From the Department of Cardiovascular and Metabolic
Diseases and the § Department of Genetic Technologies,
Pfizer Global Research and Development, Groton, Connecticut 06340
Received for publication, April 5, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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We have previously described osteoblast/osteocyte
factor 45 (OF45), a novel bone-specific extracellular matrix protein,
and demonstrated that its expression is tightly linked to
mineralization and bone formation. In this report, we have cloned and
characterized the mouse OF45 cDNA and genomic region.
Mouse OF45 (also called MEPE) was similar to
its rat orthologue in that its expression was increased during
mineralization in osteoblast cultures and the protein was highly
expressed within the osteocytes that are imbedded within bone. To
further determine the role of OF45 in bone metabolism, we generated a
targeted mouse line deficient in this protein. Ablation of
OF45 resulted in increased bone mass. In fact, disruption
of only a single allele of OF45 caused significantly increased bone mass. In addition, knockout mice were resistant to
aging-associated trabecular bone loss. Cancellous bone histomorphometry revealed that the increased bone mass was the result of increased osteoblast number and osteoblast activity with unaltered osteoclast number and osteoclast surface in knockout animals. Consistent with the
bone histomorphometric results, we also determined that OF45 knockout osteoblasts produced significantly more
mineralized nodules in ex vivo cell cultures than did wild
type osteoblasts. Osteoclastogenesis and bone resorption in ex
vivo cultures was unaffected by OF45 mutation. We
conclude that OF45 plays an inhibitory role in bone formation in mouse.
The constant modulation of the balance between skeletal strength
and mineral availability in bone is effected by competing cell types in
response to physiological needs. Osteoblasts produce, organize, and
mineralize bone matrix in forming bone. Osteoclasts break down matrix
by forming a lytic pocket in which bone is degraded and calcium is
released. The generation and activity of these cell types is
tightly regulated to provide equilibrium between formation and
resorption and, thereby, an appropriate balance of strength and mineral
release. Under certain conditions, such as aging, postmenopausal
estrogen deficiency, or some pathophysiological states, there can exist
an imbalance between bone resorption and bone formation. As a result,
skeletal mass and strength are compromised and osteoporotic fractures
can occur in the afflicted individuals.
Bone is produced by the organization and mineralization of the
extracellular matrix produced by osteoblasts. The major component of
the extracellular matrix of these cells is Type I collagen, which
functions as a scaffold for new bone. In addition, non-collagenous matrix proteins have been identified that influence the operations of
bone turnover, formation, and repair. These proteins are generally acidic and highly post-translationally modified by phosphorylation, glycosylation, or sulfation (1).
Targeted deletion of extracellular matrix genes in mice has been a
useful method to determine the in vivo functions of several matrix proteins. For example, osteocalcin is an abundant gamma carboxyl
glutamic acid-containing bone matrix protein shown to be highly
expressed in osteoblasts and is a biochemical marker of the bone
remodeling process. Osteocalcin deletion in mice results in increased
cortical bone thickness due to increased osteoblast activity,
indicating that osteocalcin has negative effects in vivo on
osteoblasts and bone formation (2). Another gene of this category,
matrix glutamic acid protein (known as
"MGP"),1 was deleted in
mice, resulting in extensive cartilage calcification and in the
inappropriate calcification of arteries, leading to blood vessel
rupture, and lethality in homozygotes (3). Ablation of the
extracellular matrix protein osteonectin causes reduced bone formation
with a decrease in both osteoblasts and osteoclasts resulting in a net
loss of trabecular bone compared with wild type controls (4). Targeted
disruption of osteopontin, an RGD-containing protein, results in a
defect in the in vitro differentiation of osteoclasts;
increased mineral content and maturity in long bones; and resistance to
ovariectomy-induced, parathyroid hormone-induced, and mechanical
unloading-induced bone resorption in mice (5-8). The functions of
matrix proteins can be further elucidated through the characterization
of mice deficient in combinations of genes. For example, mice deficient
in both osteopontin and MGP exhibit more vascular calcification than
the MGP knockout mice, demonstrating that osteopontin can be an
inhibitor of ectopic calcification in vivo (9).
In an effort to further characterize the process of bone metabolism and
identify the proteins involved, we screened RNA of rat bone marrow cell
cultures for novel transcripts specific to bone mineralization (10).
This screen produced a novel clone that we designated
osteoblast/osteocyte factor 45 (OF45). Rat OF45
encodes an RGD protein having 45% homology to the recently cloned
human MEPE as well as loose homology to
AG-1/DMP1. In rat, OF45 is highly
expressed in the tibial shaft and metaphysis as well as in osteoblasts
of induced bone marrow cultures, calvaria, and the UMR106 osteoblastic
cell line. Immunohistochemistry in rat tibia revealed abundant OF45
protein in the osteocytes. Newly embedded rat osteocytes were also
positive for OF45 in a marrow ablation model. Interestingly, the
temporal expression pattern of OF45 differs from other
well-characterized bone markers such as osteocalcin, osteonectin, and
osteopontin in both in vitro and in vivo bone
growth models (10). In this report we have cloned and characterized the
mouse OF45 cDNA. With the exception of some additional
sequence we report at the 5-prime end, the sequence reported here was
identical to that recently published as MEPE (11). To
demonstrate the functional significance of an elimination or reduction
of OF45 expression, we have cloned the mouse OF45
genomic DNA, generated a mouse line with a targeted disruption of the
OF45 gene, and subjected these mice to phenotypic analysis.
Cloning of Mouse OF45--
A 1255-bp probe encoding most of the
rat OF45 cDNA sequence, excluding the 3'-untranslated
repeat, was used to screen a mouse 129 genomic lambda phage library
(12). A 15-kb clone found to include exon 3 was used to generate PCR
primers to clone the complete cDNA, the BAC clone, as well as the
targeting construct. The 5'-end of the mRNA was determined by RNA
ligase-mediated RACE as described (13). Briefly, 50 µg of total RNA
from mouse tibia was dephosphorylated with calf intestinal alkaline
phosphatase (Roche Molecular Biochemicals, Indianapolis, IN) and the
5'-cap was removed using tobacco acid pyrophosphatase (Epicentre
Technologies, Madison, WI). The RNA linker generated from the plasmid
pGbx-1 (Gift from Dr. M. A. Frohman) was ligated to the
decapped RNA using T4 RNA ligase (Epicentre Technologies, Madison, WI).
The RNA was converted to cDNA by using Superscript II reverse
transcriptase and p(dT)12-18 primer (Invitrogen,
Gaithersburg, MD). PCR was used to amplify the 5-end of the cDNA
using primer pair NRC-1-288A and 36387.233B and primer pair
NRC-1-288B and 36387.233A in the first and second round, respectively.
A single PCR product of ~550 bp was obtained in the second round PCR.
A control sample in which the pyrophosphatase step was omitted did not
yield a PCR product, indicating that the ~550-bp product likely
resulted from full-length mRNA that had been 5'-capped. The second
round PCR fragment was isolated and ligated using a TA cloning kit
(Invitrogen, Carlsbad, CA). Insert-containing plasmids were sequenced.
The 3'-end of the mRNA was determined using a 3' RACE kit
(Invitrogen, Gaithersburg, MD) as suggested by the manufacturer. Briefly, 5 µg of mouse tibia total RNA was reverse-transcribed using
the AP primer provided. The PCR reaction amplified the sequence between OF45 nucleotide 527 (primer 36387.233E) and the AUAP
primer provided. Two fragments close in size were cloned with
the TA cloning kit (Invitrogen) and sequenced. The sequences of the two fragments were identical except for a short stretch of additional sequence at the 3'-end.
The gene-specific primers were as follows: 36387.233A,
5'-TGTGTCAGGTAGTGAGTGCTCC-3'; 36387.233B,
5'-ACTGCCACCATGTCCTTCTC-3'; 36387.233C, 5'-CCAGCAGATGTCAATGATGC-3';
36387.233D, 5'-TTGGCAGCATCTGTGTATCC-3'; 36387.233E,
5'-CCCAAGAGCAGCAAAGGTAG-3'; and 36387.233F,
5'-TGCGTGATATTTCTGAGGAGG-3'. The pGbx-1 RNA linker primers were,
NRC-1-288A, 5'-CCAAGACTCACTGGGTACTGC-3'; NRC-2-288B,
5'-CTAGAGGGGCCTGTTGAACC-3'; NRC-3-288C, 5'-GGGAGAGGCCAGCGTATTCC-3'; RC
NRC-1-30A, 5'-GCAGTACCCAGTGAGTCTTGG-3'; RC NRC-2-30B,
5'-GGTTCAACAGGCCCCTCTA-3'; and RC NRC-3-30C,
5'-GGAATACGCTGGCCTCTCCC-3'.
Isolation of BAC Clone for Mouse OF45--
A 124-bp probe for
the 5'-end of the cDNA was generated by PCR with primers to amplify
bases 12-135 of the cDNA sequence using primers 36393.80A
(5'-TTTCAGCAAATGCCCAGAG-3') and 36393.80B
(5'-CCAGGTCATACTGAAGAGGAGC-3'). This probe was sent to Genome
Systems, Inc. (St. Louis, MO) for screening of a mouse ES-129/SVIII BAC
library. A single clone was identified and characterized. Chromosomal
localization was determined by fluorescence in situ
hybridization using the BAC clone by Genome Systems, Inc. according to
their protocols. Intron/exon boundaries were determined by alignment of
BAC clone sequence with the OF45 cDNA sequence.
Mammalian Expression of Mouse OF45--
The 1340 bp containing
the coding region of the mouse OF45 cDNA (bases
53-1392) was amplified using primers 36393.44C
(5'-TTTCCTGAAGGTGAATGACG-3') and 36393.44H
(5'-CTAGTCACCATGACTCTCACTAG-3') and subcloned in the cytomegalovirus
mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA) for
transfection into CHO cells using LipofectAMINE Plus (Invitrogen).
Antibody Generation and Western Blot Analysis--
High titer
polyclonal antiserum was generated by immunization of rabbits with
bacterial expressed full-length OF45 (Zymed Laboratories
Inc., South San Francisco, CA). OF45 antibody was affinity-purified by chromatography on OF45-coupled agarose beads by
standard methods (14). Protein samples were separated by electrophoresis on Novex (San Diego, CA) 10% NuPAGE gels and
transferred to nitrocellulose using a semi-dry transfer. Blots were
blocked using 1% Western Blocking Reagent from Roche Molecular
Biochemicals (Mannheim, Germany) for 1 h and 1o
antibody diluted in 0.5% block for 1 h. After washing, goat
anti-rabbit peroxidase 2o antibody (Roche Molecular
Biochemicals) diluted into 0.5% block was applied to the blot for
1 h. Signal was detected using an ECL detection kit (Amersham
Biosciences, Buckinghamshire, England).
Northern Blot, RT-PCR, and Southern Blot
Hybridization--
Multiple tissue Northern blots
(Clontech, Palo Alto, CA) were hybridized with the
full-length OF45 cDNA. RNA isolated from mouse tissues
was converted to cDNA by using Superscript II reverse transcriptase
and poly(dT)12-18 primer (Invitrogen, Gaithersburg, MD).
PCR was used to amplify an internal 483 bp from OF45
mRNA bases 986-1468 using primers 36499-143A
(5'-ACTATCCACAAGTGGCCTCG-3') and 36499-143B
(5'-CTGTTGGCTTGCTCAGTTCC-3'). The cDNA was then hybridized with a
radiolabeled cDNA probe specific to bases 160-1020.
Gene Targeting, ES Cell Culture, and Microinjection--
Genomic
DNA fragments from the 15-kb genomic clone were subcloned into the JNS2
targeting plasmid (15) E14Tg2a embryonic stem (ES) cell line derived
from the 129sj mouse strain was used for gene targeting of the
OF45 locus (16). Electroporation, selection, expansion, and
microinjection of ES cells into C57BL/6 embryos were as described
previously (17). Out of 70 neomycin and gancyclovir-resistant clones,
four were positive for the desired recombination event, resulting in a
targeting frequency of 1 in 17. Chimeric animals were mated with
C57BL/6 males and females. An 850-bp SpeI/MscI
probe, homologous to a region directly 3' of the targeted mutation, was
used to genotype agouti offspring by Southern blot analysis. Digestion
of tail DNA with BamHI/BglII double digest
resulted in a 10-kb hybridizing band in the wild type allele and a 9-kb
hybridizing band in the targeted allele.
Animals--
F2 or F3 129/Bl6 mix heterozygote mice were bred to
generate successive populations of littermates used for in
vivo experimentation at either 4 months or 1 year of age. The same
parent animals were used to generate both 4-month and 1-year age
groups. 10-month-old males backcrossed onto a C57/Bl6 background for
nine generations were used for dynamic histomorphometry studies.
Animals were maintained on a 12-h light/12-h dark cycle and were
provided food and water ad libitum. 12 and 2 days prior to
sacrifice, mice were given subcutaneous injection of the fluorochrome
calcein at 10 mg/kg (Sigma Chemical Co., St. Louis, MO). Animals were
euthanized by cervical dislocation, and the femurs were placed in 70%
ethanol for later pQCT and histomorphometric analysis. The experiment was conducted according to Pfizer Animal Care and Use-approved protocols, and the animals were maintained in accordance with the ILAR
(Institute of Laboratory Animal Research) Guide for the Care and Use of
Laboratory Animals.
Serum Mineral Content--
Calcium and inorganic phosphate
levels were measured from blood serum at DNX Transgenic Sciences
(Cranbury, NJ) using the ACE Clinical Chemistry System (Alfa
Wassermann Inc., West Caldwell, NJ) by the manufacturer's
protocols. One-year-old animals were used for this study. Sera from 12 male animals of each genotype were used. Seven wild type and seven
knockout females were also studied.
High Resolution X-ray--
Femurs were examined at 2× and 3×
magnification on a Faxitron model MX-20 Specimen Radiography System
(Buffalo Grove, IL) with Kodak Min-R 2000 Mammography film in Min-R
2000 cassettes with an intensifying screen. Magnification was
calculated by SID/SOD = IS/OS where SID is source to image
distance, SOD is source to object distance, IS is image size, and OS is
object size. The femurs of 12 to 27 animals of each sex, age, and
genotype were examined.
Peripheral Quantitative Computerized Tomography
Analysis--
Excised femurs were scanned by a pQCT x-ray machine
(Stratec XCT Research M, Norland Medical Systems, Fort Atkinson, WI)
with software version 5.40. A 1-mm-thick cross-section of the femur metaphysis was taken at 2.5 mm proximal from the distal end with a
voxel size of 0.07 mm. Cortical bone was defined and analyzed using contour mode 2 and cortical mode 4. An outer threshold setting of
340 mg/cm3 was used to distinguish the cortical shell from
soft tissue and an inner threshold of 529 mg/cm3 to
distinguish cortical bone along the endocortical surface. Trabecular
bone was determined using peel mode 4 with a threshold setting of 655 mg/cm3 to distinguish (sub)cortical from cancellous bone.
An additional concentric peel of 1% of the defined cancellous bone was
used to ensure (sub)cortical bone was eliminated from the analysis. Volumetric content, density, and area were determined for both trabecular and cortical bone. Using the above setting, we have determined that the ex vivo precision of volumetric content,
density, and area of total bone, trabecular, and cortical regions
ranged from 0.99% to 3.49% with repositioning (18). Number of animals examined: females, 4 months, 12 WT, 20 Het, 18 KO; females, 1 year, 12 WT, 17 Het, 27 KO; males, 4 months, 18 WT, 21 Het, 18 KO; and
males, 1 year, 21 WT, 19 Het, 21 KO.
Bone Histomorphometry--
Following pQCT analysis, the
distal half of the femur from each animal of 4-month-old and 1-year-old
animals was dehydrated and embedded in methyl methacrylate. 4-µm
longitudinal sections were prepared with a Reichert-Jung Polycut S
microtome (Leica, Deerfield, IL) and stained with modified Masson's
Trichrome stain. Trabecular bone volume, trabecular number, and
trabecular thickness were determined on the distal femoral metaphysis
for these 4-month-old and 1-year-old animals (19, 20). To understand
the changes in osteoblasts and osteoclasts on the cancellous
bone, a group of wild type (n = 8) and knockout
(n = 10) male mice were necropsied at 10 months of age.
The left distal half of the femur from these 10-month-old animals was
decalcified, and 4-µm longitudinal sections were prepared and stained
with Toluidine Blue (Sigma, St. Louis, MO). These decalcified sections
were used to determine the trabecular bone volume (BV/TV), percent
osteoblast surface (Ob.S/BS), number of osteoblast per millimeter of
bone surface (N.Ob/BS), percent osteoclast surface (Oc.S/BS), and
number of osteoclast per millimeter of bone surface (N.Oc/BS) on the
same area of the distal femoral metaphysis as described above (19, 20).
The right distal half of the femur from 10-month-old animals was
dehydrated and embedded in methyl methacrylate. 10-µm longitudinal
sections were prepared and left unstained for the determination of
mineral apposition rate, bone formation rate/bone surface referent
(BFR/BS), and bone formation rate/tissue volume referent (BFR/TV) on
the distal femoral metaphysis (19, 20). An Image Analysis System
(Osteomeasure, Inc., Atlanta, GA) was used for all histomorphometric
analysis. Histomorphometric measurements were performed on cancellous
bone tissue of the distal femoral metaphyses between 0.5 and 2 mm
proximal to the growth plate-epiphyseal junction and extended to the
endocortical surface in the lateral dimension (21).
Bone Marrow Harvest and Culture--
Long bones were isolated
from 3- to 6-month-old animals. Femur and tibia bone marrow cells
harvested by centrifugation were plated at a density of 15 × 106 cells per 100-mm plate in Calvaria Harvest and Culture--
Calvaria were dissected from
postnatal day 3 mice. 0.2 mg/ml collagenase P/0.25% Trypsin digestions
were performed as described previously (23). The cells liberated in the
second digest were plated at 2 × 104
cells/cm2. At confluence, cultures were supplemented with
50 µg/ml L-ascorbic acid and 10 mM
Osteoclast Cultures--
Bone marrow cultures were established
as described above, plated at 1 × 106 cells per well
in a 24-well plate. Cells were stimulated with 10 nM
1,25-dihydroxyvitamin D3 and TRAP stained as
described (26). Bone resorption was assessed by culturing bone marrow
cells on bone slices for 21 days and counting the resorption pits as
has been described by Grasser et al. (27).
Statistical Methods--
Statistical significance of in
vivo and in vitro parameters was determined by
two-tailed Student's t test. A p value of less than 0.05 was considered statistically significant.
OF45 cDNA Cloning and Characterization--
Mouse
OF45 was cloned using low stringency hybridization of a rat
OF45 sequence to a murine 129 strain lambda phage genomic library. The full-length cDNA was then cloned by RT-PCR and 5'- and
3'-rapid amplification of cDNA ends (RACE) from mouse tibia RNA.
The complete cDNA was 1741 bp in length. An additional 1679-bp cDNA was cloned, which was identical to the longer, more abundant message with the exception of a shorter 3'-extension. Three early methionine codons allow alternative start sites for the OF45 protein, the first of which would result in a 441-amino acid protein. A Kyte-Doolittle hydrophilicity plot revealed that the peptide sequence contained a hydrophobic leader sequence followed by a hydrophilic protein (Fig. 1A). Analysis of
the amino acid sequence with PSORTII software, which predicts the
subcellular localization sites of proteins from their amino acid
sequences, indicated that this cDNA likely encodes an extracellular
protein (28-30). The predicted site of cleavage would be between
Ala-24 and Ala-25, yielding a 417-amino acid secreted peptide
with a calculated molecular mass of 44247 Da. Experimental
confirmation of this analysis was obtained by Western blot analysis of
media from transiently transfected CHO cells. An OF45-specific antibody
detected a secreted protein of ~44 kDa (Fig. 1B). An OF45
peptide product of lower molecular weight was also detected in variable
amounts, suggesting that proteolytic processing or degradation occurred
in the media. The predicted amino acid composition of the basic OF45
peptide (predicted pI 9.17) was rich in serine, glycine, and charged
residues. Several consensus protein kinase C, casein kinase II,
tyrosine kinase, and cAMP-dependant kinase phosphorylation sites, one
N-glycosylation site, and a SDGD glycosaminoglycan binding
site offer the potential for post-translational modifications to
increase the acidic character of the protein.
Comparison of OF45 Sequence to Known Genes--
Based on sequence
and expression analysis, we believe that the mouse OF45
cDNA reported here represents the mouse orthologue of the rat
OF45 gene (10) (GenBankTM accession number
260922). Alignment with the rat OF45 amino acid sequence demonstrated
67% identity. Importantly, key structural features were conserved.
Both proteins were rich in serine, glycine, and charged amino acids and
included an amino-terminal hydrophobic signal sequence that targeted
the peptides for secretion. The RGD sequence at amino acids 183-185 is
an element traditionally involved in cell-matrix interactions through
integrin binding and signaling and was also conserved between species.
Comparison of the OF45 cDNA sequence to
GenBankTM using the BLAST 1.4 algorithm (31) showed 45%
amino acid identity to the recently identified human matrix
extracellular phosphoglycoprotein (MEPE) gene (32). The
macaque MEPE gene showed similar identity to OF45
(GenBankTM accession number AB05025). The homology between
mouse OF45 and human MEPE was distributed throughout the sequences and
the RGD motif was conserved. MEPE was cloned as a candidate
gene for a tumor-secreted phosphaturic factor responsible for
tumor-induced osteomalacia. Like MEPE, OF45 shares structural features
common to a class of extracellular matrix phosphoglycoproteins that
includes osteopontin, dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein II. These genes are highly expressed in bone
or dentin and play significant roles in mineralization (33-35). The
mouse OF45 sequence was also 100% identical to the mouse
MEPE cDNA recently published by Argiro et al.
(11), although the cloned OF45 cDNA we report contained
additional sequence at both the 5'- and 3'-ends. Comparison of mouse
OF45, rat OF45, macaque MEPE, and human MEPE show several tightly
conserved regions across all four species. These include the RGD motif,
the signal sequence cleavage site, the putative
N-glycosylation site, the SGDG glycosaminoglycan binding
site, and several putative phosphorylation sites. Based on the sequence
comparison and the restricted tissue expression, we concur with Argiro
et al. that the sequence we report as mouse OF45
is the orthologue of human MEPE.
OF45 Gene Expression--
The expression of mouse OF45
was analogous to that reported for rat OF45 (10). Mouse
OF45 mRNA was induced during mineralization in primary
mouse calvarial cultures, appearing at 2 weeks and maintained
throughout 3 weeks of culture (Fig. 1C). Expression correlated with increasing differentiation of the osteoblast lineage. Mouse OF45 mRNA expression was highly bone-specific. A
high level of OF45 message was detected in total RNA
prepared from mouse tibia, but all other tested tissues were negative,
even by Northern blot analysis of poly(A)+ mRNA (Fig.
1D). Using highly sensitive RT-PCR, low levels of OF45 mRNA were detected in brown fat, white fat, testes,
brain, and aorta (Fig. 1E).
Localization of OF45 protein was determined by immunohistochemical
analysis of longitudinal tibia sections (Fig.
2). Mouse OF45 protein was
expressed in all osteocytes, which are cells of osteoblast
lineage embedded within bone matrix. In contrast to the OF45
mRNA expression observed in cell culture, osteoblasts and lining
cells observed in the bone sections were negative for OF45
immunostaining. There was no specific staining detected in osteoclasts,
chondrocytes, or periosteal cells. Also, OF45 was not detected in
hematopoietic cells of the marrow compartment.
OF45 Genomic Organization and Targeting--
The genomic DNA
encoding mouse OF45 was subcloned from a hybridizing mouse
129 BAC clone and mapped. The mouse OF45 gene was comprised
of three exons, two small 76- and 78-bp exons separated from the third
1597-bp exon by 10.5 kb (Fig.
3A). Fluorescence in
situ hybridization analysis was used to determine that the OF45 gene is located on mouse chromosome 5, region
5E3-E5.
A targeting vector was constructed to delete the third and largest exon
of OF45 and replace it with the neomycin-selectable marker
gene (Fig. 3A). This deletion removed the DNA-encoding Met-27 through the end of the translated sequence, including the final
Asp-441. This targeting construct was electroporated into E14Tg2a
embryonic stem cells and then screened for positive recombinants. Blastocyst microinjection of a targeted cell line resulted in chimeric
animals that were subsequently bred to C57/Bl6 mice to yield
mixed-background offspring heterozygous for the OF45
mutation. Heterozygote intercrosses produced litters with Mendelian
ratios of wild type, heterozygous, and homozygous pups (Fig.
3B). Northern analysis of mRNA prepared from tibia
detected no OF45 message in knockout animals and reduced
expression in heterozygous animals (Fig. 3C).
Immunohistochemistry of tibia sections confirmed the absence of OF45
protein expression in osteocytes of homozygous mutant mice (Fig.
2).
Phenotypic Analysis of OF45 Knockout Animals--
To examine the
phenotypic effects of OF45 mutation, heterozygote parents
were bred to generate groups of at least 12 animals of each sex and
genotype to be examined at 4 or 12 months of age. Mice heterozygous and
homozygous for the OF45 mutations were normal, fertile, and
healthy, with no obvious pathology. Serum chemical analysis revealed no
statistically significant alterations in blood phosphate or calcium
content (Table I). Whole-body x-rays revealed normal skeletal patterning and structure (not shown). However,
high resolution x-rays of femurs revealed a pronounced increase in the
amount of trabecular bone in both heterozygote and knockout animals at
1 year (Fig. 4). This difference was
observable with high resolution x-rays at 4 months but was less
dramatic (not shown). Quantitative analysis of the distal femoral
metaphysis by peripheral quantitative computerized tomography (pQCT)
demonstrated significantly increased volumetric trabecular bone content
and density in both males and females (Table
II). There was no consistent change in
the volumetric cortical bone content at this site. The periosteal and
endosteal circumference was increased in the knockout animals with no
significant change in cortical thickness (Table II). Caliper
measurement revealed no difference in femur length between genotypes
(Table III).
To further investigate the trabecular bone structure of these mice, we
performed cancellous bone histomorphometric analysis at the distal
femoral metaphysis (19). We observed a 50% increase in trabecular bone
volume (BV/TV(%)) in 4-month-old female knockout mice compared with
wild type mice (Fig. 5). In males, there
was a 32% greater trabecular bone volume in knockout mice at 4 months. The effect of OF45 knockout mutation was more pronounced at
1 year with >2-fold and >3-fold more trabecular bone volume in male and female knockout mice, respectively. The increased trabecular bone
volume was reflective of both increased trabecular number and increased
trabecular thickness (Fig. 5).
The loss of even one OF45 allele and the resulting reduced
gene expression (Fig. 3C) caused significant phenotypic
effects in bone. As measured by both pQCT and histomorphometry,
heterozygous animals exhibited the phenotypic effect of increased
trabecular bone volume. Trabecular bone volume in 4-month males and
1-year females was equivalent in knockout and heterozygous animals with both being significantly increased over wild type mice. Heterozygote phenotype appears to be influenced by sex and age, appearing in only
older females, but in both young and old males.
An additional important observation was that both heterozygous and
knockout mice exhibited less aging-associated bone loss than wild type
animals. The male and female 1-year-old wild type groups possessed
~40 and 70%, respectively, less trabecular bone volume than did the
matched 4-month-old groups (Fig. 5). In knockout animals, however,
males exhibited no trabecular bone loss at 1 year. Female trabecular
bone volume was reduced by only 15% in the 1-year-old mice. Thus,
OF45 mutation appeared to have protected against age-related
bone loss. This protective effect of OF45 mutation was also
observed in heterozygote animals.
The net increase in trabecular bone volume observed in the heterozygous
and knockout animals could theoretically arise from either 1) decreased
osteoclastic bone resorption, 2) increased osteoblast-mediated bone
formation, or 3) a combination of osteoblast and osteoclast effects. To
investigate the effect of OF45 mutation on bone resorption
and bone formation, we performed static and dynamic bone
histomorphometric analysis of distal femoral metaphyseal cancellous
bone using wild type and knockout male mice at 10 months of age.
Percentage of bone surface occupied by osteoblasts (Ob.S/BS) and the
osteoblast number per millimeter of bone surface (N.Ob/BS) were
significantly increased by ~2-fold in knockout animals compared with
wild type controls (Table IV). A trend
toward slightly but non-significantly decreased osteoclast surface
(Oc.S/BS) and osteoclast number (N.Oc/BS) was observed (Table IV).
Dynamic histomorphometry measurements utilizing calcein double-labeled
bones to quantitatively measure the bone formation rate within a 10-day
labeling interval demonstrated significantly increased mineral
apposition rate (an index of osteoblast activity), bone formation
rate/bone surface reference (BFR/BS), and bone formation rate/tissue
volume referent (BFR/TV) in knockout animals (Table IV). These data
demonstrate that the increased cancellous bone mass in OF45
knockout mice is a result of increased osteoblast-mediated bone
formation with unchanged osteoclast-mediated bone resorption.
Ex Vivo Cell Culture--
To further examine the cellular
mechanisms behind the increased trabecular bone volume in heterozygote
and knockout animals, we examined the in vitro
mineralization and osteoclastogenesis potential of cultures derived
from wild type and mutant animals. Primary cultures of bone marrow
cells from all three genotypes were grown in the presence of ascorbate
and
To determine if a deficiency in the formation or activity of
osteoclasts contributed to the increased bone phenotype, bone marrow
cultures from wild type and knockout animals were established under
osteoclast promoting conditions. In multiple cultures, the ability of
marrow cultures to form multinucleated tartrate-resistant acid
phosphatase (TRAP)-positive cells was unimpaired (Fig.
7, A and C). In
fact, knockout bone marrow cultures exhibited a trend toward higher
numbers of TRAP-positive multinucleated cells than wild type-derived
cultures. The osteoclasts generated in the marrow culture system had
the ability to resorb bone when cultured on bone slices (Fig.
7B). Resorption pits appeared to be identical between cells
from wild type and knockout mice. No difference in the number of
resorption pits was observed (Fig 7C).
We successfully generated a strain of mice with a deletion in the
OF45 locus and ablated OF45 protein expression. This
mutation did not affect the gross skeletal morphology or overall health of these animals. Refined examination of long bone revealed
significantly increased cancellous bone mass in both male and female
knockout animals, with differences in magnitude based on sex and age.
These differences were visible by high resolution x-ray and
quantifiable by both pQCT and histomorphometry. In addition, analysis
of sex-matched populations at 4 months and 1 year revealed that
deletion of OF45 prevented the loss of trabecular bone
characteristic of aged mice. The bone loss normally observed in aged
mammals is thought to be due to the reduced abundance and reduced
survival of osteoblasts, with no change in the abundance or activity of
osteoclasts (37-39). The increased bone mass in
OF45-ablated mice was due to increased abundance and
activity of osteoblasts as indicated by the increased N.Ob/BS and
BFR/BS levels observed relative to wild type mice. Ex vivo
cell culture from bone marrow indicated increased number osteoblast
precursors in OF45 knockout animals. Although not
specifically addressed in this study, it is possible that
OF45 deletion also affected osteoblast survival or lifetime.
Heterozygous mice also exhibited significantly increased trabecular
bone. Therefore, even a reduction of OF45 levels markedly impacted bone
structure suggesting that OF45 is a limiting control point in the
regulation of bone. The haploinsufficiency nature of OF45 suggests that
genetic variation in OF45 expression levels could result in
variation of peak bone mass or aging-related bone loss in individuals.
It will be of interest to determine if polymorphisms in the
OF45 gene contribute to differences in bone mass within the
human population.
The spectrum of tissue expression of mouse OF45 closely
matched that of rat, with abundant production restricted to bone. In
differentiating calvarial osteoblast cultures, OF45
expression increased with progressive differentiation and maintained a
steady state of expression in mature cultures. Immunohistochemistry of tibial bone sections revealed abundant staining of the embedded osteocytes. We did not observe OF45 immunostaining of osteoblasts in
either rats (10) or mice despite the abundant mRNA expression in
osteoblast-rich ex vivo cultures. This discrepancy could be explained as a technical artifact in the immunostaining methods. For
example, perhaps only the embedded cells accumulate sufficient amounts
of this secreted protein to allow detection. However, as we previously
reported, Northern blot analyses of mRNA isolated from the rat
marrow ablation model support the conclusion that OF45
mRNA is expressed only by cells embedded within bone and not by the
osteoblastic cells that are not imbedded (10). Taking these data
together, we believe that OF45 protein is expressed in vivo
in mature osteoblasts during the process of embedding in new bone and
maintained throughout osteocyte development.
In addition to bone expression, we report low mRNA levels in other
organs, including testes, fat, and the aorta. The significance of these
low levels of mRNA is unclear. Adipose tissue expression may be
meaningful given the common mesenchymal stem cell origin of both
adipocytes and osteoblasts. Expression of OF45 in the aorta
may also have significance, especially under pathophysiological conditions of vascular calcification (40). A recent report has demonstrated OF45 mRNA expression in odontoblasts, the
tooth cells analogous to osteoblasts (41). Furthermore, the human
tissue expression of OF45 (called MEPE) was
reported in brain as well as bone (32). We have not been able to detect
OF45 mRNA in mouse brain, although we have been able to
confirm relatively abundant OF45 expression in human brain
mRNA samples (T.A.B., not shown). To determine if the differences
in expression pattern between humans and mice translate to additional
functions for OF45 in humans will require further investigation.
The OF45 sequence described here is the mouse orthologue of
the human MEPE gene. The human MEPE gene was
cloned as a candidate for the long sought after phosphaturic factor
secreted by tumors causing oncogenic osteomalacia (32). A parallel
field of research on a phenotypically similar condition, X-linked
hypophosphatemic rickets, has suggested that the phosphaturic factor is
processed or degraded by the PHEX endoprotease. However, subsequent
investigation has implicated FGF23 as a phosphaturic factor in both
tumor-induced osteomalacia and autosomal-dominant hypophosphatemic
rickets (42, 43). Furthermore, it has been demonstrated that FGF23 is a
substrate for the PHEX endoprotease (42). It has been reported that
MEPE, in contrast to FGF23, does not exhibit phosphaturic activity on renal cells (42) and is not a direct substrate of the PHEX endoprotease (44). Our observation that OF45 knockout animals exhibited
no statistically significant differences in serum calcium or phosphate also argues against MEPE being a critical phosphaturic factor. Nonetheless, it is still significant that OF45/MEPE was identified due
to its overexpression by osteomalacia-inducing tumors, and there
remains a link between PHEX and OF45/MEPE. Although Bowe et
al. (44) have claimed that OF45/MEPE is not a direct substrate of
PHEX, Argiro et al. (11) have demonstrated that the
osteoblasts of PHEX-deficient Hyp mice do express markedly higher
levels of MEPE mRNA than do those from wild type mice. We
have demonstrated that co-transfection of OF45 and
PHEX resulted in decreased OF45 protein detected in the
media than observed with OF45
alone.2 Conversely, in a
cell-free system, Guo et al. (45) demonstrated that PHEX
inhibits the cleavage of OF45/MEPE. It seems increasingly clear that
FGF23 and PHEX are members of a phosphate regulatory pathway. MEPE may
be a downstream target of this pathway and a member of a bone
regulatory branch of this system.
In summary, we have determined that reduction or total ablation of the
OF45 gene in mouse resulted in increased bone mass. Although
the molecular mechanism of OF45 action is unknown, the etiology of this
increased bone mass cannot be attributed to a failure in the
differentiation or abundance of osteoclasts. Bone histomorphometry and
primary cell culture experiments did not detect any impairment in the
osteoclast cells that mediate bone resorption. The increased osteoblast
number and activity (mineral apposition rate) observed by bone
histomorphometry and the mineralization potential observed in both
marrow and calvarial cultures indicate that the phenotypic effect in
the knockout animals resulted from an increased number and activity of
osteoblasts and osteoblastic precursors. One interpretation of these
results is that OF45 functions in vivo as a negative
regulator of osteoblast number and activity. The concept that
regulation of bone mass can be controlled by negative regulators has
recently been demonstrated with the characterization of
osteocalcin-deficient (2) and leptin-deficient mice (46). Further experimentation will be needed to determine if OF45 acts directly on osteoblasts or through indirect mechanisms. The expression of OF45 within osteocytes, the terminally differentiated osteoblasts that become encased in the mineralized matrix during bone formation (47), opens the possibility that secreted OF45 may act locally on
osteoblasts and osteoblast precursors. Osteocytes can communicate through extensive dendritic processes and have been theorized to be the
primary sensors of mechanical strain within bone (48-51). OF45 may
function as an integral component of the intimate connection between
the sensing and signaling of osteocytes and the osteoblast response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal essential
medium (Invitrogen), with 10% fetal bovine serum and 50 µg/ml
Gentamicin (Invitrogen). 50 µg/ml L-ascorbic acid, and 10 mM
-glycerophosphate were added at day 10 of culture.
Cultures were fed three times per week, maintained for 4 weeks, and
then stained by the Von Kossa method (22).
-glycerophosphate. Cells were later harvested for RNA or Alizarin
Red staining (24). Stain solubilization and quantitation were as
described (25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (29K):
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Fig. 1.
OF45 encoded a secreted protein
and was expressed primarily in bone. A, a
Kyte-Doolittle hydrophilicity plot of the predicted amino acid sequence
of OF45 indicated a hydrophobic leader sequence followed by a
hydrophilic peptide. The complete cDNA nucleotide sequence was
deposited as GenBankTM accession number AF298661.
B, Western blot analysis of OF45 expression in the media
from CHO cells transfected with pcDNA3-OF45 expression vector.
Media from CHO cells transfected with the pcDNA3 vector control are
also shown. The major band corresponded to ~44 kDa as predicted from
the cDNA sequence. A lower molecular weight species, likely the
product of proteolysis, were also detected in media from
OF45-transfected cells. C, Northern blot analysis of 20 µg
of total RNA from mouse calvarial bone cultures stimulated to
mineralize with ascorbate and -glycerophosphate. RNA samples were
taken over a time course of 1 day to 3 weeks as indicated and analyzed
for OF45 mRNA expression. D, tissue-specific
expression of OF45 mRNA as measured by Northern blot
analysis of 5 µg of poly(A)+ RNA from various mouse
tissues (Clontech) and 20 µg of total RNA from
mouse tibial metaphysis. Top panel, mouse OF45;
bottom panel, glyceraldehyde-3-phosphate dehydrogenase
control probe. E, tissue-specific expression of
OF45 mRNA as measured by Southern blot analysis of
cDNA amplified from poly(A)+ RNA. Top panel,
Southern blot of RT-PCR with OF45-specific primers.
Bottom panel, ethidium bromide stain of control PCR reaction
with actin-specific primers.
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Fig. 2.
OF45 protein localized to osteocytes.
Immunohistochemical analysis of OF45 protein localization in
bone sections of mouse tibia indicated OF45 expression in
the osteocytes embedded in mineralized bone of wild type animals.
Arrows indicate positive staining osteocytes with the
cortical bone of a wild type mouse. OF45 immunoreactivity was absent in
bones from knockout mice. IgG control with secondary antibody alone
indicated the absence of nonspecific signal in bone derived from either
wild type or knockout mice. Bar, 200 µm. CB,
cortical bone; MC, marrow cavity.
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Fig. 3.
Genomic organization and targeting
of OF45. A, schematic representation
of genomic organization and targeting strategy, including schematic of
targeted locus. Exon sizes and intron/exon boundaries of the mouse OF45
gene are diagrammed in the top figure. Exons are indicated by
gray boxes. Recombination of the targeting vector with the
OF45 locus resulted in the replacement of the third and largest exon
with the neomycin selectable marker gene. A BamHI site
is introduced via neomycin integration. Successful targeting was
detected with both external (probe 1) and internal
(probe 2) probes. X, XbaI;
Bg, BglII; Sp, SpeI;
M, MscI; B, BamHI.
B, detection of successful OF45 targeting. Left
panel, Southern blot analysis of tail DNAs digested with
BamHI and BglII and hybridized with probe
2. A 10-kb band is detected at a wild type allele, and a 9-kb band is
detected at a targeted allele. C, Northern blot analysis of
20 µg of total RNA prepared from tibia of wild type, heterozygous,
and knockout animals. Note the absence of OF45 expression in the RNA
from knockout mice and the reduction of expression in the RNA from the
heterozygous mice.
Serum mineral content in 1-year-old animals
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Fig. 4.
High resolution x-ray indicated that
OF45 mutant mice had increased trabecular bone.
Femurs from male and female mice at 1 year of age were subjected to
high resolution x-ray imaging. In the magnification of the distal femur
increased trabecular bone can be observed in both heterozygous and
knockout mice when compared with the wild type mice. The increased
trabecular bone was evident in both males and females at 1 year of
age.
Selected parameters from pQCT analysis of distal femoral metaphysis
Femur length in 1-year-old animals
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Fig. 5.
OF45 mutant mice had increased trabecular
bone volume, trabecular number, and trabecular thickness.
Graphical representation of trabecular parameters measured by
histomorphometry of stained sections of the distal femur. Trabecular
bone volume, trabecular number, and trabecular thickness are shown for
each sex, age, and genotype. 10 animals of each sex and genotype were
processed for histomorphometry. Error bars denote standard
error. *, statistical significance at p < 0.05.
Selected parameters from distal femoral metaphyseal cancellous bone
histomorphometry
-glycerophosphate to induce osteoblast differentiation and
promote bone nodule formation. After 3 weeks, mineralization was
detected by Von Kossa stain and quantitated by image analysis (Fig.
6A). In several separate experiments, heterozygote- and knockout-derived cultures gave rise to
significantly more bone nodules than those from wild type bone marrow,
suggesting that there is more osteoblastic precursor cells present in
the marrow (36). In addition, the mineralized area per nodule was
increased in knockout cultures. A second culture system derived from
mouse calvarial bone of 3-day-old mice produced comparable results over
multiple experiments. In this more highly osteoblast-enriched system,
calvarial cultures from OF45 knockout mice produced 60%
more mineralized matrix as assayed by quantitation of solubilized
Alizarin Red stain (Fig. 6B). Importantly, this experiment
indicated that the osteoblastic cells derived from knockout mice at
only 3 days after birth already exhibited increased mineralization
potential.
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Fig. 6.
Increased mineralization in OF45
mutant ex vivo cell cultures. A,
primary bone marrow cultures from wild type, heterozygote, and knockout
animals were cultured under mineralizing conditions with ascorbate and
-glycerophosphate. Mineralization was detected by Von Kossa
staining. The black-stained mineralized nodules were
quantified using Optimas image analysis software. Graphs depict
quantitation of nodule number, total nodule area, and average area per
nodule. B, primary calvarial cultures derived from wild type
and knockout animals at 3 days postnatal were cultured under
mineralizing conditions with ascorbate and
-glycerophosphate.
Alizarin Red stain was used to detect mineralization. The
graph depicts concentration of alizarin red stain
quantitated by stain solubilization and optical density reading.
Error bars denote standard error. *, statistical
significance at p < 0.05.
View larger version (28K):
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Fig. 7.
Bone marrow cells from OF45
mutant mice were fully capable of osteoclast formation and
osteoclast-mediated bone resorption. A,
osteoclastogenesis in bone marrow cells derived from wild type and
knockout mice. Tartrate-resistant acid phosphatase (TRAP) cytochemical
stained cultures from wild type and knockout animals. TRAP-positive
cells with three or more discrete nuclei were considered osteoclasts.
B, bone-resorptive activity of OF45 wild type and
knockout osteoclasts. Representative resorption pits formed on bone
slices by osteoclasts from wild type and knockout animals are shown.
C, osteoclastogenesis was assessed by counting TRAP-positive
multinucleated cells. Bone resorption was assessed by counting the
number of resorption pits per bone slice. Error bars denote
standard error. No statistically significant differences were
observed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF298661.
¶ To whom correspondence should be addressed: Box 8118W-215, Groton Laboratories, Pfizer Global Research and Development, Eastern Point Rd., Groton, CT 06340. Tel.: 860-441-3127; Fax: 860-686-0170; E-mail: thomas_a_brown@groton.pfizer.com.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M203250200
2 T. A. Brown, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: MGP, matrix glutamic acid protein; TRAP, tartrate-resistant acid phosphatase; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; ES, embryonic stem; pQCT, peripheral quantitative computerized tomography; WT, wild type; KO, knockout; BV, bone volume; TV, tissue volume; BS, bone surface; BFR, bone formation rate; CHO, Chinese hamster ovary; Het, heterozygote; BAC, bacterial artificial chromosome; MEPE, matrix extracellular phosphoglycoprotein; PHEX, phosphate-regulating endopeptidose, X-linked.
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