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Mol Biol Cell. 1998 July; 9(7): 1903–1918. | PMCID: PMC25433 |
Copyright © 1998, The American Society for Cell Biology Osteoblastic Responses to TGF-β during Bone Remodeling Adrian Erlebacher,*†§ Ellen H. Filvaroff,* Jian-Qin Ye,*‡ and Rik Derynck*¶§‖ *Departments of Growth and Development, †Biochemistry and Biophysics, ¶Anatomy, and ‡Pediatrics, §Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, California 94143 Monitoring Editor: Carl-Henrik Heldin, Monitoring Editor Received November 14, 1997; Accepted April 21, 1998. Bone remodeling depends on the spatial and temporal coupling of
bone formation by osteoblasts and bone resorption by osteoclasts;
however, the molecular basis of these inductive interactions is
unknown. We have previously shown that osteoblastic overexpression of
TGF-β2 in transgenic mice deregulates bone remodeling and leads to an
age-dependent loss of bone mass that resembles high-turnover
osteoporosis in humans. This phenotype implicates TGF-β2 as a
physiological regulator of bone remodeling and raises the question of
how this single secreted factor regulates the functions of osteoblasts
and osteoclasts and coordinates their opposing activities in vivo. To
gain insight into the physiological role of TGF-β in bone remodeling,
we have now characterized the responses of osteoblasts to TGF-β in
these transgenic mice. We took advantage of the ability of alendronate
to specifically inhibit bone resorption, the lack of osteoclast
activity in c-fos−/− mice, and a new
transgenic mouse line that expresses a dominant-negative form of the
type II TGF-β receptor in osteoblasts. Our results show that TGF-β
directly increases the steady-state rate of osteoblastic
differentiation from osteoprogenitor cell to terminally differentiated
osteocyte and thereby increases the final density of osteocytes
embedded within bone matrix. Mice overexpressing TGF-β2 also have
increased rates of bone matrix formation; however, this activity does
not result from a direct effect of TGF-β on osteoblasts, but is more
likely a homeostatic response to the increase in bone resorption caused
by TGF-β. Lastly, we find that osteoclastic activity contributes to
the TGF-β–induced increase in osteoblast differentiation at sites of
bone resorption. These results suggest that TGF-β is a
physiological regulator of osteoblast differentiation and acts as a
central component of the coupling of bone formation to resorption
during bone remodeling. During development and adult life, bone undergoes continuous
remodeling through the coordinated processes of bone formation and bone
resorption. Bone is formed by osteoblasts, which are of mesenchymal
origin, and is resorbed by osteoclasts, which are derived from the
hematopoietic system. In the adult skeleton, constant bone mass is
maintained through the close microanatomical coupling of osteoblastic
and osteoclastic activities (for review, see Parfitt, 1994  ).
Deregulation of this coupling underlies the pathological loss of bone
mass seen in osteoporosis and other metabolic bone diseases. Since new
bone formation requires the continuous generation of new osteoblasts,
osteoclastic resorption is not only coupled to the activity of
osteoblasts, but also to the differentiation of osteoblasts from
osteoprogenitor cells. In spite of their importance for our
understanding of normal bone metabolism and the pathogenesis of
metabolic bone diseases, the molecular mechanisms that govern the
coordination of these processes are largely unknown. One secreted factor that modulates the differentiation of osteoblasts
and the proliferation of osteoprogenitor cells is transforming growth
factor-β (TGF-β) (for reviews see Bonewald and Dallas, 1994 ;
Centrella, et al., 1994 ). Both osteoblasts and osteoclasts
secrete TGF-β, and all TGF-β isoforms (TGF-β1, -β2, and -β3)
are present in their latent form within bone matrix ( Seyedin et
al., 1985 ; Robey et al., 1987 ; Sandberg et
al., 1988 ; Pelton et al., 1991 ). Since bone explants
release TGF-β during bone resorption ( Pfeilschifter and Mundy, 1987 )
and osteoclasts have the ability to activate latent TGF-β ( Oreffo,
et al., 1989 ; Oursler, 1994 ), it has been suggested that
TGF-β plays a role in the coupling of bone formation to bone
resorption. Thus, TGF-β deposited in bone matrix by osteoblasts may
be released and activated at sites of resorption by osteoclasts, which
in turn leads to the induction of nearby osteoblastic differentiation.
However, this model requires experimental verification in vivo, which
so far has been difficult. For example, it is not clear whether TGF-β
directly induces osteoblastic differentiation in the adult skeleton,
and there have been no good in vivo experimental systems to detect the
release of TGF-β from bone matrix during bone resorption or to assess
the physiological relevance of bone matrix-derived TGF-β for
osteoblastic differentiation. We have previously generated transgenic mice that overexpress TGF-β2
from the osteocalcin promoter, which is osteoblast-specific ( Erlebacher
and Derynck, 1996 ). Transgenic mice showed a dramatic, age-dependent
loss of bone mass similar to that seen in osteoporosis and
hyperparathyroidism, yet showed relatively few defects in skeletal
development or growth. In the transgenic line with the highest level of
TGF-β2 expression, i.e., the D4 line, the phenotype was associated
with three major histological alterations consistent with increased
rates of bone remodeling: an increase in the density of bone
matrix-embedded osteocytes, an increase in the rate of bone formation
by osteoblasts, and an increase in the rate of bone resorption by
osteoclasts. These transgenic mice provided us with a unique model with which to
characterize the regulation of osteoblast and osteoclast function by
TGF-β during bone remodeling. We focused on the two endpoint
osteoblastic responses, i.e., the increase in osteocyte density and the
increase in bone formation. Through a combination of anatomical,
genetic, and pharmacological approaches, we found that the increase in
bone formation, contrary to expectation, was a secondary consequence of
increased bone resorption. In contrast, the increase in osteocyte
density resulted from a direct stimulation of osteoblastic
differentiation by TGF-β2. This effect was greatly enhanced by
osteoclastic activity, suggesting that TGF-β activity is functionally
increased at sites of bone resorption in vivo. Our results suggest that
TGF-β is a physiological regulator of osteoblast differentiation and
a key mediator of the coupling of osteoblast differentiation to
osteoclastic bone resorption required for skeletal homeostasis. Transgenic Mice The generation of D4 mice that overexpress TGF-β2 from the
osteocalcin promoter has been described ( Erlebacher and Derynck, 1996 ).
The generation and characterization of E1 mice, which express a
cytoplasmically truncated type II TGF-β receptor from the osteocalcin
promoter, will be described elsewhere (Filvaroff et al.,
unpublished data). Both lines were generated and maintained on a
(DBA/2 × C57BL/6J) F 1 background (Jackson
Laboratories, Bar Harbor, ME). D4 mice were identified by the distinct
appearance of their calvariae (Erlebacher and Derynck, unpublished
observations) or by PCR of tail DNA using the primers
5′-GTGCTGGTTGTTGTGCTGCTC-3′ within the β-globin sequences of the
transgene, and 5′-CCTTGGCGTAGTACTCTTCGTC-3′ within the TGF-β2 cDNA
( Erlebacher and Derynck, 1996 ). E1 mice were genotyped by PCR of tail
DNA as described (Filvaroff et al., unpublished data). D4/E1
mice were generated by crossing D4 hemizygotes with E1 hemizygotes or
D4/E1 hemizygotes, and offspring were genotyped by PCR of tail DNA.
TGF-β2 expression levels were measured in bone powder extracts
prepared from mice at day 35 as described ( Erlebacher and Derynck,
1996 ) using an ELISA that is TGF-β2 specific and does not recognize
TGF-β1 or -β3 (RD Systems, Minneapolis, MN). Mice homozygous for
the D4 transgene were embryonic lethal ( Erlebacher and Derynck, 1996 )
and were therefore not present in these analyses. Mice heterozygous for an inactivated allele of c- fos
( Johnson et al., 1992 ) were generously provided by Randall
Johnson and were on a (129/SvJ × C57BL/6J) F 1
background. The inactivated c -fos allele was tracked by PCR
of tail DNA as described ( Johnson et al., 1992 ). To generate
c -fos−/− and
c -fos−/−/D4 mice,
c -fos+/− mice were crossed with D4 mice, and
the resultant F 1 c-fos+/−/D4 mice
and c-fos+/− mice were then intercrossed. All
day 16 measurements of osteocyte density, mineral apposition rate,
serum calcium and phosphorus levels, and growth rate were performed on
the litter mates of this cross. Mice homozygous for the targeted
c -fos allele were identified by their failure to undergo
tooth eruption ( Johnson et al., 1992 ; Wang et
al., 1992 ), and the D4 transgene was detected by PCR of tail DNA.
c -fos−/− mice and control litter mates
maintained past weaning were fed a liquid diet of dissolved powdered
milk and rice cereal. Serum calcium and phosphorus levels were
determined from retro-orbital bleeds using colorimetric assays (Sigma
Chemical, St. Louis, MO). Scanning Electron Microscopy Femurs from 16- and 35-d mice were deorganified in 5.25% sodium
hypochlorite (Chlorox), coated with gold, and viewed at 10 kV with a
Jeol JSM-840A scanning electron microscope (Jeol, Tokyo, Japan). At
least three D4 and three wild-type mice were analyzed at each time
point. Osteocyte Density Measurements Osteocyte density was determined using hematoxylin and
eosin-stained 5-μm sections of bones that were fixed in 2%
paraformaldehyde/PBS, decalcified, and embedded in paraffin as
described previously ( Erlebacher and Derynck, 1996 ). Osteocyte numbers
in cortical bone for each mouse were determined in the dorsal, ventral
and medial aspects of the femoral diaphysis from two cross-sections
spaced 200 μm apart at the level of the third trochanter. Osteocyte
numbers in epiphyseal cancellous bone were measured in two longitudinal
sections separated by 200 μm. Sectioned bone surfaces were scanned
into a computer using Adobe Photoshop (Mountain View, CA), and their
surface areas were measured using NIH Image (Wayne Rasband, National
Institutes of Health). Osteocyte densities (osteocytes per
mm 2) for each mouse were converted into three-dimensional
densities as described ( Sissons and O’Connor, 1977 ), assuming a
15-μm diameter for an osteocyte. Mineral Apposition Rate Measurements The mineral apposition rate was determined from 4.5-μm
sections of undecalcified bone fixed in 70% ethanol, stained en bloc
in Villanueva bone stain (osteochrome stain, Polysciences, Niles, IL),
and embedded in methylmethacrylate. For analyses at day 16, mice were
injected with 10 mg/kg calcein (Sigma) on day 12 (or on day 10 for some
c-fos−/− and
c-fos−/−/D4 mice), and on day 15 with 25 mg/kg
tetracycline (Sigma). For day 35 analyses, mice were injected on day 30
with calcein and at day 34 with tetracycline. The mineral apposition rate was measured from photomicrographs of
sections of bone viewed under UV light. The periosteal mineral
apposition rate of each mouse was measured along the dorsal and medial
aspects of the femur from at least two cross-sections spaced 200 μm
apart at the level of the third trochanter. The epiphyseal mineral
apposition rate in the femur of each mouse was determined in at least
two longitudinal sections spaced 200 μm apart. Individual
measurements (50–100 per mouse) were taken along double-labeled
surfaces at a spacing of about 40 μm. The mineral apposition rate for
each mouse was calculated as the average of the distances between the
fluorochrome labels divided by the time between their injection. The
SEM of these measurements per mouse was always lower than 10% their
average value. Mineralization lag time was calculated as the average of
individual measurements of the osteoid seam width divided by the
mineral apposition rate measured at the same location. Alendronate Treatment and Parathyroidectomy Alendronate was generously provided by Gideon Rodan (Merck
Research Laboratories, West Point, PA) or prepared as the soluble
component of Fosamax (Alendronate Sodium Tablets, Merck & Co., West
Point, PA), dissolved in PBS. Both sources gave identical results. Mice
were intraperitoneally injected with 0.3 mg/kg alendronate or PBS every
other day from days 15 to 35. Alendronate treatment during this period
of rapid growth caused a mild osteopetrosis, slightly reduced serum
phosphorus levels, but no effect on serum calcium levels, and thus may
have resulted in a mild hyperparathyroidism due to an increased demand
by the growing bones for calcium. Parathyroidectomy or sham operations were performed on day 21, after
weaning. Mice were anesthetized with intramuscular injections of 100
mg/kg ketamine (Sigma), 5 mg/kg xylazine (Sigma), and 1.25 mg/kg
acetopromazine (Sigma), and the parathyroid glands were removed by
blunt dissection as described previously ( Meyer et al.,
1989 ). Incision sites were sutured shut and sealed with collodion
(Mallinckrodt Baker, Paris, KY). Sham operation mimicked the entire
operation without the actual removal of the parathyroid glands.
Untreated mice were fed a normal diet containing 0.7–0.8% calcium and
0.6% phosphorus; sham-operated and parathyroidectomized mice were fed
a high-calcium diet containing 1.46% calcium and 0.99% phosphorus
(Purina Test Diets, Purina Mills, Richmond, IN) after surgery to
minimize the risk of hypocalcemia. Mice were intraperitoneally injected
on day 30 with calcein and on day 34 with tetracycline for analysis of
the mineral apposition rate. Mice were fasted overnight on day 34 and
retro-orbital bleeds were taken immediately before mice were killed on
day 35. Bones were dissected and fixed and stored in 70% ethanol at
4°C. For analysis of the osteocyte density, bones were refixed in 2%
paraformaldehyde/PBS before further processing (see above). Day 35 serum calcium and phosphorus levels, determined by colorimetric
assay (Sigma), were used to score for successful parathyroidectomies.
The prior overnight fast was included to minimize the dietary
absorption of calcium. Sham-operated, fasted mice had a serum calcium
of 9.5 ± 0.4 mg/dl (n = 21) and a serum phosphorus of
7.6 ± 1.0 mg/dl (n = 21); we defined a successful
parathyroidectomy as one resulting in a serum calcium level 2 SDs below
the mean, and a serum phosphorus level 1 SD above the mean. Thus, only
mice with a serum calcium less than 8.7 mg/dl and a serum phosphorus
more than 8.6 mg/dl were included for further analysis. These mice
formed a clearly defined group relative to sham-operated mice and mice
in which the surgery was unsuccessful. Analysis of Osteoblast Differentiation with Bromodeoxyuridine
(BrdU) Mice were given two intraperitoneal injections of BrdU
(Boehringer Mannheim, Indianapolis, IN) spaced 8 h apart on day 12
or day 16. Mice injected on day 12 were killed 96 h after the
second injection, and mice injected on day 16 were killed 4 h
after the second injection. Bones were fixed overnight at 4°C in 4%
paraformaldehyde/PBS, decalcified for 5 d in 10% EDTA, 0.1 M
Tris, pH 7.0, at 4°C, and embedded in paraffin. Cross-sections (5
μm) were taken at the level of the third trochanter and stained for
labeled nuclei using the BrdU staining kit (Zymed Laboratories, South
San Francisco, CA). Periosteal osteoblasts were identified as cuboidal
cells abutting the bone surface. For each mouse, labeled periosteal
osteoblast and subperiosteal osteocyte nuclei were counted from six
sections spaced at least 50 μm apart, covering the dorsal, ventral,
and medial aspects of the diaphysis. Three hundred to 800 osteoblasts
were scored per mouse. Effects of Alendronate on Plasma TGF-β Levels Before the plasma level of TGF-β2 was measured at day 35, mice
were injected intraperitoneally with 0.3 mg/kg alendronate or PBS
vehicle every other day for a total of three injections before
retro-orbital blood collection at day 35. Heparinized tubes were used
to collect plasma, and samples were acid treated as described
( Erlebacher and Derynck, 1996 ) before TGF-β2 measurement by a
TGF-β2-specific ELISA (RD Systems). To measure the plasma level of
TGF-β2 at 3 mo, mice were injected for 3 consecutive days with 3
mg/kg alendronate or PBS vehicle before the collection of plasma and
TGF-β2 ELISA. Statistical Analysis and Derivation of the Osteocyte Formation Rate The statistical significance of all comparisons of wild-type,
D4, E1, and D4/E1 mice, or measurements of individual and combined
effects of alendronate and parathyroidectomy, was determined by
analysis of variance followed by the Bonferroni t test for
multiple comparisons. p < 0.0083 was used as the criterion for
significance for each comparison, giving a final significance level of
p < 0.05 for the six comparisons per set of four experimental
groups. All other comparisons were pairwise using Student’s
t test at a significance level of p < 0.05. The
osteocyte formation rate was calculated as the mathematical product of
the mean epiphyseal osteocyte density with the mean epiphyseal mineral
apposition rate, with errors propagated as described ( Taylor, 1997 ). Increased Osteocyte Density in Mice with Osteoblastic
Overexpression of TGF-β2 Does Not Require Osteoclastic Bone
Resorption Because of the close functional relationships between osteoblastic
differentiation, bone formation, and osteoclastic bone resorption,
we first assessed whether the increased osteocyte density in D4 mice
was a direct effect of overexpressed TGF-β2 on osteoblasts or whether
it depended on bone resorption by osteoclasts. This evaluation was
pursued using anatomical and genetic approaches. As an anatomical approach, we assessed the osteocyte density of
bone at a location that is naturally devoid of osteoclastic activity,
i.e., under the periosteum of the diaphysis of a long bone. More
specifically, we measured the osteocyte density of subperiosteal
cortical bone in the diaphysis of the femur of 16-d-old mice in the
region opposite to the third trochanter (Figure
C). The bone surface at this location
undergoes intramembranous ossification in the absence of osteoclastic
bone resorption, which is easily verified by scanning electron
microscopy ( Boyde, 1972 ). Thus, the diaphyseal surfaces of both
wild-type and D4 mice clearly lacked characteristic resorption pits
(lacunae), in contrast to the continuous resorptive surface of the
distal metaphysis (Figure , A, B, and D).
Scanning electron micrographs also showed that the diaphyseal osteocyte
density, as assessed by the density of surface osteocyte lacunae, was
clearly higher in D4 bones than in normal bones at 16 d of age
(Figure , A and B). This increase was also histologically apparent in
cross-sections of diaphyseal bone (Figure
, A and B). In these sections, we
quantitated the osteocyte density in the subperiosteal 25 μm of
cortical bone (bracketed in Figure A). From our analysis of the
mineral apposition rate (see below), we know that the outer 25 μm of
cortical bone corresponds to ~5.8 d of radial growth up to day 16. As
shown in Figure E, the subperiosteal osteocyte density in D4 mice was
about 1.6-fold higher than in wild-type controls. A similar increase in
osteocyte density was also observed at day 35 (see below).
To genetically evaluate the role of osteoclastic bone resorption in the
increase of osteocyte density caused by TGF-β2 overexpression, we
crossed D4 mice with c -fos−/− mice, which have
an osteopetrotic phenotype due to a complete block in osteoclastic
differentiation ( Grigoriadis et al., 1994 ). Despite the
absence of osteoclasts in c -fos−/−/D4 mice,
their osteocyte density in cortical bone was still increased when
compared with c -fos−/− and wild-type controls
(Figure , C–E). These results are consistent with our anatomical and
histological analyses. Taken together, our findings strongly suggest
that the increase in osteocyte density in D4 transgenic mice does not
require osteoclastic bone resorption. Increased Osteocyte Density in D4 Bones Requires Osteoblastic
Responsiveness to TGF-β To conversely test whether the increase in osteocyte density
depended on osteoblastic responsiveness to TGF-β, we used a newly
generated line of transgenic mice that overexpress a cytoplasmically
truncated version of the type II TGF-β receptor from the osteocalcin
promoter (the E1 line, Filvaroff et al., unpublished data).
Overexpression of this truncated receptor in cell culture has been
shown to interfere with endogenous TGF-β signaling in a dominant
negative manner ( Chen et al., 1993 ), and since the
osteocalcin promoter is osteoblast-specific ( Baker et al.,
1992 ), we expected this transgenic line to have impaired TGF-β
signaling in osteoblasts. We crossed our D4 mice with the E1 transgenic mice to generate double
transgenic D4/E1 mice that overexpress both the TGF-β2 and the
truncated type II TGF-β receptor transgenes. Expression of the
truncated receptor transgene in D4/E1 mice did not inhibit expression
of the TGF-β2 transgene, because the high level of TGF-β2 in the
bone matrix of D4 mice was not reduced in D4/E1 mice (data not shown).
However, expression of the truncated receptor in D4/E1 mice
dramatically reduced the high osteocyte density seen in D4 mice to
almost wild-type levels. This effect was seen in both cortical bone
where resorption is absent (Figure A),
as well as in the femoral epiphyses where resorption is present (Figure
B). Furthermore, the osteocyte density of E1 mice was mildly (25%)
reduced below the wild-type level in the femoral epiphysis, yet was not
significantly different from wild-type mice in cortical bone (Figure ,
A and B). In contrast to the decrease in osteocyte density in D4/E1
mice, expression of the truncated receptor did not significantly affect
the increased bone formation rate (see below) or the overall loss of
cancellous bone mass caused by TGF-β2 overexpression. Furthermore,
the hypoplastic clavicles and patent anterior fontanels noted in D4
mice ( Erlebacher and Derynck, 1996 ; our unpublished observations) were
also still present in D4/E1 double transgenic mice. Thus, these results
strongly suggest that osteoblastic responsiveness to TGF-β is
required for both the increase in osteocyte density caused by
osteoblastic overexpression of TGF-β, as well as for the generation
of wild-type osteocyte density at some anatomical sites.
Alendronate Reduces Epiphyseal Osteocyte Density and Plasma
TGF-β2 Levels From the experiments described above, we conclude that the
increase in osteocyte density caused by TGF-β2 overexpression was a
direct effect of TGF-β on osteoblasts that did not require
osteoclastic bone resorption. However, these analyses do not rule out
the possibility that osteoclastic bone resorption might contribute to
the increased osteocyte density at sites where active resorption
occurs. We therefore treated mice with alendronate, a bisphosphonate
that potently inhibits osteoclastic bone resorption in vivo (for a
review see Rodan and Fleisch, 1996 ), for 3 wk before animals were
killed at day 35. We then measured the osteocyte density in the femoral
epiphysis, a site of ongoing resorption where osteocyte density was
1.9-fold higher in D4 mice than in wild-type mice. Since this treatment
produced a mild osteopetrosis and changes in calcium and phosphorus
serum levels consistent with secondary hyperparathyroidism (data not
shown), we included parallel experimental groups of mice that had
undergone parathyroidectomy at day 21. As shown in Figure
A, alendronate treatment resulted in a
moderate decrease in epiphyseal osteocyte density in D4 mice, but not
in wild-type mice. Parathyroidectomy did not significantly affect
osteocyte density. In contrast to its effects in the femoral epiphysis,
and consistent with our results on the diaphyseal osteocyte density,
alendronate did not affect the subperiosteal osteocyte density (Figure
B). Thus, alendronate decreased the osteocyte density in D4 mice only
at sites of osteoclastic bone resorption.
These results suggested that bone resorption somehow locally enhanced
the increase in osteocyte density caused by TGF-β2 overexpression.
One possible mechanism, as previously suggested ( Pfeilschifter and
Mundy, 1987 ), is that osteoclastic bone resorption causes the release
and activation of bone matrix-bound TGF-β and thereby increases its
local concentration on nearby bone surfaces. Since the level of
TGF-β2 in bone matrix of D4 mice is considerably higher than the
TGF-β2 level in wild-type bone ( Erlebacher and Derynck, 1996 ), its
release from the matrix might lead to dramatic increases in the local
TGF-β concentration at bone surfaces. To assess this possibility, we
measured the effect of alendronate on the plasma level of TGF-β2 in
D4 mice. This level was not significantly reduced by alendronate
treatment for 1 wk prior to day 35 and at the same dose as in our
experiments above (data not shown). However, three daily injections of
a higher dose of alendronate in 3-mo-old mice resulted in reduced
plasma TGF-β2 levels (Figure ). At
this age, the direct contribution of the TGF-β2 secreted by
osteoblasts to the plasma level of TGF-β2 is likely to be less than
at day 35, when bone growth and modeling are much more active. These
results suggest that osteoclastic bone resorption can lead to the
release of TGF-β from bone matrix and contribute to the elevation of
plasma TGF-β levels.
Kinetics of Osteoblast Differentiation To gain further insight into the cause of the osteocyte density
increase in D4 mice, we analyzed the kinetics of osteoblast
differentiation using in vivo BrdU incorporation. Twelve-day-old mice
were injected twice with BrdU, which, due to its short half-life in
vivo ( Packard, et al., 1973 ), resulted in two short periods
of mitotic cell labeling. We then determined at day 16 the labeling
index of mature periosteal osteoblasts on the femoral diaphysis, where
resorption is absent. Mature osteoblasts, which are generally
postmitotic in vivo ( Young, 1962 ), were identified as the monolayer of
cuboidal cells abutting the bone surface ( Young, 1962 , Kimmel and Jee,
1980 ; see Figure , A and B). These cells are actively engaged in bone
matrix synthesis, with the same rate in wild-type and D4 mice (see
below). Fibroblastic osteoprogenitor cells, which are premitotic, were
located more peripherally. As shown in Figure , the percentage of
BrdU-labeled periosteal osteoblasts after the 4-d chase period was
about 2.4-fold increased in D4 mice compared with wild-type mice.
Furthermore, the number of labeled subperiosteal osteocytes per section
was increased eightfold from 0.5 (±0.6) in wild-type mice (n = 4)
to 4.1 (±1.0) in D4 mice (n = 4), which is proportionally much
greater than would have been expected from the 1.6-fold increase in
subperiosteal osteocyte density. Labeled osteoblasts and osteocytes
could conceivably have been derived from osteoblasts dividing on day 12
that had either remained on the bone surface or subsequently matured
into osteocytes. Alternatively, they could have been derived from
osteoprogenitor cells dividing on day 12 that had subsequently
differentiated. To distinguish between these possibilities, mice were
given two pulses of BrdU and killed the same day. With this schedule,
D4 mice showed a low percentage of osteoblast labeling (1.6 ± 1.3
[n = 4]) that was not significantly different from wild-type
mice (0.45 ± 0.03 [n = 2]), and sections showed no labeled
osteocytes. This result is consistent with previous observations that
mature osteoblasts rarely divide in vivo ( Young, 1962 ). Furthermore, D4
and wild-type mice killed 2 d after injection also showed low
levels of osteoblast labeling (data not shown), indicating that a
longer time period was required to generate appreciable numbers of
labeled mature osteoblasts. Thus, the majority of BrdU-labeled
osteoblasts and osteocytes after 4 d represent cells that had
newly differentiated from osteoprogenitor cells that were dividing on
day 12. Subtracting the same-day labeling index from the 4-d labeling
index, we estimate that the percentage of osteoblast labeling
attributable to new cell differentiation over 4 d was increased
about 2.2-fold, i.e., from 3.5 (±1.4) in wild-type bone to 7.6 (±1.9)
in D4 bone.
In parallel to the experiments described above, we also analyzed the
kinetics of osteoblast differentiation in our transgenic mice that
overexpress the truncated type II TGF-β receptor in osteoblasts.
After BrdU labeling at day 12 and analysis at day 16, the osteoblastic
labeling index (Figure ) and the number of labeled subperiosteal
osteocytes per section (data not shown) were similar in E1 mice and
wild-type mice. In D4/E1 double transgenic animals, these values were
intermediate between wild-type and D4 mice but showed variability
between individual animals. Significantly, both results are in
agreement with the effect of truncated receptor overexpression on the
subperiosteal osteocyte density in wild-type and D4 mice (Figure ).
Taken together, these kinetic studies are consistent with the notion
that increased TGF-β2 expression in osteoblasts stimulates the rate
of osteoblastic differentiation, and that this stimulation underlies
the increased osteocyte density in D4 mice. Increased Mineral Apposition Rate in D4 Transgenic Mice Does Not
Require Osteoblastic TGF-β Receptor Function, Yet Is Inhibited by
Alendronate The rate of bone deposition is often measured by the mineral
apposition rate. This rate is determined through sequential injection
of two fluorochromes that incorporate into bone matrix at sites of
ongoing mineralization. Injection of these fluorochromes with an
interval of several days results in the histological visualization of
two parallel lines at sites of bone formation. The mineral apposition
rate is then measured as the distance between the fluorochrome labels
divided by the time between their injection ( Parfitt et al.,
1987 ). We have previously shown that the mineral apposition rate at endosteal
surfaces in the tibia was increased ~70% in D4 mice compared with
wild-type mice ( Erlebacher and Derynck, 1996 ). Consistent with this
observation, the mineral apposition rate at endosteal surfaces in the
femoral epiphysis at day 35 was increased 80% in D4 mice (Figure
). To evaluate whether this increase
required TGF-β signaling in osteoblasts, we again used D4/E1 double
transgenic mice that overexpress a cytoplasmically truncated type II
TGF-β receptor in osteoblasts. In contrast to the dramatic inhibitory
effect of this truncated receptor on the osteocyte density increase,
D4/E1 mice showed the same 80% increase in mineral apposition rate as
D4 mice, and E1 mice showed no difference in their mineral apposition
rate compared with wild-type (Figure ). Thus, dominant negative
inhibition of TGF-β receptor signaling in osteoblasts did not affect
the mineral apposition rate, which suggests that its increase in D4
mice does not require osteoblastic responsiveness to TGF-β.
To assess whether the increased mineral apposition rate in D4 mice
depended on osteoclastic bone resorption, we again tested the effect of
alendronate administered over a 3-wk period before animals were killed
on day 35. In parallel, we included experimental groups that underwent
parathyroidectomy at day 21. Consistent with its inhibitory effect on
bone resorption and, as a consequence, on overall bone formation ( Rodan
and Fleisch, 1996 ), alendronate decreased the percentage of
fluorochrome-labeled bone surfaces from ~60% in the femoral
epiphyses of both wild-type and D4 mice to ~30%. In addition, as
shown in Figure , treatment with
alendronate dramatically reduced the epiphyseal mineral apposition rate
in D4 bones to a level similar to wild-type, yet did not affect
the mineral apposition rate in wild-type mice, which is consistent with
previous results ( Rodan and Fleisch, 1996 ). The decrease in mineral
apposition rate in D4 mice was not due to impaired bone mineralization,
since alendronate did not increase the lag time between osteoid
deposition and its mineralization in wild-type or D4 mice (data not
shown). Parathyroidectomy did not affect the mineral apposition rate in
D4 or wild-type mice with or without alendronate treatment. Taken
together, our observations suggest that the increase in mineral
apposition rate in D4 mice does not depend on the responsiveness of
osteoblasts to TGF-β, yet requires osteoclastic bone resorption.
The Mineral Apposition Rate in D4 Mice Is Not Increased on
Periosteal Surfaces Lacking Bone Resorption The conclusion that the increased mineral apposition rate in
D4 mice depends on osteoclastic activity predicts that bone surfaces
that lack osteoclastic resorption would not have an increased mineral
apposition rate. We therefore measured the mineral apposition rate on
the periosteal surface of the femoral diaphysis, a site devoid of
osteoclastic activity. As shown in Figure
, this rate was the same in D4 as in
wild-type at both days 16 and 35. The higher periosteal mineral
apposition rate at day 16, compared with day 35, is consistent with the
faster growth rate of younger mice. Fluorochromes injected at this time
formed continuous concentric rings parallel to the periosteum, showing
that radial growth was continuous (data not shown). Consistent with the
absence of osteoclastic activity on the femoral diaphyseal periosteum,
alendronate treatment and parathyroidectomy did not affect the mineral
apposition rate in wild-type or D4 mice on periosteal surfaces (Figure
A).
Surprisingly, increased TGF-β2 expression in
c -fos−/−/D4 mice led to an increased
periosteal mineral apposition rate compared with
c -fos−/− controls (Figure B).
c -fos−/−/D4 and
c -fos−/− mice also showed lower serum calcium
and phosphorus levels than wild-type mice (Table
1). However, the
increase in periosteal mineral apposition rate in
c -fos−/−/D4 mice over
c -fos−/− mice could not be ascribed to a
differential defect in bone mineralization, since the mineralization
lag time, i.e., the lag time between osteoid deposition and its
mineralization (Table 1), was not significantly different between the
two types of mice, although it was dramatically increased over normal.
Thus, increased osteoblastic expression of TGF-β2 resulted in
increased periosteal bone deposition in a
c -fos−/− background, in contrast to the
unaltered mineral apposition rate in a wild-type background. We were
unable to accurately assess the mineral apposition rate of endosteal
surfaces in c -fos−/− and
c -fos−/−/D4 mice because these surfaces lacked
double fluorochrome labeling. The density of the slightly ossified
calcified cartilage that fills the bone marrow cavities of
c -fos−/− mice was unchanged in
c -fos−/−/D4 mice (data not shown).
| Table 1Growth parameters, serological values, and
periosteal mineralization lag times in wild-type, D4,
c-fos−/−, and
c-fos−/−/D4 at day
16 |
Whereas TGF-β2 overexpression increased the periosteal
mineral apposition rate in a c -fos−/−
background, mineral apposition rates in
c -fos−/− mice were generally reduced compared
with wild-type mice (Figure B), consistent with their smaller size and
dramatically reduced growth rates ( Johnson et al., 1992 ;
Table 1). TGF-β2 overexpression in osteoblasts itself did not
affect the weights and growth rates of wild-type or
c -fos−/− mice (Table 1). Lastly,
c -fos−/−/D4 mice evaluated at day 35 had
a dramatically increased incidence of long bone fractures. As
previously observed, D4 mice occasionally showed spontaneous fractures
( Erlebacher and Derynck, 1996 ); however, five of nine
c -fos−/−/D4 mice showed tibial fractures (with
bilateral fractures observed in four cases), whereas none of the six
c-fos−/− mice examined showed hind limb
fractures. Various studies have shown that TGF-β affects the activity and
differentiation of osteoblasts and osteoclasts ( Bonewald and Dallas,
1994 ; Centrella et al., 1994 ), but the complexity of these
data does not allow us to infer the role of skeletal TGF-β in bone
development and remodeling. In the present study, we used our
transgenic mice that overexpress TGF-β2 in osteoblasts to
characterize the responses of osteoblasts to TGF-β during bone
remodeling. We focused on the two endpoint osteoblastic responses to
increased TGF-β2 expression, i.e., the increases in osteocyte density
and bone formation. Increased Osteocyte Density Results from a Direct Effect of TGF-β
on Osteoblasts We have previously shown that increased TGF-β2 expression in
osteoblasts results in increased osteocyte density ( Erlebacher and
Derynck, 1996 ). Our data now strongly suggest that this increase does
not depend on osteoclastic activity. First, TGF-β overexpression
increases the osteocyte density in the subperiosteal cortical bone of
the diaphysis, a site devoid of osteoclasts. Second, the increase in
osteocyte density persists in a c -fos−/−
background, which lacks osteoclasts. Furthermore, the normal and
increased osteocyte densities both require osteoblastic responsiveness
to TGF-β, since dominant negative inhibition of TGF-β receptor
function decreases the osteocyte density in both wild-type and D4 mice,
respectively. The TGF-β–induced positive regulation of osteocyte
density therefore results most likely from a direct, autocrine effect
on osteoblasts and occurs even at endogenous levels of TGF-β
expression. Although this parameter can be conveniently assessed, the increased
osteocyte density in response to TGF-β does not accurately reflect
the rate of differentiation from osteoblast to osteocyte. For example,
increased osteocyte density could result from decreased bone formation
without a change in osteocyte differentiation. Therefore, the rate of
osteocyte formation should take into account the final osteocyte
density and the rate of bone deposition, i.e., by multiplying the
osteocyte density (cells/mm3) with the mineral apposition
rate (mm3/mm2/day). This index in
cells/mm2/day may therefore be a better measure of the
effects of TGF-β on osteocyte differentiation. Using this index, the 1.6-fold increase in subperiosteal osteocyte
density in D4 mice compared with wild-type translates into a 1.6-fold
increase in osteocyte formation rate, since the mineral apposition rate
was unchanged. In contrast, the 1.9-fold increase in osteocyte density
in the femoral epiphysis of D4 mice corresponds to a 3.3-fold increase
in osteocyte formation rate, because of the 1.7-fold increase in
mineral apposition rate at that site (Figure
A). In addition, while the truncated
type II TGF-β receptor reduced the epiphyseal osteocyte density of D4
mice to the wild-type level (Figure ), the osteocyte formation rate at
that site in D4/E1 mice is still elevated 1.7-fold over normal (Figure
A) because of the increased local mineral apposition rate (Figure ).
This partial inhibition is consistent with the competitive nature of
dominant negative interference. Lastly, the modest inhibition of the
epiphyseal osteocyte density in D4 mice by alendronate (Figure A)
underestimates a dramatic inhibition of the osteocyte formation rate
(Figure B), since alendronate strongly inhibited the mineral
apposition rate increase in D4 epiphyses (Figure ). Alendronate,
however, did not affect the periosteal osteocyte formation rate, since
it had no effect on the periosteal mineral apposition rate or the
subperiosteal osteocyte density.
The Increased Mineral Apposition Rate Depends on Osteoclastic
Activity and Not on the Direct Response of Osteoblasts to TGF-β In addition to the increase in osteocyte density, TGF-β2
overexpression in osteoblasts also increased bone formation as measured
by the mineral apposition rate ( Erlebacher and Derynck, 1996 ). However,
several observations suggest that this is a secondary consequence of
increased bone resorption and not a direct effect of TGF-β2 on
osteoblasts. First, osteoblastic overexpression of a truncated type II
TGF-β receptor did not affect the mineral apposition rate in the
femoral epiphyses of wild-type or D4 mice, even though it reduced their
osteocyte density. Second, TGF-β2 overexpression increased the
osteocyte density of subperiosteal cortical bone, which forms in the
absence of bone resorption, but did not affect the periosteal mineral
apposition rate. Lastly, inhibition of bone resorption by alendronate
prevented the TGF-β2–induced increase in mineral apposition rate at
sites of bone resorption, but not on nonresorbing surfaces. This effect
was not due to inhibition of TGF-β signaling, since alendronate did
not affect the increase in subperiosteal osteocyte density. The dependence of the mineral apposition rate on osteoclast activity
stands in contrast to the osteoblast-mediated effects of TGF-β2 on
osteocyte density. Considering the differential inhibition of distinct
TGF-β responses by dominant negative receptors ( Chen et
al., 1993 ; Derynck and Feng, 1997 ), the increases in mineral
apposition rate and osteocyte density might result from distinct
responses of osteoblasts to TGF-β2 occurring at different thresholds.
The inhibition of the increase in epiphyseal osteocyte density, but not
the increase in the mineral apposition rate at that site, would then
imply that higher TGF-β concentrations are required for the former
than for the latter response. This, however, is difficult to reconcile
with the findings that TGF-β increases the subperiosteal osteocyte
density without changing the periosteal mineral apposition rate, and
that the epiphyses of alendronate-treated D4 mice have a normal mineral
apposition rate but an elevated osteocyte density. Instead, our results are consistent with the interpretation that
increased TGF-β2 expression leads to an enhanced epiphyseal mineral
apposition rate as a secondary response to its stimulation of bone
resorption, independent of the direct effects of TGF-β2 on
osteoblasts. Since dominant negative interference with TGF-β receptor
signaling in osteoblasts did not affect the mineral apposition rate,
and TGF-β2 overexpression did not increase the fraction of total bone
surface undergoing mineralization, our results suggest that TGF-β
does not directly regulate the rate of bone formation during normal
bone remodeling. The previously observed increases in bone formation
after subperiosteal injections of TGF-β ( Noda and Camilliere, 1989 ;
Joyce et al., 1990 ; Centrella et al., 1994 ) may
reflect a microfracture repair process consistent with the role of
TGF-β in wound healing. The increased mineral apposition rate in D4 mice may reflect a
homeostatic response that maintains bone integrity past a critical
threshold of resorption. Similarly, the increase in mineral apposition
rate by high doses of parathyroid hormone also results from stimulation
of bone resorption ( Hock and Gera, 1992 ; Uzawa et al.,
1995 ). Such a response may be sensitive to impaired mechanical and
structural properties of bone. Thus, mice with osteogenesis imperfecta
show increased periosteal bone formation as a compensatory response to
impaired skeletal integrity ( Bonadio et al., 1993 ; Pereira
et al., 1995 ). This mechanism may also explain the increase
in periosteal mineral apposition rate in
c -fos−/−/D4 mice when compared with
c -fos−/− mice, even though D4 and wild-type
mice have the same periosteal mineral apposition rate. TGF-β2
overexpression probably adds to the inherent structural defects of
osteopetrotic c -fos−/− bones by decreasing
their matrix quality, since c -fos−/−/D4 mice
have a dramatic increase in fracture incidence over
c -fos−/− mice, even though both mice have the
same total bone mass. TGF-β Increases the Rate of Osteoblastic Differentiation By labeling differentiating osteoblasts using BrdU, we showed a
2.2-fold higher labeling index of periosteal osteoblasts in D4 mice
than in wild-type mice. However, the surface density of mature
osteoblasts in D4 bone remained at the wild-type level ( Erlebacher and
Derynck, 1996 ; Figure , A and B). These findings suggest that TGF-β
increases the steady-state rate of osteoblastic differentiation. This
may be due, in part, to an acceleration of the maturation rate of
already committed osteoprogenitor cells; however, the normal to
increased number of osteoprogenitors in D4 bone (Figure ; Erlebacher
and Derynck, 1996 ) strongly suggests that this effect is largely due to
increased differentiation of osteoprogenitor cells coupled to increased
osteoprogenitor cell proliferation. Thus, the increased birth rate of
osteoblasts in D4 bone explains the 1.6-fold increase in periosteal
osteocyte formation rate and the high number of labeled subperiosteal
osteocytes. Overexpression of TGF-β2 may also affect the rate of
apoptosis of osteoprogenitors and osteoblasts; however, we did not
detect any differences in the very low level of apoptotic cells, as
assessed by 4,6-diamidino-2-phenylindole staining of the femoral
periosteum at day 16 (data not shown). Consistent with the direct osteoblastic effect of TGF-β on osteocyte
density, the increased labeling of osteoblasts and osteocytes induced
by TGF-β2 overexpression occurred in the absence of bone resorption
and was partially inhibited by dominant negative interference with
TGF-β signaling in osteoblasts. Thus, the increased rate of
osteoblastic differentiation also reflects a direct effect of TGF-β
on osteoblasts. Since the osteocalcin promoter used to drive expression
of the truncated TGF-β receptor is activated only after mature
osteoblasts have stopped dividing ( Bronckers et al., 1985 ;
Groot et al., 1986 ), this effect may be to induce the
secretion of a second signal that stimulates osteoprogenitor cell
proliferation and differentiation in a paracrine manner. In addition,
TGF-β may directly regulate other aspects of osteoprogenitor cell
physiology before activation of the osteocalcin promoter. We chose the femoral periosteum to analyze the kinetics of osteoblast
differentiation to avoid concurrent effects of TGF-β2 on bone
resorption and formation. However, TGF-β is likely to increase
osteoblast differentiation at other sites as well, e.g., in the femoral
epiphysis where the D4 osteocyte formation rate is 3.3-fold higher than
wild-type. In addition, the decrease in epiphyseal osteocyte density in
E1 mice with no change in mineral apposition rate suggests that
endogenous TGF-β signaling is most likely required to maintain the
normal rate of epiphyseal osteoblastic differentiation. In contrast,
periosteal osteocyte differentiation and cortical osteocyte density
were not significantly reduced by interfering with TGF-β signaling in
wild-type osteoblasts. These results may reflect a lower level of
endogenous TGF-β activity on periosteal surfaces compared with
endosteal surfaces. Differences in distribution of TGF-β activity in
bone may at least partially explain the higher rate of osteoblastic
differentiation on endosteal surfaces compared with periosteal surfaces
( Young, 1962 ). Accordingly, increases in TGF-β activity may be
involved in the increased osteocyte density seen in several metabolic
bone diseases such as osteoporosis ( Mullender et al., 1996 ),
hyperparathyroidism ( Malluche and Faugere, 1990 ), and osteogenesis
imperfecta ( Bonadio et al., 1993 ; Whyte, 1996 ). Since
osteocytes may mediate skeletal responses to mechano-sensation ( Aarden
et al., 1994 ), the regulation of their density by TGF-β
may significantly affect bone metabolism. The developmental phenotype of D4 mice ( Erlebacher and Derynck, 1996 )
strikingly resembles the phenotype of mice heterozygous for an
inactivated allele of Cbfa1, a transcription factor required
for normal ossification and osteoblast differentiation ( Ducy et
al., 1997 ; Komori et al., 1997 ; Otto et al.,
1997 ). Both mice show the hypoplastic clavicles, patent anterior
fontanels, and a general delay in ossification, characteristic of
cleidocranial dysplasia. This similar phenotype suggests that TGF-β
may down-regulate the embryonic expression of Cbfa1. Since
the cleidocranial phenotype in D4 mice does not require osteoblastic
responsiveness to TGF-β, its underlying mechanism is likely to be
distinct from the direct stimulatory effects of TGF-β2 on the rate of
osteoblastic differentiation. Osteoclasts Contribute to the TGF-β–induced Increase in
Osteoblastic Differentiation Although the increases in osteoblastic differentiation rate and
osteocyte density are direct effects of TGF-β on osteoblasts and do
not require bone resorption, alendronate dramatically reduced the
osteocyte formation rate in D4 mice in the femoral epiphysis, a site of
bone resorption (see above and Figure B). This effect was associated
with decreased osteoclastic activity and was not due to inhibition of
TGF-β signaling, since alendronate did not alter the increased
periosteal osteocyte formation rate. Thus, at sites of bone resorption,
osteoclastic activity augments the direct effects of TGF-β2 on the
rate of osteocyte formation and osteoblastic differentiation. The
similarly drastic inhibition of the osteocyte formation rate at these
sites by overexpression of the truncated type II TGF-β receptor
therefore suggests a substantial contribution of osteoclasts to the
local level of TGF-β activity. Osteoclasts may activate latent
TGF-β ( Oreffo et al., 1989 ; Oursler, 1994 ) or lead to the
release of bone matrix-bound TGF-β. This latter possibility is
suggested by experiments in organ culture ( Pfeilschifter and Mundy,
1987 ) and is consistent with the ability of alendronate to decrease the
plasma level of TGF-β2 in D4 mice. The coupling of osteoblastic differentiation to osteoclastic
activity is one of the central tenets of bone remodeling, yet the
molecular regulation of this process remains poorly understood. Since
the direct stimulation of osteoblastic differentiation by TGF-β can
be augmented by osteoclastic activity, TGF-β may be an important
mediator of the coupling of osteoblastic differentiation to sites of
bone resorption. Since overexpression of TGF-β in transgenic mice
also leads to increased bone resorption ( Erlebacher and Derynck, 1996 ),
TGF-β may be involved in regulating and coordinating the activities
of osteoblasts and osteoclasts during bone remodeling. Deregulation of
skeletal TGF-β expression, activation, and responsiveness in humans
may have important physiological consequences and contribute to
pathological bone-remodeling states. We thank Ilse Sauerwald and Nilda Ubana for their assistance
with the histological analyses and Margaret Mayes for her assistance
with the scanning electron microscopy. We also thank Gideon Rodan for
the alendronate and Randall Johnson for the
c-fos−/− mice. We are grateful to Deborah
Zimmerman, Alfred Kuo, Steve Gitelman, Bernard Halloran, Gideon Rodan,
and Zena Werb for helpful discussions, and Jill Helms for use of the
fluorescence microscope used in the kinetic analyses. This research was
supported by National Institutes of Health grants DE-10306 and AR-41126
(to R.D.) and a postdoctoral fellowship from the American Heart
Association (to E.H.F.). A.E. is a member of the Medical Scientist
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