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Department of Research (R.D.D., Z.D., V.B., E.C.), Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105-1299; The University of Connecticut School of Medicine (Z.D., E.C.), Farmington, Connecticut 06030; and Laboratorio de Fisiopatologia Renal (V.J.), Universidade de Sao Paulo, Sao Paulo, Brazil
Address all correspondence and requests for reprints to: Ernesto Canalis, M.D., Department of Research, Saint Francis Hospital and Medical Center, 114 Woodland Street, Hartford, Connecticut 06105-1299. E-mail: ecanalis{at}stfranciscare.org.
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Abstract |
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Introduction |
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In view of its postulated stimulatory effects on bone cell growth, IGFBP-5 has been studied extensively. IGFBP-5 expression is coordinated with that of IGF-I, suggesting important interactions among the two peptides. Agents that stimulate IGF-I mRNA expression, such as IL-6, also stimulate IGFBP-5 transcription (10, 11). IGFBP-5 is secreted intact, and then can be fragmented by the action of matrix metalloproteinases and serine proteinases, and its fragments could have distinct biological activities (10, 12, 13). IGFBP-5 has been reported to stimulate cell proliferation and selected markers of osteoblastic function in vitro and in vivo (9, 14). It is not certain whether the postulated anabolic effects are due to interactions between IGF-I and IGFBP-5, although direct anabolic effects of IGFBP-5 are possible because they are also observed in cells from IGF-I null mice (14). In addition, interactions of IGFBP-5 with other extracellular matrix proteins, such as vitronectin, modulate the responsiveness to IGF-I and the anabolic effect of IGFBP-5 (15).
Although IGFBP-5 stimulates cell growth and selected parameters of osteoblastic function, such as alkaline phosphatase activity and osteocalcin, its actual effects on bone formation have not been established (14). Knowledge of these effects is necessary since isolated changes in osteocalcin and alkaline phosphatase activity do not necessarily represent changes in bone formation (16). The intent of this study was to investigate the direct effects of IGFBP-5 on bone remodeling. For this purpose, we created transgenic mice overexpressing IGFBP-5 under the control of the osteoblast-specific osteocalcin promoter, and determined their skeletal phenotype by histomorphometric analysis.
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Materials and Methods |
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).
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Bone histomorphometric analysis
Static and dynamic histomorphometry was carried out in mice following calcein, 20 mg/kg, injections 7 d and 2 d before the mice were killed by CO2 asphyxiation. Femurs were dissected and fixed in 70% ethanol. After dehydration, bone samples were embedded in methyl methacrylate and 5-µm sections were cut (Microm, Richard-Allan Scientific, Kalamazoo, MI) and stained with toluidene blue. Static and dynamic parameters of bone structure, bone formation and resorption were measured at a standardized site 195 µm below the growth plate using an OsteoMeasure morphometry system (Osteometrics, Atlanta, GA). Trabecular bone volume/density (BV/TV, %) and relative osteoid volume and surface (%) were calculated, osteoid width (µm), trabecular thickness (Tb.Th, µm), spacing (µm) and number (Tb.N, per mm) were measured. Osteoblast number/trabecular area/mm2, osteoclast surface/bone surface (Oc.S/BS, %) and osteoclast number (Oc.N/BS, mm2) were determined. For dynamic histomorphometry, mineral apposition rate (µm/d) was measured on unstained sections under UV light, mineralizing surface was expressed as a percentage of total BS (MS/BS %), and bone formation rate (µm3/µm2/d) was calculated. The terminology and units used are those recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (22).
X-ray analysis and bone mineral density
Radiography was performed on mice anesthetized with tribromoethanol (Sigma, St. Louis, MO) on a Faxitron x-ray system (model MX 20, Faxitron X-Ray Corp., Wheeling, IL). Total, femoral, and vertebral bone mineral density (BMD) was measured on anesthetized mice using the PIXImus small animal dual energy x-ray absorptiometry system (GE Medical Systems/Lunar Corp., Madison, WI) (23). Calibrations were performed with a phantom of a defined value, and quality assurance measurements were performed daily. The coefficient of variation for total BMD is less than 1% (n = 9).
Bone marrow stromal cell cultures
Femurs were aseptically removed from 7-wk-old mice, following CO2 asphyxiation, and dissected free of adhering tissues. The ends were removed and cells were recovered by centrifugation (24). Cells were plated at a density of 106 cells/cm2 and cultured in 
-MEM (Invitrogen, Rockville, MD) containing 15% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT) at 37 C in a humidified 5% CO2 incubator. Half the volume of the culture medium was replaced, after 4 d of culture, with fresh medium. At confluence (7–9 d of culture), the medium was changed to -MEM supplemented with 15% fetal bovine serum, 50 µg/ml ascorbic acid and 8 mM ß-glycerophosphate (mineralizing medium) (Sigma). The medium was replaced twice a week for the duration of the culture, and cells were harvested 3 d after the last change of medium. For cytochemical analysis, cells were fixed with 4.5 mM citric acid, 2.25 mM sodium citrate, 65% acetone, and 8% formalin and stained for the presence of mineralized nodules using 2% alizarin red (Sigma).
Northern blot analysis
Total femoral and calvarial bone RNA was isolated by the acid guanidium thiocyanate-phenol-chloroform extraction method (25) followed by purification with an RNeasy column (QIAGEN, Valencia, CA). Total cellular RNA was isolated using an RNeasy kit per manufacturer’s instructions (QIAGEN). RNA was quantitated by spectrometry, and equal amounts of RNA were loaded on a formaldehyde-agarose gel following denaturation. The gel was stained with ethidium bromide to visualize RNA standards and ribosomal RNA, and to confirm equal RNA loading of the samples. The RNA was blotted onto GeneScreen Plus charged nylon (Perkin-Elmer, Norwalk, CT), and uniformity of transfer was confirmed by revisualization of ethidium bromide-stained ribosomal RNA. A 2.5-kb rat alkaline phosphatase cDNA (Merck and Co., West Point, PA), a 2.5-kb core binding factor 1 (Cbfa-1) murine cDNA (Ito, Y., Kyoto, Japan), a 1.6-kb rat type I collagen cDNA (Kream, B., Farmington, CT), a 0.5-kb osteocalcin genomic rat DNA containing no sequence homology with the osteocalcin promoter driving IGFBP-5 (Lian, J., Worcester, MA), a 330-bp rat IGFBP-5 cDNA (Shimasaki, S., La Jolla, CA), and a 0.75-kb 18S cDNA (American Type Culture Collection, Manassas, VA) were purified by agarose gel electrophoresis (19, 26, 27, 28). DNAs were labeled with deoxy (d) CTP and [-32P]-dATP (50 µCi each at a specific activity of 3000 Ci/mmol; Perkin-Elmer) using the random hexanucleotide-primed second-strand synthesis method (29) or labeled with Ready-To-Go DNA labeling beads (dCTP) kit (Amersham Pharmacia Biotech, Piscataway, NJ), in accordance with manufacturer’s instructions. Hybridizations were carried out at 42 C for 16–72 h, followed by two posthybridization washes at room temperature for 15 min in 1x saline sodium citrate (SSC), and a wash at 65 C for 20–30 min in 0.5x or 1x SSC. The bound radioactive material was visualized by autoradiography on Kodak X-AR5 film (Eastman Kodak Co., Rochester, NY), employing Cronex Lighting Plus (Perkin-Elmer) or Biomax MS (Eastman Kodak Co., Rochester, NY) intensifying screens. Relative hybridization levels were determined by densitometry. Northern analyses shown are representative of two to three samples.
RT-PCR
One microgram of deoxyribonuclease I-treated total RNA from stromal cell and bone extracts was reverse-transcribed with murine Moloney leukemia virus reverse transcriptase (Invitrogen) in the presence of 20 µM reverse primer corresponding to an 18-bp fragment of bovine GH polyadenylation sequence present in the IGFBP-5 vector. One-microliter of reverse transcription reaction was amplified by 30 cycles of PCR at 64 C annealing temperature in the presence of 20 µM 5' to 3' primer spanning bp +459 to +479 of IGFBP-5 coding sequence, [-32P]-dATP and 2.5 U of Taq polymerase to yield a product of a predicted size of 416 bp. The products of the PCR were resolved by electrophoresis on a 6% polyacrylamide gel and exposed to Kodak X-AR5 film overnight and developed.
Western immunoblot analysis
Culture medium from transfected ROS 17/2.8 cells, concentrated under vacuum by centrifugation, protein extracts from homogenized femurs or serum aliquots from control and transgenic mice were fractionated by polyacrylamide gel electrophoresis (30). Proteins were transferred to Immobilon P membranes (Millipore Corp., Bedford, MA), blocked with 2% BSA and exposed to rabbit antiserum against human IGFBP-5 (Upstate Biotechnology, Inc., Lake Placid, NY) overnight. Blots were exposed to goat antirabbit IgG antiserum conjugated to horseradish peroxidase, and developed with a chemiluminescence detection reagent (DuPont, Wilmington, DE) (31). IGFBP-5 was identified by comigration with recombinant purified human IGFBP-5 (Upstate Biotechnology, Inc.).
Serum IGF-I, osteocalcin, and alkaline phosphatase activity
Serum immunoreactive IGF-I was measured, following removal of IGFBPs by acidification and silica chromatography, using a commercial RIA kit for human IGF-I in accordance with manufacturer’s instructions (DiaSorin, Inc., Stillwater, MN). Values are expressed in ng/ml of human IGF-I equivalents. Serum immunoreactive osteocalcin was measured using a commercial RIA kit in accordance with manufacturer’s instructions (Biomedical Technologies, Inc., Stoughton, MA). Values are expressed in ng/ml. Serum alkaline phosphatase activity was measured using an Ortho Clinical Diagnostic (Rochester, NY) enzymatic assay that determines the conversion of p-nitrophenyl phosphate to p-nitrophenol. Values are expressed in international units per liter.
Statistical analysis
Data are expressed as means ± SEM. Statistical significance for BMD and histomorphometric and serum-levels data were determined by Student’s t test.
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Results |
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, upper panel). High levels of transgene mRNA were detected in 4-wk-old transgenic mice, coinciding with the high level of activity of the osteocalcin promoter (17). IGFBP-5 mRNA expression in bone extracts declined with age, in accordance with a decline in the reported activity of the osteocalcin promoter. To confirm that the overexpression of IGFBP-5 was due to expression of the transgene in bone, RT-PCR was carried out in RNA extracted from bones of IGFBP-5 transgenic mice and wild-type controls. RT-PCR demonstrated the presence of IGFBP-5 transgene in bone from both transgenic lines and not in that from wild-type animals (Fig. 2, middle and right lower panels). To confirm that transgenic mice overexpressed IGFBP-5 in bone, Western immunoblot analysis of extracted femurs was performed and demonstrated detectable levels of IGFBP-5 in 4-wk-old transgenic (line 1) and not in wild-type controls (n = 3) (Fig. 2, left lower panel).
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, Fig. 3). At 5 wk of age, transgenic mice had about 40% less trabecular bone volume, and this appeared to be secondary to a reduction in trabecular number and thickness, although trabecular thickness was significantly lower in line 1, but not in line 2, P < 0.056. The number of osteoblasts was unchanged, but bone mineral apposition rate was 30–50% lower in transgenic than in wild-type mice, as determined by double calcein labeling, indicating impaired osteoblastic function (Table 1, Fig. 3). Bone formation rate was reduced in line 2, but not in line 1. Osteoclast number and bone resorption were not altered in transgenic IGFBP-5 mice. Consistent with the activity of the osteocalcin promoter, most of the phenotypic impact of IGFBP-5 overexpression, as determined by bone histomorphometry, occurred in the first weeks of life (Table 2). At 9 wk of age there was a modest, possibly compensatory, increase in trabecular thickness and mineral apposition rate in IGFBP-5 transgenics. The effect was transient, whereas a decrease in trabecular number was sustained for 9–24 wk (Table 2).
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). At 4 wk of age, the decrease in BMD in transgenic mice was 16% for total BMD, 19–27% for femoral BMD and 14 to 22% for vertebral BMD in both transgenic lines examined. As observed with changes in bone formation, the effect on BMD was transient and not sustained after 8–24 wk (Table 3).
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Stromal cell cultures and osteoblastic gene markers
Northern blot analysis of RNA extracted from mouse femurs and calvariae did not reveal a consistent decrease in the expression of the osteoblastic gene markers alkaline phosphatase, osteocalcin, Cbfa-1, and type I collagen in homozygous transgenic mice (line 1) compared with wild-type control mice (not shown). To investigate further the impact of IGFBP-5 on osteoblastic function, stromal cells from 7-wk-old homozygous transgenic mice overexpressing IGFBP-5 (line 1) and from age matched wild-type control mice were cultured for up to 2 wk following confluence. Stromal cells from IGFBP-5 transgenic, but not from wild-type mice, expressed the IGFBP-5 transgene, as determined by RT-PCR, at confluence and 1 and 2 wk after confluence (Fig. 4). Mineralized nodule formation, as determined by alizarin red staining, was reduced in stromal cell cultures from transgenic mice (not shown). Northern blot analysis confirmed impaired osteoblastic maturation or function in stromal cells from IGFBP-5 transgenic mice, and revealed decreased expression of alkaline phosphatase, osteocalcin, Cbfa-1, and type I collagen transcripts, relative to their expression in cultures from wild-type mice (Fig. 4). Except for osteocalcin and type I collagen, the decrease was mostly apparent 1 wk after confluence, when the expression of the IGFBP-5 transgene was more prominent. As the cells from wild-type mice matured and mineralized, there was a decline in Cbfa-1, alkaline phosphatase, and type I collagen transcripts, also contributing to a lesser difference in transcript expression between cells from wild-type and transgenic mice 2 wk after confluence.
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Discussion |
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The mechanism of the impaired osteoblastic function in IGFBP-5 transgenics could involve the binding and sequestering of IGF-I and II by IGFBP-5 in the bone microenvironment. This would be in agreement with the known effects of IGFs on bone formation. IGFs have modest mitogenic activity in bone and have a more prevalent impact on the differentiated function of the osteoblast as manifested by increased collagen expression in vitro and increased mineral apposition rates in transgenic mice overexpression IGF-I (2, 3). Because IGFBP-5 transgenics have decreased mineral apposition rates, the mechanism is likely sequestration of IGF-I. However, IGFBP-5 effects independent of IGFs are possible and were not excluded. These studies also demonstrate that a decrease in trabecular bone volume and osteoblastic function by IGFBP-5 overexpression can translate into significant and generalized osteopenia in the mouse, as determined by BMD.
Previous work has shown that IGFBP-5 stimulates bone cell growth in vitro and selected parameters of osteoblastic function in vivo (9, 14, 32). Increases in bone formation in vivo have been explored and observed in ovariectomized mice but are more evident with the administration of the truncated IGFBP-5 1–169 form than with intact IGFBP-5 (33). The present results could be considered inconsistent with the reported in vivo effects following the systemic administration of IGFBP-5 to mice on serum levels of osteocalcin and alkaline phosphatase activity (14). However, by themselves these changes do not necessarily represent changes in bone formation, and they were not confirmed by bone histomorphometric analysis. Furthermore, animals with null mutations for the osteocalcin gene display increased, and not decreased, bone formation confirming that serum markers are not sufficient to establish the presence of an anabolic effect (16). It is possible that the differences in the results observed are related to different strains of mice examined because anabolic effects and changes in bone mass are highly dependent on strain (34). Another possibility is that the inhibitory effects of IGFBP-5 found in transgenic mice are the result of excessive IGFBP-5 in the bone microenvironment, and sequestering of IGFs, and at lower levels IGFBP-5 might be anabolic. Similar discrepancies have been reported for TGF-ß. In vivo administration of TGF-ß can result in increased bone formation, whereas transgenic mice overexpressing TGF-ß under the control of the osteocalcin promoter develop osteopenia (18, 35). It is, however, important to note that TGF-ß inhibits the osteoblastic differentiation induced by bone morphogenetic protein-2, indicating direct inhibitory actions of TGF-ß on cells of the osteoblastic lineage and confirming the results observed in TGF-ß transgenics (36). The validity of the transgenic model used also can be substantiated by the demonstration of an increase in bone formation by overexpressing IGF-I under the control of the osteocalcin promoter, and confirmation of the known anabolic actions of IGF-I in vitro (2, 3).
Confirming the observations in vivo, stromal cells from transgenic mice overexpressing IGFBP-5 did not fully differentiate into cells expressing the osteoblastic phenotype. However, it is possible that, due to delayed activation of the osteocalcin promoter, stromal cells underwent partial differentiation and that IGFBP-5, by trapping IGFs, inhibited the differentiated function of partially mature cells. The possibility that IGFBP-5 has no impact on immature cells is in accordance with the known in vitro effects of IGF-I on human stromal cells. In this culture system, IGF-I does not alter the differentiation of immature stromal cells into mature cells, but stimulates osteoblast phenotypic markers following cell maturation (37).
The dual inhibitory and stimulatory effects of some IGFBPs are not limited to IGFBP-5. Previous studies have demonstrated that under selected conditions IGFBP-4, which is an inhibitory binding protein, can stimulate parameters of osteoblastic function (8, 38). Consequently, it is possible that both IGFBP-4 and 5 can have stimulatory and inhibitory effects on bone cell function. Recently, the amino terminal fragment of IGFBP-5 1–169 was shown to have anabolic effects in bone in vivo (33). This would suggest that IGFBP-5 fragments and not intact IGFBP-5 are anabolic and that under conditions of IGFBP-5 overexpression, necessary and controlled protein fragmentation might not have occurred. Another possibility could be lack of association to critical extracellular matrix proteins, such as vitronectin, under conditions of IGFBP-5 overexpression because these associations enhance the anabolic effect of IGF-I in the presence of IGFBP-5 (15).
In conclusion, our studies demonstrate that IGFBP-5 overexpression in the bone microenvironment causes a transient decrease in trabecular volume and osteopenia due to impaired osteoblastic function.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMD, Bone mineral density; BS, bone surface; Cbfa-1, core binding factor 1; d, deoxy; GFP, green fluorescent protein; IGFBP, IGF binding protein.
Received February 1, 2002.
Accepted for publication June 25, 2002.
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I and 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 23:6210–6216[CrossRef][Medline]
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