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Endocrinology Vol. 140, No. 12 5719-5728
Copyright © 1999 by The Endocrine Society

ARTICLES

Effects of Recombinant Insulin-Like Growth Factor-Binding Protein-4 on Bone Formation Parameters in Mice1

Naohisa Miyakoshi, Charmaine Richman, Xuezhong Qin, David J. Baylink and Subburaman Mohan

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4), one of the most abundant IGFBPs produced by bone cells, is a potent inhibitor of IGF actions in vitro. To evaluate the modulation of IGF actions on bone formation in vivo by IGFBP-4, we produced intact and fragment (50- to 100-fold reduced IGF affinity) forms of BP-4 and examined their local and systemic effects using biochemical markers. Local administration of IGF-I over the right parietal bone significantly increased bone extract alkaline phosphatase activity; this was completely blocked by an equimolar dose of intact IGFBP-4, but not IGFBP-4 fragment. A single sc administration of IGF-I (2 µg/g BW) significantly increased bone formation markers in both serum and skeletal extracts; surprisingly, so did intact IGFBP-4, but not fragment IGFBP-4. Subcutaneous administration of an equimolar dose of IGFBP-4 along with IGF-I did not significantly block the IGF-I effect. Administration of intact IGFBP-4 significantly increased the serum 50-kDa IGF pool and decreased the 150-kDa IGF pool without significantly changing total IGF-I. We postulate that the increase in the 50-kDa IGF pool might enhance IGFs bioavailability via a mechanism involving IGFBP-4-specific protease. 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factors (IGFs) stimulate proliferation and differentiation of chondrocytes in the epiphyseal plate (1) and are therefore essential for longitudinal bone growth. IGFs also stimulate both proliferation and differentiation of osteoblasts (2), thus playing an important role in trabecular and cortical bone formation. In vitro IGFs have been shown to increase the production of several bone matrix proteins and decrease collagen degradation in osteoblasts (3). In vivo administration of IGF-I in several human studies has been shown to cause an acute increase in bone formation marker proteins in serum (4). Bone formation is severely retarded in mice lacking functional IGF-I or IGF-II genes (5, 6, 7, 8, 9) and in an adolescent human male lacking a functional IGF-I gene who had bone mineral density significantly (>5 SD) less than bone mineral density of similar age boys as reported in the literature (10, 11). These in vitro and in vivo studies show that IGFs play an important role in the regulation of bone formation.

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).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The human osteosarcoma cell line, MG63, was purchased from American Type Culture Collection (Manassas, VA). DMEM and calf serum were purchased from Mediatech, Inc. (Herndon, VA) and HyClone Laboratories, Inc. (Logan, UT), respectively. Recombinant human IGF-I was a gift from Upjohn/Pharmacia (Stockholm, Sweden). IGF-I antiserum was a gift from Dr. Parlow (National Hormone and Pituitary Program, Torrance, CA).

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of intact and fragment forms of recombinant IGFBP-4
The intact and fragment forms of IGFBP-4 each produce a single major band of the expected mol wt on SDS-PAGE followed by sliver staining (data not shown). The IGF-binding activity of the fragment form of IGFBP-4 was 50–100 times less than that of intact IGFBP-4 (Fig. 1Go). 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. 2Go). 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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  		Figure 1. IGF-binding activity of intact and N-terminal fragment forms of IGFBP-4 using 125I-IGF-II tracer.

 


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  		Figure 2. Effect of IGFBP-4 on IGF-I-induced (A) or IGF-II-induced (B) MG63 cell proliferation. The values are the mean ± SEM (n = 8–16). **, P < 0.01; ***, P < 0.001 (compared with IGF-induced cell proliferation). The control cultures had 700.8 ± 15.9 and 699.4 ± 16.0 fluorescence units at 590 nm, respectively, in 2A and 2B.

 
To evaluate whether IGFBP-4 inhibits basal MG63 cell proliferation, serum-free cultures of MG63 cells were incubated with vehicle or intact or fragment IGFBP-4 for 48 h before determination of cell number by the alamarBlue assay. Intact IGFBP-4 at 160 ng/ml (97.2 ± 1.3% of control) or fragment IGFBP-4 at 640 ng/ml (99.9 ± 1.4% of control) did not inhibit basal cell proliferation significantly. The lack of an inhibitory effect of IGFBP-4 on basal MG63 cell proliferation is not surprising, as MG63 cells under the culture conditions used in this study produce very little IGF-I or IGF-II (26).

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 1Go). 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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  		Table 1. Bone ALP activity in the calvaria and femora of C3H mice after local administration of IGF-I and/or IGFBP-4

 
Local administration of IGF-I increased ALP activity (milliunits per mg dry wt) both on the right (injected) and left (noninjected) sides of the parietal bone in Exp 2; this is probably due to the fact that twice the treatment volume was injected, and therefore the treatment diffused across the midline, affecting the contralateral parietal bone (Fig. 3Go and Table 1Go). 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. 3Go). 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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  		Figure 3. Effect of IGFBP-4 local injection on ALP activity in the ipsilateral parietal bone extract. The values are the mean ± SEM (n = 7). **, P < 0.01; ***, P < 0.001 (compared with the ipsilateral vehicle control).

 
Systemic effect of IGF-I and IGFBP-4 on bone formation parameters
Systemic administration of IGF-I increased serum levels of bone formation markers after both one and two injections. Serum osteocalcin levels were significantly increased 24 h and 5 days after a single IGF-I administration (Fig. 4aGo). 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. 4bGo). 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. 4cGo and Table 2Go).



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  		Figure 4. Effect of systemic IGF-I injection on bone formation parameters. The mice were given either one or two systemic injections of IGF-I (2 µg/g mouse BW). The single injection group received IGF-I on day 0 (arrow), and the double injection group received IGF-I on days 0 and 2 (arrows). A, Serum osteocalcin levels after one or two injections. *, P < 0.05; **, P < 0.01 (compared with vehicle-treated control at each time point). The vehicle-treated control values are 110.5 ± 12.9, 113.2 ± 16.0, 106.2 ± 7.3, and 86.3 ± 3.4 ng serum osteocalcin/ml serum for one-injection-treated animals at baseline, day 1, day 3, and day 5, respectively. The vehicle-treated control values are 107.8 ± 6.7, 108.3 ± 4.0, and 88.7 ± 9.8 ng serum osteocalcin/ml serum for two-injection-treated animals at baseline, day 3, and day 5, respectively. B, Serum alkaline phosphatase activity after one or two injections. *, P < 0.05; **, P < 0.01 (compared with vehicle-treated control at each time point). The vehicle-treated control values are 68.4 ± 8.9, 68.3 ± 11.4, 55.3 ± 7.4, and 42.0 ± 6.2 mU ALP activity/ml serum for one-injection-treated animals at baseline, day 1, day 3, and day 5, respectively. The vehicle-treated control values are 56.1 ± 6.3, 48.5 ± 5.5, and 41.3 ± 5.2 mU ALP activity/ml serum for two-injection-treated animals at baseline, day 3, and day 5, respectively. The control values for serum osteocalcin or ALP activity are less on day 5 compared with earlier time points due to the serum being collected by decapitation at the end of the study vs. tail bleeding during the study. C, ALP activity in the femoral bone extract after one or two injections. *, P < 0.05 compared with vehicle-treated control. The values are expressed as a percentage of the vehicle-treated control value and are the mean ± SEM (n = 6).

 

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  		Table 2. Bone ALP activity in the calvaria and femora of C3H mice after systemic administration of IGF-I and/or IGFBP-4

 
Surprisingly, a single administration of intact IGFBP-4 produced a significant increase in serum osteocalcin levels both 1 and 5 days after administration (Fig. 5aGo). 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. 5aGo). In contrast to intact IGFBP-4, the IGFBP-4 fragment had no significant effect on serum osteocalcin levels (Fig. 5aGo). Similar to serum osteocalcin, serum ALP activity was increased significantly after a single administration of intact IGFBP-4 (Fig. 5bGo). ALP activity was significantly increased in both calvarial and femoral bone extracts after treatment with intact IGFBP-4 (Fig. 5cGo and Table 2Go). In contrast to intact IGFBP-4, fragment IGFBP-4 had no significant effect on ALP activity in either calvarial or femoral bone extracts (Fig. 5cGo).



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  		Figure 5. Effects of systemic administration of IGF-I and/or IGFBP-4 (fragment or intact) on bone formation parameters. Treatment was administered on day 0. A, Serum osteocalcin levels. Values are expressed as a percentage of the vehicle-treated control value and are the mean ± SEM (n = 7 or 8 mice/group). The serum osteocalcin levels of vehicle-treated controls were 51.2 ± 7.0 ng osteocalcin/ml serum on day 1 and 45.9 ± 5.9 ng/ml on day 5. B, Serum alkaline phosphatase activity. Values are expressed as a percentage of the vehicle-treated control value and are the mean ± SEM (n = 7 or 8 mice/group). The serum ALP activity of vehicle-treated controls was 66.5 ± 7.0 mU/ml serum on day 1 and 51.3 ± 6.0 mU/ml serum on day 5. C, ALP activity in bone extracts from femora or calvaria. The values are the mean ± SEM (n = 7 or 8). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with vehicle-treated control).

 
Effect of systemic administration of IGFBP-4 on serum IGF-I and IGFBP-4 levels
Serum IGFBP-4 levels were increased by more than 100-fold 30 min after systemic administration of intact forms of IGFBP-4 (data not shown). In contrast, serum levels of total IGF-I were not significantly altered after administration of intact IGFBP-4 (Table 3Go). 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 3Go). 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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  		Table 3. Serum IGF-I levels after systemic administration of IGFBP-4

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first time that the in vivo effects of recombinant IGFBP-4 on bone formation parameters have been studied. Consistent with the previously published in vitro inhibitory effect of IGFBP-4 on IGF-induced bone cell proliferation (20, 29), we show that the local administration of IGFBP-4 in vivo completely blocks the IGF-I-induced increase in ALP activity in the parietal bones of mice. To our surprise, however, we found that systemic administration of intact IGFBP-4 alone significantly increased bone formation parameters in both serum and bone extracts in mice. Thus, these data provide the first experimental evidence that systemic and local administration of IGFBP-4 may have different effects on bone formation parameters in mice.

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 {alpha}-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. 6Go). 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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  		Figure 6. A model for the modulation of IGF bioavailability by IGFBP-4 systemic administration.

 
Alternatively, systemic administration of IGFBP-4 might lead to an acute decrease in the circulating level of free IGF-I. Because free IGF-I has been implicated in the suppression of GH release (42), IGFBP-4-induced reduction of free IGF-I could cause a rebound increase in GH secretion. The increase in GH could increase bone formation via both its direct effects and its indirect effects mediated through increased production of stimulatory IGF-I and IGFBP-5 in bone. Further studies are needed to evaluate the mechanism by which systemic administration of IGFBP-4 increases bone formation parameters in mice.

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.


   Acknowledgments
 
The authors acknowledge the expert technical assistance provided by Tuan Pham, Daniel Bruch, and Joe Rung-Aroon.


   Footnotes
 
1 This work was supported by funds from the NIH (AR-31062), the V.A., and Loma Linda University. Back

Received June 14, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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