone reconstruction images and evaluation of cancellous bone structures was accomplished by in vivo measurements of the proximal tibia metaphysis or ex vivo measurements of the distal femur metaphysis and lumbar vertebra L3 using a Scanco vivaCT40 system (voxel size: 10.5 µm, high resolution; Scanco Medical, Bruttisellen, Switzerland). For in vivo measurements, the animals were placed laterally under inhalation narcosis (isoflurane, 2.5%), and the left leg was stretched and fixed. A threshold of 275 was applied to determine the mineralized bone fraction. A Gaussian filter was used (s = 0.7, support = 1) to remove noise. In case of the tibia, 125 slices were evaluated and 50 slices were used for evaluations of vertebral and femoral bone parameters.
All mice received fluorochrome markers by subcutaneous injection 10 days (alizarin complexone, 20 mg/kg, Merck, Whitehouse Station, NJ, USA) and 3 days before necropsy (calcein, 30 mg/kg, Fluka, Buchs, Switzerland) to evaluate bone formation dynamics. The left femur was fixed for 24 hours in ice-cold 4% phosphate-buffered paraformaldehye, dehydrated and defattened at 4°C, and embedded in methylmethacrylate resin. Per animal, a set of 5-mm nonconsecutive longitudinal sections was cut in the frontal midbody plane (RM2155 microtome, Leica Microsystems, Buffalo Grove, IL, USA). Fluorochrome marker-based dynamic bone parameters were determined using a Leica DM microscope fitted with a SONY DXC-950P camera and adapted Quantimet 600 software (Leica). Microscopic images were digitized and evaluated semiautomatically on screen (200-fold magnification) as previously described.31 Bone surface, single- and double-labeled bone surface, and interlabel width were measured in the cancellous bone compartment of the distal femur metaphysis. Mineralizing surface (MS/BS = [dLS + sLS/2]/BS [%]) and mineral apposition rate (MAR [mm/day], corrected for section obliquity) were calculated and the daily bone formation rate (BFR/BS [mm/d]) was derived. Osteoblast number and surface were determined on toluidine blue–stained sections using a Merz grid. Osteoclast surface was measured on sections stained for tartrate-resistant acid phosphatase (TRAP) 5b activity for Mef2c cKO mice or on toluidine blue–stained sections for heterozygous Sost KO mice using a Merz grid. Bone histomorphometric nomenclature was applied as recommended by Parfitt and colleagues.32
Serum bone biomarkers
Blood was collected after terminal CO2 exposure by heart puncture. Serum TRAP 5b activity was determined using a species-specific ELISA kit (Immunodiagnostic Systems, Boldon, UK), and serum osteocalcin concentration was quantified using an immunoradiometric assay (IRMA) kit (Immutopics, San Clemente, CA, USA).
All data represent the mean plus standard error of the mean (SEM). Statistical analyses were performed using unpaired Student's t tests (two-tailed) or one-way analysis of variance (ANOVA).
Generation of 9.6-kb Dmp1-Cre; Mef2c mutant mice and cortical bone expression analyses
To investigate the role of Mef2c in osteocytes in vivo, we crossed floxed Mef2cloxP/loxP mice29 with 9.6-kb Dmp1-Cre transgenic mice, which express Cre recombinase in bone predominantly in osteocytes under control of a 14-kb regulatory fragment containing 9.6 kb of the murine Dentin matrix protein 1 (Dmp1) gene promoter,28 to generate Dmp1-Cre; Mef2cloxP/loxP mutant mice, hereafter designated as Mef2c cKO mice, as well as control littermates. Mef2c cKO mice were born at normal Mendelian frequency and showed a normal life span with no obvious phenotypical abnormalities (data not shown). At late-stage skeletal growth, i.e., 3.5 months of age, Mef2c expression was selectively decreased by 61% and 50% in osteocyte-enriched cortical bone of Mef2c cKO male and female mice relative to control littermates, respectively (Fig. 1A,B). In contrast, Mef2a and Mef2d expression were not changed in mutant mice of either sex. During skeletal aging, Mef2c transcript levels were further reduced by 83% in cKO males and 71% in mutant females relative to controls (Fig. 1C,D). At this stage, Mef2a expression was slightly decreased by about 20% to 25% in Mef2c cKO mice (Fig. 1C,D).
Next, we assessed whether decreased Mef2c expression in osteocytes affected Sost expression, since we previously found that Mef2 transcription factors are required for endogenous Sost expression in vitro.18Sost expression was significantly reduced by 40% in 3.5-month-old mutant males and nonsignificantly by 32% in Mef2c mutant females compared with controls (Fig. 1E,F). In skeletally aging animals, Sost expression was even more strongly reduced by about 70% in both sexes, consistent with the more pronounced decrease in cortical Mef2c expression at this age (Fig. 1E,F).
Because Cre recombinase activity in 9.6-kb Dmp1-Cre transgenic mice, although most strongly expressed in osteocytes, was previously shown to be also present in some bone surface–adhering osteoblasts,28, 33 we next determined whether Mef2c deletion was indeed osteocyte-specific. To this end, we subjected femoral diaphyses from 2-month-old Mef2c cKO and control female mice to sequential enzymatic digestions to isolate osteoblast- and osteocyte-enriched cell fractions. Based on the expression profile of the known osteoblast marker genes alkaline phosphatase (ALP) and osteocalcin as well as Dmp1 and Sost as osteocyte markers, fractions 2 and 6 were found to be most strongly enriched in osteoblasts or osteocytes, respectively (Fig. 2H and data not shown). Relative to control littermates, Mef2c expression was significantly reduced by 72% in the osteocyte but not in the osteoblast cell fraction of Mef2c cKO mice (Fig. 1G). Similarly, Sost expression was significantly decreased by 26% in Mef2c mutant compared with control females (Fig. 1H).
Together, these data demonstrate that 9.6-kb Dmp1-Cre–mediated ablation of Mef2c in bone occurs selectively in osteocytes and that osteocyte Mef2c deletion is associated with decreased Sost expression in adult cortical bone in vivo.
CT analyses of the appendicular skeleton
To assess the bone phenotypic consequences of 9.6-kb Dmp1-Cre–directed Mef2c loss-of-function, we monitored the long bone phenotype in the distal femur metaphysis of mutant and control mice during skeletal growth and aging starting at 1 month of age by longitudinal in vivo pQCT analyses. Mef2c cKO mice displayed normal body weight (Fig. 2A,B) and long bone length (data not shown) at all ages analyzed. From 2 to 3 months of age onward, femoral cortical thickness was significantly increased in Mef2c cKO mice ranging from 17% to 65% in mutant males (Fig. 2C) and 18% to 24% in mutant females (Fig. 2D). Similarly, from 2 months of age onward, femoral cancellous BMD was significantly elevated by 10% to 30% in Mef2c cKO males (Fig. 2E) and 10% to 15% in mutant females (Fig. 2F) relative to controls. Consistent with the stronger decrease in cortical Mef2c expression in mutant male compared with female mice (Fig. 1A–D), the overall bone gain was more pronounced in mutant male than female mice.
To further characterize the nature of the increased cancellous BMD, we next performed high-resolution µCT analyses in the proximal tibia and the distal femur metaphysis at late-stage skeletal growth and skeletal aging. The tibial relative cancellous bone volume was significantly increased by 58% to 101% in Mef2c cKO males and by about 35% in cKO females at late-stage skeletal growth and during aging, respectively (Fig. 3A,B). In the distal femur metaphysis, the relative cancellous bone volume was similarly elevated by 49% to 134% and 57% to 121% in 3.5-month-old and aging Mef2c cKO males and females compared with controls, respectively (Fig. 3C,D). In mutant males, this was related to about 13% elevated trabecular thickness at both ages (Fig. 3E) and a significantly higher trabecular number by 33% at 6 months of age (Fig. 3G). In contrast, Mef2c cKO females did not show any major differences in trabecular number (Fig. 3H) but displayed increased trabecular thickness by 14% to 29% at 3.5 and 5 months of age, respectively (Fig. 3F), as also revealed by the representative 3D µCT images (Fig. 3I).
CT analyses of the axial skeleton
Next, we analyzed the bone phenotype in the axial skeleton by performing ex vivo high-resolution µCT measurements of lumbar vertebra L3 at late-stage skeletal growth and during aging. Consistent with the findings in the appendicular skeleton, vertebral cancellous BMD was significantly greater by 42% to 68% in Mef2c cKO males and by 25% to 40% in mutant females relative to control littermates at 3.5 months of age and during aging, respectively (Fig. 4A,B). Similarly, the vertebral relative cancellous bone volume was significantly increased by 66% to 96% in cKO males and 35% to 55% in Mef2c cKO females at 3.5 months of age and during skeletal aging, respectively (Fig. 4C,D). The increase in vertebral cancellous bone mass was caused by comparable elevations in trabecular thickness and number. Vertebral trabecular thickness was significantly higher by about 20% to 25% in mutant males and by 14% to 18% in Mef2c cKO females relative to controls at 3.5 months of age and skeletal aging (Fig. 4E,F). Similarly, vertebral trabecular number was increased by 22% to 48% in cKO males and by 13% to 20% in mutant females at late-stage skeletal growth and during aging (Fig. 4E,F).
Thus, 9.6-kb Dmp1-Cre–mediated Mef2c loss-of-function results in elevated femoral cortical thickness and increased femoral and vertebral cancellous bone mass and density because of increased trabecular thickness and number.
CT analyses of the appendicular and axial skeleton of Sost heterozygous mice
Given that the increased bone mass phenotype was significant from 2 to 3 months of age, we focused our further analyses at late-stage skeletal growth. Because at this age 9.6-kb Dmp1-Cre–induced Mef2c deletion resulted in decreased Sost expression by up to 40%, we sought to compare the increased bone mass phenotype of Mef2c cKO mice to the bone phenotype of 4-month-old constitutive heterozygous Sost KO male and female mice showing 52% to 63% decreased Sost expression. In the distal femur metaphysis as well as at the midshaft region, cortical thickness was greater in heterozygous Sost–deficient compared with wild-type mice (Table 1). Similarly, Sost heterozygosity was associated with significantly increased cancellous bone volume and density relative to wild-type mice of either gender (Table 1). However, with exception of cortical thickness at the distal metaphysis, the relative increases in femoral structural parameters were smaller in heterozygous Sost KO than Mef2c cKO males, whereas the reverse was true for Sost KO females (Table 2). In both sexes, heterozygous Sost deficiency was linked to significantly elevated trabecular thickness by 13% to 17% (Tables 1 and 2), which was comparable to Mef2c cKO mice (Table 2). Similar to Mef2c cKO mice, femoral trabecular number was not significantly different in heterozygous Sost KO male mice, but there was a small significant increase of 13% in heterozygous Sost KO females (Tables 1 and 2).
Table 1. Femoral Bone Structure Indices of 4-Month-Old Wild-Type and Heterozygous Sost KO Mice
Bone structure parameter
Note: Data were acquired by ex vivo pQCT (Ct.Th, Ct.Th mid) or high-resolution µCT imaging and represent mean ± SEM for cortical thickness at the distal metaphysis (Ct.Th) and midshaft (Ct.Th mid) region, cancellous BMD (cBMD), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) in the distal femur metaphysis, respectively. Group size: n = 6 to 7 (males), n = 13 to 14 (females). Statistical significances were determined by unpaired Student's t test and are designated as follows: *p < 0.05; **p < 0.01.
0.36 ± 0.02
0.44 ± 0.03
0.33 ± 0.01
0.39 ± 0.01**
Ct.Th mid (mm)
0.29 ± 0.00
0.31 ± 0.01
0.30 ± 0.01
0.34 ± 0.00**
184.6 ± 9.1
234.2 ± 19.7*
114.1 ± 8.1
185.0 ± 10.9**
13.1 ± 1.0
18.4 ± 1.8*
6.8 ± 0.7
13.0 ± 1.1**
44.6 ± 1.1
50.2 ± 2.0*
48.9 ± 1.0
57.3 ± 1.1**
5.1 ± 0.1
5.5 ± 0.1
3.5 ± 0.1
3.9 ± 0.1*
Table 2. Comparison of Femoral Bone Phenotypes of 3.5-Month-Old Mef2c cKO and 4-Month-Old Heterozygous Sost KO Mice
Bone parameter (% difference)
Note: Data represent mean % differences versus control ± % SEM for cortical Sost expression by qPCR analyses (Sost), cortical thickness at the distal metaphysis (Ct.Th) and midshaft (Ct.Th mid) region as determined by ex vivo pQCT, as well as cancellous BMD (cBMD), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) as measured by ex vivo µCT, respectively. Group size: n = 12 to 21 (Mef2c cKO), n = 3 to 14 (Sost+/−). Statistical significances were determined by unpaired Student's t test and are designated as follows: *p < 0.05; **p < 0.01.
−39.9 ± 6.5**
−52.0 ± 11.6**
−31.9 ± 14.9
−62.9 ± 4.7*
16.6 ± 7.7
21.9 ± 8.7
18.0 ± 4.8**
15.9 ± 3.9**
9.0 ± 1.8**
5.7 ± 2.4
3.9 ± 1.2*
10.8 ± 1.5**
31.9 ± 2.0**
27.7 ± 10.5*
29.2 ± 9.2*
62.1 ± 9.5**
49.2 ± 11.4**
41.5 ± 13.6*
56.6 ± 14.5**
91.4 ± 15.8**
12.7 ± 0.3**
12.5 ± 4.5*
13.6 ± 3.0**
17.1 ± 2.3**
5.9 ± 0.2
6.9 ± 3.0
5.9 ± 4.2
13.2 ± 3.0*
In lumbar vertebra L3, heterozygous Sost KO mice displayed mildly increased cancellous bone volume (about +20%) and density (14% to 17%) relative to wild-type littermates (Table 3). Trabecular thickness was significantly higher by 11% in heterozygous Sost KO males and 22% in mutant females (Tables 3 and 4). These relative phenotypic differences in the axial skeleton were about 1.4- to 3-fold smaller in Sost heterozygotes than in Mef2c cKO mice with the exception of trabecular thickness in heterozygous Sost KO females, which displayed 80% greater relative increases than Mef2c cKO female mice compared with their respective control littermates (Table 4). In contrast to Mef2c cKO mice, which showed 13% to 22% increases in trabecular number relative to control mice (Table 4), trabecular number was normal in heterozygous Sost KO mice of either sex (Tables 3 and 4).
Table 3. Bone Structure Indices in the Cancellous Bone Compartment of the Lumbar Vertebra 3 of 4-Month-Old Wild-Type and Heterozygous Sost KO Mice
Bone structure parameter
Note: Data were acquired by high-resolution µCT imaging and represent mean ± SEM for cancellous BMD (cBMD), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), respectively. Group size: n = 5 to 8 (males), n = 3 to 14 (females). Statistical significances were determined by unpaired Student's t test and are designated as follows: *p < 0.05; **p < 0.01.
303.6 ± 8.4
345.5 ± 10.6*
405.6 ± 32.3
475.1 ± 30.5
26.6 ± 0.9
32.0 ± 1.2**
39.6 ± 3.6
47.1 ± 3.5
43.6 ± 1.0
48.5 ± 1.4*
35.4 ± 2.1
44.3 ± 2.8*
7.8 ± 0.2
7.6 ± 0.1
7.6 ± 0.2
7.7 ± 0.3
Table 4. Comparison of Vertebral Bone Phenotypes of 3.5-Month-Old Mef2c cKO and 4-Month-Old Heterozygous Sost KO Mice
Bone parameter (% difference)
Note: Data were acquired by high-resolution µCT imaging of lumbar vertebra L3 and represent mean % differences versus control ± % SEM for cancellous BMD (cBMD), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), respectively. Group size: n = 12 to 21 (Mef2c cKO), n = 3 to 14 (Sost+/−). Statistical significances were determined by unpaired Student's t test and are designated as follows: *p < 0.05; **p < 0.01.
42.3 ± 5.1**
13.8 ± 3.5*
24.8 ± 3.4**
17.1 ± 7.5
65.7 ± 7.8**
20.4 ± 4.4**
35.4 ± 5.0**
18.9 ± 9.0
25.1 ± 3.5**
11.2 ± 3.1*
14.1 ± 2.1**
25.3 ± 8.0*
21.9 ± 2.6**
−1.9 ± 1.3
13.4 ± 3.2**
1.8 ± 3.6
Together, these data indicate that despite some overlap in the overall increased bone mass phenotype, clear differences exist between the two mutant mouse strains.
Bone histomorphometric and serum bone biomarker measurements
To address the cellular mechanisms underlying the increased bone mass phenotype of Mef2c cKO mice compared with heterozygous Sost KO animals, we next performed static and dynamic histomorphometry in the cancellous bone compartment of the distal femur metaphysis. In 3.5-month-old mice, osteoblast number and surface were similar in Mefc2 cKO and control littermates of either sex (Table 5). Likewise, Mefc2 cKO mice displayed normal osteoblastic mineral apposition and bone formation rates (Table 5 and Fig. 5A). In Mefc2 mutant female but not male mice, the mineralizing bone surface was mildly elevated by 11% compared with control littermates (Table 5). Serum osteocalcin levels as a measure for osteoblast activity were, however, normal in Mefc2 cKO mice, although there was a small 15% reduction in Mefc2 cKO males compared with control littermates (Fig. 5B).
Table 5. Histomorphometric Indices in the Cancellous Bone Compartment of the Distal Femur Metaphysis of 3.5-Month-Old Control and Mef2c cKO Mice
Note: Data represent mean ± SEM for osteoblast number (Ob.N), osteoblast surface (Ob.S), and mineralizing surface (MS) per bone surface (BS), as well as mineral apposition rate (MAR), respectively. Group size: n = 18 to 19 (control), n = 10 (cKO). Statistical significances were determined by unpaired Student's t test and are designated as follows: *p < 0.05.
5.3 ± 0.7
3.7 ± 1.1
8.2 ± 0.9
8.7 ± 1.1
9.9 ± 1.5
9.4 ± 3.2
14.0 ± 1.7
12.5 ± 2.1
42.9 ± 1.2
39.6 ± 1.7
38.3 ± 1.1
42.4 ± 1.1*
1.30 ± 0.05
1.23 ± 0.04
1.90 ± 0.05
1.80 ± 0.08
In heterozygous Sost KO male mice, mineralizing surface and osteoblastic bone formation rate were also comparable to those of wild-type littermates (Table 6). However, we observed a small but significant 27% increase in the osteoblastic mineral apposition rate relative to control mice (Table 6), which was not seen in Mef2c cKO mice (Table 5), indicating elevated osteoblast activity in Sost heterozygotes but not Mef2c mutant mice. In line with this observation, serum osteocalcin levels were also mildly albeit nonsignificantly increased by 10% in heterozygous Sost KO males and 17% in mutant females (data not shown).
Table 6. Histomorphometric Indices in the Cancellous Bone Compartment of the Distal Femur Metaphysis of 4-Month-Old Wild-Type and Heterozygous Sost KO Mice
Note: Data represent mean ± SEM for mineralizing surface per bone surface (MS/BS), mineral apposition rate (MAR), bone formation rate (BFR), and osteoclast surface (Oc.S) per bone surface (BS), respectively. Group size: n = 6 to 7. Statistical significances were determined by unpaired Student's t test and are designated as follows: *p < 0.05.
44.6 ± 2.7
45.3 ± 2.9
0.49 ± 0.02
0.62 ± 0.04*
0.22 ± 0.02
0.29 ± 0.03
0.9 ± 0.4
1.4 ± 0.6
To assess whether the observed increase in bone mass in Mef2c cKO mice could be linked to decreased osteoclastic bone resorption given that bone formation parameters were normal, we next determined the relative osteoclast surface in the cancellous bone compartment of the distal femur metaphysis. The relative osteoclast surface was significantly decreased by 39% in Mef2c mutant males and by 32% in mutant females compared with controls, respectively (Fig. 5C,E). To further support this finding, we measured osteoclastic serum TRAP 5b activity. In line with decreased osteoclast numbers in Mef2c cKO mice, serum TRAP 5b activity was significantly reduced by 38% in Mef2c cKO males but only nonsignificantly by 8% in mutant females (Fig. 5D). In contrast, the relative osteoclast surface was unchanged in heterozygous Sost KO mice (Table 6), consistent with previously reported normal serum TRAP 5b activity and relative osteoclast surface in homozygous Sost–deficient mice compared with wild-type littermates.11
Together, these findings indicate that the elevated bone mass in Mef2c cKO mice is not caused by an increase in osteoblast number or activity but by a decrease in osteoclastic bone resorption.
Cortical bone gene expression analyses
Osteoclasts differentiate from precursors of the monocyte-macrophage lineage under control of two major cytokines, macrophage colony-stimulating factor 1 (MCSF/Csf1) and receptor activator of NF-κB ligand (RANKL), both of which are expressed in cells of the osteoblast lineage and bone marrow stromal cells.34 The pro-osteoclastogenic activity of RANKL is tightly regulated by its secreted decoy receptor osteoprotegerin (OPG), which is also expressed in osteoblasts and osteocytes. To determine whether the observed decrease in osteoclast surface in Mef2c cKO mice was related to changes in MCSF, RANKL, and/or OPG expression, we performed quantitative gene expression analyses in femoral cortical bone samples of 3.5-month-old mice. Amongst the pro-osteoclastogenic factors, neither expression of MCSF nor of RANKL was significantly altered in Mef2c cKO mice of either sex (Fig. 6A,B). In contrast, expression of the anti-osteoclastogenic factor OPG was significantly increased by 70% in Mef2c cKO male mice (Fig. 6A). However, cortical OPG expression was not changed in mutant female mice (Fig. 6B).
Because OPG is a target gene of canonical Wnt/β-catenin signaling35–37 and 9.6-kb Dmp1-Cre–mediated Mef2c deficiency results in decreased expression of Sost, an inhibitor of canonical Wnt/β-catenin signaling,4 we next analyzed expression of the canonical Wnt/β-catenin target genes axin238–41 and lymphoid enhancer binding factor1 (Lef1).42, 43 No significant differences were observed in cortical axin2 expression in Mef2c cKO mice (Fig. 6C,D). In contrast, Lef1 expression was mildly but significantly upregulated by 35% in Mef2c mutant males and by 41% in mutant females relative to controls, respectively (Fig. 6C,D). Thus, overall Wnt/β-catenin signaling does not appear to be strongly activated in cortical bone of Mef2c cKO mice.
Next, we determined whether any of these factors were differentially expressed in cortical bone of 4-month-old heterozygous Sost KO mice. None of the analyzed osteoclastogenesis regulators or Wnt/β-catenin target genes was significantly altered in heterozygous Sost mutant male mice (Fig. 6E,G). There was, however, a mild increase in Lef1 expression by 35% that was comparable in magnitude to the observed upregulation of Lef1 in Mef2c cKO mice but remained nonsignificant in heterozygous Sost KO males (Fig. 6G). In contrast, neither axin2 nor Lef1 expression was increased in heterozygous Sost KO female mice (Fig. 6H). Surprisingly, we observed a small but significant decrease in cortical OPG expression by 35% in heterozygous Sost KO females, whereas no other changes in osteoclastogenic regulators were present (Fig. 6F,H). Thus, cortical OPG expression might be differentially regulated in a sexually dimorphic manner not only in Mef2c cKO but also in heterozygous Sost KO mice (Fig. 6A,B).
Together, these data demonstrate that conditional Mef2 deficiency in male but not female mice results in a decreased cortical RANKL/OPG expression ratio likely contributing to the decreased osteoclast surface observed upon 9.6-kb Dmp1-Cre–mediated Mef2c loss-of-function. However, because OPG expression was not elevated in mutant female mice, additional, so far unknown factors appear to contribute to the decrease in osteoclast surface in Mef2c cKO mice.
Finally, to assess whether inhibitors of Wnt/β-catenin signaling other than Sost are differentially expressed in Mef2c cKO mice, we analyzed expression of known secreted Wnt antagonists. Neither expression of dickkopf homolog 1 (Dkk1) nor of Wnt inhibitory factor 1 or 2 (Wif1 and 2) was altered in Mef2c cKO mice (data not shown). Similarly, cortical expression of the secreted frizzled-related protein (Sfrp) family members Sfrp1, 4, and 5 was unchanged (data not shown). However, we found a selective strong upregulation by 3.7- and 2.5-fold of Sfrp2 in Mef2c cKO male and female mice, respectively (Fig. 7A,B). Furthermore, Sfrp3 expression was significantly downregulated by 61% in mutant males and 69% in mutant females (Fig. 7A,B). In contrast, no significant changes in cortical Sfrp2 or Sfrp3 expression were observed in heterozygous Sost KO male and female mice (Fig. 7C,D).
In summary, these data reveal a critical role for Mef2c in regulating adult bone metabolism that appears to be independent of Sost downregulation and may involve differential regulation of Sfrp2 and Sfrp3 expression in cortical osteocytes.
Here, we describe a conditional knockout approach deleting the Mef2 transcription factor family member Mef2c by virtue of the Cre-loxP system to analyze the consequences of osteocyte Mef2c deficiency on Sost expression and bone mass regulation in vivo. After 9.6-kb Dmp1-Cre–mediated Mef2c deletion, Mef2c expression levels were significantly reduced by about 50% to 60% in femoral diaphyses of Mef2c cKO mice at late-stage skeletal growth. This comparatively modest reduction in Mef2c expression suggests that either transgenic 9.6-kb Dmp1-Cre expression might have been insufficient to induce homozygous recombination of both Mef2c alleles in all newly generated osteocytes at this stage44 or that contaminating Mef2c-positive but 9.6-kb Dmp1-Cre–negative bone surface–adhering cells such as osteoblasts were not entirely removed during sample preparation. Consistent with both interpretations, we observed a distinctly stronger reduction in cortical Mef2c expression during skeletal aging compared with late-stage skeletal growth when osteoblast number is high and osteocytes are still being newly generated from bone matrix–producing osteoblasts. In addition, the 5-kb-upstream region of the Mef2c promoter was shown to contain 67 conserved MEF2 response elements,45 indicating that Mef2c could be subject to transcriptional control by other Mef2 family members and/or autoregulation. Interestingly, autoregulation was shown for Drosophila Mef2,46 mammalian Mef2a,47 and mouse Mef2c, which positively regulates its own expression during mouse embryogenesis.48 Incomplete deletion of only one instead of both Mef2c alleles might thus have caused a compensatory upregulation of Mef2c expression from the intact allele resulting in overall only partially reduced Mef2c expression levels. In line with this hypothesis, 3.5-month-old heterozygous Mef2c mutant mice did not at all or only displayed mildly reduced cortical Mef2c expression by about 25% (data not shown).
Given the important role of Mef2c in skeletal muscle development and recent observations indicating skeletal muscle to bone and vice versa crosstalk as well as some reported ectopic 9.6-kb Dmp1-Cre activity in skeletal muscle,28, 33 we also analyzed expression of Mef2 family members in slow- (soleus) and fast-twitch (gastrocnemius) skeletal muscle of 2-month-old Mef2c cKO mice. Depending on the respective genetic background, skeletal muscle-specific deletion of Mef2c in mice results in a reduction in slow oxidative muscle fibers required for endurance and resistance to fatigue or even in perinatal lethality because of abnormal sarcomere organization.49, 50 Whereas Mef2a and Mef2d expression were comparable to control littermates, Mef2c expression was strongly and significantly decreased in either analyzed skeletal muscle type of 9.6-kb Dmp1-Cre; Mef2c cKO males and females (Supplemental Fig. S1). Despite the reduction in muscle Mef2c expression, none of the tested known markers for slow- and fast-twitch myofibers Myh1, 2, 4, and 7 encoding different myosin heavy-chain subtypes was altered in Mef2c cKO mice (Supplemental Fig. S1). Together with the normal life span of Mef2c cKO mice, these data suggest that the decreased Mef2c expression in skeletal muscle is most likely not contributing to the observed increased bone mass phenotype.
Previously, we demonstrated that Mef2 family members are required for activity of the Sost bone enhancer and for endogenous Sost expression in vitro.18 Consistent with a requirement for Mef2c in control of Sost expression in vivo, 9.6-kb Dmp1-Cre–mediated Mef2c deletion resulted in a significant partial reduction in cortical Sost expression by about 32% to 40% at late-stage skeletal growth and by about 70% during skeletal aging. However, because Sost expression was not reduced to a similar extent as Mef2c, additional factors are required or can functionally compensate for Mef2c loss-of-function to partly maintain Sost expression at least during skeletal growth. During skeletal aging, the relative suppression of Mef2c and Sost was comparable in magnitude, suggesting that functional compensation for loss of Mef2c could be less effective at this stage. Indeed, we and others demonstrated that Mef2a and Mef2d family members are expressed in bone tissue, albeit at lower levels than Mef2c.18, 26 However, their temporal expression pattern in bone remains to be studied in detail. Our previous in vitro studies also revealed functional redundancy and synergistic activity between Mef2a, c, and d in control of endogenous Sost expression in UMR-106 cells.18 Moreover, functional redundancy between different Mef2 family members was shown for terminal chondrocyte differentiation and hypertrophy during embryonic skeletal development.26 Hence, at present we cannot exclude functional compensation by other Mef2 family members in maintaining Sost expression in Mef2c cKO mice. However, at least during skeletal aging, Mef2c appears to play a predominant role in the regulation of Sost expression in osteocytes.
The 9.6-kb Dmp1-Cre–mediated Mef2c deletion was associated with increased bone mass and density in both genders from about 2 to 3 months of age onward. Because conditional Mef2c deletion results in partially decreased Sost expression at late-stage skeletal growth, we compared the bone phenotype of Mef2c cKO mice to that of 4-month-old heterozygous Sost KO mice. Overall, compared with control mice, the relative increases in bone mass and density were similar in the appendicular skeleton of both mutant mouse strains. In the axial skeleton, the relative changes were, however, distinctly smaller in heterozygous Sost KO than in Mef2c cKO mice. Moreover, in contrast to Mef2c cKO female mice, which generally showed smaller changes than mutant male mice, heterozygous Sost KO female mice mostly showed greater increases than heterozygous Sost KO males independent of the skeletal parameter and site analyzed. These differences cannot be explained by the slightly differing levels in cortical Sost expression in the two mouse strains, indicating that the underlying mechanisms contributing to the increased bone mass phenotypes in either mutant strain must be different.
In line with this interpretation, bone histomorphometric analyses revealed clear differences in the underlying cellular bone phenotype. Contrary to heterozygous Sost KO mice, femoral osteoclast surface was significantly decreased in Mef2c cKO mice, and correspondingly, serum TRAP 5b activity was reduced in mutant males relative to control littermates. Osteoblast number and surface were unchanged in both Mef2c cKO and heterozygous Sost KO mice. In contrast, the mineral apposition rate reflecting osteoblast activity was significantly elevated in Sost heterozygotes and serum osteocalcin levels were nonsignificantly increased, both of which was not observed in Mef2c cKO mice. These findings further corroborate that significant differences exist between Mef2c cKO and Sost mutant mice. Consistent with this notion, Dean and colleagues45 recently indentified several evolutionary conserved MEF2 response elements in a number of genes that are selectively expressed in osteocytes compared with osteoblasts, indicating that Mef2 transcription factors regulate many more genes besides the osteocyte marker Sost.
When analyzing known regulators of osteoclastogenesis to address the molecular basis underlying the decreased osteoclast surface in Mef2c cKO mice, we found that OPG expression was selectively elevated in cortical bone of mutant male but not female mice, whereas no changes were observed in RANKL expression. Thus, in Mef2c cKO males, the concomitant decrease in cortical RANKL/OPG expression ratio may be sufficient or at least contribute to the decrease in osteoclast number. In line with such a hypothesis, we and others demonstrated that osteocytes express OPG at similar levels as osteoblasts.35, 51–54 However, because Mef2c cKO females also display decreases in osteoclast surface, it seems likely that additional, so far unknown mechanisms exist, which contribute to or underlie the reduction in osteoclast surface in Mef2c cKO mice.
Surprisingly, as observed in Mef2c cKO female compared with cKO male mice, heterozygous Sost KO female and male mice also display similarly sexually differential cortical OPG expression, as OPG expression was selectively reduced in Sost mutant female but not in male mice compared with wild-type littermate controls. Thus, gender-specific differences in the regulation of cortical OPG expression could exist, which might contribute to the lack of cortical OPG upregulation in Mef2c cKO female mice opposed to cKO males. OPG expression seems indeed to be regulated in a complex manner that can be influenced by sex steroids.55, 56 Interestingly, the estrogen receptor 1 (alpha) (Esr1) gene was shown to contain functional MEF2 binding sites in the promoter region and to be a direct MEF2 target gene in the heart.57 However, we failed to detect any changes in cortical expression of Esr1 or the related family member estrogen receptor 2 (beta) (Esr2) in Mef2c cKO mice (data not shown). Recently, a genomewide ChIP-on-chip study using primary human skeletal muscle myoblasts revealed that androgen receptor (AR) and MEF2 DNA binding sites are in part overlapping at MEF2c target genes, suggesting that MEF2c and AR might functionally interact to regulate genes involved in skeletal muscle development.58 Whether similar interactions occur in 9.6-kb Dmp1-Cre–induced Mef2c cKO mice remains to be addressed in future studies.
Although we detected increases in bone mass and density in the appendicular and axial skeleton of Mef2c cKO mice of either sex, the relative bone phenotypic changes were mostly smaller in Mef2c cKO females compared with mutant male mice. This was observed in the cortical and cancellous bone compartment and at all stages analyzed. At late-stage skeletal growth, the relative increase in femoral midshaft cortical thickness was 2.3-fold greater in Mef2c cKO males. At this age, the relative differences in cancellous bone structure parameters of the axial skeleton were similarly elevated by about 60% to 90% in Mef2c mutant male compared with cKO female mice. Likewise, the relative reduction in femoral cortical Sost expression was 25% greater in Mef2c cKO males than in female mice. The most striking difference between both genders was the lack of increase in cortical OPG expression in Mef2c cKO females discussed above. Whether these observed gender-specific phenotypic differences, however, truly reflect sexually dimorphic functions of Mef2c transcription factors or whether they are also related to the slightly lower efficiency of the 9.6-kb Dmp1-Cre–mediated Mef2c deletion in female than in male mice, as revealed by the about 20% smaller relative decrease in cortical Mef2c expression in mutant female compared with male mice, remains to be resolved. For technical reasons, we were not able to quantitatively measure gene expression in the cancellous bone compartment where we performed our histomorphometric analyses. Therefore, some of the described inconsistencies between the cellular bone phenotype in the cancellous bone compartment and the observed gene expression might be related to the fact that the latter was examined in cortical bone. However, because at the tissue level overall similar patterns in bone gain were present in both bone compartments in Mef2c cKO male and female mice, it seems unlikely that different cellular mechanisms might account for the observed increases in bone mass in each bone compartment. Moreover, in contrast to Sost heterozygotes in which serum osteocalcin levels were mildly but nonsignificantly elevated by about 10% to 15%, we failed to detect any increase in serum osteocalcin levels in Mef2c cKO mice arguing against a putative osteoblast-driven bone phenotype in the cortical compartment.
OPG is not only a potent anti-osteoclastogenic factor but also a known target gene of canonical Wnt/β-catenin signaling in osteoblasts.36, 37 We recently found that 9.6-kb Dmp1-Cre–induced loss of β-catenin, the central mediator of canonical Wnt signaling, results in a dramatic low bone mass phenotype because of increased bone resorption that was associated with a selective decrease in osteocytic OPG expression.35 Therefore, osteocyte OPG appears to be of major importance for suppression of osteoclastic bone resorption and overall bone homeostasis. To determine whether canonical Wnt/β-catenin signaling was altered in Mef2c cKO mice given that OPG was upregulated in Mef2c cKO males and expression of Sost, an inhibitor of canonical Wnt signaling, was suppressed in Mef2c mutants of either sex, we analyzed expression of well-known canonical Wnt/β-catenin target genes, namely axin2 and Lef1. Whereas axin2 expression did not change, Lef1 expression was significantly increased by 35% to 41% in Mef2c cKO mice. These data suggest that either cortical Wnt/β-catenin signaling was only mildly activated in 9.6-kb Dmp1-Cre–directed Mef2c mutants or that Wnt signaling was selectively increased in subpopulations but not all osteocytes. Similar observations have been reported for mechanically stimulated cortical bone in which sclerostin levels were not uniformly suppressed but rather were specifically decreased in osteocytes located at sites subjected to highest mechanical strain.20
When determining whether secreted Wnt signaling antagonists other than Sost were differentially expressed in 9.6-kb Dmp1-Cre–mediated Mef2c-deficient mice, we found that cortical Sfrp2 expression was selectively and strongly increased in Mef2c cKO mice, whereas Sfrp3, albeit being only weakly expressed (data not shown), was significantly downregulated. In contrast, neither Sfrp2 nor Sfrp3 expression was significantly changed in heterozygous Sost KO mice, suggesting that differential regulation of cortical Sfrp expression in Mef2c cKO mice occurs independent of Sost suppression. Sfrp family members function as modulators of canonical and noncanonical Wnt signaling, which depending on the cellular context can either block or enhance Wnt signaling activity.59 In contrast to sclerostin, which has been shown to inhibit canonical Wnt/β-catenin signaling by binding to the Wnt coreceptors, low-density lipoprotein receptor-related proteins 5 and 6,60, 61 Sfrps can block Wnt signaling by directly sequestering Wnt ligands.59 It is thus tempting to speculate that the increased expression of Sfrp2 might counteract stimulatory effects on canonical Wnt signaling, thus resulting in overall only mildly activated canonical Wnt signaling in Mef2c mutant mice.
In summary, here we describe a novel role for Mef2c in control of adult bone homeostasis. 9.6-kb Dmp1-Cre–mediated Mef2c deficiency results in increased bone mass and density that is related to decreased osteoclastic bone resorption at late-stage skeletal growth. In male but not female mice, this was associated with increased cortical OPG expression and a concomitant reduction in the RANKL/OPG expression ratio. Moreover, we show that Mef2c is required to maintain normal Sost expression levels in vivo. However, the observed partial reduction in Sost expression was not related to a strong general increase in Wnt/β-catenin target gene expression in osteocyte-enriched cortical bone. This might be related to the differential expression of other pathway regulators such as the Wnt signaling inhibitor Sfrp2, highlighting the complex nature of gene expression regulation by Mef2c to control adult bone homeostasis in vivo.
All the authors are employees of the Novartis Institutes for BioMedical Research.
This study was partially supported by the Novartis Institutes for BioMedical Research Education Office Postdoctoral Fellowship Program (IK, SB).
The authors thank Profs Stefan Mundlos and John J Schwarz for providing floxed Mef2c mice and Profs Jian Q Feng and Lynda F Bonewald for sharing Dmp1-Cre transgenic mice. The authors greatly appreciate the excellent technical assistance received from Heinz Anklin for serum biomarker marker analyses, Tanja Grabenstaetter for bone cell fractionation experiments, Heidi Jeker for quantitative gene expression analyses, Charles Moes for mouse strain maintenance and genotyping, Marcel Merdes and Marco Pegurri for performing necropsies and tissue collection, Keiko Petrosky for ex vivo µCT analyses, Anne Studer for embedding and microtome sectioning of bone samples, and Andrea Venturiere for osteoclast histomorphometric analyses.
Authors' roles: Study design: IK and MK. Study conduct and data collection: IK and SB. Data analysis and interpretation: IK, SB, CH, HK, and MK. Drafting manuscript: IK and SB. Revising manuscript content: IK, HK, and MK. Approving final version of manuscript: IK, SB, CH, HK, and MK. IK and MK take responsibility for the integrity of the data analysis.