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Endocrinology. May 2008; 149(5): 2051–2061.
Published online Feb 14, 2008. doi:  10.1210/en.2007-1068
PMCID: PMC2329262

Gender-Specific Changes in Bone Turnover and Skeletal Architecture in Igfbp-2-Null Mice

Abstract

IGF-binding protein-2 (IGFBP-2) is a 36-kDa protein that binds to the IGFs with high affinity. To determine its role in bone turnover, we compared Igfbp2−/− mice with Igfbp2+/+ colony controls. Igfbp2−/− males had shorter femurs and were heavier than controls but were not insulin resistant. Serum IGF-I levels in Igfbp2−/− mice were 10% higher than Igfbp2+/+ controls at 8 wk of age; in males, this was accompanied by a 3-fold increase in hepatic Igfbp3 and Igfbp5 mRNA transcripts compared with Igfbp2+/+ controls. The skeletal phenotype of the Igfbp2−/− mice was gender and compartment specific; Igfbp2−/− females had increased cortical thickness with a greater periosteal circumference compared with controls, whereas male Igfbp2−/− males had reduced cortical bone area and a 20% reduction in the trabecular bone volume fraction due to thinner trabeculae than Igfbp2+/+ controls. Serum osteocalcin levels were reduced by nearly 40% in Igfbp2−/− males, and in vitro, both CFU-ALP+ preosteoblasts, and tartrate-resistant acid phosphatase-positive osteoclasts were significantly less abundant than in Igfbp2+/+ male mice. Histomorphometry confirmed fewer osteoblasts and osteoclasts per bone perimeter and reduced bone formation in the Igfbp2−/− males. Lysates from both osteoblasts and osteoclasts in the Igfbp2−/− males had phosphatase and tensin homolog (PTEN) levels that were significantly higher than Igfbp2+/+ controls and were suppressed by addition of exogenous IGFBP-2. In summary, there are gender- and compartment-specific changes in Igfbp2−/− mice. IGFBP-2 may regulate bone turnover in both an IGF-I-dependent and -independent manner.

IGF-BINDING PROTEIN-2 (IGFBP-2) is a 36-kDa member of a highly conserved family of six IGFBPs that circulate in relatively high concentrations. The IGFBPs have high affinity and specificity for IGF-I and IGF-II and compete with the type I and II IGF receptors for ligand binding. Each of the IGFBPs can deliver the ligand to its receptor, or block its access, depending on its concentration, the target tissue, the relative amount of local IGFs, the glycosylation of the particular IGFBP, and the activity of various proteases that can cleave IGFBPs and reduce binding affinity. Yet, despite their similarities, the IGFBPs also possess unique properties, which allow for a complex pattern of delivery, binding, and activity, alone or in combination with their respective IGF ligand (1,2,3).

The Igfbp2 gene is located on mouse chromosome 1 at approximately 36 Mb (NCBI Build 36) and is in a tail-to-head configuration with Igfbp5 (4). IGFBP-2, like IGFBP-5, has a strong affinity for components of the extracellular matrix such as heparin and hydroxyapatite (3). Unlike IGFBP-5, IGFBP-2 levels decline during neonatal and pubertal growth and increase with advancing age in humans. Malnutrition, certain malignancies (e.g. glioblastoma, prostate, and breast carcinoma), and catabolic states are all associated with high serum IGFBP-2 levels (5,6,7,8,9,10,11,12,13,14). Administration of GH suppresses IGFBP-2, whereas IGF-I infusions can increase circulating IGFBP-2 (15).

Igfbp2 is expressed in several mammalian tissues (16). The transcriptional regulation of Igfbp2 is less well established than Igfbp5, although there are several regulatory elements in the 22.4-kb intergenic region between Igfbp2 and Igfbp5, including a cAMP response element. Although Igfbp2 and Igfbp5 have structural similarities and lie adjacent to each other in the genome, most studies have concluded that IGFBP-2 is an inhibitory binding protein, in contrast to the dual nature of IGFBP-5 as an agonist or antagonist for IGF-I (2,17).

Recently, the role of IGFBP-2 in the skeleton has received additional attention. Previously, IGFBP-2 was thought to be inhibitory in respect to bone size and growth (18). But, Khosla et al. (19) reported increased serum IGFBP-2 in the syndrome of hepatitis C-associated osteosclerosis, and this increase was associated with a large form of IGF-II. Subsequently, this group demonstrated that IGF-II and IGFBP-2 could stimulate bone formation, even during states of mechanical unloading, and the combination could protect rats against ovariectomy-induced bone loss (20). Amin et al. (21) also noted that serum IGFBP-2, of all the measurable IGF system components, was most closely related to markers of bone resorption in men and postmenopausal women. Yet, despite these recent observations, the mechanisms that underlie this relationship have not been clarified. In this study, we hypothesized that IGFBP-2 regulated bone turnover, and thus, any perturbation in its expression would affect peak skeletal acquisition. To test this hypothesis, we examined the skeletal phenotype of the Igfbp2-null mice and analyzed the cellular and molecular consequences that resulted from global deletion of the Igfbp2 gene (22).

Materials and Methods

Mice

The original mixed background strain, B6;129-Igfbp2<tm1Jep> (22), was backcrossed to C57BL/6J 10 times to create the B6.129- Igfbp2<tm1Jep> congenic strain before being imported to The Jackson Laboratory (Bar Harbor, ME) from the laboratory of Dr. Terri Wood. All mice used in this study were produced and maintained in our research colony at The Jackson Laboratory. All analyses were conducted using mutant mice and same-sex colony controls. For each experiment, the numbers of mutant and control mice used are provided in Results. All experimental studies and procedures involving mice were reviewed and approved by the Institutional Animal Care and Use Committee of The Jackson Laboratory.

Body weights and lengths

Body weights were measured on a standard balance in grams postmortem. Nose-to-tail length was measured using digital calipers and reported in millimeters. Organs were then dissected out and weighed immediately.

Measurements of IGFs and IGFBPs

Serum IGF-I was measured by RIA (ALPCO, Windham, NH) as reported previously (23,24,25). The sensitivity of the assay is ±0.01 ng/ml IGF-I, and the intraassay coefficient of variation was 4.5%. All samples were analyzed within the same assay. Serum IGFBP-2 was measured by an ELISA (ALPCO) validated for mouse IGFBP-2. The sensitivity of the assay is ±0.04 ng/ml IGFBP-2, and the intraassay coefficient of variation was 5%. The other mouse IGFBPs were examined by ligand blot, except IGFBP-5, which was also assessed by immunoblotting as described previously (26,27).

Serum osteocalcin

Serum osteocalcin was measured by immunoradiometric assay (ALPCO) specific for mouse. The sensitivity of the osteocalcin assay is ±0.1 ng/ml, and the intraassay coefficient of variation for osteocalcin was 4.6%. Uncarboxylated osteocalcin was measured using a hydroxyapatite assay in the laboratory of Dr. Caren Gundberg (28).

Quantitative real-time PCR

RNA was extracted from the livers of four 6-wk-old Igfbp2−/− and Igfbp2+/+ male mice, as well as from femurs of four 8-wk-old Igfbp2−/− and Igfbp2+/+ male mice as described previously (23,25). Briefly, liver RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) per the manufacturer’s instructions. Femurs were isolated and snap frozen in liquid nitrogen, RNA was then isolated using the Total RNA isolation system (Promega, Madison, WI) as per the manufacturer’s instructions. DNA was then removed from the RNA samples using The DNA-free DNase treatment and removal reagents (Ambion, Inc., Austin, TX). RNA quality and quantity were assessed using an Agilent bioanalyzer (Caliper Technologies Corp., Hopkinton, MA). Four hundred nanograms of RNA were then converted to cDNA in a RT reaction using the MessageSensor RT Kit (Ambion, Inc., Austin, TX) and random decamers as primers. The cDNA was then diluted 1:5 with water. For each PCR, 2 μl of diluted cDNA was added to 16 μl SYBR green with ROX mix (Invitrogen, Carlsbad, CA) and 100 nm of each forward and reverse primer in a total reaction volume of 20 μl. Cycling conditions were 2 min hold at 50 C, 10 min hold at 95 C, and 40 cycles of 95 C for 15 sec and 60 C for 1 min, and all reactions were run on the ABI 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). Expression of Igf1, Igfbp1-6, Igf2, Irs1, Irs2, Igfals, Pparg, Csf1 (Mcsf), and Csf1r (Cfms) was assessed by comparing the expression of each to the normalizer Actb (β-actin) using the ΔΔCt method as previously described (23). Fold changes were adjusted for primer efficiencies using the pfaffl-spread program (http://pathmicro.med.sc.edu/pcr/pfaffl-spread.xls). Each experiment was run in triplicate with different cDNA preps from the same mice. Primer sequences are reported in Table 11.

Table 1
Quantitative real-time PCR primer sequences

Dual-energy x-ray absorptiometry (DXA) for areal bone mineral density (aBMD) and body composition

DXA scanning was done by the PIXImus (GE-Lunar, Madison, WI). We used PIXImus to assess whole-body aBMD and body composition (lean mass and percent body fat) in Igfbp2−/− and Igfbp2+/+ mice at 8 and 16 wk of age. A phantom standard provided by the manufacturer was assessed each day for instrument calibration. Bone mineral content by PIXImus is highly correlated with mineral content of hydroxyapatite standard of known density (r2 = 0.997) (25).

Peripheral quantitative computed tomography (pQCT) for volumetric bone densitometry

pQCT was used to measure volumetric BMD (vBMD) on the left femur of female and male Igfbp2−/− and Igfbp2+/+ mice at 8 and 16 wk of age. Isolated femur lengths were measured with digital calipers (Stoelting, Wood Dale, IL), and then femurs were measured for density using the SA Plus densitometer (Orthometrics, White Plains, NY). Calibration of the SA Plus instrument was accomplished with a manufacturer-supplied phantom and with hydroxyapatite standards of known density (50–1000 mg/mm3) with cylindrical dimensions (2.4 mm diameter × 24 mm length) that approximate mouse femurs. Accuracy of linear measures was checked with defined thickness aluminum foils. The bone scans were analyzed with thresholds of 710 and 570 mg/cm3, using Orthometrics software version 5.50 yielding cortical bone areas that were consistent with histomorphometrically derived periosteal values. Mineral content was determined with thresholds of 220 and 400 mg/cm3, selected so that mineral from most partial voxels (0.07 mm) would be included in the analysis. Precision of the SA Plus for repeated measurement of a single femur was found to be within 1.2–1.4%. Isolated femurs were scanned at seven locations at 2-mm intervals, beginning 0.8 mm from the distal ends of the epiphyseal condyles. Total vBMD values were calculated by dividing the total mineral content by the total bone volume and expressed as milligrams per cubic millimeter. Cortical thickness was obtained at the midshaft scan (29).

Microcomputed tomography

Distal trabecular bone volume fraction (BV/TV) and cortical architecture were measured by micro-CT (MicroCT40; Scanco Medical AG, Bassersdorf, Switzerland) analysis. This instrument provides high-resolution data for trabecular bone volume as well as trabecular number, thickness, and spacing. Femurs from female and male Igfbp2−/− and Igfbp2+/+ control mice at 8 and 16 wk of age were scanned for microarchitecture in the metaphyseal region of the distal femur. In addition, cortical thickness was obtained at the midshaft. The femurs were scanned at low resolution, energy level of 45 keV, and intensity of 177 μA. The distal trabecular scan started about 0.6 mm proximal to the growth plate and extended proximally 1.5 mm. One hundred fifty cross-sectional slices were made at 12-mm intervals at the distal end beginning at the edge of the growth plate and extending in a proximal direction, and then 100 contiguous slices were selected for analysis. These were contoured inside the endosteal edge of the cortical shell to obtain the total volume (TV) of the space, followed by analysis of the trabecular bone volume (BV) with the Scanco software version 5.0. The scans for midshaft cortical thickness were obtained by 18 slices at the exact midpoint of the femur. These slices were contoured by user-defined thresholds for cortical bone and iterated across slices using the Scanco software.

Glucose and insulin tolerance tests

Both Igfbp2−/− males and females along with Igfbp2+/+ controls were tested at 8 wk of age. Blood glucose levels were measured using the OneTouch Ultra Blood Glucose Monitoring System (LifeScan, Inc., Milpitas, CA) per the manufacturer’s instructions. After a 16-h fast, basal glucose levels were measured. For the glucose tolerance test, a 200-mg/kg dose of glucose was administered ip, and at h 1, 2, and 4 after injection, blood glucose levels were measured. For the insulin tolerance test, mice were injected ip with 20 mU insulin in a volume of 0.10 ml. At h 1, 2, and 4 after injection, glucose levels were measured.

Histomorphometry

Static and dynamic histomorphometry were performed by double labeling as described previously (29,30). Briefly, Igfbp2−/− and Igfbp2+/+ mice were injected ip with 20 mg/kg calcein at 15 wk and 50 mg/kg demeclocycline 7 d later. Mice were killed 48 h after the last injection. Muscle was then removed from the femurs and fixed in 70% ethyl alcohol. Femurs were dissected and embedded in methyl methacrylate. Longitudinal sections, 5 μm thick, were cut on a Micron microtome (Richard-Allan Scientific, Kalmazoo, MI) and stained with 0.1% toluidine blue (pH 6.4). Static parameters of bone formation and resorption were measured in a defined area between 725 and 1270 μm from the growth plate using an Osteomeasure morphometry system (Osteometrics, Atlanta, GA). For dynamic histomorphometry, mineralizing surface per bone surface and mineral apposition rate were measured in unstained sections under UV light as described previously. The terminology and units used are those recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (31).

Osteoblast and osteoclast cultures

Bone marrow cells were harvested from femurs and tibias of three 8-wk-old Igfbp2−/− and Igfbp2+/+ control mice, pooled, and plated at 20 × 106 per well on six-well plates in α-MEM and 10% fetal calf serum (FCS). Media were changed on d 1 and 3. Differentiation media containing α-MEM, 10% FCS, 8 mm β-glycerophosphate, and 50 μg/ml ascorbic acid was added at d 7, and media were subsequently changed every 2 d until the termination of the culture. At 18 and 21 d in culture, adherent osteoblast progenitor cells were fixed with 4% paraformaldehyde and identified by alkaline phosphatase staining [alkaline phosphatase-positive colony-forming units (CFU-ALP)] using a commercially available kit (86-R ALP staining kit; Sigma Chemical Co., St. Louis, MO). The cells were then counterstained with von Kossa for mineral deposits [CFU-osteoblast (CFU-OB)].

Osteoclast-like cells were generated by plating bone marrow stromal cells at 1 × 106 cells per well in 48-well plates in α-MEM supplemented with 10% FCS, macrophage colony-stimulating factor (M-CSF) (30 ng/ml; PeproTech Inc, Rocky Hill, NJ), and soluble receptor activator of nuclear factor-κB ligand (RANKL) (50 ng/ml; PeproTech). Media were changed at d 3 and 6. At d 7, the cells were fixed using 2.5% glutaraldehyde and stained for tartrate-resistant acid phosphatase (TRAP) using a kit from Sigma. TRAP-positive multinucleated (more than three nuclei) osteoclasts were counted using light microscopy. Data presented for these cell culture experiments correspond to three independent experiments with at least three replicate wells within each experiment.

Immunoblotting of phosphatase and tensin homolog (PTEN)

In vivo analysis.

Mouse aortas were removed and immediately washed with saline and frozen in liquid nitrogen. The frozen tissue was weighed and then homogenized in RIPA buffer using 1 g tissue/ml buffer. Insoluble proteins were removed by centrifugation at 2000 × g. The supernatants from mouse aorta were analyzed by bicinchoninic acid assay to determine total protein concentrations. Western blot analyses of the aorta were conducted with 50 mg protein loaded per lane and the proteins separated by SDS-PAGE (12.5% gel). The proteins were transferred to Immobilon filters (Immobilon-P; Millipore, Bedford, MA) and then immunoblotted for PTEN phosphatase using a 1/1000 dilution of a polyclonal anti-PTEN phosphatase antiserum (Upstate Biologicals, Lake Placid, NY). The immune complexes were visualized by enhanced chemiluminescence as described previously (26,27). The band intensities were analyzed by scanning densitometry. Four animals were analyzed for each genotype. To control for loading artifacts, duplicate aliquots of the aortic lysate were immunoblotted for α-actin. The blot was stripped and reprobed using a 1:500 dilution of α-actin antibody, and then the immune complex band intensities were analyzed as described previously (26,27).

In vitro analysis.

Another series of in vitro experiments were performed in a manner identical to what was described above for osteoblasts and osteoclasts. For the osteoblast culture experiments, 50 ng/ml IGF-I, 200 ng/ml IGFBP-2, or the combination of IGF-I and IGFBP-2 were added to media containing 10% FCS at d 1, 3, 7, 9, 11, and 13. Cells were collected at d 14 and then lysed for measurement of PTEN. For the osteoclast culture experiments, 50 ng/ml IGF-I, 200 ng/ml IGFBP-2, or the combination of IGF-I and IGFBP-2 were added to media containing 2.5% FCS at d 1, 3, and 6. At d 7 the cells were then removed, and lysates were analyzed for PTEN. PTEN levels were expressed as density units per milligram protein loaded. The percent change in PTEN levels was determined for the osteoblast and osteoclast cell cultures as density units per milligram protein in the Igfbp2−/− and Igfbp2+/+ cells for the treated condition (IGF-I, IGFBP-2, or IGF-I and IGFBP-2 treatment) divided by density units per milligram protein in the untreated control cultures (i.e. basal conditions) for each genotype, respectively.

Statistics

Data are expressed as mean ± sem in tables and figures. Statistical evaluation was performed using the JMP ANOVA software program (SAS, Cary, NC). The histomorphometry data were analyzed using Student’s t test. Differences between the Igfbp2−/− and Igfbp2+/+ mice were considered significant when P ≤ 0.05 unless otherwise noted.

Results

Body composition and glucose tolerance

There were no differences in the growth rates between the Igfbp2−/− mice and their Igfbp2+/+ control littermates. Both male and female Igfbp2−/− mice had similar nose-to-tail lengths at 8 and 16 wk as Igfbp2+/+ controls, and their body weights did not differ significantly from Igfbp2+/+ controls at 8 wk. At 16 wk, the Igfbp2−/− male mice were heavier and had greater percent body fat than Igfbp2+/+ males (P < 0.05) (Table 22).). Neither male nor female Igfbp2−/− mice were insulin resistant, and both had normal glucose homeostasis as measured by glucose and insulin tolerance tests (data not shown). At 8 and 16 wk, both male and female Igfbp2−/− mice had approximately 20% smaller spleens than the Igfbp2+/+ controls (P < 0.01) as previously reported (22). There were no differences in liver, heart, brain, or other organ weights, however, between Igfbp2−/− mutants and Igfbp2+/+ control mice.

Table 2
Body composition measured by PIXImus in Igfbp2−/− and Igfbp2+/+ males and females at 8 and 16 wk of age

Circulating IGF-I system

We confirmed the absence of IGFBP-2 in the serum of 8- and 16-wk-old Igfbp2−/− mice using a mouse-specific ELISA. The levels of IGFBP-2 in the Igfbp2+/− heterozygous mice were found to be intermediate between those of Igfbp2+/+ and Igfbp2−/− mice (Fig. 11).). Interestingly, Igfbp2+/+ serum IGFBP-2 concentrations in females were significantly less (P < 0.001) than males (females, 361.3 ± 38.2 ng/ml; males, 599.5 ± 100 ng/ml). A different picture emerged for serum IGF-I. The Igfbp2−/− male and female mice had 10% higher IGF-I than Igfbp2+/+ animals (P < 0.05) at 8 wk of age. At 16 wk of age, only the female Igfbp2−/− mice had persistently higher IGF-I levels than Igfbp2+/+ controls (Table 33).

Figure 1
Serum IGFBP-2 levels at 16 wk of age; n = 6 for each genotype and sex. *, P ≤ 0.05 for comparison of male with female Igfbp2+/+.
Table 3
Serum IGF-I levels for Igfbp2−/− and Igfbp2+/+ mice at 8 and 16 wk of age

Assessment of circulating IGFBPs and hepatic expression of the IGF regulatory system

To determine whether there were compensatory changes in the other circulating components of the IGF system, we assessed semiquantitatively serum IGFBPs by ligand blot in male Igfbp2−/− and appropriately matched Igfbp2+/+ controls at 8 wk of age. There were no apparent differences in the ligand-binding intensity for the 43-, 32-, 30-, and 24-kDa bands representing the major IGFBPs from the Igfbp2−/− mice compared with Igfbp2+/+ (data not shown). We also studied differences in hepatic IGF-I and IGFBP expression of Igfbp2−/− mice vs. Igfbp2+/+. Real-time mRNA expression of the Igfbps from 6-wk-old Igfbp2−/− males, taken as an average of at least three runs, revealed modest but significant increases in Igf1, Igfbp-1, -3, -4, -5, and -6 when compared with Igfbp2+/+ controls (Table 44).). Of particular note, Igf1 mRNA was 4-fold higher in the Igfbp2-null than the Igfbp2+/+ mice (P < 0.02), consistent with increased circulating levels of IGF-I found in the 8-wk-old Igfbp2−/− mice. Irs1 and Irs2 expression was also increased in the Igfbp2−/− mice, which may be secondary to the increased IGF-I levels. IGFBP-5 is structurally very similar to IGFBP-2 and can bind to the acid-labile subunit to form a ternary complex in the circulation. Hepatic expression of Igfbp5 was 3-fold higher in the Igfbp2−/− mice than Igfbp2+/+ controls (P < 0.001). But, when we assessed IGFBP-5 levels in the circulation using a specific mouse antibody by immunoblot, no differences were noted between male Igfbp2−/− and Igfbp2+/+ controls (data not shown), consistent with the absence of change by ligand blot. Igfals expression in the liver was not statistically different between genotypes (Table 44).). Hence, the ternary IGF circulating complex did not appear to be altered by the absence of IGFBP-2 in the circulation. Finally, we also measured hepatic Pparg expression in the liver, in part because of the absence of insulin resistance despite increased body fat, in the Igfbp2−/− male mice. Similar to what was found in expression patterns from bone and marrow (see below), Pparg mRNA was 4.3-fold higher in the Igfbp2-null mice than Igfbp2+/+ controls (P < 0.001).

Table 4
Quantitative real-time PCR of Igfbp2−/− male liver and bone expression compared with Igfbp2+/+ controls

Serum osteocalcin levels

Osteocalcin is a biochemical marker of bone turnover that principally reflects bone formation. We measured serum osteocalcin levels in groups of 20 males and 15 females of Igfbp2−/− and Igfbp2+/+ mice at 16 wk of age. The Igfbp2−/− males showed 40% lower osteocalcin levels (Igfbp2+/+, 118.5 ± 5.25 ng/ml vs. Igfbp2−/−, 74.2 ± 6.85 ng/ml; P < 0.0001) compared with controls. In contrast, the Igfbp2−/− females showed 20% higher serum osteocalcin levels compared with Igfbp2+/+ controls (Igfbp2+/+, 113.4 ± 4.64 ng/ml vs. Igfbp2−/−, 139.5 ± 5.60 ng/ml; P < 0.007). We also measured the percentage of osteocalcin that was uncarboxylated in the male mice and found that despite the reduction in total serum levels for the Igfbp2−/−, there was a greater proportion of uncarboxylated osteocalcin than in the Igfbp2+/+ mice (Igfbp2−/−, 36.3 ± 5.3% vs. Igfbp2+/+, 20.4 ± 3.5%; P < 0.03).

aBMD by DXA

At 8 wk of age, both female and male Igfbp2−/− mice had slightly greater total body aBMD than Igfbp2+/+ controls. By 16 wk of age, females did not differ from Igfbp2+/+ controls, whereas males showed a slight but significant (P < 0.05) decrease in total-body aBMD compared with Igfbp2+/+ controls (see Table 22).

vBMD by pQCT

At 8 wk of age, there were no genotypic differences in femur length. Periosteal circumference did not differ from Igfbp2+/+ controls at 8 wk of age in male Igfbp2−/− mice but was increased in female Igfbp2−/− (Table 55 and Fig. 2A2A).). Endosteal circumference in the Igfbp2−/− mice did not differ from Igfbp2+/+ controls for either gender. Interestingly, cortical thickness at 8 wk in the femur was increased in female Igfbp2−/− mice and decreased in male Igfbp2−/− mice, but total vBMD by pQCT was not different by genotype for either Igfbp2−/− null at 8 wk compared with Igfbp2+/+ controls. At 16 wk of age, female Igfbp2−/− mice had significantly greater periosteal circumferences than Igfbp2+/+ mice resulting in thicker cortices, but again there was no change in total femoral vBMD (see Table 55).). On the other hand, at 16 wk, male Igfbp2−/− mice had slightly but significantly shorter femurs than Igfbp2+/+ mice (P < 0.004) and still had thinner cortices, resulting in decreased total vBMD (P < 0.06) (Table 55 and Fig. 22).

Figure 2
Micro-CT images at 8 wk of age. A, Cortical compartment for Igfbp2−/− and Igfbp2+/+ control male and females; B, distal trabecular compartment for the Igfbp2−/− and Igfbp2+/+ control males. ...
Table 5
Femoral pQCT phenotypes for Igfbp2−/− and Igfbp2+/+ females and males at 8 and 16 wk of age

Micro-CT of the femur

At 8 wk, male Igfbp2−/− mice had significantly reduced total bone area at the midshaft (P < 0.01; see Table 66)) and thinner cortices compared with Igfbp2+/+ mice (P < 0.01), whereas female Igfbp2−/− mice had thicker cortices (P < 0.02) with greater bone area (P < 0.05), similar to what was revealed by pQCT. For the male Igfbp2−/−, at 8 wk, there was an 18% reduction in distal trabecular BV/TV compared with Igfbp2+/+ mice (P < 0.01) (Table 66 and Fig. 2B2B).). This was almost entirely due to reduced trabecular thickness (P < 0.003) rather than any change in trabecular number. In contrast, female Igfbp2−/− mice showed virtually no trabecular changes compared with Igfbp2+/+ mice (Table 66 and Fig. 22).). At 16 wk of age, the femur of male Igfbp2−/− mice showed a similar pattern for both the trabecular and cortical compartments, that is, a reduced trabecular BV/TV and decreased cortices in the male Igfbp2−/− mice and no differences in the trabecular compartment and increased cortices in the female Igfbp2−/− mice.

Table 6
Femoral MicroCT phenotypes for Igfbp2−/− and Igfbp2+/+ mice at 8 and 16 wk of age

Histomorphometry

At 16 wk, male Igfbp2−/− mice had significantly fewer osteoblasts/bone perimeter (P < 0.01) and fewer osteoclasts/bone perimeter (P < 0.05) than Igfbp2+/+ mice (Table 77).). Mineralizing surface/bone surface in Igfbp2−/− male mice was also markedly reduced (P < 0.001). Likewise, the bone formation rate/bone surface/day was reduced by 45% (P < 0.001) in the Igfbp2−/− male mice. There were also more adipocytes/total area in the marrow of male Igfbp2−/− compared with controls (Table 77).). However, the mineral apposition rate was not different by genotype for the males. For female Igfbp2−/− mice at 16 wk, the only parameters that showed differences were the bone formation rate, which was slightly but not significantly reduced (P > 0.10) in the Igfbp2−/− mice, and the mineralizing surface/bone surface, which was lower than Igfbp2+/+ mice (P = 0.03) (Table 77).

Table 7
Histomorphometry of Igfbp2−/− and Igfbp2+/+ mice at 16 wk of age in the distal femur

In vitro studies

We studied cultures from bone marrow stromal cells of both males and female Igfbp2−/− and Igfbp2+/+ animals at 8 wk of age. We found that the number of CFU-ALP+ pre-OBs was significantly lower in the male Igfbp2−/− than Igfbp2+/+ controls (Fig. 3A3A).). Similarly, the amount of mineral, measured by von Kossa staining was less in the Igfbp2−/− males than the Igfbp2+/+ controls. Interestingly, the female Igfbp2−/− cultures when compared with the Igfbp2+/+ control cultures, had reduced CFU-ALP+ pre-OBs and mineral as well (data not shown). When nonadherent marrow cells from male Igfbp2−/− and Igfbp2+/+ mice were cultured in M-CSF and RANKL, the numbers of TRAP+ osteoclasts were also significantly lower in the Igfbp2-null mice after 7 d than in Igfbp2+/+ controls (P < 0.05). In cultures of female Igfbp2−/− cells, the numbers of TRAP+ osteoclasts were also reduced but did not reach significance (P = 0.08; see Fig. 3B3B).

Figure 3
Bone marrow stromal cultures of Igfbp2−/− and Igfbp2+/+ control males. A, At d 18 in culture, adherent OB progenitor cells were identified by alkaline phosphatase staining (CFU-ALP) and were counterstained with von Kossa ...

Real-time PCR of whole bone

To understand the possible mechanisms responsible for the changes in osteoclast activity noted in vitro and the histomorphometric indices in the Igfbp2−/− male mice, we measured real-time expression of four genes from the whole femur (i.e. marrow plus cortical bone) of 8-wk-old male Igfbp2−/− mice (n = 4) compared with Igfbp2+/+ controls. Both the ligand Mcsf and its receptor Cfms mRNA levels were more than 4-fold higher in Igfbp2−/− mice than in Igfbp2+/+ controls, whereas skeletal Igf1 and Pparg mRNA were also markedly increased in the Igfbp2−/− mice compared with Igfbp2+/+ controls (Table 44).

Changes in PTEN phosphatase protein levels

IGFBP-2 has recently been reported to regulate PTEN phosphatase levels in several cancer cell lines, independent of IGF-I (32). To assess whether changes in the IGF-I signaling pathway might be affected in the male Igfbp2−/− mice, we measured PTEN phosphatase in vivo by immunoblotting extracts from aortas of null and control mice (n = 4 each). Scanning densitometry showed that the male Igfbp2−/− aortas had a significant 5.5 ± 1.7-fold increase in PTEN phosphatase compared with the Igfbp2+/+ aortas (P < 0.01; see Fig. 4A4A).

Figure 4
Immunoblot of PTEN. A, Aortas from Igfbp2+/+ controls and Igfbp2−/− males were lysed and processed. After SDS-PAGE, the amount of PTEN was determined by immunoblotting. The arrow denotes the position of the PTEN band. The ...

We then assayed PTEN levels in osteoblasts and osteoclasts in vitro to determine whether the changes observed in aortic tissues were tissue specific. Lysates of differentiated adherent marrow stromal cells were prepared at d 14 from both male Igfbp2−/− and Igfbp2+/+ male controls and examined for PTEN. We also took nonadherent marrow cells from null and Igfbp2+/+ controls and cultured them in 2.5% FCS after addition of M-CSF and RANKL. Cells were collected at d 7 and the lysates examined for PTEN. In a second series of experiments, we added back IGFBP-2 (200 ng/ml) and IGF-I (50 ng/ml) to the media of the osteoblasts and osteoclasts and then measured PTEN. In the basal state, PTEN expression was higher in the Igfbp2−/− osteoblasts than Igfbp2+/+ controls (Fig. 4B4B).). This genotypic difference was even greater in the Igfbp2−/− osteoclasts, which exhibited a 43% increase in PTEN expression compared with Igfbp2+/+ control cells. Furthermore, exogenous IGFBP-2 suppressed PTEN from the Igfbp2−/− osteoblasts by 50% and in Igfbp2+/+ osteoblasts by 40% when compared with untreated control cultures (i.e. basal conditions) for each genotype, respectively. Likewise, addition of IGFBP-2 to the Igfbp2−/− osteoclast cultures decreased PTEN expression by 50% when compared with the Igfbp2−/− control culture. Addition of IGF-I alone suppressed PTEN levels in osteoblasts from Igfbp2−/− mice by 44% and in Igfbp2+/+ osteoblasts by 67%. The combination of IGF-I plus IGFBP-2 suppressed PTEN in osteoblasts from both genotypes by 57% and 72%, respectively. Changes in PTEN expression in osteoclasts after the addition of IGF-I alone or IGF-I plus IGFBP-2 were more marked in the Igfbp2−/− osteoclasts compared with that of the osteoblasts. Thus, PTEN is up-regulated in vascular and skeletal tissues from Igfbp2−/− mice and is suppressed by IGFBP-2 with or without IGF-I.

Discussion

Based on previous studies, we hypothesized that because IGFBP-2 was an important regulator of bone turnover, any perturbation in its expression would significantly affect peak bone acquisition. In fact, we found gender-, age-, and compartment-specific differences in skeletal acquisition of the Igfbp2−/− mice compared with their Igfbp2+/+ controls. In particular, male Igfbp2−/− mice exhibited reduced trabecular bone density, whereas female Igfbp2−/− mice had increased cortical bone density. In vitro studies of bone cells from male null mice demonstrated a profound defect in both osteoblast and osteoclast differentiation, a finding consistent with our comprehensive histomorphometric data. We also detected significantly higher amounts of PTEN in the Igfbp2−/− osteoblasts and osteoclasts compared with the Igfbp2+/+ controls, and these differences were eliminated by the addition of recombinant IGFBP-2 or IGF-I plus IGFBP-2. Thus, one possible mechanism to explain the effects of IGFBP-2 on bone turnover in male mice would be changes in intracellular PTEN activity, which, in turn, would have a profound effect on a major signaling pathway for skeletal remodeling.

In most mammalian systems, when bone turnover is suppressed, the bone volume fraction in the distal femur is usually preserved, due to coupling between formation and resorption. However, in male Igfbp2−/− mice, the trabecular BV/TV was significantly reduced, suggesting that formation might be suppressed more than resorption. In support of that concept, we found nearly a 50% reduction in the bone formation rate and significantly more adipocytes in the marrow of male Igfbp2−/− mice compared with controls (Table 77).). Furthermore, when we measured gene expression by real-time PCR of the whole femur and marrow, Pparg, an essential adipocyte differentiation factor, was also markedly increased (Table 44).). Those findings, plus a reduction in the number of von Kossa forming osteoblast colonies in the male nulls (Fig 3A3A),), suggest that one of the alterations in bone turnover in the Igfbp2−/− mice is a defect in stromal cell allocation into the osteoblast lineage.

In contrast to male nulls, female Igfbp2−/− mice did not demonstrate a trabecular phenotype either by micro-CT or histomorphometry. In fact, we found that female mice had increased femoral cortical thickness (Fig. 2A2A and Table 66),), reflecting significant gender dimorphism. This increase in cortical thickness was related to greater periosteal expansion rather than a change in the endosteal envelope. A possible mechanism for periosteal expansion in the female nulls might be the persistently higher circulating IGF-I concentrations in the female Igfbp2−/− mice at 16 wk of age but not in the male Igfbp2−/− or in the Igfbp2+/+ controls. Several studies have shown that increased circulating IGF-I can stimulate periosteal growth in mice (33,34,35). Still, it is not clear why serum IGF-I is greater in the female Igfbp2−/− mice beyond the time of the pubertal growth spurt (i.e. 8 wk of age). Interestingly, IGFBP-2 is expressed in relatively high amounts in the hypothalamus and pituitary (36). Hence, it is conceivable that if this binding protein transports IGF-I locally, the absence of IGBP-2 could result in impaired presentation of IGF-I, resulting in less negative feedback within the hypothalamic-pituitary axis (37). This would eventually lead to increased GH secretion, which would be reflected by enhanced hepatic Igf-1 and Igfbp3 mRNA expression and higher circulating IGF-I levels (37,38,39).

The skeletal phenotype of male Igfbp2−/− mice is consistent with human studies by Amin and colleagues (21), who demonstrated that serum IGFBP-2 is strongly correlated with markers of bone turnover in 344 men of various ages. Using micro-CT of the distal radius, those authors also found that serum IGF-I levels were positively related to trabecular thickness in men but not in women. In osteosclerosis from hepatitis C, Khosla et al. (19) noted that increases in both IGF-II and IGFBP-2 were responsible for a phenotype that included higher bone mass, increases in alkaline phosphatase, and greater bone formation. Interestingly, most of the reported cases of hepatitis C osteosclerosis occur in men, suggesting that both ligand (IGF-I or IGF-II) and binding protein (IGFBP-2) are critical for regulating trabecular thickness. In our studies, male Igfbp2−/− mice had thinner trabeculae in the distal femur, suggesting that the delivery of IGF-I and/or IGF-I plus IGFBP-2 may be critical for optimizing bone formation and the thickness of trabeculae during growth. In rats, IGFBP-2, in combination with IGF-II, has been shown to stimulate bone formation and protect against ovariectomy- and unloading-induced bone loss (20). It is conceivable that trabecular thickness in these rats was maintained by a combination of excess ligand and increased binding protein. Ultimately, however, an in vivo rescue experiment with recombinant IGFBP-2 in male mice will be important to determine whether IGFBP-2 is necessary and sufficient for peak trabecular thickness.

There are two possible mechanisms to explain why turnover is altered in the Igfbp2−/− mice. First, the absence of IGFBP-2 means there is less transport of IGF-I in the intra- and extravascular spaces. Several lines of evidence from our studies suggest that the absence of IGFBP-2 alone as a carrier protein for IGF-I is a contributing factor to the skeletal phenotypes. IGFBP-2 has a strong propensity to bind to components of the extracellular matrix and therefore increases the local concentration of IGF-I that is available to bind to receptors. It is likely that the reduction in spleen size in Igfbp2−/− mice is a function of altered IGF-I transport to the spleen and/or anchoring due to the absence of IGFBP-2. Similarly in the skeleton, if IGFBP-2 is not present and cannot bind IGF-I or IGF-II, osteoblast growth and/or hard tissue deposition might be significantly altered. Finally, our studies of PTEN expression in wild-type osteoblasts suggest that the combination of IGF-I and IGFBP-2 is twice as effective in suppressing PTEN levels as IGFBP-2 alone.

Second, it is conceivable that IGFBP-2 may work in an IGF-I-independent manner. Our in vitro studies demonstrated that IGFBP-2 suppressed PTEN levels in both osteoblasts and osteoclasts. However, because suppression was further enhanced with the combination of IGFBP-2 plus IGF-I, PTEN suppression may not be entirely IGF-I independent in either osteoblasts or osteoclasts. Holly and colleagues (40) found that in MCF-7 breast cancer cells, IGFBP-2 could directly bind to an integrin receptor and suppress PTEN phosphatase activity. Therefore, the absence of IGFBP-2 would be predicted to lead to an increase in PTEN phosphatase expression and, in turn, reduced IGF-I signaling. There are several studies that show that activation of the phosphoinositide (PI)-3 kinase pathway and AKT activation are necessary for IGF-I to stimulate differentiative osteoblast function (41,42). Because PTEN inhibits AKT activation, its suppression by IGFBP-2 would be expected to enhance IGF-I actions and stimulate bone turnover. But PTEN also alters other bone signaling pathways such as the Wnt-β-catenin pathway (43,44), and bone morphogenetic protein has been shown to suppress PTEN levels in osteoblasts (45). Furthermore, inactivation of PTEN expression in osteochondrocyte precursor cells leads to epiphyseal growth plate abnormalities and skeletal overgrowth (46). Clemens and colleagues (47) recently showed that targeted deletion of PTEN phosphatase using an osteocalcin-Cre Lox P promoter system resulted in extremely high bone mass throughout adulthood. Hence, it is conceivable that IGFBP-2, either bound to extracellular matrices such as collagen and/or hydroxyapatite, or free, could interact with an integrin receptor and suppress PTEN. This would lead to enhanced IGF-I signaling through the PI-3 kinase pathway to promote osteoblast and/or osteoclast differentiation.

With respect to osteoclasts, our in vitro and in vivo studies demonstrated that osteoclastic activity in Igfbp2−/− mice was modestly suppressed. The mechanism for this effect could be either direct or indirect via cytokine release from the osteoblast. In regard to the former possibility, we found up-regulation of both c-fms and m-csf in the marrow of Igfbp2−/− mice. If IGFBP-2 is important for αvβ3 integrin activation, then up-regulation of c-fms, the receptor for m-csf, which regulates osteoclast proliferation, might provide an important compensatory mechanism for impairment of differentiated osteoclast function.

There are several limitations to the current work. First, we phenotyped Igfbp2−/− mice at 8 and 16 wk of age; hence, definitive conclusions about skeletal acquisition will require developmental studies at earlier time points. Second, as noted above, we still cannot say for certain whether IGFBP-2 acts alone or in concert with IGF-I in bone to affect turnover. This will require rescue studies in vivo using recombinant IGFBP-2 with or without IGF-I. Third, changes in PTEN phosphatase in the vascular bed and bone of the Igfbp2−/− mice are provocative but will require more detailed analysis of the PI-3 kinase pathway. Also, more studies are needed to analyze the extent of insulin sensitivity in the Igfbp2−/− mice, particularly in response to high-fat feeding. Finally, it remains to be determined why female Igfbp2−/− are protected from the trabecular changes seen in male Igfbp2−/− mice.

In summary, we found that IGFBP-2 is critical for optimal trabecular bone acquisition in male mice. In contrast, the absence of IGFBP-2 in females resulted in increased cortical bone acquisition with minimal effects on the trabecular compartment. One possible mechanism that underlies skeletal changes in the male Igfbp2−/− mice may be the marked changes in PTEN found in three distinct cell types. These studies highlight the complex nature and gender-specific characteristics of IGFBP-2 actions in the skeleton.

Acknowledgments

We thank Dr. Kenneth Johnson and Dr. Lenny Shultz for critical review of this manuscript. We also thank Dr. Caren Gunburg for running the uncarboxylated osteocalcin samples. Furthermore, we thank Krista Delahunty, Julie Burgess, Marianne Lagerklint, Carolina Evsikova, Cheryl Ackert-Bicknell, and Ilka Pinz for their technical assistance and expertise.

Footnotes

Address all reprint requests to: Victoria E. DeMambro, The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609. E-mail: gro.xaj@gev.

This work was supported by National Institutes of Health Grants AR45433, AR54664, DK042424, and AG02331.

Disclosure Statement: The authors have nothing to declare.

First Published Online February 14, 2008

See editorial p. 2048.

Abbreviations: aBMD, Areal bone mineral density; BV, bone volume; CFU-ALP, alkaline phosphatase-positive colony-forming units; CFU-OB, CFU-osteoblastoid; DXA, dual-energy x-ray absorptiometry; FCS, fetal calf serum; IGFBP-2, IGF-binding protein-2; M-CSF, macrophage colony-stimulating factor; PI, phosphoinositide; pQCT, peripheral quantitative computed tomography; PTEN, phosphatase and tensin homolog deleted on chromosome 10; RANKL, receptor activator of nuclear factor-κB ligand; TRAP, tartrate-resistant acid phosphatase; TV, total volume; vBMD, volumetric BMD.

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