JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M702435200 on September 19, 2007

J. Biol. Chem., Vol. 282, Issue 48, 35318-35327, November 30, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/48/35318    most recent
M702435200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yeo, H.
Right arrow Articles by Zayzafoon, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yeo, H.
Right arrow Articles by Zayzafoon, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Conditional Disruption of Calcineurin B1 in Osteoblasts Increases Bone Formation and Reduces Bone Resorption*

Hyeonju Yeo{ddagger}, Lauren H. Beck{ddagger}, Sunnie R. Thompson§, Mary C. Farach-Carson, Jay M. McDonald{ddagger}, Thomas L. Clemens{ddagger}, and Majd Zayzafoon{ddagger}1

From the Departments of {ddagger}Pathology and §Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-2182 and the Department of Biological Sciences, University of Delaware, Newark, Delaware 19716

Received for publication, March 21, 2007 , and in revised form, September 13, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently reported that the pharmacological inhibition of calcineurin (Cn) by low concentrations of cyclosporin A increases osteoblast differentiation in vitro and bone mass in vivo. To determine whether Cn exerts direct actions in osteoblasts, we generated mice lacking Cnb1 (Cn regulatory subunit) in osteoblasts ({Delta}Cnb1OB) using Cre-mediated recombination methods. Transgenic mice expressing Cre recombinase, driven by the human osteocalcin promoter, were crossed with homozygous mice that express loxP-flanked Cnb1 (Cnb1f/f). Microcomputed tomography analysis of tibiae at 3 months showed that {Delta}Cnb1OB mice had dramatic increases in bone mass compared with controls. Histomorphometric analyses showed significant increases in mineral apposition rate (67%), bone volume (32%), trabecular thickness (29%), and osteoblast numbers (68%) as well as a 40% decrease in osteoclast numbers as compared with the values from control mice. To delete Cnb1 in vitro, primary calvarial osteoblasts, harvested from Cnb1f/f mice, were infected with adenovirus expressing the Cre recombinase. Cre-expressing osteoblasts had a complete inhibition of Cnb1 protein levels but differentiated and mineralized more rapidly than control, green fluorescent protein-expressing cells. Deletion of Cnb1 increased expression of osteoprotegerin and decreased expression of RANKL. Co-culturing Cnb1-deficient osteoblasts with wild type osteoclasts demonstrated that osteoblasts lacking Cnb1 failed to support osteoclast differentiation in vitro. Taken together, our findings demonstrate that the inhibition of Cnb1 in osteoblasts increases bone mass by directly increasing osteoblast differentiation and indirectly decreasing osteoclastogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone is a highly dynamic structure that is constantly renewing through a process called remodeling (1). This process is critical for maintaining healthy bones and is mainly controlled by the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. The presence of these two opposing cell types with contrasting activities in close proximity requires tight regulation to maintain healthy and strong bones. Bone resorption is attained by the action of osteoclasts, which are specialized macrophages whose differentiation is primarily regulated by receptor activator of NF{kappa}B ligand (RANKL)2 and osteoprotegerin (OPG) (2). Osteoblasts originate from multipotent mesenchymal progenitors that replicate as undifferentiated cells but have the potential to differentiate into different lineages of mesenchymal tissues including bone, cartilage, fat, muscle, and marrow stroma (3, 4). Osteoblasts control bone formation not only by synthesizing bone matrix proteins and regulating mineralization but also by orchestrating the process of bone resorption through the modulation of RANKL and OPG expression (2, 4).

We have recently shown that the pharmacologic inhibition of calcineurin (Cn) by low concentrations of cyclosporin A (CsA) increases osteoblast differentiation and bone formation (5). We also demonstrated that this response was associated with an increase in the expression and activation of Fra-2, an activator protein-1 family member that plays an important role in osteoblast differentiation and bone formation (5). Cn is a Ca2+/calmodulin-dependent serine/threonine-protein phosphatase that regulates several physiological processes (6). Cn exists as a heterodimeric protein complex consisting of two subunits, the 61-kDa calmodulin binding, catalytic subunit A (CnA) and the 19-kDa Ca2+ binding, regulatory subunit B (CnB) (7). Three mammalian isoforms of CnA ({alpha}, β, and {gamma}) and two B isoforms (1 and 2) have been identified. The isoforms CnA{alpha},-β, and -B1 are ubiquitously expressed, whereas CnA{gamma} and B2 are specifically expressed in the testis (8). The CnA subunit contains the phosphatase domain, a CnB binding domain, a calmodulin binding domain, and an autoinhibitory loop. In resting cells the autoinhibitory domain obscures the phosphatase domain and is displaced upon the binding of CnB and Ca2+/CaM to CnA, resulting in the full activation of Cn (8). Nuclear factors of activated T cells (NFAT) are members of a family of transcription factors that are known to be dependent on Cn activation. In resting cells NFAT proteins are highly phosphorylated and reside in the cytoplasm. Upon Cn activation, NFAT proteins are dephosphorylated and translocate to the nucleus where they regulate the transcription of NFAT-dependent genes (6).

Cn and NFAT are key promoters of osteoclast differentiation and bone resorption (9-12). In contrast, the role of Cn and NFAT in osteoblast differentiation and bone formation is not well defined. It has been reported that the Cn/NFAT signaling pathway is also a positive regulator of osteoblast differentiation and bone formation (13-15). The conclusions of these reports were based on data from murine animal models where the Cn/NFAT signaling pathway was disrupted either globally and/or in multiple tissues and was not limited to differentiated osteoblasts. These reports did not consider that Cn and NFAT are also expressed in a variety of tissues such as vasculature, T-cells, skeletal muscles, chondrocytes, and adipocytes which can indirectly alter the bone phenotype of these animals (16, 17).

In contrast to the notion that Cn/NFAT is a positive regulator of bone formation, we discovered that the pharmacologic inhibition of Cn by low concentrations of CsA increased osteoblast differentiation in vitro and bone mass in vivo (5). The increase in bone mass was associated with enhanced bone formation and a decrease in bone resorption (5). Therefore, we hypothesized that Cn signaling negatively regulates osteoblast differentiation and bone mass. To examine our hypothesis and to determine whether Cn directly impacts osteoblast differentiation, we utilized Cre-mediated recombinase technology to create mice lacking the calcineurin regulatory subunit (Cnb1) specifically in osteoblasts. Our results demonstrate that a block in the Cn/NFAT signaling pathway, when restricted to osteoblasts, leads to an increase in bone mass by directly increasing osteoblast differentiation and indirectly decreasing osteoclastogenesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of {Delta}Cnb1OB Mice—Mice expressing the Cre recombinase under the control of an osteoblast-specific human osteocalcin promoter (OC-Cre) were provided by Dr. Thomas L. Clemens (University of Alabama at Birmingham) (18). Mice expressing loxP-flanked calcineurin b1 (Cnb1f/f) were generously provided by Dr. Gerald Crabtree (Stanford University) (19). Cnb1f/f mice were crossed with OC-Cre mice to generate OC-Cre/{Delta}Cnb1OB progeny, which were used in subsequent mating. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. For mice genotyping, DNA was prepared from tail biopsies. PCR-based strategies were then used to genotype these mice according to the manufacturer's recommendations (Sigma). The sequences for the primers that were used for genotyping were previously published (18, 19). The animals that had a genotype of Cre-/-/Cnb1f/f were considered control, Cre+/-/Cnb1f/- were considered heterozygote, and Cre+/-/Cnb1f/f) were considered {Delta}Cnb1OB.

Histology and Histomorphometry—Tibiae and femora were harvested from 3-month-old male mice (control and {Delta}Cnb1OB). Bones were fixed in 10% (v/v) buffered formalin, decalcified in EDTA, embedded in paraffin, sectioned, and then stained for tartrate-resistant acid phosphatase (TRAP). Also, bones were fixed, embedded in methyl methacrylate, sectioned, and stained with Goldner's Trichrome. A region of interest, an area at least 0.5 mm below the growth plate (excluding the primary spongiosa and trabecular-connected cortical bone), was selected and remained constant for all animals regardless of the shape of the section (5). Standard bone histomorphometry as described by Parfitt et al. (20) was performed using the Bio-Quant image analysis software (R & M Biometrics, Nashville, TN) in the Histomorphometry and Molecular Analysis Core Laboratory of the University of Alabama at Birmingham Bone Center (5, 21).

Dual Energy X-ray Absorptiometry (DXA) and Microcomputed Tomography (µCT) Analysis of Bone—Non-cephalic bone mineral density (g/cm2) was determined by DXA, which was measured by a Lunar PIXImus densitometer (GE Lunar Corp., Madison, WI) (5). After euthanizing the animals, tibiae were removed and dissected free of soft tissue. The bones were then fixed in 10% (v/v) buffered formalin for 24 h and analyzed by a µCT system using the manufacturer's included three-dimensional analysis software (µCT 40, Scanco Medical, Basserdorf, Switzerland). The region of interest analyzed from each mouse was the metaphysis of the proximal tibia. The trabecular bone in the tibia was scanned using a 12-µm slice increment on 100 slices, starting 360 µm distal to the growth plate and extending for 120 µm (5).

Adenovirus-Green Fluorescent Protein (GFP) and -Cre Infection—Primary calvarial osteoblasts were isolated from 1-day-old Cnb1f/f mice as previously described (5). Briefly, mice calvariae were subjected to three sequential digestions in an enzymatic solution containing 0.1% (w/v) collagenase (Worthington, Lakewood, NJ) and 0.05% (v/v) trypsin containing 0.53 mM EDTA at 37 °C. Osteoblasts were collected from the second and third digestions. Cells were maintained in minimum essential medium Eagle-{alpha} modification (Sigma) containing 10% fetal bovine serum (Atlanta Biologicals), 100 units/ml penicillin G, and 100 µg/ml streptomycin (Invitrogen) at 37 °C with 5% CO2. Adenovirus-GFP and -Cre were purchased from Vector Biolabs (Philadelphia). At confluency, cells were infected for 48 h with adenovirus-GFP and -Cre at an multiplicity of infection of 100 (22). Osteoblastic induction was performed by supplementing the culture medium with 5 mM β-glycerophosphate and 250 µM ascorbic acid 2-phosphate (5, 21).

Immunohistochemistry—Tissues were deparaffinized and rehydrated followed by antigen retrieval using 10 mM citrate buffer, pH 6. Endogenous peroxidase activity was quenched using 1% hydrogen peroxide. Samples were then blocked for 1 h in Fc receptor blocker (Innovex Biosciences, Richmond, CA). Anti-Fra-2 (Santa Cruz Biotechnology) was diluted in Fc Blocker solution and applied to the sections overnight at 4 °C. Biotin-conjugated secondary antibodies were then used followed by incubation with avidin-biotin enzyme reagents. Finally, specimens were incubated in peroxidase substrate for 30 s. Tissues were counterstained in Gill's hematoxylin for 10 s, dehydrated, cleared, and mounted. Negative controls were processed alongside the examined tissue using rabbit IgG instead of the primary antibody. At least 10 randomly selected microscopic fields were examined using 10x and 40x objectives. Photos were taken using a Nikon digital camera (23).

Osteoblasts/Osteoclasts Co-culture—Primary osteoclast precursor cells were isolated by flushing bone marrow cells from the long bones of 2-month-old control and {Delta}Cnb1OB mice. Cells were plated and cultured overnight in minimum essential medium Eagle-{alpha} modification containing heat-inactivated 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 100 units/ml penicillin G, and 100 µg/ml streptomycin (Invitrogen). Suspended cells were collected and layered on a Ficoll (Amersham Biosciences) then centrifuged at 340 x g for 15 min at room temperature. Bone marrow macrophage precursor cells were then collected from the interface. Primary adenovirus-GFP and -Cre-infected Cnb1f/f osteoblasts (105/well) were co-cultured with bone marrow cells (106/well) in minimum essential medium Eagle-{alpha} modification containing 10% fetal bovine serum, 10-7 M dexamethasone, and 10-8 M 1{alpha}, 25(OH)2D3-dihydroxyvitamin D3 in 24-well plates. Culture media were changed every 3 days for 10 days. At the end of the study cultures were stained for TRAP using a TRAP staining kit (Sigma) (24).


Figure 1
View larger version (65K):
[in this window]
[in a new window]

  		FIGURE 1.
Generation of {Delta}Cnb1OB mice. A, reverse transcription-PCR analysis was performed using different tissues that were harvested from {Delta}Cnb1OB mice to examine specific deletion of the Cnb1 gene. As indicated, bands represent Cnb1 gene alleles that are either flanked with a loxP site (Cnb1f) or deleted ({Delta}). B, Cnb1 and NFATc1 protein levels were determined by Western blot analysis using protein extracts from primary calvarial osteoblasts and liver tissues that were harvested from control, heterozygote (f/{Delta}Cnb1OB), and homozygote Cnb1-deleted mice ({Delta}Cnb1OB). CnA{alpha} and actin were used as controls. NFATc1 antibody detects total NFATc1 protein regardless of its phosphorylation state. Please note that the shown decrease in NFATc1 mobility shift is indicative of an increase of the protein phosphorylation due to inactivation, and the increase in intensity is a result of an accumulation of NFATc1 protein in the cytoplasmic compartment due to an inhibition of its nuclear translocation. Photographs are representative of three different experiments using cells, and tissues are from three different animals.

 
Real-time PCR Analysis—Total RNA was extracted by the Trizol method as recommended by the manufacturer (Invitrogen) (5). One µg of RNA was reverse-transcribed, and the equivalent of 10 ng was used for real-time PCR as previously described (25). Expression of actin was used for the normalization of gene expression values. The sequences for the specific primers used in this study were previously described: alkaline phosphatase, osteocalcin, collagen I, osteoprotegerin, RANKL, and actin (21, 22, 26).

Whole Cell Protein Extraction and Western Blot Analysis—Cells were lysed in 0.5% Nonidet P-40 lysis buffer supplemented with protease and phosphatase inhibitors (23). Samples were then centrifuged at 14,000 x g for 30 min at 4 °C, and the supernatant protein concentration was measured by the Bio-Rad DC protein assay (23). Protein extracts (20 µg/lane) were separated by SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane, Immobilon-P (Millipore Co.), using a Bio-Rad wet transfer system. Membranes were then blocked with TBS-Blotto B (Santa Cruz) for 1 h at room temperature and subsequently incubated overnight with antibodies directed against calcineurin β-subunit (anti-CnB1, Sigma C0581), calcineurin A-subunit (anti-PP2BA, Santa Cruz, SC-9070), total NFATc1 (NFATc1, Santa Cruz, SC-7294), the C terminus of actin (actin, Santa Cruz, SC-1615), and the N terminus of Fra-2 (Fra-2, Santa Cruz, SC-604). Signals were detected using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (ECL, Amersham Biosciences) (21, 25).

Enzyme Serum Measurements—Serum was collected from the blood of control and {Delta}Cnb1OB animals immediately after euthanasia. OPG and RANKL serum levels were measured using the mouse OPG/TNFSRSF11B and mouse TRANCE immunoassay according to manufacturer's protocol (R&D Systems, Minneapolis, MN).

Statistical Analysis—All statistical analyses were performed using the Microsoft Excel data analysis program for Student's t test analysis. Experiments were repeated at least three times unless otherwise stated. Values are expressed as the mean ± S.E. or ±S.D. as indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To determine whether Cnb1 exerts direct actions in osteoblasts, we generated mice lacking Cnb1 in osteoblasts ({Delta}Cnb1OB) using Cre-mediated recombination. Transgenic mice expressing the Cre recombinase driven by the human osteocalcin promoter were crossed with homozygous mice that express loxP-flanked Cnb1 (Cnb1f/f). To confirm that Cnb1 deletion in {Delta}Cnb1OB mice is indeed specific to osteoblasts, we performed PCR analysis using a combination of primers that specifically detect floxed Cnb1 alleles (Cnb1f) and null alleles (Cnb1{Delta}). Genomic DNA was extracted from several non-skeletal tissues such as brain, kidney, lung, liver, heart, and muscle as well as skeletal tissues including calvarium, femur, and spine from the {Delta}Cnb1OB offspring. Fig. 1A demonstrates that Cre-mediated recombination (Cnb1{Delta}) occurred exclusively in tissues that contain osteoblastic cells, whereas non-skeletal tissues retained the Cnb1-floxed alleles (Cnb1f). Non-osteoblastic cells in skeletal tissues such as chondrocytes, adipocytes, and myocytes also retained their Cnb1f alleles (Fig. 1A).

We then assessed the specific deletion of Cnb1 in osteoblasts by examining the levels of Cnb1 protein in primary calvarial osteoblasts that were harvested from 1-day-old control, f/{Delta}Cnb1OB, and {Delta}Cnb1OB pups. Primary calvarial osteoblasts were cultured for 30 days in the presence of β-glycerophosphate and ascorbic acid to induce osteoblast differentiation and ensure the activation of the osteocalcin promoter in vitro and, thus, the expression of Cre-recombinase. {Delta}Cnb1OB osteoblasts completely lacked Cnb1 as compared with controls and heterozygotes (f/{Delta}Cnb1OB) (Fig. 1B, left panel). Loss of one allele of Cn (f/{Delta} Cnb1OB) in osteoblasts resulted in a slight decrease in the dephosphorylation of NFATc1 demonstrated by a slower mobility shift of NFATc1 due to the increase in its phosphorylation and an increase in its intensity because of NFATc1 cytoplasmic accumulation in response to inactivation (Fig. 1B, left panel). Because of the potential compensation of other Cn isoforms in response of Cnb1 deletion, we examined the expression of the major Cn catalytic subunit (CnA{alpha}) as well as the dephosphorylation of NFATc1, which is dependent on Cn activation. The deletion of Cnb1 isoform (regulatory subunit) in osteoblasts did not affect the levels of the CnA{alpha} (catalytic subunit), and it was sufficient to inhibit the dephosphorylation of NFATc1 as demonstrated by the accumulation of highly phosphorylated NFATc1 proteins (slow mobility shift) in the cytoplasmic compartment (increase in intensity). This suggests that the deletion of Cnb1 in osteoblasts is not compensated for by other Cn isoforms (Cn A{alpha},Aβ,A{gamma}, and B2). Furthermore, protein levels of Cnb1, CnA{alpha}, and NFATc1 were not affected in non-osteoblastic tissues such as the liver (Fig. 1B, right panel). Taken together, these data confirm that the {Delta}Cnb1OB mice represent a suitable model for determining the direct role of Cnb1 in osteoblasts on bone formation and bone mass.


Figure 2
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 2.
Bone volume is increased in osteoblast-specific {Delta}Cnb1OB mice. A, µCT three-dimensional reconstruction images of proximal metaphysis of tibia from control and {Delta}Cnb1OB mice are shown. Sagittal and transverse views were reconstructed. Images were obtained from control mice (n = 4) and {Delta}Cnb1OB mice (n = 6). B, bone mineral density was determined by using DXA. Values represent the mean ± S.D. of {Delta}Cnb1OB mice (n = 10) compared with control (n = 8); *, p ≤ 0.05. C, mean values of structure model index, calculated by µCT of tibiae, demonstrating a conversion of trabecular bone in the {Delta}Cnb1OB mice to plate-like rather than rod-like structures as shown in control mice. D-E, quantitative analysis of histomorphometrical indices was performed; D, trabecular bone volume/tissue volume (BV/TV); E, trabecular thickness (Tb.Th); F, trabecular number (Tb.N); G, trabecular separation (Tb.Sp). Values represent the mean ± S.D. of {Delta}Cnb1OB mice (n = 6) compared with control (n = 4); *, p ≤ 0.05.

 
There were no gross abnormalities in the skeletal development of the f/{Delta}Cnb1OB or {Delta}Cnb1OB mice as compared with controls. To examine the effects of Cnb1 ablation in osteoblasts on bone architecture, tibiae from 3-month-old male {Delta}Cnb1OB and control mice were compared by µCT analysis. Three-dimensional images revealed a pronounced increase in the trabecular bone volume in the {Delta}Cnb1OB mice as compared with control mice using sagittal and transverse scanning of the metaphysis of the proximal tibia (Fig. 2A). There was no difference between heterozygote and control animals even though Cnb1 was somewhat deleted in these animals (data not shown). Non-cephalic whole body bone mineral density measured by DXA is significantly increased in the {Delta}Cnb1OB mice as compared with control mice (Fig. 2B). This microarchitectural increase in trabecular bone volume was further substantiated by a decrease in the structure model index (Fig. 2C). This indicates the conversion of trabecular bone to plate-like rather than rod-like structures because of an increase in bone connectivity and a decrease in perforation. Static histomorphometric analyses of distal femora of 3-month-old male mice confirmed the µCT findings. Mutant mice that have had Cnb1 specifically deleted in osteoblasts demonstrated a 32% increase in trabecular bone volume/tissue volume, a 29% increase in trabecular thickness, a 10% increase in trabecular number, and a 17% decrease in trabecular separation when compared with controls (Figs. 2, D-G). The combination of these microarchitectural and histomorphometrical results indicates that Cnb1 in osteoblasts negatively regulates bone mass. These changes were limited to trabecular bone, as we did not find a statistical difference in the cortical bone thickness between {Delta}Cnb1OB and control mice when measured by µCT histomorphometry (data not shown).


Figure 3
View larger version (65K):
[in this window]
[in a new window]

  		FIGURE 3.
Conditional deletion of Cnb1 in osteoblasts increases bone formation in vivo. Distal femora from 3-month-old male control and {Delta}Cnb1OB mice were harvested; tissues were fixed, embedded in methyl methacrylate, sectioned, and examined. A, upper panel, unstained sections were examined under a florescence microscope to visualize and measure the distance between the two calcein layers (white arrow) which reflects the mineral apposition rate. A, lower panel, tissues were stained with Trichrome to distinguish the mineralized bone (green/blue), and osteoid that lines bone (red). Representative pictures are shown from four control and six {Delta}Cnb1OB mice. B-E, quantitative analysis of histomorphometrical indices was performed and shows osteoid perimeter (mm) (B), mineral apposition rate (MAR) (µm/day) (C), osteoblast surface/bone surface (Ob.S/BS) (D), and number of osteoblasts/bone surface (N.Ob/BS) (E). Values represent the mean ± S.D. of {Delta}Cnb1OB mice (n = 6) compared with control (n = 4); *, p ≤ 0.05.

 
The increased bone volume seen in the {Delta}Cnb1OB mice was accompanied by an increase in bone formation measured histomorphometrically. We show dynamic changes in bone remodeling by injecting calcein twice at 7-day intervals before euthanizing the animals. The distance between two consecutive labels in the trabecular bone of the femur was dramatically greater in the {Delta}Cnb1OB mice than in the controls (Fig. 3A, upper panel). Goldner's Trichrome staining showed that {Delta}Cnb1OB mice had an increase in bone volume (green/blue) and newly formed bone (red) (Fig. 3A, lower panel). These parameters were quantitated using Bio-Quant image analysis system and demonstrated that {Delta}Cnb1OB mice had an increase in the osteoid perimeter (131%), mineral apposition rate (12%), osteoblast surface (70%), and osteoblast number (68%) when compared with the values from control mice (Figs. 3, B-E). Furthermore, all of the bone mass and bone formation indices were similar between heterozygote and control animals (data not shown). Taken together, these results demonstrate that the increase in bone mass in the {Delta}Cnb1OB mice is associated with an increase in bone formation.

To determine the role of Cnb1 in osteoblast differentiation, we first examined the levels of osteocalcin gene expression, which is a late marker of osteoblast differentiation. Primary ex vivo calvarial osteoblasts that were harvested from 1-day-old control, f/{Delta}Cnb1OB, and {Delta}Cnb1OB pups were cultured for 30 days in the presence of β-glycerophosphate and ascorbic acid to induce osteoblast differentiation. The deletion of Cnb1 in primary osteoblasts significantly increases osteocalcin gene expression (220%) as compared with control and heterozygote mice (Fig. 4A). Furthermore, we examined the effects of the in vitro deletion of Cnb1 in primary calvarial osteoblasts. Primary osteoblasts were harvested from mice carrying Cnb1 floxed alleles (Cnb1f/f) and then infected with either adenovirus expressing Cre recombinase (Cre+) or a control adenovirus directing the expression of GFP (Cre-). Cells were cultured in osteogenic differentiation media (in the presence of ascorbic acid and β-glycerophosphate) for 14 days. To confirm the efficiency of the adenovirus-cre in deleting Cnb1, proteins were extracted, and the levels of Cnb1 protein were examined by Western blotting. Here we show that infecting primary osteoblasts harvested from Cnb1f/f mice with adenovirus-Cre (Cre+) completely abolished Cnb1 protein, as compared with adenovirus-GFP-infected cells (Cre-). The deletion of Cnb1 in these cells also inhibited the activation of Cn-downstream targets, as we show that Cre+ cells have a dramatic increase in the levels of the phosphorylated and inactive form of NFATc1 protein (Fig. 4B). Cre+ and Cre- primary osteoblasts were then cultured for 14 days under osteogenic conditions, and the expression of several osteoblastic differentiation markers was examined. The deletion of Cnb1 in osteoblasts (Cre+) increases the expression of osteoblast-specific genes, alkaline phosphatase (150%), osteocalcin (134%), and collagen I (155%) as compared with control cells (Cre-) (Fig. 4, C-E). We also examined the expression of Runx2 in osteoblasts and found it to be unaffected in response to the deletion of Cnb1, suggesting that early events in the osteoblast differentiation do not depend on Cn signaling (data not shown).


Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 4.
The deletion of Cnb1 in osteoblasts increases osteoblast differentiation in vitro. A, osteocalcin expression level was determined by real-time reverse transcription-PCR using RNA extract from primary calvarial osteoblasts that were harvested from control, heterozygote (f/{Delta}Cnb1OB), and homozygote Cnb1-deleted mice ({Delta}Cnb1OB) pups. Values represent the means ± S.E. of osteocalcin (OC) mRNA expression relative to actin expression; *, p ≤ 0.05 compared with control. B, primary calvarial osteoblasts from Cnb1f/f mice were transduced with adenovirus-GFP (Cre-) or adenovirus-Cre (Cre+). Proteins were extracted and separated by SDS-PAGE. Immunoblots were developed using antibodies against Cnb1, CnA{alpha}, NFATc1, and actin. C-E, RNA was extracted, and real-time reverse transcription-PCR was performed for alkaline phosphatase (C), osteocalcin (D), collagen I and actin (E). Values represent the mean ± S.E. of alkaline phosphatase and osteocalcin mRNA expression relative to actin expression; *, p ≤ 0.05 compared with control. All studies were repeated three times using cells harvested from three different animals.

 
We have previously shown that the pharmacologic inhibition of Cn by CsA and the specific silencing of NFATc1 by small interfering RNA increases the expression of Fra-2, an activator protein-1 family member that is known to play a critical role in the differentiation of osteoblasts (5, 27, 28). Therefore, we examined whether the deletion of Cnb1 increases Fra-2 expression both in vivo and in vitro. Osteoblasts lining the bone in {Delta}Cnb1OB mice express higher levels of Fra-2 than osteoblast in control mice (Fig. 5A). Furthermore, the deletion of Cnb1 in primary osteoblasts in vitro (Cre+) also increases the levels of Fra-2 protein compared with control cells (Cre-) (Fig. 5B).

Bone mass is known to be controlled by the activities of boneforming osteoblasts and bone-resorbing osteoclasts. Therefore, we next examined bone resorption in the {Delta}Cnb1OB mice. TRAP staining demonstrated that the activity and the number of bone-resorbing osteoclasts are dramatically decreased in the {Delta}Cnb1OB as compared with control mice (Fig. 6A). This is confirmed by a quantitative static histomorphometrical analysis showing that {Delta}Cnb1OB mice have a decreased osteoclast surface (50%) and osteoclast number (42%) when compared with the values from control mice (Figs. 6, B and C). These results indicate that the deletion of Cnb1 in osteoblasts not only induces bone formation but also decreases bone resorption. This observation that {Delta}Cnb1OB mice exhibit a decrease in bone resorption parameters could be due to a disruption in the OPG-RANKL axis. To further explore this possibility, we measured serum levels of OPG (the osteoclast inhibitory factor) and RANKL (the major osteoclast differentiation factor) using mouse OPG and RANKL immunoassays. Our results demonstrate that the levels of OPG protein in the serum of {Delta}Cnb1OB mice were 193% increased when compared with control mice, whereas RANKL was 78% decreased (Fig. 7, A and B). Furthermore, we examined the gene expression of RANKL and OPG in primary osteoblasts that were Cnb1-deficient. Our results show that differentiated osteoblasts that lack Cnb1 exhibit a 155% increase in OPG expression accompanied by a 76% decrease in the expression of RANKL when compared with control cells (Figs. 7, C and D). Finally, to determine whether Cnb1-deficient osteoblasts are capable of supporting osteoclast differentiation, bone marrow macrophages from control and {Delta}Cnb1OB mice were co-cultured with Cnb1f/f primary calvarial osteoblasts that had been infected with either Cre- or Cre+ adenovirus in the presence of dexamethasone and 1,25-vitamin D3 for 10 days. Cultures were then stained for TRAP activity to identify TRAP-positive, multinucleated osteoclasts. Control primary calvarial osteoblasts that lack Cre recombinase were able to support osteoclasts generated from bone marrow macrophages collected from either control or {Delta}Cnb1OB mice, whereas Cre+ osteoblasts that lack Cnb1 failed to do so (Fig. 7E). Taken together, these results demonstrate that the inhibition of Cnb1 in osteoblasts increases bone mass by directly increasing the activation of bone-forming osteoblasts and indirectly decreasing the formation of bone-resorbing osteoclasts.


Figure 5
View larger version (91K):
[in this window]
[in a new window]

  		FIGURE 5.
Deletion of Cnb1 in osteoblasts increases Fra-2 protein levels. A, distal femora from 3-month-old male control and {Delta}Cnb1OB mice were harvested; tissues were fixed, decalcified with EDTA, embedded in paraffin, and sectioned. Immunohistochemical staining was performed using an antibody against Fra-2 (brown), and tissues were counterstained with hematoxylin (blue). Arrows indicate bone lining osteoblasts. Photomicrographs were taken at 400x magnification and are representative of four different mice in each group. B, primary calvarial osteoblasts from Cnb1f/f mice were transduced with adenovirus-GFP (Cre-) or adenovirus-Cre (Cre+). Proteins were extracted and separated by SDS-PAGE. Immunoblots were developed using antibodies against Fra-2 and actin. All studies were repeated three times using cells harvested from three different animals.

 


Figure 6
View larger version (102K):
[in this window]
[in a new window]

  		FIGURE 6.
Conditional deletion of Cnb1 in osteoblasts decreases bone resorption in vivo. A, femora were harvested from control and {Delta}Cnb1OB mice at 3 months old. Tissues were fixed, decalcified with EDTA, embedded in paraffin, sectioned, and stained for TRAP (red). Arrows indicate TRAP-positive, multinucleated osteoclasts. Representative pictures are shown from four control and six {Delta}Cnb1OB mice. B and C, quantitative analysis of histomorphometrical indices was performed and shows osteoclast surface/bone surface (Oc.S/BS) (B) and number of osteoclasts/bone surface (N.Oc/BS) (C). Values represent the mean ± S.D. of {Delta}Cnb1OB mice (n = 6) compared with control (n = 4); *, p ≤ 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have recently reported that the pharmacologic inhibition of Cn by low concentrations of CsA increased osteoblast differentiation in vitro and bone mass in vivo (5). However, these findings alone do not confirm that this bone-anabolic response is due to the specific inhibition of Cn in osteoblasts. This is because Cn is ubiquitously expressed and CsA in the blood could also be acting elsewhere. To determine whether Cn directly impacts osteoblast differentiation and is indeed a negative regulator of osteoblast differentiation and bone formation, we utilized Cre-mediated recombinase technology to create mice lacking the calcineurin regulatory subunit (Cnb1) specifically in osteoblasts. We discovered that in response to Cnb1 deletion, when limited to osteoblasts, bone formation is enhanced, and bone resorption is diminished, which ultimately led to an increase in bone mass.

We generated mutant mice that have Cnb1 gene deleted only in osteoblasts ({Delta}Cnb1OB). Mice lacking Cnb1 in osteoblasts developed normally. Mice were viable and showed no significant differences in size, weight, and survival rate (data not shown). However, mice that lacked Cnb1 in osteoblasts developed an increase in bone volume and bone mass. This is consistent with our previous findings where we reported that low concentrations of the Cn antagonist cyclosporin A increase osteoblast differentiation and bone mass in vitro and in vivo (5). Furthermore, we have previously shown that that anabolic response of CsA is due to the inhibition of NFATc1 activation by decreasing its dephosphorylation (5). Here we also show, similar to the pharmacologic inhibition of Cn, the genetic deletion of Cnb1 in osteoblasts decreases the dephosphorylation of NFATc1 and ultimately increases osteoblast differentiation and bone mass. We have also shown that the deletion of Cnb1 (in vitro and in vivo) results in a complete inhibition of the dephosphorylation of NFATc1. It has been shown by others that several Cn isoforms are expressed in osteoblasts (14). However, it has been demonstrated that whereas there is redundancy and compensation in response to the inhibition of different CnA catalytic subunits (29), the deletion of the Cnb1 regulatory subunit is sufficient to eliminate all Cn activity in several biological systems (19, 30, 31). Our results are consistent with these reports and demonstrate that Cnb1 deletion in osteoblasts is not compensated by any of the other Cn isoforms that were reported to be expressed in osteoblasts (Cn A{alpha},Aβ,A{gamma}, and B2).

The {Delta}Cnb1OB mice revealed a significant increase in bone formation, as demonstrated by an increase in calcein double labeling, implying accelerated osteogenesis. The osteoid perimeter and mineral apposition rate, which are markers of osteoblastic bone formation, were significantly increased in {Delta}Cnb1OB mice as compared with the control mice. Finally, osteoblast surface/bone surface as well as number of osteoblasts were significantly higher in {Delta}Cnb1OB mice, confirming that bone formation is greatly increased in response to Cnb1 deletion in osteoblasts. However, the increase in osteoblast number in the {Delta}Cnb1OB mice could not be explained by an increase in osteoblast proliferation. In this mouse model we deleted Cnb1 in mature osteoblasts by expressing Cre-recombinase that is driven by the osteocalcin promoter. It is known that osteocalcin promoter is activated at a late stage of osteoblast differentiation, long after osteoblasts exit the cell cycle. Therefore, it is unlikely that osteoblast proliferation was increased in response to Cnb1 deletion during the late stage of osteoblast differentiation. This notion was supported by examining the proliferation of primary cells that lack Cnb1 in vitro. Cre-mediated deletion of Cnb1 in primary calvarial osteoblasts showed no significant changes in osteoblast proliferation, as compared with control (data not shown). Thus, the increase in the number of osteoblasts as a result of deleting Cnb1 in osteoblasts could be due to an increase in the recruitment of new osteoblasts to the bone surface, suggesting a role of Cnb1 in osteoblasts in regulating an autocrine or paracrine growth factor in the bone microenvironment that increases osteoblast numbers in vivo. This hypothesis is supported by reports demonstrating that Cn is a negative regulator of epidermal growth factor receptor signaling (32). Interestingly, epidermal growth factor signaling has been shown to play an important role in recruiting osteoblastic lineage cells from mesenchymal stem cells (33).


Figure 7
View larger version (42K):
[in this window]
[in a new window]

  		FIGURE 7.
Osteoblasts lacking Cnb1 cannot maintain osteoclastogenesis. A and B, serum was collected from the blood of control and {Delta}Cnb1OB 3-month-old mice immediately after euthanasia. Protein levels of OPG (A) and RANKL (B) in serum were measured using OPG and RANKL serum enzyme-linked immunoassay. Values were obtained from four mice in each control and {Delta}Cnb1OB group and represent the mean ± S.E.; *, p ≤ 0.05. C and D, primary calvarial osteoblasts were harvested from Cnb1f/f mice and transduced with adenovirus-GFP (Cre-) or adenovirus-Cre (Cre+). Cells were cultured in osteogenic differentiation media (in the presence of ascorbic acid and β-glycerophosphate) for 7 days. RNA was extracted and real-time reverse transcription-PCR was performed for OPG (C) and RANKL (D). Values represent the mean ± S.E. of OPG and RANKL mRNA expression relative to actin expression; *, p ≤ 0.05 compared with control. All studies were repeated three times, each repeated in triplicate. E, Cre- and Cre+ primary calvarial osteoblasts from Cnb1f/f mice were co-cultured with bone marrow macrophages from control and {Delta}Cnb1OB mice in the presence of 10-8 M 1,25-vitamin D3 and 10-7 M dexamethasone. After 10 days, cells were fixed and stained for TRAP to detect multinucleated osteoclasts (indicated by black arrows). Representative pictures from two separate experiments are shown.

 
The increase in bone mass may result from enhancing bone formation and/or decreasing bone resorption. The deletion of Cnb1 in osteoblasts increases the differentiation of osteoblasts and enhances their activity and their function. The increase in the levels of unmineralized osteoid is an indication of the increase in activity of osteoblasts rather than a decrease in the mineralization of new bone. This is supported by our data showing that the {Delta}Cnb1OB when compared with control mice have an increase in trabecular bone volume as shown by µCT and histology, an increase in mineral apposition rate, and an increase in bone mineral density. Interestingly, the increase in bone volume and formation were limited to trabecular bone, as we did not find a statistical difference in the cortical bone thickness between {Delta}Cnb1OB and control mice when measured by µCT histomorphometry (data not shown). Nevertheless, these findings do not conclusively prove that the remodeling of cortical bone is independent of Cnb1. It is well known that the rate of cortical bone remodeling is significantly slower than that of the cancellous bone. Furthermore, our studies specifically examine the skeleton of 3-month-old mice which might not reflect the true state of a fully mature skeleton and of cortical bone in particular. Therefore, to unequivocally determine the role of Cnb1 in cortical bone remodeling, more studies are required, such as examining the cortical bone volume and mineral apposition rate in older animals that have more mature skeletons.

Furthermore, mice lacking Cnb1 in osteoblasts develop a decrease in bone resorption demonstrated by a decrease in TRAP activation and TRAP-positive osteoclasts. This decrease in osteoclast number appears to be secondary to the deletion of Cnb1 in osteoblasts for several reasons. First, monocytes or macrophages from either {Delta}Cnb1OB mice or control mice differentiated into multinucleated osteoclasts when cultured with control osteoblasts, indicating that osteoclasts harvested from {Delta}Cnb1OB mice are normal. Second, deletion of Cnb1 in osteoblasts rendered these cells incapable of inducing osteoclastogenesis. Finally, {Delta}Cnb1OB mice have reduced osteoclast surface and numbers.

It is has been shown that decreases in bone resorption could result from dysregulation in the OPG-RANKL axis. In the process of examining this possibility, we discovered that OPG protein levels were indeed increased in {Delta}Cnb1OB mice as compared with control mice, whereas RANKL levels were dramatically decreased. This was also confirmed by demonstrating that the deletion of Cnb1 in osteoblasts increased the expression of OPG and decreased that of RANKL. It is not completely clear how Cn regulates the expression of RANKL and OPG in osteoblasts. This could be through a direct binding of different transcription factors that are regulated by Cnb1 that leads to an activation of the OPG promoter and inhibition of RANKL. Alternatively, Cnb1 could be controlling the expression and/or activation of some unidentified factor(s) that indirectly regulates the expression of OPG and RANKL. Nevertheless, our data demonstrate that the absence of Cnb1 in osteoblasts indirectly regulates osteoclastogenesis in {Delta}Cnb1OB mice in vivo and in vitro. Together, our findings demonstrate that the deletion of Cnb1 in osteoblasts increases trabecular bone volume and bone mineral density by not only increasing osteoblastic bone formation but also reducing bone resorption. Therefore, it is not possible to accurately deduce from these data if either osteoblastic or osteoclastic bone remodeling is predominant in this apparent bone phenotype.

The precise mechanism by which Cnb1 regulates the expression of osteogenic gene expression is currently not known. However, transcription factors, namely Fra-2, which is an activator protein-1 family member, is known to play a critical role in the differentiation of a variety of cells, including osteoblasts (27, 28). We have previously shown that the pharmacologic inhibition of Cn by CsA and the specific silencing of NFATc1 by small interfering RNA increase the expression of Fra-2 (5). Similarly, we found that the levels of Fra-2 protein are dramatically increased in mature osteoblasts lining the bone in the {Delta}Cnb1OB mice as compared with control mice. Furthermore, we also show that the deletion of Cnb1 in osteoblasts in vitro also increases the levels of Fra-2 protein compared with control cells. Therefore, it is possible that the up-regulation of osteogenic gene expression in the {Delta}Cnb1OB mice is due an increase in fra-2 expression. Nevertheless, we cannot rule out the potential direct involvement of other transcription factors as well as the cooperation between NFATc1 and factors that are involved in osteoblast differentiation in response to Cnb1 deletion.

Our results are in contrast to three recent reports describing the Cn/NFAT signaling pathway as a positive regulator of osteoblast differentiation and bone formation (13-15). In these studies this conclusion was based on data from murine animal models where the Cn/NFAT signaling pathway was globally altered and/or expressed in multiple tissues. Together, this makes the global genetic alteration of Cn/NFAT signaling unreliable for examining the direct roles of Cn and NFAT in osteoblast differentiation and bone mass. For example, the reported decrease in bone mass in response to the global loss of CnA{alpha} could be an indirect response to an alteration in skeletal muscle phenotype (14, 34). Also, the global loss of CnA{alpha} is known to alter T cell responses and activation that may indirectly affect the bone phenotype (35). Furthermore, boneforming osteoblasts are known to originate from the osteogenic differentiation of mesenchymal stem cells. These bone marrow stem cells are pluripotent and capable of differentiating into several lineages such as chondrocytes, myocytes, and adipocytes in addition to osteoblasts (3). Interestingly, Cn/NFAT signaling has been shown to play a critical role in the lineage decision of stem cells to differentiate into chondrocytes, myocytes, and adipocytes (34, 36, 37). Indeed, the global deletion of NFATc2 in mice has been shown to increase the expression of chondrocytic-specific genes and chondrogenesis, suggesting that NFATc2 is a negative regulator of chondrocyte growth and differentiation (37). Global NFATc2/c4 double knock-out mice exhibit defects in fat accumulation and reduced adiposity, suggesting that NFAT signaling is positive regulator of adipogenesis (38). Finally, it has been shown that the number of primary myofibers and muscle size are decreased in the global NFATc3-deficient mice (39). Taken together, it is likely that the global modulation of Cn or NFAT expression in murine animal models does not provide a reliable tool for examining osteoblast differentiation and bone mass, as Cn/NFAT signaling is globally expressed and is involved in the lineage decision of mesenchymal stem cells.

The work described here is consistent with our previous reports that describe the effects of the pharmacologic inhibition of Cn by low concentrations of CsA on bone formation (5). Indeed, the skeletal phenotypes of mice that were treated with a low dose of CsA and osteoblast-specific Cnb1 deleted are similar. Both had increased bone mass and osteoblastic bone formation and showed a decrease in osteoclastogenesis and bone resorption. Clinically, this work provides a better understanding of the mechanisms by which Cn/NFAT regulates osteoblastic bone formation and may have important implications in the development of new anabolic drugs that target the Cn/NFAT pathway in osteoblasts to treat osteoporosis and bone loss.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AR050235 and CA109119 (to J. M. M.), P01-CA098912 (to M. Z.), and R01-AR053898 (to M. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: University of Alabama at Birmingham, 813 Shelby Biomedical Research Bldg., 1825 University Blvd., Birmingham, AL 35294-2182. Tel.: 205-934-5574; Fax: 205-996-6119; E-mail: mzayzafo{at}uab.edu.

2 The abbreviations used are: RANKL, receptor activator of NF{kappa}B ligand; OPG, osteoprotegerin; Cn, calcineurin; CsA, cyclosporin A; NFAT, nuclear factors of activated T cells; TRAP, tartrate-resistant acid phosphatase; GFP, green fluorescent protein; DXA, dual energy x-ray absorptiometry; µCT, microcomputed tomography. Back


   ACKNOWLEDGMENTS
 
We thank Patty Lott in the Histomorphometry and Molecular Analysis Core Laboratory for help with the histology and histomorphometry and Dr. Xu Feng in the Human Bone Cell Production Core in the University of Alabama at Birmingham, Center for Metabolic Bone Disease (supported by National Institutes of Health Grant P30-AR46031). We also thank Dr. Gerald Crabtree for providing the Cnb1f/f mice and the Clinical Nutrition Research Unit, Small Animal Phenotyping Core (supported by National Institutes of Health Grant P30-DK56336), for help with the DXA and µCT. Finally, we thank Jennifer Paige-Robinson for the critical reading of the manuscript and Drs. Kaiyu Yuan and Jiangzhong Liu for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Karsenty, G., and Wagner, E. F. (2002) Dev. Cell 2, 389-406[CrossRef][Medline] [Order article via Infotrieve]
  2. Teitelbaum, S. L. (2000) Science 289, 1504-1508[Abstract/Free Full Text]
  3. Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. (1999) Science 284, 143-147[Abstract/Free Full Text]
  4. Aubin, J. E. (1998) J. Cell. Biochem. Suppl. 30-31, 73-82[Medline] [Order article via Infotrieve]
  5. Yeo, H., Beck, L. H., McDonald, J. M., and Zayzafoon, M. (2007) Bone (NY) 40, 1502-1516
  6. Hogan, P. G., Chen, L., Nardone, J., and Rao, A. (2003) Genes Dev. 17, 2205-2232[Free Full Text]
  7. Klee, C. B., Crouch, T. H., and Krinks, M. H. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 6270-6273[Abstract/Free Full Text]
  8. Shibasaki, F., Hallin, U., and Uchino, H. (2002) J Biochem. (Tokyo) 131, 1-15[Abstract/Free Full Text]
  9. Zhu, L. L., Zaidi, S., Moonga, B. S., Troen, B. R., and Sun, L. (2005) Biochem. Biophys. Res. Commun. 326, 131-135[CrossRef][Medline] [Order article via Infotrieve]
  10. Komarova, S. V., Pereverzev, A., Shum, J. W., Sims, S. M., and Dixon, S. J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 2643-2648[Abstract/Free Full Text]
  11. Matsuo, K., Galson, D. L., Zhao, C., Peng, L., Laplace, C., Wang, K. Z., Bachler, M. A., Amano, H., Aburatani, H., Ishikawa, H., and Wagner, E. F. (2004) J. Biol. Chem. 279, 26475-26480[Abstract/Free Full Text]
  12. Hirotani, H., Tuohy, N. A., Woo, J. T., Stern, P. H., and Clipstone, N. A. (2004) J. Biol. Chem. 279, 13984-13992[Abstract/Free Full Text]
  13. Koga, T., Matsui, Y., Asagiri, M., Kodama, T., de Crombrugghe, B., Nakashima, K., and Takayanagi, H. (2005) Nat. Med. 11, 880-885[CrossRef][Medline] [Order article via Infotrieve]
  14. Sun, L., Blair, H. C., Peng, Y., Zaidi, N., Adebanjo, O. A., Wu, X. B., Wu, X. Y., Iqbal, J., Epstein, S., Abe, E., Moonga, B. S., and Zaidi, M. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 17130-17135[Abstract/Free Full Text]
  15. Winslow, M. M., Pan, M., Starbuck, M., Gallo, E. M., Deng, L., Karsenty, G., and Crabtree, G. R. (2006) Dev. Cell 10, 771-782[CrossRef][Medline] [Order article via Infotrieve]
  16. Crabtree, G. R., and Olson, E. N. (2002) Cell 109, (Suppl. 1) 67-79
  17. Horsley, V., and Pavlath, G. K. (2002) J. Cell Biol. 156, 771-774[Abstract/Free Full Text]
  18. Zhang, M., Xuan, S., Bouxsein, M. L., von Stechow, D., Akeno, N., Faugere, M. C., Malluche, H., Zhao, G., Rosen, C. J., Efstratiadis, A., and Clemens, T. L. (2002) J. Biol. Chem. 277, 44005-44012[Abstract/Free Full Text]
  19. Neilson, J. R., Winslow, M. M., Hur, E. M., and Crabtree, G. R. (2004) Immunity 20, 255-266[CrossRef][Medline] [Order article via Infotrieve]
  20. Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Kanis, J. A., Malluche, H., Meunier, P. J., Ott, S. M., and Recker, R. R. (1987) J. Bone Miner. Res. 2, 595-610[Medline] [Order article via Infotrieve]
  21. Zayzafoon, M., Fulzele, K., and McDonald, J. M. (2005) J. Biol. Chem. 280, 7049-7059[Abstract/Free Full Text]
  22. Liu, X., Bruxvoort, K. J., Zylstra, C. R., Liu, J., Cichowski, R., Faugere, M. C., Bouxsein, M. L., Wan, C., Williams, B. O., and Clemens, T. L. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 2259-2264[Abstract/Free Full Text]
  23. Zayzafoon, M., Abdulkadir, S. A., and McDonald, J. M. (2004) J. Biol. Chem. 279, 3662-3670[Abstract/Free Full Text]
  24. Feng, X., Novack, D. V., Faccio, R., Ory, D. S., Aya, K., Boyer, M. I., McHugh, K. P., Ross, F. P., and Teitelbaum, S. L. (2001) J. Clin. Investig. 107, 1137-1144[Medline] [Order article via Infotrieve]
  25. Zayzafoon, M., Gathings, W. E., and McDonald, J. M. (2004) Endocrinology 145, 2421-2432[Abstract/Free Full Text]
  26. Shin, C. S., Her, S. J., Kim, J. A., Kim, D. H., Kim, S. W., Kim, S. Y., Kim, H. S., Park, K. H., Kim, J. G., Kitazawa, R., Cheng, S. L., and Civitelli, R. (2005) J. Bone Miner. Res. 20, 2200-2212[CrossRef][Medline] [Order article via Infotrieve]
  27. Wagner, E. F. (2002) Ann. Rheum. Dis. 61, Suppl. 2, 40-42
  28. McCabe, L. R., Banerjee, C., Kundu, R., Harrison, R. J., Dobner, P. R., Stein, J. L., Lian, J. B., and Stein, G. S. (1996) Endocrinology 137, 4398-4408[Abstract]
  29. Klee, C. B., Ren, H., and Wang, X. (1998) J. Biol. Chem. 273, 13367-13370[Free Full Text]
  30. Heit, J. J., Apelqvist, A. A., Gu, X., Winslow, M. M., Neilson, J. R., Crabtree, G. R., and Kim, S. K. (2006) Nature 443, 345-349[CrossRef][Medline] [Order article via Infotrieve]
  31. Chang, C. P., McDill, B. W., Neilson, J. R., Joist, H. E., Epstein, J. A., Crabtree, G. R., and Chen, F. (2004) J. Clin. Investig. 113, 1051-1058[CrossRef][Medline] [Order article via Infotrieve]
  32. Sullivan, K. M., and Rubin, G. M. (2002) Genetics 161, 183-193[Abstract/Free Full Text]
  33. Kratchmarova, I., Blagoev, B., Haack-Sorensen, M., Kassem, M., and Mann, M. (2005) Science 308, 1472-1477[Abstract/Free Full Text]
  34. Parsons, S. A., Wilkins, B. J., Bueno, O. F., and Molkentin, J. D. (2003) Mol. Cell. Biol. 23, 4331-4343[Abstract/Free Full Text]
  35. Zhang, B. W., Zimmer, G., Chen, J., Ladd, D., Li, E., Alt, F. W., Wiederrecht, G., Cryan, J., O'Neill, E. A., Seidman, C. E., Abbas, A. K., and Seidman, J. G. (1996) J. Exp. Med. 183, 413-420[Abstract/Free Full Text]
  36. Neal, J. W., and Clipstone, N. A. (2003) J. Biol. Chem. 278, 17246-17254[Abstract/Free Full Text]
  37. Ranger, A. M., Gerstenfeld, L. C., Wang, J., Kon, T., Bae, H., Gravallese, E. M., Glimcher, M. J., and Glimcher, L. H. (2000) J. Exp. Med. 191, 9-22[Abstract/Free Full Text]
  38. Yang, T. T., Suk, H. Y., Yang, X., Olabisi, O., Yu, R. Y., Durand, J., Jelicks, L. A., Kim, J. Y., Scherer, P. E., Wang, Y., Feng, Y., Rossetti, L., Graef, I. A., Crabtree, G. R., and Chow, C. W. (2006) Mol. Cell. Biol. 26, 7372-7387[Abstract/Free Full Text]
  39. Kegley, K. M., Gephart, J., Warren, G. L., and Pavlath, G. K. (2001) Dev. Biol. 232, 115-126[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 IBMS BoneKEyHome page
K. K. VanKoevering and B. O. Williams
Transgenic Mouse Strains for Conditional Gene Deletion During Skeletal Development
IBMS BoneKEy, May 1, 2008; 5(5): 151 - 170.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available