Parathyroid hormone (PTH) is a potent anabolic agent for bone, but the mechanism(s) by which it works remains imperfectly understood. Previous studies have indicated that PTH stimulates insulin-like growth factor (IGF) I production, but it remains uncertain whether IGF-I mediates some or all of the skeletal actions of PTH. To address this question, we examined the skeletal response to PTH in IGF-I-deficient (knockout [k/o]) mice. These mice and their normal littermates (NLMs) were given daily injections of PTH (80 μg/kg) or vehicle for 2 weeks after which their tibias were examined for fat-free weight (FFW), bone mineral content, bone structure, and bone formation rate (BFR), and their femurs were assessed for mRNA levels of osteoblast differentiation markers. In wild-type mice, PTH increased FFW, periosteal BFR, and cortical thickness (C.Th) of the proximal tibia while reducing trabecular bone volume (BV); these responses were not seen in the k/o mice. The k/o mice had normal mRNA levels of the PTH receptor and increased mRNA levels of the IGF-I receptor but markedly reduced basal mRNA levels of the osteoblast markers. Surprisingly, these mRNAs in the k/o bones increased several-fold more in response to PTH than the mRNAs in the bones from their wild-type littermates. These results indicate that IGF-I is required for the anabolic actions of PTH on bone formation, but the defect lies distal to the initial response of the osteoblast to PTH.
Although the ability of parathyroid hormone (PTH) to stimulate bone formation is now well established,(1) the mechanism by which it does so is unclear. PTH, given intermittently in vivo, results in a rapid increase in the mRNA levels for osteocalcin, alkaline phosphatase, and collagen.(2,3) These data support a direct effect of PTH on the activity of the osteoblast, a cell known to have the PTH receptor. When bone marrow stromal cells (BMSC) are evaluated in vitro, more colony-forming units and an increased percentage of alkaline phosphatase positive colonies are observed when the cells are taken from animals treated in vivo with PTH.(4,5) These results suggest that PTH also may promote the proliferation and/or differentiation of osteoblast precursors in addition to stimulating the function of the mature osteoblast. In addition, PTH has been shown to reduce apoptosis of bone cells,(6) suggesting yet another mechanism for its anabolic effects.
Insulin-like growth factor (IGF) I has been postulated to mediate the anabolic actions of PTH on bone. PTH increases the mRNA and protein levels of IGF-I in bone or bone cells both in vivo(7,8) and in vitro.(9–11) When PTH is administered to fetal rat calvariae for 24h it increases collagen synthesis over the subsequent 48h in a manner blocked by antibodies to IGF-I.(11) PTH also stimulates proliferation of the periosteal layer of fetal rat calvariae, but this action is not blocked by antibodies to IGF-I.(11) The anabolic action of PTH on bone in vivo is blunted in hypophysectomized rats but is restored with growth hormone (GH) perhaps in part because of the regulation by GH of IGF-I production in bone.(12)
Thus, although there is substantial evidence that PTH can increase IGF-I levels in bone and bone cells and that antibodies to IGF-I can block at least some responses of bone to PTH, there remains uncertainty regarding the actual role(s) that IGF-I plays and the importance of that role(s) in mediating the anabolic effects of PTH on bone. To address this issue, we studied the skeletal response to PTH in a mouse model in which IGF-I production was eliminated.(13) Our results indicate that IGF-I is critical for the skeletal response to PTH.
MATERIALS AND METHODS
IGF-I-deficient mice were provided as a gift from Dr. Lyn Powell-Braxton (Genentech, San Francisco, CA, USA). They were developed as previously described,(13) and then backcrossed into a CD-1 genetic background. The construct used for the homologous recombination contained a neomycin cassette inserted into exon 3 of the IGF-I gene. Genotyping of the offspring used polymerase chain reaction (PCR) of tail DNA with the appropriate primers to identify either the intact exon 3 or the neomycin insertion as described.(13) The mice were raised for 3 months on standard mouse chow. To the extent possible, each IGF-I-deficient mouse was matched to two wild-type and two heterozygous littermates of the same sex for each experiment. The male mice responded better to PTH than did the female mice, so only the results from the male mice are reported. Fourteen wild-type, 15 heterozygotes, and 6 IGF-I-deficient animals received either 80 μg of PTH/kg body weight (BW) or vehicle by subcutaneous injection every day for 2 weeks beginning on day 120. To determine bone formation during the period of PTH administration, the mice were injected subcutaneously with demeclocycline on day 1 of PTH administration followed in 12 days by calcein (each 15 mg/kg) to label the mineralization fronts of the bone. Two days later, the mice were killed. The animals were weighed; blood was obtained for IGF-I determinations; the right tibias were obtained for fat-free weight (FFW), peripheral quantitative computerized tomography (pQCT), and microcomputer tomography (μCT); the left tibias were obtained for mineral appositional rate (MAR) and bone formation rates (BFRs); and the right femurs were obtained for mRNA determinations. These studies were approved by the Animal Use Committee of the San Francisco Veterans Affairs Medical Center where the animals were raised and studied.
FFW of bone
The tibias were cleared of adherent soft tissue and extracted in ethanol and diethyl ether using a Soxhlet apparatus (Fisher Scientific, Pittsburgh, PA). Then, the bone was dried at 100°C and weighed.
MAR and BFRs at the tibiofibular junction were performed as follows. Diaphyseal segments of the tibias were dehydrated, defatted in acetone followed by ether, and then embedded in bioplastic (Tap Plastics, Dublin, CA, USA). After polymerizing overnight, the blocks were sectioned at a thickness of 60 μm using a Leica SP 1600 circular bone saw (Leica Inc., Deerfield, IL, USA). The section containing the tibiofibular junction was digitized with a Hamamatsu video camera (Carl Zeiss Inc., Thornwood, NY, USA) coupled to a Leica DMR microscope, and periosteal MAR and BFR were determined using the National Institutes of Health (NIH) Image program.
Tomographic measurements of trabecular, cortical, and total bone density were performed using a Stratec XCT-960A from Norland Medical Systems (Fort Atkinson, WI, USA) according to previously described methods.(14) One-millimeter-thick sections were obtained throughout the length of the tibia beginning just distal to the growth plate. To compensate for the differences in bone size between the IGF-I-deficient animals and their wild-type and heterozygous littermates, the data from the first section below the growth plate is reported as representing the proximal tibia, and the data from the next to the last slice is reported as representing the distal tibia.
The tibias were analyzed with a Scanco Medical AG μCT apparatus (Scanco Medical AG, Basserdorf, Switzerland) as previously described.(15) Three-dimensional (3D) information was obtained by stacking successively measured slices on top of each other. The voxel size was 9 μm in all three spatial dimensions. One hundred twenty-eight slices were measured in each sample, covering a total of 1.15 mm of the metaphysis. To analyze the trabecular part of the tibias, the compact part of the bone was masked out in the following way: with a 3D box-shaped low-pass filter applied to the original gray-scale CT images, an artificial partial volume effect was created, which blurs out the individual trabeculae but leaves the dense compact shell intact. The cortex mask is then extracted with a simple thresholding operation with a fixed threshold of 20.0% of the maximal gray-scale value. To analyze the cortex, a low-pass Gaussian filter (s = 3.0) was applied to the original images, with a threshold of 10.0%. The axial position and extent are identical with the trabecular volume. The trabecular region is masked out, and a 3D component labeling of the cortex is performed additionally to extract only the main connected component. Then, the cortex is evaluated with the direct distance transformation method to calculate its thickness. Bone volume (BV) and bone surface (BS) are calculated using a tetrahedron meshing technique generated with the marching cubes method. Total volume (TV) is calculated from the volume of the conforming voxels of interest (VOI).
Serum levels of IGF-I
IGF-I in serum was measured as previously described.(16) Acid ethanol extraction was used to remove the IGF-binding proteins (IGFBPs), and the extracted samples were assayed for IGF-I with an radioimmunoassay (RIA) kit from Nichols Institute (San Juan Capistrano, CA, USA). The detection limit is this assay was 22 ng/ml.
mRNA levels in bone
The right femurs were cleaned of adherent tissue and then frozen in liquid nitrogen and stored at −80°C until processed. The bones from each experimental group were pooled to form four or five pools in wild-type and heterozygous animals and three pools in IGF-I-deficient animals. Bone pools were pulverized in a steel mortar and pestle cooled in liquid nitrogen, and then extracted by means of an RNA Stat-60 kit (Tel-Test, Friendswood, TX, USA). For each pool, 500 ng of total RNA was reverse-transcribed in 100 μl of a reaction mixture that contained 10 mM of Tris-HCl (pH 8.3); 50 mM of KCl; 7.5 mM of MgCl2; 1 mM each of deoxynucleoside triphosphate (dNTP); 5 μM of random primers (Gibco BRL, Rockville, MD, USA); 0.4 U/μl of RNAse inhibitor (Roche, Indianapolis, IN, USA); and 2.5 U/μl of Moloney murine leukemia virus (M-MLV) reverse transcriptase (GIBCO BRL, Rockville, MD, USA) at 25°C for 10 minutes, 48°C for 40 minutes, 95°C 5 minutes, and 4°C (stored). The sequences of the PCR primers and probes are listed (Table 1). These primers and probes were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). Primers were synthesized by the Biomolecular Resource Center (University of California, San Francisco, CA, USA). Probes were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA). The internal probe was labeled at the 5′ end with the reporter dye 6-carboxyfluorescein (FAM) and at the 3′ end with the quencher dye 6-carboxy-tetramethyl rhodamine (TAMRA). During PCR, the 5′-3′ nuclease activity of Taq DNA polymerase releases the reporter fluorescence.(17) The fluorescence intensity is proportional to the accumulation of PCR product and was detected with an ABI 7700 Prism (Applied Biosystems). The sequence of the probe or primers for each target gene spans an exon/exon boundary to minimize the signal generated by genomic DNA that may be contaminating the RNA sample, thereby eliminating the need to DNAse treat the sample.
Table Table 1.. Primers and Probes for Real-Time PCR
PCR was carried out in triplicate with 50-μl reaction volumes of 1× PCR buffer A (Applied Biosystems), 5.5 mM of MgCl2, 1 mM each of dNTP, 500 nM each of primer, 200 nM of probe, and 0.025 U/μl of AmpliTaq Gold DNA polymerase (Applied Biosystems) with 4 μl of cDNA template. The PCR reaction was performed in an ABI 7700 Prism using the following cycle parameters: 1 cycle of 95°C for 12 minutes and 45 cycles of 95°C for 15 s and 60°C for 1 minute. Analysis was carried out using the sequence detection software supplied with the ABI 7700 Prism. The number of PCR cycles (threshold cycles [Ct]) required for the FAM intensities to exceed a threshold just above background was calculated for the test reactions.(18) The Ct values were determined for three test reactions in each sample and averaged. The ΔCt values were obtained by subtracting the GAPDH (as endogenous control) Ct values from the target gene Ct values of the same samples. The relative quantification of the target genes was given by 2−ΔCt.
The data were analyzed by multivariate ANOVA and Student's t-test as appropriate using programs in Sigma-Stat (Jandel Scientific). The value of p < 0.05 was considered significant. All data are expressed as mean ± SD.
Serum IGF-I levels
The serum levels of IGF-I in the wild-type mice were 280 ± 92 ng/ml. The levels in the heterozygotes were 223 ± 53 ng/ml, whereas the levels in the IGF-I-deficient mice were undetectable. The gene dosage effect on serum IGF-I was highly significant (p = 0.001). PTH did not significantly alter the IGF-I levels.
Bone mass and body mass
The IGF-I-deficient animals were 18% the size of their wild-type littermates, whereas the heterozygotes were 82% the size of the wild-type mice (Table 2). When analyzed by ANOVA, the gene dosage effect on BW was highly significant (p < 0.0001). PTH did not significantly alter BW. The FFW of the tibia paralleled BW, although when normalized to BW (FFW/BW), a significant gene dosage effect was observed (p = 0.02 by ANOVA), with the IGF-I-deficient animals having a 60% greater FFW/BW than the wild-type mice (p < 0.05). PTH significantly increased FFW (p = 0.03 by ANOVA) and FFW/BW (p = 0.01 by ANOVA). When considering the different genotypes PTH stimulated FFW/BW 33% in the wild-type animals (p < 0.05), 16% in the heterozygotes (p < 0.1), but not at all in the k/o mice.
Table Table 2.. FFW of the Tibia
Bone mineral content and density
The total mineral content of the proximal and distal tibia paralleled that of bone weight with the IGF-I-deficient animals having 25% and 23% of the total mineral of their wild-type littermates in the proximal and distal tibia, respectively, with the heterozygotes being 89% and 90% that of wild-type littermates for the proximal and distal measurements, respectively (Table 3). The gene dosage effect for these measurements was highly significant (p < 0.0001). The bone density of the proximal and distal tibia also tended to be less in the IGF-I-deficient animals, but this trend achieved significance only in the proximal tibia (p = 0.02). PTH administration increased total mineral content and density of the proximal tibia (p = 0.01 and 0.05, respectively, by ANOVA). However, when each genotype was considered separately, this effect of PTH was significant only in the heterozygotes.
Table Table 3.. Tibial Bone Mineral and Density
pQCT does not have sufficient resolution to distinguish trabecular and cortical bone in these small animals. For this purpose, we used μCT to evaluate the proximal tibia (Table 4). The TV of the proximal tibia in the k/o animals was 19% that of the wild-type animals (p < 0.01), whereas the heterozygotes were 84% that of the wild-type animals (NS). Similarly, total trabecular BV was significantly less in the k/o mice (24% of wild-type; p < 0.01). However, as we(15) previously reported, when BV is normalized to TV (BV/TV), the trabecular bone volume (BV/TV) of the k/o animals is significantly greater than the wild-type animals. This is because of the increased trabecular number in the k/o animals.(15) Surprisingly, PTH had opposite effects on trabecular and cortical bone in that it decreased BV (p = 0.02 by ANOVA) while increasing cortical thickness (C.Th; p = 0.05 by ANOVA) in the wild-type animals. The reduction in BV and BV/TV is caused by a reduction in trabecular number with little effect on trabecular thickness (data not shown). When expressed as a ratio (BV/C.Th) PTH reduced BV/C.Th 56% in the wild-type animals (p = 0.05) and 14% in the heterozygotes (NS) but not at all in the k/o animals. These disparate actions of PTH on cortical and cancellous bone explain the more subtle changes seen by pQCT in that this measurement assessed total bone mineral and density.
Table Table 4.. μCT Determination of the Structure of the Proximal Tibia
The periosteal bone formed during the period of PTH administration is illustrated with representative samples in Fig. 1 and quantitated for all samples in Fig. 2. Basal MARs are well maintained in the IGF-I-deficient animals when compared with their wild-type controls. However, because of the smaller-size bone, the calculated BFR in the IGF-I-deficient animals is ∼50% that of the wild-type controls. The response of these bones to PTH is quite different. PTH stimulated MAR 178% and BFR 166% in the wild-type animals. The responses of the heterozygous bone were less, being 64% and 62% for MAR and BFR, respectively. These responses were all significant. The bones from the IGF-I-deficient animals were totally unresponsive to PTH with respect to MAR and BFR.
Bone mRNA levels
The mRNA levels as determined by quantitative real-time PCR of α1-collagen, alkaline phosphatase and osteocalcin in the femur after 2 weeks of PTH or vehicle administration are shown in Fig. 3 and Table 5. These values are expressed as a percentage of the wild-type control (vehicle treated). All values have been normalized to GAPDH mRNA levels in the same samples. The basal mRNA levels of these osteoblast differentiation markers are quite reduced in the IGF-I-deficient animals (being 8, 45, and 4% of wild-type controls for α1-collagen, alkaline phosphatase, and osteocalcin, respectively). The mRNA levels of α1-collagen and alkaline phosphatase in the heterozygous animals tended to be lower but not significantly lower. In contrast to its failure to increase BFR in the IGF-I-deficient animals, PTH increased the mRNA levels of α1-collagen, alkaline phosphatase, and osteocalcin by 18-, 3.4-, and 25-fold, respectively. This compares with the more modest and insignificant increases in these mRNA levels in the wild-type and heterozygous bones after PTH administration.
Table Table 5.. mRNA Levels in the Whole Femura
We also examined the mRNA levels of the receptors for IGF-I and PTH. These results are found in Table 5. The IGF-I-deficient animals had a 69% increase in IGF-I receptor mRNA levels compared with wild-type controls. The heterozygous animals had a 33% increase, but this was not significant. However, the gene dosage effect by ANOVA was highly significant (p = 0.005). The mRNA levels for the PTH receptor were comparable in all genotypes. PTH administration did not significantly alter IGF-I receptor mRNA levels in any of the genotypes but did significantly increase the PTH receptor mRNA levels in the IGF-I-deficient animals by twofold.
As the anabolic actions of PTH on bone become more appreciated and its therapeutic potential comes closer to being realized,(19) interest in the mechanism(s) by which this anabolic effect is achieved increases. In vivo, the increase in bone formation after PTH administration has been attributed to increased production of osteoprogenitors,(4,5,20) differentiation of osteoblasts from an existing pool of osteoprogenitors,(2,3,21) and decreased apoptosis of preexisting osteoblasts.(6) IGF-I is an attractive candidate as a mediator for some or all of these actions of PTH on bone in that PTH stimulates IGF-I production by bone cells,(9–11) and IGF-1 can reproduce the effects of PTH on bone cell proliferation,(4) bone cell differentiation,(22) and bone cell survival.(23) Furthermore, several of the actions of PTH on bone can be blocked by antibodies to IGF-I.(11,24) Nevertheless, discrepancies among studies in vitro and the difficulty in using antibodies to IGF-I to block PTH action in vivo have handicapped the effort to provide a definitive answer to the role of IGF-I in PTH action on bone.
To address this question, we evaluated a mouse model in which IGF-I production was knocked out by homologous recombination.(13) Previous studies with this mouse model(15,25) and other IGF-I-deficient mouse models(26–28) have clearly shown the need for IGF-I in normal growth including growth of the skeleton, but a careful assessment of the mechanisms involved at least in bone has not been reported. These mice are born at ∼60% of normal weight. Postnatal growth is markedly retarded, such that by the time they reach adulthood (3–4 months) they are ∼20% of normal weight. This suggests that IGF-I may be more important for postnatal development than for in utero development. Our previous observations(29) that in utero both IGF-I and IGF-II are expressed in bone but that after birth IGF-I becomes the dominant IGF in rodents and the observations that IGF-II k/o animals, although small at birth, grow normally postnatally,(30) support this conclusion. The heterozygotes are much less affected than the k/o animals, reaching 80–85% normal size. This suggests some degree of compensation with respect to IGF-I production, in that the serum levels of IGF-I in the heterozygotes are also 80% of wild-type controls. The bones of these animals are reduced in proportion, although when expressed as a function of BW the tibial weight/BW of the IGF-I-deficient mice is greater than their wild-type controls. This is caused by at least in part the greater density of their trabecular bone.
The major question posed by this study is whether PTH would have an anabolic effect in the absence of IGF-I. The clearest answer came when we assessed bone formation. The IGF-I-deficient mice completely failed to respond to PTH with an increase in BFR, unlike their wild-type controls, and the heterozygotes had only a partial response. In an earlier study we(15) had determined that the bones of these mice have a robust (supranormal) response to IGF-I with respect to BFR, indicating that the cellular machinery to make bone is in place; it is just not turned on by PTH in the absence of IGF-I. When we looked at the effects of PTH on FFW and total bone density by pQCT, we were disappointed to see only modest changes in the wild-type animals. Part of the problem may be the limited duration (2 weeks) of PTH administration. However, unlike the anabolic response of trabecular bone to PTH in rats using a comparable protocol,(31) PTH administration to these mice reduced trabecular bone while increasing cortical bone in the proximal tibia. These disparate actions would obscure the effects of PTH when total bone mass (FFW) or density (by pQCT) were evaluated. Nevertheless, whether the catabolic effect of PTH on trabecular bone or the anabolic effect on cortical bone was measured, the k/o mice failed to respond suggesting that IGF-I mediates both the catabolic and the anabolic actions of PTH.
When we explored the nature of this resistance to PTH at the molecular level, we were surprised to find that PTH markedly increased the mRNA levels for markers of bone formation—α1-collagen, alkaline phosphatase, and osteocalcin—in the IGF-I-deficient mice. This stimulation was much greater than that observed in the wild-type or heterozygous bones. Part of this may reflect the increase in PTH receptor levels after PTH in these IGF-I-deficient mice (as assessed by mRNA levels) but also could mean that IGF-I reduces the ability of PTH to stimulate gene expression by some as yet unrecognized mechanism. These mRNA levels are quite reduced in the IGF-I-deficient bone compared with wild-type animals, suggesting that IGF-I is required for their basal expression, but PTH administration clearly is able to overcome whatever inhibition of expression exists in the absence of IGF-I. Furthermore, the mRNA levels for the PTH receptor are normal in IGF-I-deficient bone and appear to increase in response to PTH by a mechanism(s) we do not yet understand. Thus, IGF-I does not appear to be required for PTH to activate at least some functions of the osteoblast. However, the increase in mRNA levels may not lead to increased protein levels. In a study published after our study was originally submitted for publication, Mikakoshi et al.(32) indicated that PTH failed to increase serum osteocalcin and serum and bone alkaline phosphatase activity in the IGF-I-deficient mouse, suggesting that a disconnect may exist between mRNA levels and functional proteins in these animals. Now, the problem is in determining the link and how IGF-I affects that link between the proximal events of osteoblast activation by PTH and the distal events resulting in new bone formation.
We acknowledge the administrative support of Victoria Lee and Vivian Wu and the financial support from the NIH (grant RO1 DK54793) and National Aeronautics and Space Administration (NASA; grant NAG 2-1371).