The authors have no conflict of interest.
Paracrine Overexpression of IGFBP-4 in Osteoblasts of Transgenic Mice Decreases Bone Turnover and Causes Global Growth Retardation†
Article first published online: 1 MAY 2003
Copyright © 2003 ASBMR
Journal of Bone and Mineral Research
Volume 18, Issue 5, pages 836–843, May 2003
How to Cite
Zhang, M., Faugere, M.-C., Malluche, H., Rosen, C. J., Chernausek, S. D. and Clemens, T. L. (2003), Paracrine Overexpression of IGFBP-4 in Osteoblasts of Transgenic Mice Decreases Bone Turnover and Causes Global Growth Retardation. J Bone Miner Res, 18: 836–843. doi: 10.1359/jbmr.2003.18.5.836
- Issue published online: 2 DEC 2009
- Article first published online: 1 MAY 2003
- Manuscript Accepted: 1 NOV 2002
- Manuscript Revised: 9 SEP 2002
- Manuscript Received: 18 JUL 2002
- insulin-like growth factor-1;
- insulin-like growth factor binding proteins;
- transgenic mice;
- bone histomorphometry
Insulin-like growth factor binding protein 4 (IGFBP-4) is abundantly expressed in bone and is generally believed to function as an inhibitor of IGF action. To investigate the function of locally produced IGFBP-4 in bone in vivo, we targeted expression of IGFBP-4 to osteoblasts using a human osteocalcin promoter to direct transgene expression. IGFBP-4 protein levels in calvaria of transgenic (OC-BP4) mice as measured by Western ligand blot were increased 25-fold over the endogenous level. Interestingly, levels of IGFBP-5 were decreased in the OC-BP4 mice, possibly because of a compensatory alteration in IGF-1 action. Morphometric measurements showed a decrease in femoral length and total bone volume in transgenic animals compared with the controls. Quantitative histomorphometry at the distal femur disclosed a striking reduction in bone turnover in the OC-BP4 mice. Osteoblast number/bone length and bone formation rate/bone surface in OC-BP4 mice were approximately one-half that seen in control mice. At birth, OC-BP4 mice were of normal size and weight but exhibited striking postnatal growth retardation. Organ allometry (mg/g body weight) analysis revealed that, whereas most organs exhibited a proportional reduction in weight, calvarial and femoral wet weights were disproportionally small (∼70% and 80% of control, respectively). In conclusion, paracrine overexpression of IGFBP-4 in the bone microenvironment markedly reduced cancellous bone formation and turnover and severely impaired overall postnatal skeletal and somatic growth. We attribute these effects to the sequestration of IGF-1 by IGFBP-4 and consequent impairment of IGF-1 action in skeletal tissue.
The insulin-like growth factors (IGF-1 and IGF-2) control somatic growth and exert profound anabolic effects in most tissues, including bone.(1) Studies in vitro have shown that IGFs stimulate osteoblast replication,(2, g3) enhance differentiation of osteoblasts,(4) promote bone matrix deposition,(5–7) and retard osteoblast apoptosis.(8, 9) The activity of IGF is tempered by a family of at least six IGF binding proteins (designated IGFBP-1 to -6).(3) However, the precise functions of individual IGFBPs are still poorly understood. Some seem to act as carrier proteins to prolong the half-life of IGFs, whereas others are thought to act in an autocrine/paracrine fashion to inhibit IGF actions by preventing access to the receptors or stimulate IGF actions by providing a stable source of available IGFs.(10) Still other data suggest that several of the IGFBPs may exert actions independent of IGF-1.(11)
Among the IGFBPs, IGFBP-4 and IGFBP-5 are major products of the osteoblast. IGFBP-4 is of particular interest because its abundance is regulated by a number of bone active agents, including parathyroid hormone (PTH)(12, 13) and 1,25-(OH)2D3,(14) and thereby might be mechanistically involved in their actions in bone. Although most studies suggest that IGFBP-4 inhibits IGF binding to cell surface receptors(15) and antagonizes IGF's mitogenic effects,(16–19) other reports suggest that IGFBP-4 facilitates delivery of IGF-1.(20, 21) The definition of the mode of action of IGFBP-4 is complicated by the existence of specific proteases that seem to have evolved to attenuate its ability to bind IGF-1.(22–25) Thus, the magnitude and duration of IGF-1 action in a particular tissue seems to depend on the production of IGF-1, the level and complement of IGFBPs, and the extent of proteolysis of the secreted IGFBP.(10) The complexity of the IGF-1 system presents a challenge to experimentalists wishing to define the functions of this growth factor, particularly in vivo.
To investigate the function of locally produced IGFBP-4 in bone in vivo, we targeted overexpression of IGFBP-4 to osteoblasts of transgenic mice using a human osteocalcin (OC) promoter that has been shown to direct transgene expression specifically in osteoblasts and osteocytes.(26) In this way, the actions of IGFBP-4 could be examined within the context of the complex three-dimensional structure of bone tissue to better understand the putative roles of IGFBP-4 in the skeleton.
MATERIALS AND METHODS
Generation of osteocalcin-BP4 mice and examination of transgene expression
The osteocalcin-BP4 chimeric gene (OC-BP4) was constructed by fusing a segment of the human osteocalcin promoter into a construct encoding the second intron of the rabbit β-globin gene upstream of the rat IGFBP-4 cDNA with an SV40 poly A signal. The OC-BP4 transgene was excised by NotI and KpnI digestion and microinjected into the pronuclei of fertilized eggs from the FVB-N mouse strain at the transgenic core facility of the University of Cincinnati (Cincinnati, OH, USA). Transgenic founders were identified by Southern analysis and bred with wild-type FVB-N mice to produce mice heterozygous for the transgene. For routine genotyping of progeny, the IGFBP-4 transgene was detected by polymerase chain reaction (PCR; 94°C 1 minute, 53°C 1 minute, 72°C 1 minute, for 30 cycles) with the following primers: 5′-CAAATAGCCCTGGCAGATTC-3′ (forward) and 5′-TGATACAAGGGACATCTTCC-3′ (reverse) to generate a 260-bp product corresponding to a segment of the OC promoter and the rabbit β-globin intron. All animals received humane care in compliance with the local Institutional Animal Care and Use Committee.
RNA isolation and Northern blot analysis
Total RNA was isolated from tissues by a single-step acid guanidinium thiocyanate-phenol-chloroform extraction. Northern analyses were performed as described.(26) Briefly, 10 μg of tissue total RNA were gel-separated, transferred to a nylon membrane, and hybridized with random primed rat IGFBP-4 cDNA. 18s rRNA was used for comparisons between different tissues.
Western ligand blotting
To determine the skeletal and serum content of IGFBPs, Western ligand blotting was performed as previously described.(27) Femurs were pulverized in liquid nitrogen with mortar and pestle and then homogenized in Trizol reagent (Life Technologies, Rockville, MD, USA). The organic phase was processed following the manufacturer's instructions, and the resultant extract resuspended in guaninidium HCl. Protein concentration was determined by micro-BCA assay (Pierce, Rockford, IL, USA), and 100 μg of total protein (per lane) was fractionated by nonreducing SDS-PAGE. Proteins were then transferred to a nylon membrane and probed with125I-labeled IGF-1 overnight at 4°C. The blot was then washed, and radio-labeling was assessed by phosphor-imaging (PhosphorImager; Molecular Dynamics, Sunnyvale, CA, USA).
Volumetric assessment of bone morphology
Peripheral quantitative computed tomography (pQCT) was performed as described previously.(28) Briefly, femurs were analyzed by a Stratec XCT(960 M; Norland Medical Systems, Ft. Atkinson, WI, USA) for both total femur density and diaphyseal cortical density. A threshold of 1.300 attenuation units differentiated mouse bones from water, adipose tissue, muscle, and tendon; a threshold of 2.000 differentiated high-density cortical bone from low-density bone. Calibration of the densitometer was routinely performed with a set of hydroxyapatite standards. The precision of the XCT 960M for repeated measurements of the femur and vertebrae was 1.2% and 1.4%, respectively. The coefficients of variation in the femoral parameters from 4-month-old animals was 3% for bone mineral density (BMD), 9% for mineral, 7% for volume, 3% for mid-diaphyseal periosteal circumference, and 4% for cortical thickness. Femoral periosteal circumference and cortical thickness were calculated at the midpoint of the diaphysis.
Mineralized bone histology and bone morphometry
Distal femurs were fixed in 100% ethanol. After dehydration, bone samples were embedded in methyl methacrylate,(29) and 4-μm sections were cut with a heavy-duty microtome (Microm; Zeiss, Thornwood, NY, USA). This thickness allows analysis of the same histological features in slides prepared for normal and fluorescent microscopy. Four bone sections were stained using the modified Masson-Goldner trichrome technique,(30) and four other sections serial to the stained sections were left unstained for fluorescent microscopy.
Static and dynamic parameters of bone structure, formation, and resorption were measured using a semiautomatic method (Osteoplan II; Kontron, Munich, Germany) as described previously.(26, 31) Briefly, the semiautomatic computerized system involves the use of a microscope equipped with a drawing tube overlying a digitalized tablet. The histological field is projected on the tablet, which is linked to a cursor. Using the cursor, the operator circuits, traces, and counts histological profiles. Data in two dimensions, that is, area, length, distance, are then transformed in three dimensions as volume, surface, and thickness based on stereological principles. Measurements were made at a magnification of 500× and were confined to the secondary spongiosa of the distal femur to ensure that only remodeling sites were analyzed. For dynamic endpoints, 3- and 6-week-old animals were injected with calcein peritoneally on days 1 and 4 and killed 2 or 3 days later. In the present study, the following parameters of bone formation were obtained: osteoid volume/bone volume (OV/BV; %), osteoid surface/bone surface (OS/BS; %), osteoid thickness (O.Th; μm), osteoblast surface/bone surface (Ob.S/BS; %), and number of osteoblasts/perimeter length (Nob/BPm; #/100 mm). The parameters of bone erosion encompassed erosion surface/bone surface (ES/BS; %), osteoclast surface/bone surface (Oc.S/BS; %), and number of osteoclasts per bone perimeter (NOc/BPm: #/100 mm). Parameters of bone dynamics included mineral apposition rate (MAR; μm/day), mineralizing surface/bone surface (MS/BS; %), bone formation rate per bone surface (BFR/BS; mm3/cm2/year), bone formation rate per osteoblast (BFR/Ob; mm2/ob/year), and mineralization lag time (Mlt; days). All parameters comply with the recommendations of the Histomorphometry Nomenclature Committee of the American Society of Bone and Mineral Research.(32)
Body weight and organ allometry
Transgenic mice and their nontransgenic littermates were weighed weekly for up to 14 weeks postnatally. Groups of mice were killed by CO2 asphyxiation. After determining the body weight, blood was collected by cardiac puncture, and serum was stored at −80°C. Major organs were collected and weighed. The contents of the stomach were flushed out with PBS before weighing. Calvaria and femurs were trimmed of soft tissue, weighed, and then fixed in 95% ethanol for histomorphometric analysis. Femur length was measured using calipers.
Histomorphometry data are expressed as mean ± SE and tests were two-sided. An assigned significance level of 0.05 was used. Comparability of the two groups at any given time was assessed using the Mann-Whitney U test or unpaired t-test. Comparability of data from a group at different time points was done using the Kruskal-Wallis H test. Computations were performed using the SPSS software package for Windows release 7.5 (SPSS, Chicago, IL, USA). All other results were analyzed using Student's t-test performed with GraphPad Prism version 3.02 (GraphPad Software Inc., San Diego, CA, USA).
Expression of the OC-BP4 transgene
Five OC-BP4 founder mice were obtained and propagated. The level of expression of the IGFBP-4 transgene mRNA in calvaria from 3-week-old OC-BP4 mice varied among these five lines as shown in Fig. 1B. Two lines with high levels of mRNA expression in bone (12 and 13) were selected for further analysis. The tissue distribution of endogenous and transgene IGFBP-4 mRNA in line 12 is shown in Fig. 1C. The endogenous IGFBP-4 mRNA transcript was detected in the liver and kidney. The transgenic mRNA transcript was expressed at high levels in bone, including calvaria, femur, and vertebrae, and was undetectable in other tissues. Similar results were obtained in tissues from line 13 (data not shown).
The level of IGFBP-4 protein in tibias from 6-week-old mice of line 12 measured by Western ligand blot was increased 25-fold over that observed in control mice (Fig. 2). Interestingly, the increased IGFBP-4 protein levels in bone were associated with decreased levels of IGFBP-5. Tibia from line 13 showed a 5-fold increase of IGFBP-4 compared with control (data not shown).
Overexpression of IGFBP-4 in osteoblasts decreases femoral size, volume, and cortical bone density
To investigate the impact of IGFBP-4 overexpression on bone morphology, pQCT measurements were made on femurs from groups of 6-week-old OC-BP4 (line 12) and control nontransgenic littermates. Total femur length (determined using calipers), volume, and mineral content were similarly reduced in both male and female transgenic mice (Table 1), as was total cortical volume and mineral content. There was no significant change in BMD of the whole femur, although the total cortical density was significantly reduced in female transgenic mice. Measurements at the central diaphysis revealed a significant decrease in periosteal circumference, cortical thickness, and density in the OC-BP4 transgenic mice compared with controls (Table 1). There were no statistical differences in morphometric parameters between male and female mice.
Overexpression of IGFBP-4 in osteoblasts decreases bone formation rate
Static and dynamic histomorphometric measurements were performed at the distal femur on groups of female OC-BP4 (n = 5) and control mice (n = 6) at 6 weeks of age (Table 2). Bone formation rate and mineral apposition rate were dramatically reduced in transgenic mice (Table 2) compared with controls. These changes were associated with a significant reduction in osteoid volume, osteoid surface, osteoblast surface, and the number of osteoblasts. Mineral apposition rate was significantly (p < 0.05) reduced in femurs from the second (line 13) transgenic line (2.10 ± 0.7 for transgenic vs. 2.52 ± 1.3 μm/day for controls, n = 6). No statistical differences were observed in any other histological parameter in femurs of mice from this line, likely because of the lower level of transgene expression.
Overexpression of IGFBP-4 in osteoblasts causes global growth retardation and reduced bone size
Total body size and weight was determined in mice derived from line 12 at weekly intervals for up to 14 weeks postnatally. As shown in Fig. 3A, both male and female transgenic mice exhibited markedly reduced body weight compared with nontransgenic littermates. At birth, total body weights were slightly reduced in transgenic versus control mice (Fig. 3B). When expressed as a percentage of the control mice, body weights of the transgenic mice declined by approximately 25% over the first 3 to 4 weeks of age. No further decline in body weights relative to control occurred after 4 weeks. Body weights of mice from line 13 were also consistently lower than the controls, although these differences did not reach statistical significance (data not shown).
To determine the effect of IGFBP-4 overexpression on organ weights, allometry was performed on 6-week-old animals. Femurs and calvaria were disproportionally small relative to other organs, being 70-80% of that expected for the degree of overall body size reduction (Fig. 4A). Most nonskeletal organs exhibited a proportional reduction in weight, with the exception of brain, kidney, and stomach (females only), which were disproportionally large compared with controls (Fig. 4B).
Serum levels of IGFBP-4
To determine whether overexpression of IGFBP-4 in osteoblasts altered circulating IGFBP-4, the serum levels of IGFBPs were examined by Western ligand blotting (line 12). At 3 and 6 weeks of age, IGFBP-4 levels were not significantly different between the transgenic and control mice (Fig. 5). However, 5-day-old transgenic mice had elevated serum IGFBP-4 levels.
In this study, we investigated the skeletal functions of IGFBP-4 in vivo by targeting expression to osteoblasts. Examination of the bone structure and morphology revealed that overexpression of IGFBP-4 caused a reduction in bone volume and cortical bone density consistent with the notion that osteoblast-derived IGFBP-4 inhibits IGF-1 action in bone when present in excess. The marked reduction in bone formation observed in the OC-BP4 transgenic mice is most likely the result of the decreased number and activity of osteoblasts. These observations are consistent with previous in vitro studies in which IGFBP-4 attenuated IGF-1 stimulation of osteoblast proliferation.(15) There was also a reduction (albeit not statistically significant) in osteoclasts, suggesting coupling of formation and resorptive signals emanating from osteoblasts.
Comparison of the changes in bone volume in the OC-BP4 overexpressing mice to those observed previously in mice of the same strain overexpressing an OC-IGF-1 transgene(26) provides an estimate of the magnitude of the effect of IGF-1 on mouse bone acquisition in the postnatal period. Total bone volume (TB/TV) in 6-week-old IGF-1 overexpressing mice was 11.4% (22% > controls) compared with only 2.8% (60%< control) in the IGFBP-4 transgenics. This constitutes a 4-fold difference in bone volume, underscoring the critical importance of this growth factor in postnatal bone acquisition. However, it is important to note that the changes in bone phenotype observed in these two mouse models are not diametrically opposite as might be expected. For example, the long bones from the OC-IGF-1 mice were of normal size and demonstrated an increased bone formation rate in the absence of an increase in the total number of osteoblasts.(26) It is possible that this difference in skeletal phenotype could be because of a difference in partitioning of IGF-1 and IGFBP-4 in the bone microenvironment of the two mouse models. Circulating levels of IGFBP-4 were slightly elevated in the OC-BP4 transgenic mice in the immediate postnatal period, suggesting that the binding protein escaped the local osteoblast microenvironment that might have enabled it to inhibit IGF-1 action on stromal osteoblast precursors and preosteoblasts. In contrast, circulating IGF-1 levels were not elevated in the OC-IGF-1 overexpressing mice, indicating that IGF-1 remained more restricted to osteoblasts and osteocytes. Consequently, it is reasonable to propose that the marked reduction in osteoblast number and bone formation in the OC-BP4 transgenic mice reflects a more widespread inhibition of IGF-1 action in the bone.
Also notable was the finding that overexpression of IGFBP-4 resulted in a decreased level of IGFBP-5 in bone. IGF-1 has been reported to stimulate the synthesis of IGFBP-5,(33, 34) and IGFBP-5 sequesters IGF-1, thereby stabilizing it from proteolytic degradation.(35) Therefore, the reduction of IGFBP-5 is likely a compensatory response to sequestration of IGF-1 by IGFBP-4, resulting either from reduced production or enhanced degradation of IGFBP-5. Consistent with this observation, mice with targeted overexpression of IGF-1 show an increase in IGFBP-5.(36)
The reduction in growth of the OC-BP4 transgenic mice that occurred over the first 3-4 weeks after birth was unexpected and remains incompletely explained. Whereas most nonskeletal organs were proportionally reduced in weight, all bones examined were disproportionately small. The most straightforward explanation for the growth attenuation is that the increased skeletal levels of IGFBP-4 inhibited IGF-1 action during bone growth, which in turn stunted overall growth and organ development. The concept that skeletal growth can influence general somatic growth has been suggested,(37) but precisely how such an interaction would occur remains unclear. On the other hand, it might be argued that the generalized growth retardation resulted from exposure of other tissues to IGFBP-4 that was either promiscuously expressed during development or that escaped from skeletal tissue into the circulation. However, these alternative scenarios seem unlikely. First, our recent studies of the timing and tissue distribution of the human OC-Cre transgene show that the promoter is first active at day E17, is entirely restricted to osteoblasts and osteocytes, and is not expressed in growth plate chondrocytes.(38) Furthermore, the circulating concentrations of IGFBP-4 over the first 3 weeks postnatally do not seem high enough to yield such dramatic growth retardation. In addition, serum IGFBP-4 is increased in 5-day-old OC-BP4 mice, but it is not the predominant IGFBP, and at 3 weeks, the difference is even less striking. Perhaps more interesting is the possibility that the changes in the operation of the GH/IGF axis during development altered the dynamics of response to the increase in bone IGFBP-4. It is known, for example, that mouse growth during the first 3 weeks is dependent on paracrine actions of IGF-1 and is largely independent of GH.(39) Thereafter, IGFBP-3 concentrations increase and growth is more dependent on GH action. The anti-IGF-1 effects of IGFBP-4 are therefore potentially ameliorated by either direct actions of GH, provision of increased IGF-1 through the circulation, or the altered complement of IGFBPs within bone. Clearly, however, further studies are needed to clarify the mechanisms that account for somatic growth retardation in these animals.
In summary, overexpression of IGFBP-4 in bone osteoblasts reduces bone formation and severely impairs skeletal growth. We attribute these effects to the sequestration of IGF-1 by IGFBP-4 with consequent impairment of IGF-1 actions. The creation of this transgenic mouse model should enable additional studies on the interplay between IGF-1 and IGFBP-4 in bone.
The authors thank Lisa Clayton for performing the Western ligand blots and William Stuart for help in preparing the manuscript. A Merit Review grant from the Veterans Administration to TLC supported this work.
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