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GROWTH FACTORS-CYTOKINES-ONCOGENES |
Department of Physiology and Biophysics (D.G.-K., J.K.C.), and Center for Osteoporosis and Metabolic Bone Disease (E.A., R.L.J., S.C.M.), Division of Endocrinology, Department of Medicine, Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Address all correspondence and requests for reprints to: D. Gaddy-Kurten, Department of Physiology and Biophysics University of Arkansas for Medical Sciences 4301 West Markham, Slot 505, Little Rock, Arkansas 72205. E-mail: gaddykurtendana{at}uams.edu
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Introduction |
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However, recent data have suggested that some clinical indices of increased bone turnover can first be detected in late premenopausal women with normal circulating estrogen levels (5). Thus, this increased bone turnover must be nonsex steroid-dependent. Indeed, the endocrine parameter best correlated with this increase is elevated serum FSH levels. This early rise in FSH levels in perimenopausal women is attributable to a selective decrease in inhibin B secretion. The decrease in inhibin B secretion occurs in the presence of normal levels of E2, inhibin A, GnRH, and LH (6). Because both inhibin A and inhibin B isoforms selectively inhibit pituitary FSH secretion, these data suggest that increased FSH is attributable to a loss in feed-back inhibition by gonadal inhibin B in perimenopausal women, resulting in bone loss before the loss of sex steroids. As the loss of gonadal function progresses in postmenopausal women, the well-established decreases in E2 accompany declining levels of both inhibin B and inhibin A, further increasing serum FSH (6) and markedly increasing bone loss. Thus, although the molecular mechanisms responsible for bone loss in perimenopausal women are not yet clear, we hypothesize that this loss reflects a direct role of inhibins on bone marrow cell differentiation.
Inhibin B and inhibin A are heterodimeric proteins in the TGFß
superfamily composed of 
ßB or ßA subunits, respectively.
Inhibins were originally identified based on their ability to suppress
pituitary FSH secretion (7). Suppression of FSH by the
inhibins is antagonized by the related peptide, activin A, a homodimer
composed of ßA ßA subunits that is locally produced in the
gonad (7). In addition to opposing effects on pituitary
FSH production and gonadal steroid production, inhibins and activin
exert opposing effects on erythroid (8), megakaryocyte
(9), and granulocyte-macrophage cell development
(10). Activin ßA subunit mRNA is also locally produced
in bone marrow (11); and, like TGFß (12)
and bone morphogenetic proteins (BMPs) (13), activin A is
abundantly localized in bone matrix (14). Although inhibin
-subunit expression (required for inhibin dimer formation) is very
low in human and rat bone marrow (15, 16), inhibin
accumulates in the bone marrow of 25-d-old rats within 10 min of iv
injection of [125I]-inhibin A and is retained
for at least an hour (17). These results are consistent
with the idea that the effects of inhibin on marrow cell hematopoiesis
(8, 9, 10) are attributable to inhibin derived from gonadal
sources (18).
Increasing evidence suggests the involvement of activin and inhibin in
the regulation of bone formation. In neonatal rat calvaria, activin
promotes ectopic bone formation induced by BMP (14), can
enhance proliferation of osteoblastic cells and collagen synthesis
(19), and is released from the bone matrix of calvaria
undergoing resorption (20). In fracture callus, activin
administration stimulates fracture healing (21), whereas
inhibin--subunit is expressed at sites of endochondral ossification
(15). During endochondral bone development, the
stimulatory actions of activin are regulated by changes in follistatin
expression during the transition from cartilage to bone (15, 22), and activin receptors have been localized during this
process (23). Follistatin is a natural antagonist that
binds activin with high affinity and neutralizes its biological
activities by preventing activin interaction with its membrane
receptors (24, 25). Follistatin mRNA and protein also have
been detected in osteoblasts and osteocytes in the developing mouse
mandible (16) and in fracture callus (21).
In vitro, follistatin mRNA has been detected in
calvarial-derived MC3T3-E1 osteoblast-like cells (26).
However, the expression of follistatin in bone marrow in
vivo has not been reported, and it may be that it is only
expressed in bone marrow as a feedback mechanism to limit activin
action, as in the pituitary (27, 28).
Finally, administration of activin to ovariectomized rats caused a dose-dependent blunting of ovariectomized- induced bone loss (20, 29), providing additional evidence that local activin tone may be a critical component in the regulation of bone cell differentiation.
These studies led us to postulate that changes in the inhibin/activin ratio, detected by cells within the bone marrow, contribute significantly to bone turnover. By directly altering the balance of osteoblastogenesis and osteoclastogenesis, the effect of the increased inhibin/activin ratio is to increase bone resorption in the presence of normal estrogen levels. In the current study, we tested our hypothesis in vitro, using primary murine bone marrow cells, cultured under osteogenic or osteoclastic conditions in the presence of inhibins, activin A, and the activin antagonist follistatin. Our results demonstrate direct effects of inhibin, activin, and follistatin on both osteoblast and osteoclast development. These in vitro effects support the existence of an endocrine/paracrine loop in which these factors regulate osteoblast and osteoclast development in vivo.
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Materials and Methods |
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Materials
Recombinant human activin A, follistatin-288, and inhibin-A were
obtained both from the NIH National Pituitary Hormone Program and by
purchase from R&D Systems (Minneapolis, MN). Inhibin A preparations
were also purified from porcine follicular fluid and were generously
provided by Joan Vaughan and Wylie Vale at the Salk Institute. These
preparations were all verified for bioactivity before use, and all
provided similar qualitative and quantitative results. Doses for
activin, inhibin, and follistatin used in these studies were in the
physiological range of doses used to characterize effects of these
compounds on pituitary and ovarian function (30).
1,25(OH)2 vitamin D3 was
purchased from BioWhittaker, Inc. (Walkersville, MD); BMP2
and noggin were purchased by R&D Systems and were also generously
provided by Vicki Rosen (Genetics Institute, Cambridge,
MA) and Niel Stahl (Regeneron Pharmaceuticals, Inc.,
Tarrytown, NY), respectively. Antibodies to the ßA- and -subunits
were obtained from Serotec (Raleigh, NC). Patrick Sluss
(Harvard Medical School, Massachusetts General Hospital) generously
provided the antiserum against the 288- and 315-amino acid forms of
follistatin, the sizes of which are 31-kDa and 35-kDa proteins,
respectively.
Osteoblast progenitor assays
Ex vivo bone marrow cell cultures were generated from
3- to 4-month-old Swiss Webster mice as described previously
(31). Briefly, bone marrow cells were flushed from the
femurs with MEM containing 15% FBS (HyClone Laboratories, Inc., Logan, UT), penicillin, streptomycin, and antimycotics.
Marrow cells from all mice were combined, rinsed, resuspended to obtain
a single cell suspension, and seeded in at least triplicate wells. The
numbers of colony forming unit-fibroblast (CFU-F) and fully mature
colony forming unit-osteoblast (CFU-OB) present in the bone
marrow preparations were determined as previously described
(31). Cells were cultured for osteoblastogenesis in 15%
FBS containing MEM, and supplemented with 50 µg/ml ascorbic acid
and 10 mM ß-glycerophosphate to support
mineralization. Plating density in 6-well plates was 1.5 x
106 cells/well for CFU-F development and 2.5
x 106 cells/well for CFU-OB development. Growth
factors and antagonists were diluted in 0.1 N
acetic acid, 0.1% BSA, which was used as the vehicle control in all
experiments.
The total number of CFU-F colonies represent the total number of colonies in the culture, and they were determined on d 10 and d 28 as an indicator of multipotential mesenchymal progenitors (32, 33). Cultures were fixed with neutral buffered formalin and stained with either hematoxylin or methyl green to observe all colonies (of at least 50 cells) in the dish, regardless of differentiated state.
To determine the recruitment of mesenchymal cell progenitors into the osteoblastic lineage early in the culture, the number of alkaline phosphatase (AP)-positive (AP+) colonies (AP+ CFU-F) were determined by AP staining after 10 d of culture (Sigma, St. Louis, MO). Because osteoblastogenesis is a lengthy process, requiring 3–4 wk for complete differentiation (34, 35), fully mature CFU-OB was determined on d 25–28 of culture, by the staining of mineral deposits using Von Kossa staining, as previously described (31). All multiple groups in a given experiment were harvested on the same day of culture. Similar results were obtained in osteoblastogenesis experiments harvested on d 25–28.
Osteoclast progenitor assays
Bone marrow cells were obtained from the femurs of 3- to
4-month-old mice, as described above, and cultured for
osteoclastogenesis, as previously described (31), at
densities of 1 x 106 cells/well, in 24-well
plates, in 10% FBS containing MEM supplemented with 10
nM 1,25(OH)2 vitamin
D3 to support osteoclast development. Osteoclast
formation was assessed on d 8–12 by staining for tartrate-resistant
acid phosphatase (TRAP; Sigma) and counting the
multinucleated (MNC) TRAP+ cells in each well. All multiple groups in a
given experiment were harvested on the same day of culture. Similar
results were obtained in osteoclast development experiments harvested
on d 8–12.
Statistics
Colony numbers per well and TRAP+ MNC cell numbers per well were
obtained in at least triplicate wells. Data were analyzed by one-way
ANOVA to determine significant differences between means of multiple
groups in given experiments. Post hoc analyses by Tukey’s
and Mann-Whitney U tests, where appropriate, were also
applied to verify that the significance levels were valid.
Immunoblots
Cellular extracts were prepared by cell lysis, for 15 min at 4
C, in modified RIPA buffer (36), followed by
centrifugation at 14,000 x g. Supernatants (50–200
µg) were fractionated on 10% or 12% SDS-PAGE and
electrophoretically transferred to either nitrocellulose or PVDF
membranes. Membranes were blocked with 1% nonfat dry milk in 20
mM Tris (pH 7.4), 0.15 M
NaCl containing 0.05% Tween 20 (TTBS), and subsequently incubated with
primary antisera overnight at 4C. Blots were washed extensively with
TTBS, reblocked with 1% milk-TTBS, and incubated for 1 h at room
temperature with horseradish peroxidase-conjugated goat antimouse or
goat antirabbit secondary antibody (1:30,000) (Pierce Chemical Co., Rockford, IL) before visualization using chemiluminescence
reagents (Pierce Chemical Co.).
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Only inhibin exerted an effect, which was to suppress recruitment of
mesenchymal cell progenitors to the AP+ preosteoblastic lineage.
Interestingly, follistatin did not mimic the suppressive effect of
inhibin on AP+ colony formation, even at high concentrations (200
ng/ml), suggesting that the basis of inhibin repression is not inhibin
competition with activin for activin receptors in these cultures.
Inhibin also decreased AP activity on d 10 by up to 80% within each
colony (P = 0.006 by Wilcoxon nonparametric analysis).
This result suggests that inhibin suppressed commitment of mesenchymal
progenitors recruited to the osteoblast lineage.
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). The
number of CFU-OB colonies was scored in triplicate wells for each
treatment, and the values were expressed as the number of Von
Kossa-positive colonies per well. Inhibin blocked CFU-OB formation by
up to 90% in three separate experiments (Fig. 1B). Interestingly,
although follistatin did not block AP expression (Fig. 1A), it blocked
formation of CFU-OB (Fig. 1B). This inhibitory effect of follistatin at
100 ng/ml provided the first indication that activin is produced in
ex vivo bone marrow cultures and that activin action is
required for full osteoblast maturation and formation of a mineralized
matrix (CFU-OB) in these cultures.
Activin A is produced and secreted by ex vivo bone marrow cultures
during osteoblastogenesis
We next confirmed that activin A was produced and secreted in
these cultures during osteoblastogenesis (Fig. 1C). Cells were grown in
ascorbic acid and ß-glycerolphosphate to stimulate
osteoblastogenesis. Lysates and conditioned medium were harvested on
different days of culture and analyzed for activin A content. Activin A
was expressed in lysates and secreted into the medium in a
time-dependent manner. Activin A was undetectable in lysates or
conditioned medium on d 5 of culture. However, 28-kDa activin A dimer
was detected in 100 µg lysate and 500 µg conditioned medium on d
10. In multiple experiments, although lysate production (or
accumulation) was consistently maximal sometime between d 15–20,
activin A content in conditioned medium progressively increased to d 25
of culture. These data demonstrate that CFU-OB formation in 25-d
ex vivo marrow cultures occurs in the presence of increasing
activin accumulation. In addition, activin accumulation at a time in
which follistatin inhibited CFU-OB formation is consistent with
follistatin neutralization of secreted activin A, as has been
demonstrated previously for neutralization of pituitary and erythroid
effects of activin A (37).
Activin and inhibin exert opposing effects on osteoclast
development
Osteoblastogenesis is intimately coupled with stromal cell support
of osteoclast development (38, 39). Thus, it was critical
to determine whether activin and inhibin also affected
osteoclastogenesis in primary bone marrow cultures. Therefore, the
effects of activin, inhibin, and follistatin on
1,25(OH)2 vitamin
D3-dependent osteoclastogenesis were measured in
primary bone marrow cultures by analyzing the formation of TRAP+
multinucleated cells on d 9 (Fig. 1D). Exogenous activin (50 ng/ml)
enhanced osteoclastogenesis in the presence of
1,25(OH)2 vitamin D3 (as
demonstrated previously) (40), whereas inhibin suppressed
the formation of multinucleated TRAP+ cells (Fig. 1D). Thus, activin
and inhibin were found to exert opposing actions on both
osteoblastogenesis and osteoclastogenesis. However, follistatin (100
ng/ml) had no effect on the formation of TRAP+ cells (Fig. 1D),
suggesting that activin is not produced during the 9 d of
osteoclastogenesis stimulated by 1,25(OH)2
vitamin D3. The precise target cells of activin
and inhibin action during osteoblast and osteoclast differentiation
have not yet been defined. However, the fact that inhibin suppressed
stromal cell recruitment to the osteoblastic lineage is consistent with
the idea that support of osteoclast development requires stromal cell
recruitment (2, 3, 4).
Inhibin suppression of osteoblast and osteoclast development is
maintained in the presence of exogenous activin
Previous reports of inhibin action in other tissues have been
explained by inhibin opposing an endogenous activin action through
competition of ligands for activin receptors. Further, inhibin actions
in other tissues have been shown to be mimicked by the addition of
follistatin, to antagonize activin binding to the type II receptor
serine kinase (7). Therefore, we wanted to test whether
the suppressive effects of inhibin on osteoblastogenesis and
osteoclastogenesis were maintained in the presence of activin. Bone
marrow cells were cultured in the presence of 50 ng/ml inhibin A in the
absence or presence of 100 ng/ml activin A (Fig. 2, A and
B). In the presence of the increased
activin concentration, inhibin completely suppressed osteoblastogenesis
(Fig. 2A). The number of total CFU-F colonies was unchanged by any
treatment, excluding the possibility that inhibin nonspecifically
blocked colony formation or cell proliferation per se. Follistatin, at
100 ng/ml, had no effect on osteoblastogenesis, demonstrating the
specificity of the inhibin effect.
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) was not
overcome by the addition of activin, even though activin alone
consistently stimulated at least a 3-fold increase in the formation of
TRAP+ cells (Fig. 2B). Taken together, these data provide further
evidence that the suppressive effects of inhibin on marrow cell
differentiation are not likely to be mediated solely through
competition with activin for activin receptors.
Activin substitutes for BMP and recruits cells into the
osteoblastic lineage and stimulates the formation of the full
osteoblastic phenotype in vitro (CFU-OB)
To gain an understanding of the full range of activin action on
osteoblast differentiation, we used noggin to remove the effect of
endogenous BMPs on early osteoblastogenesis (41). In
addition, we also tested whether activin stimulation of
osteoclastogenesis would be maintained in the presence of BMP
antagonism. In Fig. 3A, noggin (100
ng/ml) suppression of early and late osteoblastogenesis in murine bone
marrow cultures was overcome by treatment with activin A (30 ng/ml).
Similarly, noggin suppression of osteoclast development, as indicated
by TRAP+ multinucleated cell formation, was overcome by exogenous
activin treatment (Fig. 3B). These results are similar to our previous
data in which exogenous BMP2 overcame noggin suppression of both
osteoblast and osteoclast development (41). These data
suggest that activin can overcome BMP suppression and support both the
recruitment of mesenchymal cells into the osteogenic lineage and
support osteoclastogenesis. The capacity of activin to selectively
overcome the suppressive effects of noggin, but not inhibin, indicates
that the suppressive effects of noggin and inhibin occur via distinct
mechanisms.
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-subunit (Fig. 4A). The recombinant inhibin A standard
is a 32-kDa dimeric protein composed of the 18-kDa -subunit
and a 14-kDa ßA-subunit. The d-0 freshly isolated bone marrow
contained inhibin -subunit-containing proteins, including a band
comigrating with the inhibin A standard. Additional bands are visible
that correspond with several known species of ß dimers comprised
of uncleaved proregions of each subunit (42). However,
none of the lysates after 5, 10, or 20 d of culture produced
inhibin -subunit, either under control conditions or in response to
stimulation with activin A, noggin, or inhibin A. Thus,
osteoblastogenesis in murine bone marrow cultures proceeds in the
absence of basal or stimulated endogenous inhibin production. It is
likely that the -subunit containing inhibin proteins, found in
freshly isolated bone marrow, are the result of circulating inhibin
from the gonads.
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), we determined whether
endogenous activin accumulation was autoregulated or could be
down-regulated by inhibin, follistatin, and noggin. Lysates were
prepared from freshly isolated bone marrow (d 0), as well as from
cultured cells after 5, 10, or 20 d in the presence or absence of
50 ng/ml activin A or inhibin-A and 100 ng/ml noggin or follistatin
(Fig. 4B). As shown in Fig. 1C, endogenous activin accumulation was
most prevalent in control cultures after d 10, corresponding to the
later stages of osteoblastogenesis. Activin autostimulated its
production to higher levels. The increased production was detected as
early as d 5 of culture, and there were progressive increases
throughout the culture period (Fig. 4B). These data are consistent with
activin autoregulation serving to enhance osteoblastogenesis. One
possibility that cannot be excluded is that internalized activin A may
be contributing to the progressive activin A accumulation observed in
lysates during osteoblastogenesis. However, because a constant amount
of activin A was added to all cultures isolated on different days, the
observed progressive accumulation of activin A in lysates is more
consistent with activin autoregulation of its content. Conversely,
treatment with noggin, inhibin, or follistatin completely blocked the
production of activin A during osteoblastogenesis (Fig. 4B). Thus, in
addition to antagonizing existing activin action, the suppressive
effects of inhibin and follistatin on osteoblastogenesis are also
mediated by inhibition of the osteogenic activin synthesis in the
cultures. Furthermore, the ability of noggin to block activin
biosynthesis indicates that endogenously produced BMPs stimulate
activin production as well.
Basal follistatin production by ex vivo bone marrow cultures is not
regulated during osteoblastogenesis
In reproductive tissues, activin action is dependent on the ratio
of activin to follistatin in the local microenvironment
(7). Thus, it was important to determine the extent to
which murine bone marrow cells produced endogenous follistatin
during osteoblastogenesis. Immunoblot analyses were performed
using cell lysates (Fig. 5A) and
concentrated conditioned medium (Fig. 5B) from different days of
culture. Follistatin was not found in freshly isolated bone marrow
cells (d 0). However, it was detected in lysates from d 5, and the
levels progressively decreased throughout osteoblastogenesis.
Interestingly, the amount of follistatin secreted into the medium was
low to undetectable during the time course, suggesting that most of the
follistatin remained membrane-associated in these cultures. Together
with the observed increases in activin A production during the same
time course (Fig. 4B), these data provide further evidence of an
increasing net effect of activin during osteoblastogenesis in
vitro.
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) or osteoclastogenesis (Fig. 6B)
caused by inhibin. These results contrast with the ability of exogenous
BMPs to restore osteoblast and osteoclast development in the presence
of noggin (41) (and data not shown). Follistatin (100
ng/ml) was unable to suppress the osteogenic effect of endogenous BMPs
(Fig. 6A) that were blocked by noggin (Fig. 3A). In addition, there was
no further increase in osteoblastogenesis in the presence of exogenous
BMP2 and follistatin (Fig. 6A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Either
activin A or BMPs (such as BMP2) can recruit murine bone marrow
mesenchymal cells into the osteoblast lineage, where they express AP
(AP+). Endogenous activin accumulation contributes to development of
the full osteoblast phenotype, because follistatin blocks the later
stage of matrix mineralization. Inhibin, as well as noggin
(41), represses mesenchymal stem cell recruitment to the
osteoblast lineage. Activin stimulation can override the inhibitory
effect of noggin on osteoblast development and support full osteoblast
differentiation. However, the suppressive effect of inhibin seems to be
dominant over the activities of both activin and BMP.
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Activin stimulates (and inhibin blocks) osteoclast
development
Inhibin and activin also exert opposing effects on
osteoclastogenesis. The effect of activin on TRAP+ multinucleated
cell formation is consistent with a previous report showing that
activin stimulates osteoclast-like cell formation in vitro
(38). Although the target cell mediating the
osteoclastogenic effect of activin has not been identified, the
effects of both activin and inhibin on osteoclastogenesis are
consistent with activin and inhibin receptors residing, at least in
part, on stromal support cells.
It is now well accepted that the mechanism for stromal cell support of osteoclastogenesis is the expression of RANKL on stromal cells, which binds the receptor (RANK) on osteoclast progenitors, stimulating osteoclast development (38, 39). By preventing recruitment of stromal cells into the osteoblastic lineage, inhibin and noggin may prevent stromal cells from acquiring the ability to synthesize RANKL, thereby limiting osteoclastogenesis. Because activin can recruit stromal cells into the osteoblastic lineage, this stimulation may be sufficient for the expression of RANKL and subsequent osteoclastogenesis. However, these studies do not exclude the possibility of a direct effect of activin on osteoclasts and/or their precursors, as has been recently reported for BMPs (43).
Activin actions on bone in vitro and in vivo
Our studies are the first to suggest a direct role for activin on
mesenchymal cell differentiation in the bone marrow. However, other
direct actions of activin in vitro on osteoblastic cells
derived from calvaria cells have been reported (19, 26, 44). These results suggest that activin action in the local
environment of calvarial cell development (intramembranous bone
development) may be distinct from activin effects on bone remodeling
driven by cells in the bone marrow.
Activin has also been shown to be osteogenic in several in vivo systems, similar to the BMPs (13). Local administration of activin increases periosteal bone matrix thickness in newborn rat parietal bone (17) and enhances noncartilagenous ectopic bone formation stimulated by BMP2 (14). In addition, activin (0.4–10 µg/d) promotes fracture healing when administered locally to rat fibula (21).
However, recent studies in rats, using exogenous systemic activin administration to counteract the ovariectomy- induced vertebral bone loss, demonstrate a biphasic effect. In aged rats, in which serum activin and follistatin are diminished (20), activin administration at low doses (1–5 µg/kg) increased vertebral bone mass and mechanical properties (20). Administration of higher doses (25 µg/kg) of activin diminished the magnitude of these effects (20). These conflicting results highlight the importance of local activin tone in target tissues, such that low doses of activin may be sufficient to specifically favor bone formation over the general elevation of bone turnover.
Biological relevance of inhibin actions on bone marrow cell
differentiation
As far as we are aware, our data are the first to identify a
physiological effect of inhibin that is not mimicked by the activin
antagonist, follistatin. In addition, this inhibin effect was not
blocked by exogenous activin. This result provides functional evidence
to support the existence of a unique inhibin-specific receptor. Indeed,
the recent identification of specific binding of inhibin to both a
novel protein, p120 (45), and to betaglycan
(46) raises the possibility that these proteins may be
involved in mediating the suppressive effects of inhibin.
The inhibin-specific suppression of osteoblast and osteoclast differentiation has strong biological implications. Reproductive aging is accompanied by decreases in both inhibin A and inhibin B; the decrease in inhibin B in perimenopausal women precedes the decrease in inhibin A (47). This selective decrease in inhibin B occurs in concert with an increase in E2 and an increase in FSH (6, 47). In fact, the early increase in FSH is the only endocrine parameter that has been correlated with increased markers of bone resorption in perimenopausal women, although inhibin B levels were not determined in that study (5). Thus, our data, in conjunction with these clinical findings and the observation that circulating iodinated inhibin can accumulate in bone marrow (48), support our hypothesis that gonadal inhibin may provide endocrine input into the local regulation of osteoblastogenesis and osteoclastogenesis.
The bone loss and ensuing osteoporosis after loss of gonadal function has been previously attributed solely to estrogen deficiency. Our results are the first to directly demonstrate a role for other gonadal factors on marrow cell differentiation. The in vitro data presented here suggest a paradigm in which, before loss of sex steroids, when diminished ovarian function is indicated by selective decreases in inhibin B secretion, bone turnover may be accelerated through changes in osteoblast and osteoclast development driven by altered inhibin and activin tone. Confirmation of these in vitro effects of inhibin and activin in vivo may provide critical insight into the control of normal bone turnover in cycling women.
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Acknowledgments |
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Footnotes |
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1 Current Address: Mount Sinai School of Medicine One Gustave L. Levy
Place, Box 1055, New York, New York 10029. 
Abbreviations: AP, Alkaline phosphatase; BMP, bone morphogenetic protein; CFU-OB, colony forming unit-osteoblast; CFU-F, colony forming unit-fibroblast; MNC, multinucleated; TRAP, tartrate-resistant acid phosphatase; TTBS, 20 mM Tris (pH 7.4), 0.15 M NaCl containing 0.05% Tween 20.
Received May 4, 2001.
Accepted for publication September 13, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ßA, and ßB subunits in
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