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Endocrinology Vol. 140, No. 10 4699-4705
Copyright © 1999 by The Endocrine Society


ARTICLES

Recombinant Human Insulin-Like Growth Factor-Binding Protein-5 Stimulates Bone Formation Parameters in Vitro and in Vivo1

C. Richman, D. J. Baylink, K. Lang, C. Dony and S. Mohan

J. L. Pettis Veterans Administration Medical Center and Loma Linda University (C.R., D.J.B., S.M.), Loma Linda, California 92357; and Roche Diagnostics Boehringer Mannheim GmbH, Pharma Research, Bone Metabolism, Am Nonnenwald (K.L., C.D.), D-82372 Penzberg, Germany

Address all correspondence and requests for reprints to: Subburaman Mohan, Ph.D., Musculoskeletal Disease Center, J. L. Pettis Veterans Administration Medical Center, 11201 Benton Street (151), 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-binding protein-5 (rhIGFBP-5) is stored in bone and stimulates osteoblast cell proliferation in vitro. Bone formation is dependent on the number and activity of osteoblasts. We therefore evaluated the ability of recombinant human (rh) IGFBP-5 to increase osteoblast activity in vitro; both alkaline phosphatase (ALP) activity and osteocalcin levels showed a dose-dependent increase. In in vivo time-course studies, daily sc administration of 50 µg rhIGFBP-5/day/mouse significantly increased serum osteocalcin levels by day 7, and these levels were sustained through day 21. We further evaluated whether rhIGFBP-5 was as effective as IGF-I. Daily sc administration of rhIGFBP-5 (50 µg/day), IGF-I (13 µg/day), or IGF-I plus rhIGFBP-5 complex for 9 days increased serum osteocalcin levels by 58%, 65%, and 81% (P < 0.001 in all) and femoral bone extract ALP activity by 85% (P < 0.001), 29% (P < 0.05), and 13% (P = NS), respectively, and decreased carboxyl-terminal cross-linked telopeptide of type I collagen by 29% (P < 0.05), 20% (P = NS), and 12.5% (P = NS), respectively. One sc injection of rhIGFBP-5 (50 µg/mouse) increased serum osteocalcin and bone ALP activity by 21% (P < 0.05) and 27% (P < 0.02), respectively, after 5 days, but did not significantly increase serum IGF-I (1, 6, or 24 h/postinjection), suggesting that the effects of rhIGFBP-5 on bone are not mediated by increasing circulating IGF-I. Our data demonstrate that systemic administration of rhIGFBP-5, either alone or in combination with IGF-I, increases bone formation parameters in vivo.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN-LIKE growth factor (IGF) system has been shown to modulate growth in many tissues, such as nervous tissue, lymphoid tissue, reproductive tissue, smooth muscle, endothelium, and bone (1, 2). A number of in vitro and in vivo findings demonstrate that IGF-I and IGF-II are important regulators of bone formation. When added exogenously, both increase osteoblast cell proliferation and differentiation in vitro, and bone formation in vitro and in vivo (2, 3, 4, 5, 6, 7). Mice lacking functional IGF-I gene exhibit severe impairment of bone formation (8). In addition, an adolescent male human with a disrupted IGF-I gene had a bone mineral density significantly less than that found in other studies of healthy adolescent human males (9).

Both IGF-I and IGF-II are produced by osteoblasts, and their mitogenic effects are mediated by their binding to the IGF plasma membrane receptors. The IGF type 1 receptor, which binds both IGF-I and IGF-II, appears to be the predominate receptor involved in mediating the effects of these growth factors in most cell types, including osteoblasts (10, 11). IGF-binding proteins (IGFBPs) modulate the actions of secreted IGFs by binding to them and increase the IGF half-life in the extracellular milieu and circulation by sequestering them in this bound form. IGFBPs either enhance or inhibit IGF actions on target cells (12, 13); in bone, the individual IGFBPs either inhibit or potentiate IGF effects on osteoblasts (4, 12, 13, 14, 15, 16, 17). Of the various IGFBPs secreted by bone cells, rhIGFBP-5 has several unique features that suggest that it is a key component of the IGF system in bone. rhIGFBP-5 is the most abundant IGFBP stored in bone, having a high specific binding affinity for hydroxyapatite and extracellular matrix proteins, therefore fixing it and its bound IGFs within bone (18, 19, 20). We have previously proposed that the local release of these sequestered IGFs and rhIGFBP-5 in bone could provide the mechanism by which osteoclastic bone resorption during remodeling gives rise to a coupled increase in bone formation. Therefore, the significant age-related decline in the skeletal content of IGF-I, which is positively correlated with decreasing rhIGFBP-5, could contribute in part to the age-related impairment in the coupling of bone formation to resorption (18, 19, 21).

rhIGFBP-5 is also unique, in that it is the only IGFBP that has been shown to consistently stimulate osteoblast cell proliferation in vitro (20, 22, 23, 24), thus increasing the number of osteoblasts. Recent studies suggest that the mitogenic effects of rhIGFBP-5 may in part be independent of IGFs and mediated through rhIGFBP-5’s own signal transduction pathway (14, 25). Because bone formation is dependent not only on the number but also on the activity of osteoblasts, in this study we evaluated whether recombinant human rhIGFBP-5 (rhIGFBP-5) also increases the activity of osteoblasts in vitro. Based on our findings that rhIGFBP-5 treatment increases osteoblast cell activity [increases osteocalcin production and alkaline phosphatase (ALP) activity], we further investigated the effect of rhIGFBP-5 on bone formation in an intact animal model.


    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). Untransformed normal human bone cells were a gift from Dr. Jon Wergedal (Loma Linda, CA) (26). DMEM, calf serum, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and IGF-I was purchased from Mediatech, Inc. (Herndon, VA), HyClone Laboratories, Inc. (Logan, UT), Biomol (Plymouth Meeting, PA), and GroPep Pty. Ltd. (Adelaide, Australia), respectively. rhIGFBP-5 was expressed in Escherichia coli and purified to homogeneity as described previously (14). This preparation did not contain detectable levels of IGF-I or IGF-II as measured by specific RIA. rhIGFBP-5 has been shown to have similar IGF binding and biological activity as native human rhIGFBP-5 (14). BSA and paranitrophenol phosphate were purchased from Fluka (Buchs, Switzerland). All other chemicals were enzyme grade and purchased from Fisher Scientific (Tustin, CA) or Sigma Chemical Co. (St. Louis, MO). Of the inbred mouse strains used, BALB/c was purchased from B&K Suppliers (Fremont, CA), and C3H/HeJ was obtained from The Jackson Laboratory (Bar Harbor, ME).

In vitro experiments
Cells were grown at 37 C in humidified incubators with 5% CO2. For osteocalcin experiments, conditioned medium was collected from serum-free (DMEM-0.1% BSA) cultures of MG63 or normal human bone cells treated with various concentrations of rhIGFBP-5 and 10-8 M 1,25-(OH)2D3. This conditioned medium was frozen at -70 C until assayed for osteocalcin by specific RIA (27), the cells were extracted, and this extract was assayed for protein concentration (28).

For ALP activity experiments, serum-free (DMEM-0.1% BSA) cultures of MG63 or normal human bone cells were treated with various concentrations of rhIGFBP-5 in the presence or absence of 10-8 M 1,25-(OH)2D3. The cell lysate was assayed for ALP activity (29).

In vivo experiments
Seven-week-old BALB/c or C3H/HeJ inbred mice were used for the in vivo experiments. 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. In each experiment the treatment was injected sc at the nape of the neck of each mouse at the same time each day (0900 h). The vehicle used throughout was 150 mM arginine phosphate buffer, pH 7. When IGF-I and rhIGFBP-5 were administered together, IGF-I and rhIGFBP-5 were mixed and allowed to incubate at room temperature for 1 h before administration so as to form an IGF-I/rhIGFBP-5 complex before administration. Size separation of this IGF-I/rhIGFBP-5 complex showed that more than 80% of the IGF-I eluted in the 50-kDa fractions, suggesting that the majority of the IGF-I exists as a complex in the IGF-I/rhIGFBP-5 treatment solution. The mice were killed with ethrane and decapitated. Blood and both femurs were collected and used for biochemical measurements of bone turnover. The experimental procedures performed in this study are in compliance with the NIH Guide for the Care and Use of Laboratory Animals. All animals studies were reviewed and monitored by the animal studies subcommittee at the Jerry L. Pettis Veterans Administration Medical Center (Loma Linda, CA).

Exp 1. BALB/c mice were divided into three groups of eight animals each. Group 1 received vehicle, group 2 received 13 µg/day IGF-I, and group 3 received 50 µg/day rhIGFBP-5. All groups received treatment for 20 days. The 13 µg/20 g mouse dose of IGF-I (7.7 kDa) was determined based on previous studies of biological effects of IGF-I on body and cartilage growth in mice (30). rhIGFBP-5 (29 kDa) was administered in an equimolar dose (50 µg/20 g mouse) to that of IGF-I. The mice were tail bled on days 7 and 14. On day 21 the mice were killed.

Exp 2. C3H/HeJ mice were divided into four groups of eight animals each. Group 1 received vehicle, group 2 received 13 µg/day IGF-I, group 3 received 50 µg/day rhIGFBP-5, and group 4 received 13 µg/day IGF-I and 50 µg/day of rhIGFBP-5. All groups received treatment for 9 days. On day 10 the mice were killed.

Exp 3. C3H/HeJ mice were divided into two groups of six animals each. These animals received only one injection (day 1); group 1 received vehicle, and group 2 received 50 µg/day rhIGFBP-5. On day 6 the mice were killed.

Exp 4. C3H/HeJ mice were divided into seven groups of six animals each. Groups 2–4 received 50 µg/mouse rhIGFBP-5, and groups 5–7 received 13 µg/mouse IGF-I. Group 1 was killed at time zero as the baseline group; groups 2 and 5 were killed at 1 h, groups 3 and 6 were killed at 6 h, and groups 4 and 7 were killed at 24 h post injection.

Serum collection. Whole blood was collected in 50-ml tubes and centrifuged at 3500 rpm in a cold IEC Centra-7R centrifuge (International Equipment Co., Needham, MA), and serum was skimmed off and stored at -70 C until assayed.

Femur collection. Both femurs were dissected out of each carcass and cleaned of soft tissue, being careful not to destroy the periosteum. Each bone was sectioned in the middiaphysis and 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 ALP activity measurements.

Immunoblot. Serum from animals in Exp 4 was subjected to electrophoresis on a 12% acrylamide gel under denaturing conditions and transferred to a nitro-cellulose membrane, and the membrane was immunoblotted for rhIGFBP-5 using the same antibody as that used in the rhIGFBP-5 RIA, as described previously (31).

Gel filtration of treatment solution. To evaluate the extent of complex formation between IGF-I and rhIGFBP-5, an aliquot of the IGF-I/rhIGFBP-5 treatment solution used for these in vivo studies was subjected to size separation using Sephadex G-75. The proteins were eluted with PBS containing 0.1% BSA and 0.02% sodium azide, pH 7.2. The fractions were collected and assayed for IGF-I after separation of IGF-I from the IGFBPs by a biospin protocol (see below). Molecular mass standards were used to determine the elution positions of the 40-kDa (IGF-I/rhIGFBP-5 complex) and 7.7-kDa (IGF-I) proteins.

Biochemical assays
ALP activity assay. The ALP activity of the cell and bone extract was determined as previously described (29).

Protein assay. The protein concentrations were determined using the Lowry Folin assay (28).

IGF-I RIA. Serum IGF-I was measured by specific RIA after separation from the IGFBPs. Because IGFBPs produce artifacts in IGF RIAs, it is essential to completely separate the IGFBPs from the IGFs for the IGF-I determinations to be valid, as previously described (32). The cross-reactivity of IGF-II in the IGF-I RIA is less than 0.5%. The sensitivity of the IGF-I RIA is less than 50 ng/liter; the intra- and interassay coefficients of variation are less than 10% (33). This assay was used to measure both endogenous mouse IGF-I and administered IGF-I.

rhIGFBP-5 RIA. Serum rhIGFBP-5 was measured by specific RIA using rhIGFBP-5 as standard and tracer as previously described (34). None of the other high affinity IGFBPs cross-react with the antiserum used in this assay. The sensitivity of the rhIGFBP-5 RIA is less than 5 ng/ml; the intra- and interassay coefficients of variation are less than 8%.

Osteocalcin assay. Human osteocalcin levels in conditioned medium and serum were measured by specific RIA (27). Mouse osteocalcin standard and tracer were purchased from BTI (Stoughton, MA). The mouse osteocalcin RIA had less than 8% interassay variability. We have previously developed and validated the mouse osteocalcin RIA (35).

Carboxyl-terminal cross-linked telopeptide of type I collagen (C-telopeptide) assay. C-telopeptide was measured in the mouse serum according to the manufacturer’s instructions using a Rat ICTP RIA Double Antibody Kit purchased from DiaSorin, Inc. (Stillwater, MN). The sensitivity of this assay is 0.5 µg/liter, and the mean recovery was 108.5%. The intra- and interassay coefficients of variation are less than 11% (36).

Statistics
Results are reported as the mean ± SEM for six replicates for in vitro studies or six to eight animals per group for in vivo studies and were compared by Student’s t test, ANOVA, and Duncan’s post-hoc test as appropriate. Results were considered significantly different for P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In vitro experiments
The effect of rhIGFBP-5 on osteoblast activity was evaluated by measurement of ALP activity and osteocalcin production (37, 38). rhIGFBP-5 treatment increased osteocalcin levels in the conditioned medium of MG63 and human osteoblast cells in a dose-dependent manner with a maximal increase (184 ± 12% of control; P < 0.01) at 100 ng/ml rhIGFBP-5 in normal human bone cells and (204 ± 11% of control; P < 0.0001) at 300 ng/ml rhIGFBP-5 in MG63 cells (Fig. 1Go). To evaluate that the rhIGFBP-5-induced increase in osteocalcin level was not due to increased cell number, osteocalcin levels were standardized on the basis of cell protein to correct for changes in cell number. rhIGFBP-5 (100 ng/ml) caused a significant increase in osteocalcin levels even when the values were corrected for cellular protein (0.2 ± 0.02 ng osteocalcin/µg cell protein in control vs. 0.31 ± 0.06 ng osteocalcin/µg cell protein in rhIGFBP-5-treated cells; P < 0.01). The specific activity of ALP in cell extracts also showed a dose-dependent increase with and without 10-8 M 1,25-(OH)2D3 (178 ± 18% and 169 ± 9% of control, respectively, at 100 ng/ml rhIGFBP-5; P < 0.01; Fig. 2Go). Similar results were obtained with normal human osteoblasts (data not shown). 1,25-(OH)2D3 has been shown to be essential for the expression of osteocalcin in human osteoblasts and to increase ALP activity (39). Based on the previous findings that rhIGFBP-5 increased osteoblast cell proliferation (14, 20, 25) and the findings in this study that rhIGFBP-5 increased the differentiative functions of osteoblasts in vitro, we predicted that rhIGFBP-5 may have a stimulatory effect on bone formation parameters in vivo. We therefore tested the effect of rhIGFBP-5 on bone formation parameters in vivo using a mouse model.



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Figure 1. Effect of rhIGFBP-5 on osteocalcin production in MG63 and normal human osteoblasts. Serum-free cultures of MG63 or human osteoblasts were treated for 48 h with different concentrations of rhIGFBP-5 in the presence of 10-8 M 1,25-(OH)2D3. Values are given in nanograms of osteocalcin per ml conditioned medium and are expressed as the mean ± SEM (n = 6 wells/group). *, P < 0.01 (by Duncan’s post-hoc test).

 


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Figure 2. Effect of rhIGFBP-5 on ALP activity in MG63 cells. Serum-free cultures of MG63 were treated for 72 h with different concentrations of rhIGFBP-5 in the presence or absence of 10-8 M 1,25-(OH)2D3. ALP activity was then determined in the cell extract. Values are expressed as a percentage of the vehicle-treated control value and are the mean ± SEM (n = 6 wells/group). *, P < 0.01 (by Duncan’s post-hoc test). The specific activities of ALP in the control cultures in the absence and presence of 10-8 M 1,25-(OH)2D3 were 1.2 ± 0.27 and 3.0 ± 0.38 mU/mg protein, respectively.

 
In vivo experiments
To assess the effect of rhIGFBP-5 on bone formation and resorption parameters in vivo, we measured bone-derived ALP activity and serum osteocalcin as well as C-telopeptide.

Exp 1. Daily sc administration of rhIGFBP-5 (50 µg/day) caused a significant increase in serum osteocalcin levels as early as day 7 in BALB/c mice (Fig. 3Go); this increase was maintained up to day 21. The magnitude of the rhIGFBP-5-induced increase in osteocalcin was similar to that induced by an equimolar dose of IGF-I.



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Figure 3. Effect of rhIGFBP-5 or IGF-I on serum levels of osteocalcin in mice. BALB/c mice were treated with rhIGFBP-5 (50 µg/day), IGF-I (13 µg/day), or vehicle for 20 days. Animals were tail bled on days 7 and 14 to obtain blood in which to measure the serum osteocalcin levels. Values are expressed as a percentage of the vehicle-treated control value and are the mean ± SEM (n = 6 mice/group). *, P < 0.05 (by Duncan’s post-hoc test). The vehicle-treated control value is 437 ng osteocalcin/ml serum on day 7, 358 ng/ml on day 14, and 209 ng/ml on day 21.

 
Exp 2. To eliminate BALB/c strain-specific effects of rhIGFBP-5 on serum osteocalcin, we evaluated rhIGFBP-5 effects in a different strain of mice. Consistent with the data in BALB/c mice, 9 days of rhIGFBP-5 treatment of C3H/HeJ mice significantly increased serum osteocalcin levels (58%; P < 0.001; Fig. 4Go). As in the case of the BALB/c mice, in C3H/HeJ mice the rhIGFBP-5-induced increase in serum osteocalcin was similar to that which resulted from administration of an equimolar dose of IGF-I (65%; P < 0.001; Fig. 4Go) and the combination of IGF-I and rhIGFBP-5 (81%; P < 0.001; Fig. 4Go). rhIGFBP-5 treatment caused a significantly greater increase in ALP activity in femoral bone extract compared to an equimolar dose of IGF-I or the combination of IGF-I and rhIGFBP-5 (Fig. 5Go). To determine whether rhIGFBP-5 modulates bone resorption as well as bone formation parameters, we measured serum C telopeptide levels. These were significantly reduced by 20% and 29% of the control value (P < 0.05), respectively, in IGF-I- and rhIGFBP-5-treated animals (Fig. 6Go).



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Figure 4. Effect of rhIGFBP-5 on serum levels of osteocalcin in C3H/HeJ mice. Equimolar doses of rhIGFBP-5 (50 µg/day) and/or IGF-I (13 µg/day) were given daily for 9 days before the serum osteocalcin determination. Values are expressed as nanograms of osteocalcin per ml serum ± SEM (n = 6 mice/group). *, P < 0.001 (by Duncan’s post-hoc test).

 


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Figure 5. Effect of rhIGFBP-5 on bone ALP in C3H/HeJ mice. Equimolar doses of rhIGFBP-5 (50 µg/day) and/or IGF-I (13 µg/day) were given daily for 9 days before the determination of bone ALP activity. Values are expressed as milliunits of ALP activity per mg total protein in the bone extract ± SEM (n = 6 mice/group). *, P < 0.001 (by Duncan’s post-hoc test).

 


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Figure 6. Effect of rhIGFBP-5 on carboxyl-terminal cross-linked telopeptide (C-telo) of type I collagen in C3H/HeJ mice. Equimolar doses of rhIGFBP-5 (50 µg/day) and/or IGF-I (13 µg/day) were given daily for 9 days before the serum C-telopeptide determination. Values are expressed as nanograms of C-telo per ml serum ± SEM (n = 6 mice/group). *, P < 0.05 (by Duncan’s post-hoc test).

 
Exp 3. To determine whether a single administration of rhIGFBP-5 would cause an increase in serum osteocalcin and bone ALP activity, we administered a single injection of rhIGFBP-5 on day 1, and on day 6 collected serum and performed biochemical measurements of bone formation parameters. The time frame for this experiment is based on studies in which systemic IGF-I administration was evaluated for several days to determine the optimal time to evaluate the changes in serum levels of bone formation markers (our unpublished data). In this experiment, both serum osteocalcin (124% of control; P < 0.001) and bone ALP (184% of control; P < 0.001) were increased at the lowest dose tested (Fig. 7Go).



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Figure 7. Effect of rhIGFBP-5 on serum levels of serum osteocalcin or bone ALP in C3H/HeJ mice. rhIGFBP-5 of varying doses was given daily for 9 days before determination of serum osteocalcin or bone ALP activity. Values are expressed as a percentage of the vehicle-treated control value and are the mean ± SEM (n = 6 mice/group). *, P < 0.01 (by Duncan’s post-hoc test). The mean values for the vehicle-treated control are 198 ng osteocalcin/ml serum and 128 mU ALP/mg protein.

 
Exp 4. To evaluate whether rhIGFBP-5 increases bone formation parameters by a mechanism involving an increase in circulating levels of IGF-I, we measured serum IGF-I levels in mice after the administration of rhIGFBP-5. As a control, we administered an equimolar dose of IGF-I. Serum IGF-I levels were not significantly increased at 1, 6, or 24 h (Fig. 8Go) after rhIGFBP-5 injection; however, in the control IGF-I-treated animals, serum IGF-I levels were increased by 111% of the baseline (P < 0.01) at 1 h, indicating that the administered IGF-I had reached the circulation. In addition, serum rhIGFBP-5 levels were increased from 103 ± 24 ng/ml (at baseline) to 353 ± 52 ng/ml 1 h after rhIGFBP-5 administration and had returned to baseline value at 24 h (102 ± 40 ng/ml). To determine whether the administered rhIGFBP-5 remains in an intact form, pooled sera from baseline and rhIGFBP-5-treated groups (1 h after treatment) were subjected to immunoblot analysis. Figure 9Go shows that serum levels of both intact and fragment forms of rhIGFBP-5 were increased 1 h after rhIGFBP-5 administration.



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Figure 8. Serum levels of total IGF-I in the IGF-I- and rhIGFBP-5-treated animals. After a single administration of IGF-I or rhIGFBP-5, animals were killed at 1, 6, and 24 h postinjection, and serum was collected and used for IGF-I determination. Values are expressed as a percentage of the baseline control value and are the mean ± SEM (n = 6 mice/group). *, P < 0.001 (by Student’s t test). The mean value for the baseline control is 209 ± 17 ng IGF-I/ml serum.

 


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Figure 9. Western immunoblot analysis of pooled mouse sera before and 1 h after treatment with rhIGFBP-5. Serum proteins were separated by SDS-PAGE, transferred to a nitro-cellulose membrane, and immunoblotted with rhIGFBP-5 antiserum. The molecular markers in kilodaltons are shown on the left. Both intact (29 kDa) and fragment (21 kDa) forms of rhIGFBP-5 were increased after rhIGFBP-5 treatment.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we describe for the first time that in vivo systemic administration of rhIGFBP-5 alone or in combination with IGF-I stimulates bone formation parameters in mice over the short term. Our data also demonstrate that rhIGFBP-5 treatment alone increased the activity of osteoblast-line cells in serum-free culture in vitro (increased ALP activity and osteocalcin production). This together with previously published data showing that rhIGFBP-5 also increases osteoblast cell proliferation (14, 40) provide evidence that rhIGFBP-5 has a unique role among the IGFBPs in promoting osteoblast cell proliferation and activity, and consequently bone formation.

Osteoblasts have been shown to produce all six of the high affinity IGFBPs known to date (4, 12, 16), although the amount of each IGFBP varies from cell line to cell line (41). There have been a number of in vitro studies that examined the effects of various IGFBPs on modulating the biological effects of IGF on various cell types, including osteoblasts (4, 12, 13, 16); however, there have been very few studies on the in vivo effects of IGFBPs, but those that exist are limited to IGFBP-3. In this regard, Bagi et al. (42, 43, 44) have shown that systemic administration of IGFBP-3 in combination with IGF-I increased bone formation in ovariectomized rats in a dose-dependent manner, with optimal effects at 7.5 mg/kg IGF-I complexed to an equimolar dose of IGFBP-3. In these studies, the complex of IGFBP-3/IGF-I produced consistently greater effects than IGF-I alone in stimulating bone formation. According to the researchers, this complex has a much longer half-life in the circulation and lowers blood glucose levels to a lesser extent than IGF-I alone because the IGF-I/IGFBP-3 binary complex binds to the circulating free acid-labile subunit (ALS) in the blood to form a 150-kDa complex that does not cross the vascular endothelium.

Surprisingly, in our study the combination of IGF-I and rhIGFBP-5 was less effective than rhIGFBP-5 alone in modulating changes in biochemical measurements of bone turnover in some, but not in other, experiments. The reason for this discrepancy is not clear at this time. One potential explanation is that, similar to IGFBP-3, rhIGFBP-5 when complexed to IGF-I has been shown to bind ALS (45). It is therefore possible that upon administration of IGF-I/rhIGFBP-5, some of this complex may exist in the 150-kDa complex by binding the ALS. This large molecular mass complex may trap the administered IGF-I as well as the rhIGFBP-5 in the serum, as the 150-kDa complex is too large to cross the vascular endothelium. Further studies are needed to test this possibility.

In contrast to the IGF-I/IGFBP-3 studies, our study shows that systemic administration of rhIGFBP-5 alone increased bone formation parameters in mice. rhIGFBP-5 treatment at the dose used in this experiment (50 µg/mouse) did not significantly alter the circulating levels of total IGF-I or the relative ratio of serum IGF-I in the 150- and 50-kDa forms (data not shown). Because rhIGFBP-5 has been shown to bind to ALS only when complexed to IGF-I and because only a small amount (<1%) of total IGF-I is present in the free form, it is possible that the majority of rhIGFBP-5 administered remains in a free form and is thus freely available to target tissues. We and others have reported indirect evidence showing that rhIGFBP-5 may affect osteoblasts by an IGF-independent mechanism (14, 40), raising the possibility that the bone-forming effects of rhIGFBP-5 may in part be mediated by an IGF-independent mechanism.

Although IGFBP-5 administration did not modulate circulating levels of IGF-I, we did not determine the effects of administered rhIGFBP-5 on local production of IGF-I or other IGF system components, which could provide a mechanism for the bone-forming effects of rhIGFBP-5. In addition, rhIGFBP-5 has been shown to bind to hydroxyapatite and extracellular matrix proteins and thereby may be involved in fixing locally produced IGFs in bone. Thus, it is possible that rhIGFBP-5 treatment may increase IGF-I content in bone and bring about the increases in bone formation parameters seen in this study. IGFBP-3 has also been shown to bind to certain extracellular matrix proteins, but not to hydroxyapatite (18, 46). Because the IGFBP-3 content in bone is much less than that of rhIGFBP-5 (18), this mechanism of action may not be as important in mediating IGFBP-3 effects to increase bone formation in the presence of IGF-I. Further studies are needed to establish that the mechanism by which rhIGFBP-5 mediates its bone-forming effects in vivo is different to that of IGFBP-3.

Our present study does not identify the target cells involved in mediating the bone-forming effects of rhIGFBP-5, as we indirectly evaluated bone formation by biochemical measurements of osteoblast cell activity and osteoblast cell products and not by direct histomorphometric methods. In this regard, the findings that serum osteocalcin and ALP activity in bone were increased by day 5 after a single injection of rhIGFBP-5 suggest that rhIGFBP-5 may affect existing osteoblastic cells. Further long term studies using histomorphometric techniques are needed to evaluate the target cells of rhIGFBP-5, as biochemical markers in serum and bone extracts are only indirect measurements of osteoblast cell number and activity.

In contrast to the effects on bone formation parameters, short term administration of both rhIGFBP-5 and IGF-I decreased serum C-telopeptide levels, a marker of bone resorption. In contrast to these effects in mice, short term administration of IGF-I has been shown to increase bone resorption markers in elderly postmenopausal women (47). There are a number of potential explanations for the observed differences between IGF-I effects in mice and humans. IGF-I may have different effects in mice than in humans, Jonsson et al. (48) showed that IGF-I had no effect on 45Ca release from prelabeled neonatal mouse calvarial bones, although IGF-I pretreatment increased the formation of multinucleate tartrate-resistant acid phosphate-positive cells in murine bone marrow cultures. In addition, IGF-I treatment inhibited bone resorption-induced by PGE2 and 1,25-(OH)2D3 (48). Alternatively, the IGF-I effects on bone resorption may be different depending on estrogen status. The mice used in these studies were intact young animals (7 weeks old) as opposed to the human IGF-I studies that were performed in postmenopausal women. Further studies are needed to evaluate whether IGF-I also decreases bone resorption in estrogen-deficient (ovariectomized) older mice and, if so, what is the molecular basis for the observed differences in IGF-I effects on bone resorption between mice and humans.

In conclusion, this study demonstrates for the first time that a single administration of rhIGFBP-5 increases bone formation parameters; however, this increase in bone formation parameters is not mediated by increases in serum IGF-I levels. We also show that rhIGFBP-5 administration decreases bone resorption parameters in mice. We have shown that rhIGFBP-5 is as potent as IGF-I in stimulating bone formation parameters. Long term studies on the effects of rhIGFBP-5 on bone histomorphometry, bone density, and bone strength are essential to confirm the anabolic effects of rhIGFBP-5 on bone.


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


    Footnotes
 
1 This work was supported by NIH Research Grant AR-31062, Roche Diagnostics, the V.A., and Loma Linda University (Loma Linda, CA). Back

Received February 22, 1999.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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