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Musculoskeletal Disease Center, J. L. Pettis Veterans Administration Medical Center (N.M., X.Q., Y.K., C.R., A.K.S., D.J.B., S.M.), Loma Linda, California 92357; and Departments of Medicine (X.Q., A.K.S., D.J.B., S.M.), Biochemistry (S.M.), and Physiology (S.M.), 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}lom.med.va.gov
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Abstract |
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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 a paracrine/autocrine manner to regulate bone formation (6, 7, 8, 9). Both the endocrine and paracrine/autocrine effects of IGFs in bone are now known to be regulated by the relative amounts of stimulating and inhibiting IGF-binding proteins (IGFBPs) present in the circulation and in the extracellular milieu (10). Of the six high affinity IGFBPs that are produced by osteoblasts, IGFBP-4 has been consistently shown to inhibit IGF actions in vitro (11, 12, 13, 14). 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 (11); and 2) IGFBP-4 fragments with reduced IGF-binding activity are less potent in inhibiting IGF-induced cell proliferation (15).
Consistent with the in vitro findings that IGFBP-4 acts to inhibit IGF actions in osteoblasts primarily by an IGF-dependent mechanism, our recent in vivo study demonstrated that local administration of IGFBP-4 inhibited the IGF-I-induced increase in bone formation parameters in mice. However, to our surprise, we found that systemic administration of IGFBP-4 alone at pharmacological doses caused a significant increase in bone formation parameters and did not inhibit the IGF-I effect (16). In regard to the mechanism by which systemic IGFBP-4 administration increases bone formation parameters, we predicted, based on the past findings, that IGFBP-4 administration increases IGF bioavailability and thereby stimulates bone formation. This prediction is based on the following findings: 1) we found that systemic IGFBP-4 administration shifts IGF-I from a 150-kDa fraction into a 50-kDa IGFBP-4+IGF complex that can cross the vascular endothelium (16); and 2) IGFBP-4 protease is present in serum and produced by osteoblasts capable of cleaving the IGFBP-4+IGF complex to release IGFs to bind to IGF receptors (17). In this study we evaluated the hypothesis that proteolysis of IGFBP-4 is essential for the stimulatory effects of systemically administered IGFBP-4 by evaluating the in vitro and in vivo effects of recombinantly expressed wild-type, protease-resistant, and IGFBP-4 proteolytic fragments on bone formation parameters. To determine whether systemic administration of IGFBP-4 increased bone formation via increasing IGF bioavailability, we further evaluated the effect of IGFBP-4 in IGF-I midi mice, which exhibit extremely low levels of IGF-I in the circulation (18).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purification of recombinant IGFBP-4
Recombinant His6-tagged wild-type IGFBP-4
and IGFBP-4 mutants were expressed in Escherichia coli XL-1
blue cells (19). In this study we prepared wild-type
IGFBP-4 (-5/237), N-terminal and C-terminal IGFBP-4 fragments
(-5/135 and 142/237), and protease-resistant IGFBP-4 without the
sequence His121 to Pro141
(
121–141), as previously described (15, 17).
Recombinant IGFBP-4 proteins were purified by sequential nickel-agarose
and IGF-I affinity chromatography and quantitated by specific RIA
(15) and Bradford assays (20). The
His6 tag was not removed from IGFBP-4, because
the presence of the His6 tag at the N-terminal
end did not affect the biological activity (15). The
purity of the IGFBP-4 proteins was evaluated by SDS-PAGE, followed by
silver staining.
Osteoblast cell culture and conditioned medium
Osteoblast cells used were isolated by collagenase digestion
from calvariae of newborn C3H/HeJ mice as previously described
(21). The cells released were washed in DMEM and 10% calf
serum and plated in the same medium in 10-cm plates. Cells at passage 2
were used for the cell proliferation study.
For collection of conditioned medium (CM), osteoblast cells from calvariae at passage 2 were plated in 10-cm plates. At 70–80% confluence, culture dishes were rinsed twice with PBS and incubated with serum-free DMEM. After 48-h incubation, CM samples were collected and concentrated 50 times (50x CM) using centrifugal filter devices (Centricon YM30, Millipore Corp., Bedford, MA) at 4 C.
In vitro experiments
The biological activity of the purified IGFBP-4 preparations was
established by cell proliferation using the Alamar Blue assay (AccuMed
International, Inc., Westlake, OH). Briefly, osteoblast cells derived
from mouse calvariae were seeded into 96-well plates at 2000 cells/well
in 50 µl DMEM/0.1% BSA containing 0.1% calf serum. Twenty-four
hours later, 50 µl of 20 ng/ml IGF-I were added in DMEM/0.1% BSA
with or without different concentrations (80–1280 ng/ml) of wild-type
IGFBP-4 or IGFBP-4 analogs. The medium was replaced 48 h later
with 100 µl 10% Alamar Blue diluted in phenol red-free DMEM. Alamar
Blue is reduced by reactions innate to cellular metabolism and,
therefore, provides an indirect measure of viable cell number (AccuMed
International, Inc., Westlake, OH). The fluorescence was determined
4 h later using a fluorescent plate reader (Fluorolite 1000, Dynex
Technologies, Inc., Chantilly, VA).
In vivo experiments
Seven-week-old female C3H/HeJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Both sexes of adult IGF-I
midi mice (IGF-Im/m), which have low, but
detectable, IGF-I in their sera (18), were gifts from Dr.
Lyn Powell-Baxton. The animals were housed in a controlled environment
with 12-h light/dark cycles at 70 F with food and water ad
libitum. The IGF-I dose was determined from a previous study
reporting the effect of IGF-I local administration on bone formation in
parietal bones (16). In all experiments mice were grouped
according to 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
Jerry L Pettis V.A. Medical Center (Loma Linda, CA).
Exp 1: local effect of wild-type IGFBP-4 and IGFBP-4
analogs
On day 1, C3H/HeJ mice received 0 or 0.125 nmol IGF-I and/or an
equimolar dose of wild-type, protease-resistant (121–141),
N-terminal (-5/135), or C-terminal (142/237) IGFBP-4 (n = 8
animals/group). Each mouse received a single 20-µl aliquot of
treatment effector administered via a Hamilton syringe (Reno, NV) to
the outer periosteum of the right parietal bone (22, 23).
Before administration, the IGF-I was incubated with various IGFBP-4
analogs for 1 h at room temperature. Five days later (day 6), the
mice were euthanized.
Exp 2: local effect of protease-resistant IGFBP-4
On day 1, C3H/HeJ mice received 0 or 0.125 nmol IGF-I with 0,
0.031, 0.063, or 0.125 µmol wild-type or protease-resistant
(121–141) IGFBP-4 (n = 8 animals/group). Each mouse received
20 µl of treatment administered as described for Exp 1 (see above).
Before administration, the IGF-I was incubated with wild-type or
protease-resistant IGFBP-4 for 1 h at room temperature. On day 6,
the mice were killed.
Exp 3: systemic effect of protease-resistant IGFBP-4
On day 1, C3H/HeJ mice received 0, 0.05, 0.25, 1.25, or 6.25
nmol (0, 0.0024, 0.012, 0.06, or 0.3 nmol/g BW) of wild-type or
protease-resistant (121–141) IGFBP-4 (n = 8 animals/group).
The treatment was administered by sc injection at the nape of the neck
of each animal on day 1. On day 6, the mice were killed.
Exp 4: free IGF-I levels in serum after treatment with wild-type
IGFBP-4
C3H/HeJ mice received 0 or 1.25 nmol wild-type IGFBP-4 by sc
injection at the nape of the neck. They were killed at 30 min, 4
h, and 24 h after administration (n = 8 animals/group·time
point).
Exp 5: free IGF-I levels in serum after treatment with
protease-resistant IGFBP-4
C3H/HeJ mice received 0 or 1.25 nmol protease-resistant
(121–141) IGFBP-4 by sc injection at the nape of the neck. They
were killed 30 min after administration (n = 8 animals/group).
Serum was collected and used for free IGF-I measurements.
Exp 6: systemic effect of IGFBP-4 in IGF-I midi mice
On day 1, 9-week-old IGF-Im/m mice
received vehicle, 0.9 nmol (0.06 nmol/g BW) wild-type IGFBP-4 or 0.9
nmol (0.06 nmol/g BW) IGF-I or IGF-I (0.9 nmol) plus an equimolar dose
of IGFBP-4 (0.9 nmol) by sc injection at the nape of the neck (n =
6 animals/group). The doses of IGF-I and IGFBP-4 used in this
experiment were selected based on the results from Exp 3.
Bone collection
Femurs and right 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.
Biochemical assays
Free IGF-I RIA. Separation of free IGF-I from bound IGF-I
was performed by centrifugal ultrafiltration as previously described
(24) with the following modifications. Amicon Centrifree
UF devices with Ultracel YMT membranes were used (Millipore Corp.). Before centrifugation, filtrate cups were incubated with
1 mg/ml BSA for 30 min at 37 C to minimize nonspecific binding of IGF-I
to the plastic surface, then were washed with PBS and dried. Serum
samples were applied to the ultrafiltration chambers and incubated for
30 min at 37 C and centrifuged (1000 x g at 30 C).
IGF-I was measured by specific RIA using rabbit polyclonal antiserum
and recombinant IGF-I as standard and tracer as previously described
(25). 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%. The recovery of exogenously added IGF-I to serum by
centrifugation was 64%.
Osteocalcin RIA. Serum osteocalcin was measured by synthetic peptide-based RIA as previously described (26). The intra- and interassay coefficients of variation for mouse osteocalcin RIA for two controls were less than 10% (26).
ALP activity. The ALP activity of the serum and bone extracts was determined as previously described (27). The ALP activity of the bone extracts was expressed as milliunits per mg protein or as milliunits per mg dry weight of bone.
Total protein levels. The protein concentration was determined by Bradford assay using a commercial kit (Bio-Rad Laboratories, Inc., Richmond, CA).
Glucose levels. The serum glucose level was measured by hexokinase-glucose-6-phosphate dehydrogenase method using a commercial kit (Glucose Flex reagent cartridge, Dade Behring, Inc., Newark, DE).
Protease assay by [125I]-IGF-II Western
ligand blot analysis
To test IGFBP-4 protease activity in CM or mouse serum,
wild-type or protease-resistant (121–141) IGFBP-4 peptides were
incubated with concentrated CM or serum from 7-week-old female C3H/HeJ
mice at 37 C in the presence or absence of IGF-II. To remove the
endogenous IGFBPs, serum samples were subjected to ultrafiltration with
a 50 kDa cut-off membrane using a centrifugal filter device (Microcon
YM-50, Millipore Corp.) before protease assays. After 4-
or 24-h incubation, the assay mixtures were combined with SDS-PAGE
loading buffer, boiled for 5 min, and separated by 12% SDS-PAGE gels.
Proteins were transferred to nitrocellulose filters to perform
[125I]IGF-II Western ligand blot analysis as
previously described (28).
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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). IGF-I at 10 ng/ml increased cell
number, as measured by Alamar Blue assay, by more than 40% in mouse
osteoblasts. Wild-type IGFBP-4 at a 16-fold higher molar dose than
IGF-I was needed to block the IGF-I effect by 50% in mouse
osteoblasts. Protease-resistant IGFBP-4 (121–141), on the other
hand, was much more potent than wild-type IGFBP-4 in blocking the IGF-I
effect (equimolar dose of protease-resistant IGFBP-4 blocked IGF-I
effect by 50%). The lower potency of wild-type IGFBP-4 in blocking the
IGF-I effect in mouse osteoblasts could be ascribed to the presence of
IGFBP-4 protease capable of degrading IGFBP-4 in the conditioned
medium of mouse osteoblasts.
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). N-Terminal and C-terminal IGFBP-4 alone at 40–640 ng/ml also did
not show a significant effect on basal cell proliferation in the
absence of exogenously added IGF-I (data not shown).
We next determined whether wild-type or protease-resistant
(121–141) IGFBP-4 peptides are resistant to the protease in mouse
CM or mouse serum. Based on previous findings that exogenous addition
of IGF-II to cell-free osteoblast cell-conditioned medium increased
IGFBP-4 proteolysis (17), we evaluated IGFBP-4 proteolysis
in the absence or presence of exogenously added IGF-II. Figure 2 shows IGF-II ligand blot results after
in vitro digestion of IGFBP-4 peptides by concentrated mouse
osteoblast CM (A) or prefiltered mouse serum (B). In both the presence
and the absence of IGF-II, wild-type IGFBP-4 was cleaved after
incubation with CM or mouse serum. In contrast, no apparent degradation
was seen with an equivalent amount of protease-resistant IGFBP-4 in
both CM and serum when no exogenous IGF-II was added. There is some
evidence for degradation of protease-resistant IGFBP-4 (<20%) in the
presence of IGF-II after 24 h (Fig. 2), but not after 4 h
(data not shown). Protease-resistant IGFBP-4 bound IGF-II tracer and
blocked the IGF-I effect on cell proliferation equally well compared
with wild-type IGFBP-4, thus suggesting that a 20-amino acid deletion
did not influence IGF-dependent action of IGFBP-4.
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). IGFBP-4 alone (wild-type or various
analogs) 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
wild-type IGFBP-4 and protease-resistant IGFBP-4 (121–141). In
contrast to wild-type and protease-resistant IGFBP-4, neither
N-terminal (-5/135) nor C-terminal (142/237) IGFBP-4 fragments had any
significant effect on the IGF-I-induced increase in ALP activity in
parietal bone extract. ALP activity was not significantly different in
the 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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). Protease-resistant IGFBP-4
significantly blocked the IGF-I effect at both 0.063- and
0.125-nmol doses of IGFBP-4, whereas wild-type IGFBP-4
inhibited only at the highest dose. Similar results were obtained if
ALP activity was standardized on the basis of milligrams of protein in
calvarial bone extracts (data not shown).
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). Similarly, both femoral and calvarial
bone extract ALP activity standardized on the basis of milligrams of
dry weight of bone showed a significant increase by wild-type IGFBP-4
at 1.25 nmol and higher (Fig. 6). In
contrast to wild-type IGFBP-4, systemic administration of
protease-resistant IGFBP-4 (121–141) showed no significant effect
on bone formation markers in serum and bone extract. Similar results
were obtained if ALP activity in bone extracts was standardized on the
basis of milligrams of extractable protein (data not shown).
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). In contrast, systemic administration
of protease-resistant IGFBP-4 did not increase free IGF-I levels
compared with those in vehicle-treated controls 30 min after treatment
(5.8 ± 0.6 ng/ml in vehicle-treated control mice vs.
5.2 ± 0.7 ng/ml in protease-resistant IGFBP-4-treated mice;
mean ± SEM; n = 8/group). Because
IGF-I administration is known to cause a decrease in serum glucose, we
evaluated glucose levels in IGFBP-4-treated and control mice. We found
that glucose levels were reduced by about 10% at 30 min and 4 h
(Table 1).
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). In contrast, systemic administration
of IGF-I alone increased ALP activity in femoral bone extract
significantly, and administration of IGFBP-4 along with IGF-I
did not inhibit the IGF-I effect (Fig. 7). Similar results were
obtained using serum ALP activity measurements (data not shown). These
data from the IGF-I+IGFBP-4 restitution experiment in IGF-I midi mice
are similar to those obtained by administering IGF-I+IGFBP-4 complex to
normal mice (16).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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The lack of stimulatory effects of systemically administered protease-resistant IGFBP-4 could not be explained on the basis of loss of bioactivity of this analog due to structural modification that is caused by deletion of amino acid residues 121–141. In a previous study we found that deletion of His121-Pro141 had no effect on IGF-binding activity, but was resistant to protease produced by human osteoblasts (17). Consistent with these data, we found that wild-type, but not protease-resistant, IGFBP-4 analog was degraded by protease present in mouse osteoblast cell-conditioned medium or mouse serum. In addition, we found that protease-resistant IGFBP-4 analog was much more potent than wild-type IGFBP-4 in inhibiting IGF-I-induced cell proliferation in mouse osteoblasts, which produce IGFBP-4 protease. In contrast, there was no significant difference between the protease-resistant and wild-type IGFBP-4 in inhibiting IGF-I-induced cell proliferation in MG63 human osteosarcoma cells, which do not produce IGFBP-4 protease (17). Consistent with the in vitro data that deletion of residues 121–141 had no significant effect on IGF-binding activity, we found that local administration of protease-resistant IGFBP-4 inhibited IGF-I-induced bone formation, as expected. In addition, protease-resistant, but not wild-type, IGFBP-4 blocked the IGF-I effect at the lowest dose tested, thus suggesting that deletion of residues 121–141 in the protease-resistant IGFBP-4 analog does not affect IGF-binding activity.
Our in vivo data on the comparison of protease-resistant and wild-type IGFBP-4 in inhibiting IGF-I-induced bone formation is much less convincing than the in vitro data. One of the potential explanations of why the difference in potency of protease-resistant IGFBP-4 and wild-type IGFBP-4 is not as large in vivo compared with in vitro could be related to differences in the rate of IGFBP-4 clearance in the two models. In vitro, exogenously added IGFBP-4 remains in the medium unless it is degraded or taken up by cells. As there is no evidence for uptake of IGFBP-4 by osteoblasts, and degradation of protease-resistant IGFBP-4 is minimal, much of the added protease-resistant IGFBP-4 should remain in the medium of osteoblast cell cultures during the 48-h culture period in the cell proliferation experiment. In contrast, single local administration of protease-resistant IGFBP-4 on top of the parietal bone would be cleared rapidly in the extracellular fluid. Thus, further experiments involving continuous delivery of IGFBP-4 by minipump would be required to evaluate the in vivo potency of protease-resistant IGFBP-4 vs. wild-type IGFBP-4 in the local in vivo model.
In previous studies it has been demonstrated that proteolysis of IGFBP-3 in serum from pregnant women leads to an increase in free IGF-I levels (30). Based on the finding that mouse serum contains IGFBP-4 protease and that systemic administration of wild-type, but not protease-resistant, IGFBP-4 increased bone formation, we predicted that systemic administration of wild-type IGFBP-4 increases bone formation by increasing IGF-I bioavailability. We therefore measured free IGF-I levels in serum at different times after systemic administration of wild-type IGFBP-4. We found that free IGF-I levels were significantly elevated at 30 min after administration of wild-type, but not protease-resistant, IGFBP-4. Consistent with the increase in free IGF-I levels, we found that serum glucose levels were significantly lower at 4 h after IGFBP-4 administration. To confirm that increased IGF availability is the primary mechanism by which systemically administered IGFBP-4 increases bone formation, we evaluated the effects of IGFBP-4 on bone formation parameters in IGF-I-deficient midi mice, which are severely depleted of circulating levels of IGF-I. The IGF-I expression in homozygous insertional mutant IGF-I midi mice is reduced by more than 65%, resulting in lower serum IGF-I levels and decreased growth in these mice (18). Our findings that the systemic administration of IGFBP-4 failed to stimulate bone formation parameters in IGF-I-deficient midi mice and that administration of wild-type, but not protease-resistant, IGFBP-4 increases serum levels of free IGF-I in wild-type mice provide the first direct evidence that systemic administration of IGFBP-4 increases bone formation via an IGF-dependent mechanism.
The nature of IGFBP-4 protease in mouse serum that is involved in the proteolysis of IGFBP-4 can only be speculated upon at the present time. Lawrence et al. (31) reported that pregnancy-associated plasma protein-A (PAPP-A), a protein previously identified in serum from pregnant women, was identical to the IGF-II-dependent IGFBP-4 protease produced by human fibroblasts. PAPP-A is also the major IGFBP-4 protease present in human ovarian follicular fluid (32). In recent studies we found that IGFBP-4 proteolysis during pregnancy was accounted for mainly by the IGF-II-dependent IGFBP-4 proteolysis and that PAPP-A is the major protease present in serum of pregnant women (33). In this study we found that some degradation of protease-resistant IGFBP-4 occurred in the presence of exogenously added IGF-II after prolonged incubation in mouse serum, thus suggesting that PAPP-A and/or additional protease may cleave protease-resistant IGFBP-4, but at a considerably lower rate compared with the wild-type IGFBP-4. Further studies are needed to evaluate whether PAPP-A is the major protease that contributes to proteolysis of IGFBP-4 in mouse serum.
Based on the findings that IGFBP-4 is a potent inhibitor of IGF actions in various cell types in vitro (11) and that local administration of IGFBP-4 along with IGF-I inhibits IGF-I actions as shown in this study, we predict IGFBP-4 in the local milieu to be inhibitory, whether it is produced locally or has a systemic source. On the other hand, systemic administration of pharmacological doses of IGFBP-4 (doses that increase serum level of IGFBP-4 by 50-fold or greater) produces different effects by a mechanism that involves increased IGF bioavailability via proteolysis of IGFBP-4 in the circulation. In this model, systemically administered IGFBP-4 causes an acute increase in serum IGFBP-4 levels, which increases IGF-I in the IGFBP-4 complex. Subsequent proteolysis of IGFBP-4 leads to an increase in free IGF-I, thus increasing bone formation. Accordingly, although protease-resistant IGFBP-4 treatment would increase IGF-I in the protease-resistant IGFBP-4 complex, little or no proteolysis of protease-resistant IGFBP-4 should occur, resulting in no increase in free IGF-I in serum. The finding that IGFBP-4 proteolytic fragments had no significant effect on basal or IGF-I-induced osteoblast cell proliferation suggests that the lack of stimulatory effect of systemically administered protease-resistant IGFBP-4 is due to its inability to increase free IGF-I and not to the absence of proteolytic fragments. The mechanism that we have described for a stimulatory effect of pharmacological doses of systemically administered IGFBP-4 may also be applicable to other inhibitory IGFBPs (e.g. IGFBP-1) provided that similar proteolytic mechanisms are operative for the other inhibitory IGFBPs that could influence the free level of IGF-I. Furthermore, our data are consistent with the concept that the observed mechanism by which IGFBP-4 increases the level of free IGF-I in serum may have physiological relevance with respect to regulating the free IGF-I levels in serum. Accordingly, physiological regulation of the IGF-I in the 50-kDa pool may influence the levels of free IGF-I in serum and, thereby, the endocrine actions of IGF-I.
In conclusion, this study provides the first direct evidence that systemic administration of IGFBP-4 at pharmacological doses increases bone formation parameters in mice by increasing IGF bioavailability via an IGFBP-4 protease-dependent mechanism. The question of whether PAPA is the IGFBP-4 protease that cleaves the IGFBP-4+IGF complex to increase IGF bioavailability requires further studies.
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Acknowledgments |
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Footnotes |
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Received December 15, 2000.
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,
P < 0.05 compared with IGF-I alone.
,
Serum osteocalcin; 
, serum ALP. Seven-week-old C3H/HeJ mice received
0–6.25 nmol wild-type or protease-resistant IGFBP-4 by sc injection at
the nape of the neck. Five days later, the blood was collected, and
serum osteocalcin levels and ALP activity were determined as described
in Materials and Methods. The values are expressed as a
percentage of the vehicle-treated control value and are the mean
± SEM (n = 8). The vehicle control values are
112.0 ± 12.5 ng serum osteocalcin/ml and 157.6 ± 11.5 mU
ALP activity/ml serum. *, P < 0.05; **,
P < 0.01 (compared with vehicle-treated control).
