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Musculoskeletal Disease Center, J. L. Pettis Veterans Administration Medical Center (N.M., C.R., X.Q., D.J.B., S.M.), Loma Linda, California 92357; and the Departments of Medicine (X.Q., D.J.B., S.M.), Biochemistry (S.M.), and Physiology (S.M.) Loma Linda University, Loma Linda, California 92350
Address all correspondence and requests for reprints to: Subburaman Mohan, Ph.D., Musculoskeletal Disease Center (151), J. L. Pettis Veterans Administration Medical Center, 11201 Benton Street, Loma Linda, California 92357. E-mail: mohans{at}llvamc.va.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGFs are unique in that they act in both an endocrine and
paracrine/autocrine manner to regulate bone formation (2, 12, 13, 14). High
circulating levels of IGFs (
0.1 µmol/liter) are derived mostly
from the liver and provide a readily available IGF reservoir for their
endocrine functions (15). When IGF actions are blocked in
vitro, basal osteoblast cell proliferation is decreased by 50%,
suggesting that locally produced IGFs may play a significant
paracrine/autocrine role in the regulation of bone formation. Both the
endocrine and paracrine/autocrine effects of IGFs in bone are now known
to be regulated by the relative amounts and types of IGF-binding
proteins (IGFBPs) present in the circulation and in the extracellular
milieu (15). Of the six high affinity IGFBPs that are known to
circulate in blood and that are produced by osteoblasts, IGFBP-4 has
been proposed to play an important role in the regulation of bone
formation for a number of reasons. First, IGFBP-4, one of the most
abundant IGFBPs produced by osteoblasts, is a potent inhibitor of
IGF-induced cell proliferation in a number of cell types in
vitro (16). Second, IGFBP-4 has been shown to inhibit the growth
of embryonic chick pelvic cartilage in vitro (17). Third,
both systemic and local regulators of bone formation regulate
osteoblast cell production of IGFBP-4 (16). And fourth, serum levels of
IGFBP-4 are altered in clinical disease states (15).
In vitro studies on the mechanism by which IGFBP-4 inhibits osteoblast cell proliferation show that IGFBP-4 may inhibit IGF actions in osteoblasts by preventing the binding of IGF ligand to its membrane receptors. This binding inhibition has been proposed based on the following key findings. 1) IGFBP-4 competes with IGF receptors for IGF binding in both monolayer cell cultures and purified type I IGF receptor preparations (16). 2) IGFBP-4 fragments with reduced IGF binding activity are less potent in inhibiting IGF-induced cell proliferation (18). Although the in vitro findings to date are consistent with the idea that IGFBP-4 inhibits IGF actions in osteoblasts primarily by an IGF-dependent mechanism, the in vivo effects of IGFBP-4 on IGF-I-induced bone formation and its mechanism of action remain unknown. To evaluate whether IGFBP-4 inhibits IGF actions in vivo as it does in vitro, we measured the local and systemic effects of recombinant IGFBP-4 on bone formation parameters using a mouse model system. We also evaluated whether high affinity IGF binding is required for IGFBP-4 to block IGF-I effects on bone formation by comparing the effects of an IGFBP-4 fragment that binds IGFs with severalfold lower affinity than intact IGFBP-4 (18).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purification of recombinant IGFBP-4
Intact (His6-tagged -5/237) and fragment
(His6-tagged -5/182) forms of IGFBP-4 were expressed in
Escherichia coli XL-1 blue cells as previously described
(19). Recombinant IGFBP-4 protein was purified by sequential
nickel-agarose and IGF-I affinity chromatography and quantitated by
specific RIA (18). The His6 tag was not removed from
IGFBP-4 since the presence of His6 tag at the N-terminal
end did not affect the biological activity (18). The purity of IGFBP-4
was evaluated by SDS-PAGE followed by silver staining. The IGF-binding
activity of the purified IGFBP-4 proteins was determined using
[125I]IGF-II tracer by polyethylene glycol precipitation
assay as previously described (20).
In vitro experiments
The biological activity of the purified IGFBP-4 preparations was
established by cell proliferation using the alamarBlue assay (AccuMed
International, Inc., Westlake, OH). Briefly, MG63 cells were seeded
into 96-well plates at 2000 cells/well in 50 µl DMEM containing 0.1%
calf serum. Twenty-four hours later, 50 µl 20 ng/ml IGF-I or IGF-II
were added in DMEM-0.1% BSA with or without different concentrations
(40–2560 ng/ml) of fragment or intact forms of IGFBP-4. The medium was
replaced 48 h later with 100 µl 10% alamarBlue diluted in
phenol red-free DMEM. The fluorescence was determined 4 h later
using a fluorescent plate reader (Fluorolite 1000, Dynex Technologies,
Inc., Chantilly, VA).
In vivo experiments
Female C3H/HeJ retired breeder mice (6 months of age) were
purchased from The Jackson Laboratory (Bar Harbor, ME).
The animals were housed in a controlled environment with 12-h light,
12-h dark cycles at 70 F with food and water ad libitum. The
IGF-I dose was determined from a previously published study reporting
the effect of local administration of IGF-II on bone formation in
parietal bones (21). Intact and fragment IGFBP-4 were administered in a
dose equimolar to that of IGF-I. In all experiments the groups of mice
were made up of animals similar in size and weight. At the end of each
experiment, the mice were killed by ethrane inhalation and
decapitation; blood and bones were collected and stored at -70 C until
biochemical measurements were performed. The experimental procedures
performed in this study are in compliance with the NIH Guide for the
Care and Use of Laboratory Animals and were approved by the animal
studies subcommittee at the J. L. Pettis V.A. Medical Center (Loma
Linda, CA).
Exp 1: local effect of IGF-I
The mice were divided into three groups (n = 8
animals/group). Each group received a single 10 µl treatment
administered via a Hamilton syringe (Reno, NV) to the outer periosteum
of the right parietal bone (22, 23). Group 1 received vehicle (PBS),
group 2 received 0.2 µg IGF-I/mouse, and group 3 received 1.0 µg
IGF-I/mouse. Five days later, the mice were killed.
Exp 2: local effect of IGFBP-4
On day 0, the mice were divided into six groups (n = 7
animals/group). Each group received 20 µl treatment administered as
described in Exp 1 (see above). Group 1 received vehicle (PBS), group 2
received 1.0 µg IGF-I/mouse, group 3 received 4.0 µg intact
IGFBP-4/mouse, group 4 received 1.0 µg IGF-I/mouse and 4.0 µg
intact IGFBP-4/mouse, group 5 received 3.6 µg fragment IGFBP-4/mouse,
and group 6 received 1.0 µg IGF-I/mouse and 3.6 µg fragment
IGFBP-4/mouse. Before administration, IGF-I was added to either intact
or fragment forms of IGFBP-4 and allowed to incubate for 1 h at
room temperature. On day 5, the mice were killed.
Exp 3: systemic effect of IGF-I
The mice were divided into four groups (n = 6
animals/group). Group 1 received one injection of vehicle (PBS), group
2 received two injections of vehicle (PBS), group 3 received one
injection of 2.0 µg IGF-I/g mouse BW, and group 4 received two
injections of 2.0 µg IGF-I/g mouse BW. The treatment was administered
by sc injection at the nape of the neck of each animal on day 0 of the
experiment in one-injection groups and on days 0 and 2 in two-injection
groups. Blood samples were collected by tail bleeding at baseline (1
day before injection) and 1 and 3 days after injection in the
one-injection groups and 3 days after the first injection in the
two-injection groups. On day 5, the mice were killed.
Exp 4: systemic effects of IGFBP-4
On day 0, the mice were divided into six groups (n = 7 or 8
animals/group). Group 1 received vehicle (PBS), group 2 received 50
µg IGF-I/mouse, group 3 received 200 µg intact IGFBP-4/mouse, group
4 received 50 µg IGF-I/mouse and 200 µg intact IGFBP-4/mouse, group
5 received 160 µg fragment IGFBP-4/mouse, and group 6 received 50
µg IGF-I/mouse and 160 µg fragment IGFBP-4/mouse. Before
administration, the IGF-I was added to either intact or fragment forms
of IGFBP-4 and allowed to incubate for 1 h at room temperature.
Blood samples were collected by tail bleeding on day 1. On day 5 the
mice were killed.
Exp 5: serum IGF-I and IGFBP-4 levels after treatment with
IGFBP-4
The mice were divided into two groups (n = 5
animals/group). Group 1 received vehicle (PBS), and group 2 received
200 µg intact IGFBP-4/mouse. As serum half-lives of IGFBP-1 and
IGFBP-2 have been estimated to be on the order of 1–2 h (24), we
evaluated the effects of IGFBP-4 on the serum IGF-I level 30 min after
administration.
Bone collection
Femurs and parietal bones were dissected out of each carcass and
cleaned of soft tissue, being careful not to destroy the periosteum.
Each bone was rinsed in PBS at 4 C for 24 h, followed by
extraction in 0.01% Triton X-100 at 4 C for 72 h. This bone
extract was used for the alkaline phosphatase (ALP) activity
measurements.
Separation of 150- and 50-kDa IGF pools
The mouse serum was fractionated using gel exclusion
chromatography on a Sephadex G-75 (Pharmacia Biotech,
Piscataway, NJ) column. Briefly, 250 µl serum were mixed with 250
µl elution buffer (PBS containing 0.1% BSA and 0.02% sodium azide)
and loaded onto a Sephadex G-75 column (HR10 column from
Pharmacia Biotech) at a flow rate of 0.4 ml/min. The
proteins were eluted with elution buffer, and 1-ml fractions were
collected. The 150-kDa (12, 13, 14, 15, 16, 17, 18, 19, 20) and 50-kDa (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) fractions were
pooled and concentrated using a Centricon (Centricon-10, Amgen, Inc., Beverly, MA) centrifugal devise to a final volume of 1 ml.
The concentrated pools were biospun to separate the IGFs from the
IGFBPs, as previously described (25). This procedure, separating the
IGFs from the IGFBPs, has inter- and intraassay coefficients of
variation of less than 10%. Fifty microliters of the resulting IGF
pool were neutralized with 50 µl 1.2 M Tris base, and the
IGF-I content was determination by specific RIA (26).
Biochemical assays
IGF-I RIA. IGF-I was measured by specific RIA using rabbit
polyclonal antiserum and recombinant IGF-I as standard and tracer as
previously described (26). The inter- and intraassay coefficients of
variation were less than 10%. The cross-reactivity of IGF-II in the
IGF-I assay was less than 2%.
IGFBP-4 RIA. IGFBP-4 was measured by a specific RIA. Antibodies were raised against recombinant human IGFBP-4 in guinea pig. Recombinant human IGFBP-4 was used as standard and tracer (19). The inter- and intraassay coefficients of variation were less than 10%. The cross-reactivity of other IGFBPs in the IGFBP-4 assay was less than 2%.
Osteocalcin RIA. Serum osteocalcin was measured by specific RIA using mouse osteocalcin as tracer and standard (Biomedical Technologies, Stoughton, MA). The mouse osteocalcin RIA had interassay variability of less than 8% (27).
ALP activity
The ALP activity of the cell and bone extracts was determined as
previously described (28). ALP activity was expressed as milliunits per
mg protein or as milliunits per mg dry wt of bone.
Total protein levels
The protein concentration was determined by Bradford assay using
a commercial kit (Bio-Rad Laboratories, Inc., Richmond,
CA).
Statistical analysis
Statistical analysis of the data was performed by t
test or Fisher’s protected least significant difference method
(post-hoc test) for multiple comparisons in a one-way ANOVA
as appropriate. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Similar results were obtained
by ligand blot analysis (data not shown). The biological potencies of
intact and fragment forms of IGFBP-4 were evaluated by cell
proliferation assays in serum-free cultures of MG63 human osteosarcoma
cells. IGF-I or IGF-II at 10 ng/ml increased cell proliferation by
50%. Intact IGFBP-4 at an equimolar concentration inhibited the
IGF-I or IGF-II effect by more than 50%. The IGF-I or IGF-II effect
was completely blocked by intact IGFBP-4 at a 4-fold higher
concentration than that of IGF (Fig. 2).
In previous studies, we demonstrated that the biological activity of
human recombinant IGFBP-4 is very similar to that of native IGFBP-4
purified from bone cell-conditioned medium (19). In contrast to intact
IGFBP-4, fragment IGFBP-4 inhibited IGF-I or IGF-II-induced cell
proliferation to a much lesser extent. To block 50% of the IGF-I or
IGF-II effect, 2560 ng/ml IGFBP-4 fragment were required compared with
40 ng/ml intact IGFBP-4.
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Local effect of IGF-I and IGFBP-4 on bone formation
parameters
Bone formation was evaluated by measuring osteoblast cell products
such as ALP and osteocalcin in the serum and bone extracts. ALP
activity expressed on the basis of bone weight was significantly
increased by day 5 in the right parietal bones (injected side) in both
low (mean ± SEM, 0.88 ± 0.05 mU/mg dry wt) and
high (0.96 ± 0.1) dose IGF-I-treated groups compared with that in
vehicle-treated controls (0.64 ± 0.09). ALP activity expressed on
the basis of extractable protein was significantly increased in the
high dose IGF-I group only (Table 1). Although ALP activity showed a
slight increase in the noninjected left side, this increase was not
statistically significant. In contrast to calvaria, ALP activity was
not significantly different in the femoral bone extract (data not
shown), nor was serum ALP activity or osteocalcin level (data
not shown), suggesting that the IGF-I activity was restricted to the
injected site.
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and Table 1
). IGFBP-4 alone (intact or
fragment) had no significant effect on parietal bone extract ALP
activity. The IGF-I-induced increase in ALP activity in the parietal
bones, however, was completely blocked by an equimolar dose of intact
IGFBP-4. In contrast to intact IGFBP-4, fragment IGFBP-4 had no
significant effect on the IGF-I-induced increase in parietal bone
extract ALP activity (Fig. 3). In addition, ALP activity was not
significantly different in femoral bone extract of any of the treatment
groups compared with that in the vehicle group (data not shown). There
were also no significant differences in serum osteocalcin or ALP
activity between the treatment and control groups after local injection
(data not shown).
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). The
reason why serum osteocalcin was not increased 3 days after IGF-I
administration is not known. Serum ALP activity showed a significant
increase 3 days after IGF-I administration and remained elevated 5 days
after the injection (Fig. 4b). The magnitude of increase in serum ALP
and osteocalcin did not differ significantly after one or two IGF-I
administrations. The femoral bone extract ALP activity was also
increased after both one and two IGF-I administrations (Fig. 4c and
Table 2).
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). The increase in serum osteocalcin by
IGFBP-4 was similar to that of an equimolar dose of IGF-I. Serum
osteocalcin levels in the IGF-I plus intact IGFBP-4 group was not
different from that in the IGF-I or IGFBP-4 alone treatment group (Fig. 5a). In contrast to intact IGFBP-4, the IGFBP-4 fragment had no
significant effect on serum osteocalcin levels (Fig. 5a). Similar to
serum osteocalcin, serum ALP activity was increased significantly after
a single administration of intact IGFBP-4 (Fig. 5b). ALP activity was
significantly increased in both calvarial and femoral bone extracts
after treatment with intact IGFBP-4 (Fig. 5c and Table 2). In contrast
to intact IGFBP-4, fragment IGFBP-4 had no significant effect on ALP
activity in either calvarial or femoral bone extracts (Fig. 5c).
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). To determine whether the relative
distribution of IGF-I in the 150- and 50-kDa IGF pools was altered in
the IGFBP-4-treated mice, we subjected serum to size separation before
measurement of IGF-I. IGFBP-4 treatment caused a significant increase
in IGF-I in the 50-kDa pool compared with the control group (Table 3).
Correspondingly, the amount of IGF-I in the 150-kDa pool was
significantly less in mice treated with intact IGFBP-4 than in control
mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the local effects of IGFBP-4 on bone formation, we used a calvaria model that was established to study the in vivo effects of transforming growth factor-ß1 on bone turnover in mice (22). In this model, we found that a single local administration of IGF-I at a dose of 1 µg/mouse caused a statistically significantly increase in ALP activity at the site of injection after 5 days. This increase in ALP activity caused by IGF-I treatment is consistent with a number of previous studies that have reported the anabolic effects of IGF-I on bone formation in both animal models and humans. The systemic administration of IGF-I has been shown to increase bone formation parameters in both cortical and trabecular bone and to increase bone mineral density at various skeletal sites in ovariectomized rats (12, 30, 31). In addition, Wakisaka et al. (32) reported that local infusion of IGF-I into the right femora of old rats stimulated the expression of matrix proteins and improved trabecular bone status compared with those in vehicle-treated left femora. Thus, the IGF-I-induced increase in parietal bone extract ALP activity is likely to represent IGF-I-induced increase in bone formation.
The finding that concomitant administration of intact IGFBP-4
with IGF-I completely blocked the IGF-I-induced increase in ALP
activity is consistent with the previous in vitro findings
that IGFBP-4 is a potent inhibitor of IGF actions in a variety of cell
types, including osteoblasts (15, 33). Consistent with these data, Wang
et al. (34) have shown that overexpression of IGFBP-4 in
smooth muscle cells of transgenic mice through a smooth muscle
-actin promoter caused smooth muscle hypoplasia. As the IGFBP-4
fragment (-5/182), which binds IGFs with a 50- to 100-fold lower
affinity than intact IGFBP-4, did not have similar inhibitory activity,
we speculate that the inhibitory effect of IGFBP-4 on the IGF-I-induced
increase in bone formation marker proteins in vivo is
mediated via an IGF-dependent mechanism. It is well established that
the affinity of intact IGFBP-4 for IGF-I is an order of magnitude
higher than those of signaling type I IGF receptors (35, 36). In
previous studies, we found that the IGF-binding domain is located in
the N-terminal region of IGFBP-4, but that the C-terminal region is
also required for high affinity binding of IGF (18). Thus, deletion of
the C-terminal region (183–237) of IGFBP-4 decreased IGF binding
affinity an order of magnitude less than that of the type I IGF
receptor and thereby reduced its capacity to compete with type I IGF
receptor to bind IGF-I. Consistent with this mechanism of action,
previous studies have shown that intact IGFBP-4 blocked the binding of
IGF-I to both monolayer cell cultures and purified type I IGF receptor
preparations in vitro (16). Taken together, these findings
are consistent with the idea that locally administered IGFBP-4 blocks
the IGF-I effect by preventing the binding of IGF-I to type I IGF
receptors.
In previous studies, we found that exogenous addition of intact IGFBP-4 caused a significant inhibition of basal cell proliferation in serum-free cultures of osteoblasts (20). We therefore anticipated a reduction in ALP activity in the calvaria of mice treated with intact IGFBP-4 alone. There are a number of potential explanations for the failure of IGFBP-4 to inhibit basal ALP activity in vivo. Firstly, these studies were performed in old mice (6 months old), which have low basal ALP activity compared with young (prepubertal) mice. A single administration of IGFBP-4 may not be adequate to further reduce the already low rate of bone formation. Secondly, the levels of both serum IGF-I and skeletal IGF-I decrease with age (37); thus, the contribution of IGF-I to basal ALP activity in the calvaria of older mice may be less than that in younger mice. Thirdly, IGFBP-4 effects were evaluated using a single dose and a single administration; higher doses of IGFBP-4 and repeated administrations of IGFBP-4 may be required to cause a significant reduction in basal ALP activity in this model.
The most significant finding in this study is that systemic administration of intact IGFBP-4 alone increased bone formation parameters in mice. This finding is completely unexpected, as IGFBP-4 is the only IGFBP, among the six high affinity IGFBPs, that consistently inhibits IGF actions in vitro (15). A single administration of intact IGFBP-4 at a dose of 200 µg/mouse caused a 50% increase in serum ALP activity and osteocalcin levels after 24 h, and these levels remained elevated for 5 days. This increase in serum bone formation markers is similar to that seen with an equimolar dose of IGF-I. Furthermore, IGFBP-4 treatment caused a significant increase in ALP activity in the femoral bone extracts. This systemic effect of IGFBP-4 to increase bone formation markers appears to be specific, because the IGFBP-4 fragment, which has reduced IGF-binding activity, did not elicit similar effects.
We would like to speculate on the mechanism of action by which
systemic administration of IGFBP-4 increases bone formation parameters
in mice. The majority of IGFs (>99%) circulate in plasma bound to
IGFBPs either as a large (150-kDa) or a small (50-kDa) molecular weight
complex. Seventy-five to 80% of IGFs circulate in the 150-kDa
fraction, which consists of IGF-I or IGF-II bound to IGFBP-3 that
is then bound to a non-IGF-binding acid-labile subunit. This large
molecular weight complex cannot cross the vascular endothelial barrier
(15). The remaining 20–25% of IGFs circulate bound to one of the
other six high affinity IGFBPs, and these complexes (50 kDa) can freely
cross the vascular endothelium (38). We speculate that systemic
administration of IGFBP-4 leads to an increase in circulating levels of
IGFBP-4, which increases the amount of IGF-I in the 50-kDa complex by
shifting the IGF-I from the larger IGFBP-3/IGF-I/acid-labile subunit
complex to the smaller IGFBP-4/IGF-I complex (Fig. 6). In this regard IGFBP-4 cannot replace
IGFBP-3 in the 150-kDa complex. The ability of the IGF/IGFBP-4 complex
to cross the endothelial barrier intact may make it an important
carrier molecule, delivering IGFs to target tissues. Because the IGF in
the IGF/IGFBP-4 complex is biologically inactive, the IGF-I must be
dissociated from IGFBP-4 by proteolysis in the target tissues before
IGF is free to bind the type I IGF receptor. Serum contains and
osteoblasts produce an IGF-dependent IGFBP-4-specific protease capable
of degrading IGFBP-4 (39), and these released IGFs act in a positive
feedback mechanism to activate more of the IGF-dependent
IGFBP-4-specific protease (40, 41), thus providing a mechanism for
increased IGF bioavailability in bone. Our present study supports this
conclusion, as systemic administration of intact IGFBP-4 caused a
significant increase in IGF-I in the 50-kDa pool and a
corresponding decrease in IGF-I in the 150-kDa pool.
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Systemic administration of the combination of IGF-I/fragment IGFBP-4 was less effective in stimulating bone formation parameters than IGF-I/intact IGFBP-4. There are a number of potential explanations for this: 1) decreased binding of IGF-I to fragment IGFBP-4, 2) lack of proteolysis of fragment IGFBP-4, and 3) decreased half-life of the IGF-I/fragment IGFBP-4 complex due to its smaller size.
Further studies are needed to verify whether systemically administered IGFBP-4 stimulates bone formation parameters by increasing IGF-I bioavailability in bone. Several recent studies are consistent with this proposed model. First, we found that GH treatment in GH-deficient adults as well as in children with chronic renal failure increased serum levels of IGFBP-4 (43, 44). This increase in serum IGFBP-4 may increase IGF bioavailability by increasing the relative ratio of IGF-I in the 50-kDa pool compared with 150-kDa pool. Second, serum levels of IGFBP-4 showed significant positive correlation with bone mineral density at several skeletal sites both before and after GH therapy in GH-deficient adults (43). Third, IGFBP-4-deficient mice generated by disruption of the IGFBP-4 gene are born significantly smaller (10–15%) than their wild-type and heterozygous littermates and remain proportionally smaller throughout life (45). Based on this model, we would predict that the decreased growth rate in the IGFBP-4 knockout mice is in part due to a decrease in IGF-I in the 50-kDa complex.
In conclusion, this study demonstrates for the first time that a single local administration of IGFBP-4 inhibits IGF-I-induced increases in bone formation, whereas systemic administration of IGFBP-4 alone increases serum levels of bone formation markers. This differential local and systemic effects of IGFBP-4 on IGF-I-induced bone formation appears to be mediated by an IGF-dependent mechanism, as the IGFBP-4 fragment, which has reduced IGF-binding affinity, did not exhibit similar effects. Future long term dose-response studies using histomorphometric techniques are needed to confirm the IGFBP-4 effects on bone formation in mice.
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Acknowledgments |
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Footnotes |
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Received June 14, 1999.
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