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B Ligand in Vitro and OPG in Vivo
Emory University and Veterans Affairs Medical Center (J.R., L.Z., X.F., T.C.M., M.S.N.), Decatur, Georgia 30033; The Jackson Laboratory (C.L.A.-B., W.G.B., C.J.R.), Bar Harbor, Maine 04609; Maine Center for Osteoporosis Research and Education (W.G.B., C.J.R.), St. Joseph Hospital, Bangor, Maine 04401; and Veterans Affairs Medical Center (R.M., L.H.), Palo Alto, California 94304
Address all correspondence and requests for reprints to: Clifford Rosen, M.D., Maine Center for Osteoporosis Research and Education, St. Joseph Hospital, Bangor, Maine 04401. E-mail: . rofe{at}aol.com
Abstract
IGF-I, a ubiquitous polypeptide, plays a key role in longitudinal bone growth and acquisition. The most predominant effect of skeletal IGF-I is acceleration of the differentiation program for osteoblasts. However, in vivo studies using recombinant human (rh) IGF-I and/or rhGH have demonstrated stimulation of both bone formation and resorption, thereby potentially limiting the usefulness of these peptides in the treatment of osteoporosis. In this study, we hypothesized that IGF-I modulates bone resorption by regulating expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-
B (RANK) ligand (RANKL) in bone cells. Using Northern analysis in ST2 cells, we found that human IGF-I suppressed OPG mRNA in a time- and dose-dependent manner: 100 µg/LIGF-I (13 nM) decreased OPG expression by 37.0 ± 1.8% (P < 0.002). The half maximal inhibitory dose of IGF-I was reached at 50 µg/liter (
6.5 nM) with no effect of IGF-I on OPG message stability. Conditioned media from ST2 cells confirmed that IGF-I decreased secreted OPG, reducing levels by 42%, from 12.1–7 ng/ml at 48 h (P < 0.05). Similarly, IGF-I at 100 µg/liter (13 nM) increased RANKL mRNA expression to 353 ± 74% above untreated cells as assessed by real-time PCR. In vivo, low doses of rhGH when administered to elderly postmenopausal women only modestly raised serum IGF-I (to concentrations of 18–26 nM) and did not affect circulating OPG concentrations; however, administration of rhIGF-I (30 µg/kg•d) for 1 yr to older women resulted in a significant increase in serum IGF-I (to concentrations of 39–45 nM) and a 20% reduction in serum OPG (P < 0.05). In summary, we conclude that IGF-I in a dose- and time-dependent manner regulates OPG and RANKL in vitro and in vivo. These data suggest IGF-I may act as a coupling factor in bone remodeling by activating both bone formation and bone resorption; the latter effect appears to be mediated through the OPG/RANKL system in bone.
IN THE SKELETON, IGF-I can act as a systemic hormone or as an autocrine/paracrine growth factor IGF-I modulating growth and differentiation of osteoblasts (1, 2). In vitro studies have demonstrated that IGF-I modestly stimulates bone cell proliferation, markedly accelerates osteoblast differentiation, and enhances production of several components of bone matrix (3). IGF-I may also be important in preventing apoptosis (2, 4). In vivo, IGF-I deficiency, as noted in IGF-I null (-/-) mice and in a single human case with a point mutation in exon 5 of the IGF-I gene, results in significant growth retardation and extremely low bone mineral density (5, 6, 7). On the other hand, genetically manipulated mice, with targeted overexpression of IGF-I in mature osteoblasts, exhibit increased bone formation and enhanced trabecular and cortical bone volume (8, 9). Hence, IGF-I is considered a major anabolic factor for the growth and maintenance of bone.
Despite the overwhelming evidence that IGF-I stimulates bone formation, its role as a therapeutic agent for osteoporosis is questionable, in part because of its actions on bone resorption. For example, in mice that have targeted overexpression of IGF-I directed by either the osteocalcin or Col1A1 promoter, a striking finding is the presence of increased numbers of osteoclasts at both 6 and 8 wk of age (8, 9). In earlier studies, Middleton et al. (10) found that osteoclasts express IGF-I, IGF-II, and the type I IGF receptor mRNA. In vitro, IGF-I promotes formation of osteoclasts from mononuclear precursors, stimulates activity of preexisting osteoclasts, and increases pit area in bone cell cultures (11, 12). In vivo studies with recombinant human (rh) GH and/or rhIGF-I in GH deficiency or frail elderly have demonstrated a significant increase in both bone resorption and bone formation, especially within the first 12 months, as evidenced by histomorphometric analyses and biochemical markers (13, 14, 15, 16, 17). Nevertheless, at least in GH deficiency, beyond the first year of treatment, bone formation rates exceed resorption and bone mineral density increases (15). Recently, Friedlander et al. (18) noted that low dose rhIGF-I (30 µg/kg•d) for 1 yr in older postmenopausal women was not associated with an increase in spine or hip bone mineral density, and was accompanied by higher rates of bone turnover than in women receiving estrogen replacement along with the human IGF-I therapy. Thus, there is circumstantial evidence to support the role of IGF-I as a coupling agent in the remodeling unit, activating both resorption and formation.
The balance of two peptides produced by stromal osteoblasts, osteoprotegerin (OPG) and the ligand for receptor activator of NF-B (RANKL), are critical in the bone resorption process. RANKL stimulates osteoclast differentiation through its receptor (RANK), whereas secreted OPG, a member of the TNF receptor superfamily, acts as a soluble decoy receptor by binding RANKL and preventing RANKL-induced osteoclastic bone resorption (19). Both local and systemic factors important in bone remodeling are associated with changes in stromal OPG/RANKL ratios. For example, agents that decrease OPG/RANKL and thereby favor osteoclast recruitment include PTH, 1,25 dihydroxyvitamin D, and glucocorticoids (20, 21, 22). Conversely, those factors that increase OPG/RANKL such as estrogen, androgen and leptin reduce bone resorption and slow bone turnover (23, 24, 25). Because there is some evidence that IGF-I might be important during bone resorption, we hypothesized that IGF-I regulated the OPG/RANKL system in stromal cells. Hence we tested, in vivo and in vitro, the effects of IGF-I administration on expression of these two osteoactive peptides.
Materials and Methods
Materials
The ST2 murine stromal cell line was obtained from Riken Cell Bank in Japan. For cell culture, media was purchased from Sigma (St. Louis, MO), fetal bovine serum was from Atlanta Biologicals, Inc. (Atlanta, GA). Reagents for Northern analysis were from Life Technologies, Inc. (Rockville, MD) and Stratagene (La Jolla, CA). Vitamin D was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). IGF-I as a recombinant peptide was obtained from Nichols Institute Diagnostics (San Capistrano, CA). IGF-I was tested on ST-2 cells using concentrations in the range of 10–500 µg/liter (1.3–65 nM).
Cell culture
OPG and RANKL expression were examined in mouse ST2 stromal cells. ST2 cells were used until passage 10 when they cease to express RANKL (26). For experiments cells were plated at 75,000 per 6-well dish in 
-MEM media with 10% fetal bovine serum and penicillin/streptomycin and glutamine. One day later, when cells were subconfluent, 10 nM vitamin D were added if noted, and 24 h later IGF-I was added for specified times. IGF-I in concentrations in 10% fetal calf serum was 15 µg/liter.
Assessment of OPG and RANKL mRNA species
The Northern probe for OPG was a 370 nucleotide PCR product representing sequence from 151–521 that was amplified from ST2 mRNA. The probe for RANKL has been previously described (27). mRNA was prepared by Trizol (Life Technologies, Inc.) or RNAeasy (QIAGEN, Valencia, CA), electrophoresed through a 1.5% denaturing agarose gel and transferred as described previously. For the half-life study of OPG, cells were treated with IGF-I for 24 h before adding actinomycin at 25 µg/ml; total RNA was collected at specified times after actinomycin addition. Densities of 32P-ATP signal in labeled bands on Northern blots were measured in a Molecular Dynamics, Inc. (Sunnyvale, CA) PhosphorImager. Before analysis OPG mRNA was normalized to densitometries of ethidium stained 18S RNA in each lane.
Real-time PCR for RANKL and 18S was performed using the iCycler (Bio-Rad Laboratories, Inc., Hercules, CA). Reverse transcription (RT) of 0.5 µg total RNA was performed with random decamers (Ambion, Inc., Austin, TX) and superscript reverse transcriptase (Life Technologies, Inc.). For real time, PCR amplification reactions were performed in 25 µl containing primers at 0.5 µM and deoxy-NTPs (0.2 mM each) in PCR buffer and 0.03 U Taq polymerase (Life Technologies, Inc.) along with SYBR-green (Molecular Probes, Inc., Eugene, OR) at 1:150,000. Aliquots of cDNA from reverse-transcribed control RNA were diluted 3- to 256-fold to generate relative standard curves to which sample cDNA was compared (20). For RANKL, forward and reverse primers were 5'-CAC CAT CAG CTG AAG ATA GT and 5'-CCA AGA TCT CTA ACA TGA CG, respectively, creating an amplicon of 150 bp. For 18S, an amplicon of 345 was generated with forward primer 5'-GAA CGT CTG CCC TAT CAA CT and reverse 5'-CCA AGA TCC AAC TAC GAG CT. Standards and samples were run in triplicate. RANKL was normalized for amount of 18S in the RT sample, which was also standardized on a dilution curve from a control RT sample (28). The efficiencies of reactions for both RANKL and 18S were more than 90% (slope for RANKL = -3.29, for 18S = -3.39).
OPG ELISA
Mouse OPG in conditioned media was assayed using a Quantikine M Murine OPG ELISA kit (No. MOP00, R&D Systems, Minneapolis, MN). The standard curve was generated by serial dilution of a 2000 pg/ml stock provided by the manufacturer. After only one defrost, serum samples were diluted 1:4 with provided buffer and the assay was performed following the manufacturer’s directions. Optical density was read at 450 nm with a correction wavelength of 540 nm. The intraassay coefficient of variation, based on B6 serum pools, was 5.5% and the limit of detection was 4.5 pg/ml.
Human studies
All blood collections were performed with full informed consent of the subjects as part of two different NIA-sponsored trials for trophic factors as reported previously (17, 18). The first trial was a 1-yr randomized placebo-controlled trial of varying doses of rhGH with or without an exercise program to frail elders (17). This trial was approved by the Institutional Review Board (IRB) at Rhode Island Hospital (Providence, RI) as well as St. Joseph Hospital (Bangor, ME). The second was also a 1-yr randomized placebo-controlled study of low dose rhIGF-I to elder postmenopausal women (18). Samples were available for assay of OPG at baseline, 3 and 12 months from subjects receiving placebo, rhGH 0.005 mg/kg•d (n = 6), or 0.0025 mg/kg•d (n = 8) in the first study. In the second trial, samples were available from women receiving placebo (n = 7) or rhIGF-I, 30 µg/kg•d (n = 9).
Prior approval for the rhIGF-I trial was obtained from the Stanford University IRB and was re-reviewed by the IRB at St. Joseph Hospital before sample assay that was performed at St. Joseph Hospital (18). IRB approval from St. Joseph Hospital was also noted before assay evaluation for samples from the earlier rhGH intervention study in elders. Subjects from that trial had previously given informed consent at Rhode Island Hospital, with prior approval of the Rhode Island Hospital IRB, for serum evaluations to be performed at St. Joseph Hospital (17).
Serum OPG levels were measured in older postmenopausal women treated with placebo, graded doses of rhGH or rhIGF-I given as 30 U/kg•d in two divided doses as reported previously (17, 18). OPG was assayed using a specific commercial ELISA kit from ALPCO, Inc. (Windham, NH) for human samples. The ELISA uses two highly specific antibodies against OPG. One is the binding antibody attached to the wells of the microtiter plate; the second is the detection antibody labeled with biotin. The detection limit is 0.2 pmol/liter. The interassay coefficient of variation was 5.5%. Age of the individual sample varied between 1 and 4 yr depending on the trial. Assay performance was evaluated on older and younger samples by comparing variability within the sample by age; intraassay coefficient of variation did not significantly differ among samples by age. Human serum obtained at 12 months and 1 month from a single research technician also did not significantly vary for serum OPG, by age of the sample as long as the assay was performed on the first defrosted sample.
Statistical analysis
Differences in stromal cell expression of OPG and RANKL were analyzed by using a one-way ANOVA. Time-dependent individual changes in serum OPG in response to GH or IGF-I in human samples were analyzed using paired t test, whereas percentage differences from baseline between placebo and treated groups were assessed by the unpaired t test. Statistical significance was assigned at P < 0.05.
Results
OPG mRNA and protein are decreased in the presence of IGF-I
IGF-I was added to subconfluent ST2 cells 24 h before collecting cells for RNA lysis. Northern analysis for OPG showed that there was a dose-dependent decrease in OPG mRNA (a representative blot is shown in Fig. 1A
). Compiling of at least five separate experiments where IGF-I was dosed for 24 h before OPG mRNA assessment revealed that the half maximal inhibitory dose of IGF-I was reached at about 35–50 µg/liter (4.5–6.5 nM) IGF-I, as shown in Fig. 1B, with little further effect after 100 µg/liter IGF-I (13 nM). The suppression at 100 µg/liter was 37.0 ± 1.8% (P < 0.002) at 24 h.
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. OPG measured with a mouse sensitive OPG kit was high, 12,000 pg/ml in control cells, a value equivalent to about 3,000 pg per 100,000 cells secreted over 48 h. A 24-h exposure to IGF-I significantly decreased OPG secretion compared with control, an effect that continued to decrease to 40% by 48 h, as shown in Fig. 4.
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We wished to see if IGF-I might have reciprocal effects on RANKL, the distaff partner to OPG in bone resorption. In the absence of vitamin D or other cytokine stimulation, RANKL mRNA is nearly undetectable in ST2 cells. The effect of IGF-I was variable, but in four of six experiments was able to cause a dose-dependent increase in RANKL mRNA expression. Figure 5A shows a representative experiment where IGF-I caused a dose-dependent increase in RANKL mRNA. Although several experiments appeared to show increases in RANKL mRNA signal in the presence of IGF-I, it was difficult to quantify the degree of this effect because in at least half the experiments the signal was very weak, as expected in ST2 cells (28).
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, 100 µg/liter IGF-I caused a tripling in RANKL expression compared with control that continued to rise at the 200 µg/liter (26 nM) dose. rhIGF-I suppresses serum OPG in humans
Serum samples from two randomized placebo-controlled trials were analyzed with the OPG assay. Serum IGF-I in patients treated with 30 µg/kg•d rhIGF-I was between 300–350 µg/liter (39–45 nM) per day during the study (17). The IGF-I levels in GH-treated patients were highest in those treated with 0.005 mg/kg•d, and, although statistically higher than placebo groups, were all less than 250 µg/liter (<32 nM) by trial design (17). In both studies, at no time point did the bone mineral density, as assessed by dual energy x-ray absorptiometry, improve, nor were there any significant differences in N-telopeptide excretion (17, 18). Serum osteocalcin rose significantly in a dose-dependent manner (i.e. approximately 40%) in relation to 0.005 mg/kg•d of rhGH but not to rhIGF-I administration as described in Ref. 17 . Serum samples collected at baseline, 3, and 12 months were analyzed for OPG. In placebo and in patients treated with either dose of rhGH, the OPG concentrations did not change as noted in Table 1. In the 9 patients treated with rhIGF-I (30 µg/kg•d), however, there was a downward trend in serum OPG at both time points, although it only reached statistical significance at 12 months (Table 1). When the data were analyzed as percent change from baseline, there was a significant decrease in OPG (P < 0.05) at 12 months (Fig. 6).
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Bone is the second richest source of insulin-like growth factors, and their role in stimulating osteoblast differentiation and bone formation has long been accepted (1, 2, 3, 29). Indeed, several lines of in vitro and in vivo evidence converge to demonstrate that IGF-I, in particular, is essential for the differentiation program of osteoblasts (2, 3, 5, 29). Recent studies using genetically modified mice have established the importance of IGF-I in the acquisition and maintenance of trabecular bone mass (5, 6, 8, 9). Furthermore, it seems likely that expression of adequate skeletal IGF-I is necessary for the full anabolic effects of PTH on bone (6, 8, 30, 31). On the other hand, the role of IGF-I in bone resorption—and even as a regulator of bone remodeling—is less clear.
Early studies confirmed the presence of IGF-I type I receptors on osteoclasts, and in vitro work demonstrated that IGF-I could enhance osteoclast recruitment as well as differentiation (10, 11, 12). IGF-I both induced osteoclast formation and increased pit area in bone cell cultures, as well as enhanced vitamin D stimulation of osteoclastogenesis from hematopoietic blast cells (12). IGF-I may enhance parathyroid induction of bone turnover as treatment with anti-IGF-I antibody significantly impaired PTH-stimulated osteoclast formation in murine marrow cultures (32). Similarly, the effects of GH on bone remodeling are at least partially mediated by IGF-I, as evidenced by reduction of GH-stimulated osteoclastic resorptive activity in vitro when neutralizing antibody to IGF-I was added (33, 34). Clinical studies using rhGH or rhIGF-I in GH deficiency states and in the elderly lend further support to the tenet that IGF-I could act as a coupling factor within the remodeling unit, activating both resorption and formation simultaneously (13, 14, 17, 18). However, the mechanisms responsible for generating osteoclast activity on the remodeling equation have not been fully delineated.
In this study, we tested the hypothesis that IGF-I could stimulate bone resorption by acting on the OPG/RANKL equilibrium through stromal cell expression of these two molecules. Our data strongly support this thesis because IGF-I, in a dose- and time-dependent manner, suppressed OPG expression and protein secretion in ST-2 cells. Moreover, this effect appeared to be transcriptional in nature because IGF-I did not appreciably shorten the half-life of OPG mRNA. Equally important, IGF-I significantly increased RANKL expression. These experiments add to mounting indirect evidence that IGF-I could activate bone resorption by altering the OPG/RANKL ratio in a manner analogous to hormones known to stimulate bone remodeling such as PTH and vitamin D (20, 21).
To extend our observations in vivo, we were able to measure postfacto circulating OPG concentrations in two distinct cohorts entered in an NIH program initiative. Both of these trials were designed to test the role of anabolic therapies in enhancing bone and muscle mass in elders (17, 18). The study participants included: women receiving rhIGF-I (30 µg/kg•d) or placebo for 12 months; frail elderly men and women randomized to low doses of rhGH (0.0025 mg/kg•d or 0.005 mg/kg•d) with or without modest weight bearing exercise for 12 months. As noted in Fig. 6 and Table 1, OPG concentrations remained virtually the same in the rhGH-treated subjects at baseline, 3 and 12 months regardless of type or duration of exercise. However, women treated with rhIGF-I showed a time-dependent decrease in serum OPG, which at 12 months differed significantly from baseline as well as from 12-month placebo-treated individuals. These data suggest that IGF-I may be important in the regulation of circulating OPG, a finding that could have implications in respect to understanding and measuring the complex processes that lead to coupling of bone resorption to bone formation.
Based on in vitro studies with stromal cells, we predicted a priori that both GH and IGF-I treatment would suppress serum OPG concentrations in elderly subjects. Surprisingly, there were no differences in OPG after rhGH treatment at either dose (see Table 1). The reason for the disparate response between GH and IGF-I is not clear, especially because rhGH induces hepatic IGF-I synthesis and raises circulating levels of this peptide (1, 2, 3, 15, 29). Previous studies proved that both these hormones can increase markers of bone resorption and formation, even at relatively low doses, although for doses of rhGH and for rhIGF-I in this report, changes in N-telopeptide excretion in the cohorts were relatively modest and not statistically significant (+20% for rhGH at 0.005 mg/kg•d, +18% for rhIGF-I) (17, 18). One potentially important distinction between studies, however, relates to circulating IGF-I concentrations. Doses of rhGH were titrated in the former study to serum levels of IGF-I between 18 and 28 nM (140–220 µg/liter; mean levels 180 µg/liter), primarily for safety reasons (17). Although these concentrations are about 40–100% higher than IGF-I levels in healthy elderly women, in the rhIGF-I-treated subjects, mean serum levels exceeded 39 nM (300 µg/liter) (18). Moreover, administration of sc IGF-I may be associated with a relatively higher proportion of free IGF-I than during treatment with rhGH. Hence, there may be a threshold concentration of IGF-I required to suppress circulating OPG. Further support for this argument comes from two studies, one an earlier pilot trial using higher doses of rhIGF-I (60 µg/kg•d) in older women from the same investigative group (17). In that 7-d study, both bone resorption and formation indices rapidly increased (17). In the other trial, administration of rhIGF-I to older women (40 µg/kg•d) caused significant elevations in all resorptive and formative indices at both 3 and 6 months (35).
Comparing data from studies of rhIGF-I and rhGH to several randomized placebo controlled trials with human PTH, it seems likely that anabolic peptides affect bone remodeling through different mechanisms. Our data suggest that IGF-I activates bone resorption and formation simultaneously. In contrast, PTH, which is entering the clinical armamentarium as an anabolic agent for osteoporosis, appears to stimulate bone formation more rapidly than resorption, even though PTH enhances skeletal IGF-I synthesis and coincidentally alters OPG/RANKL (3, 30, 31, 32, 36). The disparity in bone accretion between these two anabolic peptides likely represents the sum of several factors acting locally and/or systemically on the bone remodeling unit.
There are several limitations to our report with respect to the in vivo studies. At this time, transmembranous RANKL protein cannot be measured in vivo, and the newly available assays of circulating RANKL have not been verified to represent bone turnover at this time. Thus, we are unable to examine a RANKL response to IGF-I in the human studies. As well, the human studies were performed on subsets of cohorts from selected populations that volunteered for these trials, and therefore represent the usual limitations of study populations. Larger randomized trials comparing various anabolic agents such as PTH, rhGH, and rhIGF-I will be necessary to confirm our initial suggestions that IGF-I promotes skeletal turnover at several levels. Finally, the significant changes we found in serum OPG levels in a small number of elderly women were confined to the 12-month period of the study. It would be interesting to determine whether suppression of OPG persists in women treated for 24 months with rhIGF-I, especially because it is well recognized that in GH-deficient individuals, improvements in bone mineral density require replacement with rhGH for at least 18 months (15).
In conclusion, we have noted that IGF-I strongly affects OPG/RANKL equilibrium in murine stromal cells in a manner that predisposes to osteoclast recruitment and bone resorption. Furthermore, high circulating levels of IGF-I appear to suppress serum concentrations of OPG. We believe this may at least partially account for the bone resorbing actions of IGF-I, noted in several clinical studies and in genetically engineered mice (5, 6, 7, 8, 9). Further work is needed to define the clinical importance of our findings in relation to the use of anabolic peptides to treat postmenopausal osteoporosis.
Acknowledgments
Footnotes
This work was supported by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR-42360, to J.R.; AR-45433, to C.J.R.) and by the Veterans Administration (to J.R.).
Abbreviations: IRB, Institutional Review Board; OPG, osteoprotegerin; RANK, receptor activator of nuclear factor-B; RANKL, RANK ligand; rh, recombinant human; RT, reverse transcription.
Received April 29, 2002.
Accepted June 4, 2002.
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