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Endocrinology Vol. 143, No. 5 1942-1949
Copyright © 2002 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Rescue of the Osteopetrotic Defect in op/op Mice by Osteoblast-Specific Targeting of Soluble Colony-Stimulating Factor-1

S. L. Abboud, K. Woodruff, C. Liu, V. Shen and N. Ghosh-Choudhury

Department of Pathology (S.L.A., K.W., N.G.-C.), University of Texas Health Science Center and the South Texas Veteran’s Health Care System, Audie L. Murphy Division, San Antonio, Texas 78284; and Skeletech Inc. (C.L., V.S.), Bothell, Washington 98021

Address all correspondence and requests for reprints to: Sherry L. Abboud, M.D., Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284. E-mail: . abbouds{at}uthscsa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Soluble colony-stimulating factor-1 (sCSF-1) and membrane bound CSF-1 are synthesized by osteoblasts and stromal cells. However, the precise role of each form in osteoclastogenesis is unclear. In the op/op mouse, absence of osteoblast-derived CSF-1 leads to decreased osteoclasts and osteopetrosis. To determine whether sCSF-1 gene replacement can cure the osteopetrotic defect, we took advantage of the osteoblast specificity of the osteocalcin promoter to selectively express sCSF-1 in the bone of op/op mice. Transgenic mice harboring the human sCSF-1 cDNA under the control of the osteocalcin promoter were generated and cross-bred with heterozygous op/wt mice to establish op/op mutants expressing the transgene (op/opT). The op/op genotype and transgene expression were confirmed by PCR and Southern blot analysis, respectively. High levels of human sCSF-1 protein were selectively expressed in bone. At 21/2 wk, op/opT mice showed normal growth and tooth eruption. Femurs removed at 5 and 14 wk were analyzed by peripheral quantitative computed tomography and histomorphometry. The abnormal bone mineral density, cancellous bone volume, and growth plate width observed in op/op mice was completely reversed in op/opT mice by 5 wk, and this effect persisted at 14 wk, with measurements comparable with wt/wt mice at each time point. Correction of the skeletal abnormalities in the 5-wk-old op/opT mice correlated with a marked increase in the total osteoclast number, and their number per millimeter of bone surface compared with that of op/op mutants. Osteoclast number was maintained at 14 wk in op/opT mice and morphologically resembled wt/wt osteoclasts. These results indicate that sCSF-1 is sufficient to drive normal osteoclast development and that the osteocalcin promoter provides an efficient tool for delivery of exogenous genes to the bone. Moreover, targeting sCSF-1 to osteoblasts in the bone microenvironment may be a potentially useful therapeutic modality for treating bone disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MACROPHAGE COLONY-STIMULATING factor (CSF-1) is a key regulatory molecule for cells of the mononuclear phagocyte lineage, including monocytes, tissue macrophages, microglia, and osteoclasts (1). Activation of macrophages in multiple tissues has been shown to mediate, at least in part, the diverse biological effects of CSF-1 on male and female fertility, trophoblastic implantation, mammary gland development, dermal thickness, and neural function (2, 3, 4, 5). In bone, CSF-1 stimulates the proliferation and differentiation of osteoclast progenitors and enhances osteoclast survival (6, 7). Osteoblasts and stromal cells are the main source of CSF-1 in the bone microenvironment, and these cells synthesize both the soluble form of CSF-1 (sCSF-1) and the membrane bound form (8). However, the precise biological effect of each form on osteoclastogenesis in vivo is unknown. sCSF-1 and membrane bound CSF-1 are derived by posttranscriptional processing of a common CSF-1 transcript, and the soluble form is rapidly secreted into the circulation as either a glycoprotein or proteoglycan, whereas the membrane bound form is expressed as an integral transmembrane glycoprotein (9, 10, 11).

The importance of CSF-1 in osteoclast development has been demonstrated in studies using the osteopetrotic (op/op) mouse model. In the op/op mutant, a thymidine insertion in the coding sequence of the CSF-1 gene results in CSF-1 deficiency that, in turn, leads to decreased macrophage and osteoclast production (12, 13, 14). By 10 d of age, op/op mice develop an osteopetrotic phenotype characterized by stunted growth, absence of tooth eruption, and a domed skull. Failure of bone marrow transplantation to rescue the osteopetrotic phenotype and the inability of op/op osteoblasts to support osteoclast formation in vitro indicate that the primary defect in these mice is caused by a lack of osteoblast-derived CSF-1 (15, 16). The potential of sCSF-1 to cure the osteopetrotic defect has been controversial (17, 18, 19, 20). Sundquist et al. (20) showed that restoration of physiological concentrations of circulating CSF-1 in op/op mice, from 1 d after birth, for 4 wk, only partially rescued the osteopetrosis, with persistent metaphyseal sclerosis possibly attributable to inadequate delivery of recombinant human CSF-1 to this site or to a lack of membrane bound CSF-1. Alternatively, the discrepancy between this and other studies may be attributable to differences in the dose or duration of CSF-1 administration. When Wiktor-Jedrzejczak et al. (19) administered high concentrations of recombinant human CSF-1 to 6-d-old op/op mice for 2 months, circulating CSF-1 was restored to normal levels, body weight was partially corrected, and osteopetrosis was resolved. However, osteopetrosis relapsed 1 month after cessation of CSF-1 therapy, and it could not be reversed in mice treated after 7 d of age, suggesting that early and sustained postnatal CSF-1 protein expression in the bone microenvironment is required for complete remission of the osteopetrotic defect.

Osteoblasts play a key role in modulating osteoclast function via the release of cytokines and through cell-cell interaction (21, 22). Therefore, they provide a useful in vivo target for delivering exogenous genes that regulate osteoclastogenesis. In the present study, we took advantage of the tissue specificity of the osteocalcin promoter to selectively target sCSF-1 to osteoblasts in the bone microenvironment (23, 24). Osteocalcin is an abundant bone matrix protein expressed by mature osteoblasts but not by osteoprogenitor cells. To determine whether sCSF-1 can rescue the osteopetrotic defect, op/op mice carrying an osteocalcin promoter-driven human sCSF-1 transgene (op/opT) were generated. At 5 and 14 wk, resolution of osteopetrosis was assessed using peripheral quantitative computed tomography (pQCT) scanning, tartrate resistant acid phosphatase (TRAP) staining of osteoclasts on histologic preparations, and histomorphometric analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Transgenic mice were prepared using the B6C3 strain, which is the same background as the op/op mouse. These mice and heterozygote mice for the op/op mutation (op/wt) were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained and used according to the principals outlined in the Guide for the Care and Use of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, National Research Council.

Generation of op/op mice expressing sCSF-1
The transgene consists of the osteocalcin promoter linked to the sCSF-1 cDNA. The 1.8-kb 5'-flanking sequence of the rat osteocalcin promoter in sp64 (Genentech, Inc., San Francisco, CA) was digested with SmaI-HindIII, subcloned into the HincII site of pUC7, and then inserted into the BAMHI site of the pBSpKCR3 vector (24). The 1.8-kb CSF-1 cDNA that encodes the full-length human sCSF-1 was excised from p3ACSF-RI (Genetics Institute, Cambridge, MA) with XhoI-EcoRI, subcloned into the HincII site of pUC7 and then inserted into the EcoRI-digested pBSpKCR3 (obtained from Dr. Windle, University of Texas Health Science Center, San Antonio, TX) (25). In this vector, the sCSF-1 cDNA is inserted into a fragment of the rabbit ß-globin gene that provides an intron and polyadenylation signal required for efficient expression of the transgene. Transgenic mice were generated, according to standard methods, at the San Antonio Cancer Institute Transgenic Facility, University of Texas Health Science Center, San Antonio (26). The ClaI-NotI-digested fragment was microinjected into fertilized eggs derived from the mating of B6C3 mice and implanted into the oviducts of pseudopregnant CD-1 foster females. Offspring were screened for the transgene by Southern blot hybridization of tail DNA with the osteocalcin-CSF-1 injection fragment. Three founders were identified with 4–5 copies of the transgene per haploid genome. Transgenic lines were established by breeding founders to B6C3 mice. One line, showing the highest CSF-1 protein levels in bone lysates, was selected for breeding with heterozygous op/wt mice to generate CSF-1 transgenic op/wt (op/wtT) mice. These mice were then interbred to establish op/op mice expressing the transgene (op/opT). At 5 and 14 wk, op/opT mice were killed and weighed, and bone and plasma CSF-1 protein levels were determined. Age-matched wt/wt and op/op littermates served as normal and mutant controls, respectively. Femurs and tibias were excised, and a portion was frozen in liquid nitrogen for CSF-1 protein analysis. The remaining bones were fixed in 10% formalin for 2 d before pQCT, histologic, and histomorphometric analysis. Four to five femurs were analyzed at each time point, and results are expressed as mean ± SE

Analysis of transgene expression
Human CSF-1 protein levels were measured in plasma, bone, and tissue extracts. Two tibias were rapidly dissected and frozen in liquid nitrogen before being crushed and homogenized in TENES V buffer [50 mM Tris-HCl (pH 7.4), 1% NP-40, 2 mM EDTA, 100 mM NaCl, 10 mM sodium oxyvanadate] containing proteinase inhibitors (1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin). Tissues harvested from mice were frozen and then homogenized in the same buffer. After centrifugation, bone, tissue, and plasma samples were diluted 1:2–1:6 and assayed using the human Quantikine enzyme-linked immunoassay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. CSF-1 concentrations were calculated from a standard curve (31.2–2000 pg/ml) using log-log linear regression. This assay specifically detects human CSF-1 and shows negligible species cross-reactivity. The sensitivity of the test is less than 9 pg/ml.

PCR detection of op genotype
The op allele was identified using PCR to amplify the segment containing the op mutation on chromosome 3, as previously described (27). Genomic DNA from tails was extracted using the QIAmp Tissue Kit (QIAGEN, Chatsworth, CA). Two primers (5'-TG TGTCCCTTCCTCAGATTACA-3' and 5'-GGTCTCATCTATTATGTCTTGTACCAGCCAAAA-3'), designed to generate PCR products of 195 bp (wt allele) or 196 bp (op allele), were used. A 2-bp mismatch in the 3'-antisense primer (underlined) introduced a second BglI site into the PCR product spanning the extra base of the op mutation that was absent from the PCR product from the wild-type template. Conditions for PCR reactions were 3 min at 94 C; 1 min at 94 C, 2 min at 62 C, and 2 min at 72 C for 40 cycles; and 10 min at 72 C. Twenty microliters of PCR product was then digested with 1 µl BglI for 2 h. The fragments were separated by electrophoresis in a 4% Metaphor agarose gel and visualized by ethidium bromide staining.

Bone mineral density (BMD) measurement
BMD of the femurs was measured by pQCT (RM 3000 pQCT, Norland Medical Systems, Inc., Fort Atkinson, WI). Quantitative readings were obtained at the metaphysis (12% from the distal end) and diaphysis (50% from the distal end). At each site, a 0.5-mm segment was scanned, and the BMD was calculated using small-animal software.

Histology and histomorphometric measurements
The distal half of each femur was decalcified in 10% sodium EDTA in 0.1 M phosphate buffer (pH 7.0) at 4 C for 4 d. The samples were dehydrated through standard graded alcohol solutions and embedded in glycol methacrylate using a JB-4 embedding kit (Polysciences, Inc., Warrington, PA). Tissues were sectioned longitudinally, at 4 mm, using a Jung Ultracut microtome (Reichert-Jung, Heidelberg, Germany), and the sections were stained for tartrate-resistant acid phosphatase activity followed by thionin green counterstaining (28). Static parameters were measured in a 2-mm square, 1 mm distal to the lowest point of the growth plate in the secondary spongiosa. Bone and osteoclast surfaces were traced; and cancellous bone volume (BV/TV), trabecular measurements, osteoclast numbers, and surfaces were calculated using Osteomeasure software (Osteometrics, Atlanta, GA), as we have previously described (29). The growth plate width was the average of the measurements at four equal distances along the growth plate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation and identification of op/op mice expressing the sCSF-1 transgene (op/opT)
Transgenic mice harboring the full-length human sCSF-1 cDNA driven by a 1.8-kb osteocalcin promoter were generated (CSF-1T). Three founders were identified; however, only one transgenic line was shown to express sCSF-1. By 14 wk of age, high human sCSF-1 levels were detected in bone extracts of these transgenic animals (1,588 pg/mg protein), whereas plasma levels were 247 pg/ml. To further confirm tissue specificity of the transgene, CSF-1T tissue extracts were assayed for human sCSF-1 protein. Little or no sCSF-1 was detected in tissues other than bone, with results (in pg/mg protein) showing: 1.19 in brain, 1.81 in heart, not detected in lung, 4.51 in thymus, 0.69 in liver, 2.98 in spleen, 0.84 in kidney, 0.55 in gut, and 5.42 in muscle.

These CSF-1T mice were then used to generate op/opT mice, and the op/op genotype was confirmed by PCR analysis as shown in Fig. 1Go (left panel). An ethidium bromide-stained 4% agarose gel of BglI-digested PCR products shows a 96-bp fragment in all cases and fragments diagnostic of either the op allele (70 and 30 bp) or wt allele (99 bp). DNA isolated from transgenic mice in lanes 1 and 2 shows the wt/wt genotype, whereas DNA in lanes 7 and 8 shows the op/op genotype. DNA from op/wt mice in lanes 3–6 shows the expected 99- and 96-bp fragments, along with the 70- and 30-bp fragments, which stain less intensely than those observed in op/op mice because of the presence of only a single op allele. Ten days after birth, op/opT mice began to show tooth eruption; and, by 21/2 wk, eruption of the upper and lower incisors was comparable with wt/wt littermates, whereas op/op mice remained toothless (Fig. 1Go, right panel).



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  		Figure 1. Identification of the op genotype using PCR analysis (left panel) and correction of tooth eruption in op/opT mice (right panel). Ethidium bromide-stained 4% agarose gel of BglI-digested PCR products generated from wt/wt (lanes 1 and 2), op/wt (lanes 3–6), and op/op (lanes 7 and 8) templates shows fragments diagnostic of the op allele (70 and 30 bp) and wt allele (99 bp). At 21/2 wk, op/opT mice show eruption of upper and lower incisors comparable with those of wt/wt littermates, whereas op/op mice are toothless.

 
Analysis of CSF-1 transgene expression and body weight of op/opT mice
To determine the level of transgene expression in op/opT mice, CSF-1 protein levels were measured in bone extracts, and plasma samples were obtained from wt/wt and op/opT mice at 5 and 14 wk. High levels of human sCSF-1 protein were selectively expressed in the bone and plasma of 5-wk-old op/opT mice (1,570 pg/mg protein and 1,362 pg/ml, respectively). This effect persisted in bone at 14 wk; however, plasma levels declined to 315 pg/ml. At 5 wk, the weight of op/op mice was approximately half that of the wt littermates. Expression of sCSF-1 in op/op mice was sufficient to correct their growth rate, with the body weight of op/opT mice similar to that of the wt/wt controls at 5 and 14 wk.

Radiographic and BMD measurements in op/opT mice
Skeletal growth and development in op/opT mice were significantly improved, compared with op/op mice. As shown in Fig. 2Go, x-rays of op/op mice at 5 and 14 wk show marked skeletal sclerosis, with short and thickened long bones. There is dense, radiopaque bone in the iliac crest, and in the tibial and femoral metaphysis, with obliteration of marrow spaces that normally appear radiolucent. The caudal vertebrae of the tail also appear sclerotic. In contrast, op/opT mice show resolution of metaphyseal sclerosis by 5 wk and radiolucent marrow spaces in the iliac crest, caudal vertebrae, tibial, and femoral metaphyses comparable with wt/wt controls at 5 and 14 wk.



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  		Figure 2. Comparative radiographic findings in wt/wt, op/opT, and op/op mice at 5 and 14 wk. At each time point, op/op mutants show skeletal sclerosis with dense, radiopaque bone in the iliac crest, tibial, and femoral metaphysis (arrows) and loss of marrow spaces. In contrast, 5- and 14-wk op/opT mice lack metaphyseal sclerosis and show radiolucent marrow spaces comparable with wt/wt controls.

 
To more accurately evaluate the skeletal changes in op/opT mice, BMD measurements of the femurs, using pQCT, were performed. pQCT images of the metaphysis and diaphysis of femurs, isolated from wt/wt, op/opT, and op/op mice at 5 and 14 wk, are shown in Fig. 3AGo. At 5 and 14 wk, the metaphysis in op/op mice shows an osteopetrotic pattern characterized by lack of a clear cortical envelope and calcified cartilage in all four quadrants. The diaphysis at 5 wk shows an abundant amount of unresorbed bone with marked narrowing of the marrow cavity. By 14 wk, there is early expansion of the marrow cavity, with this region containing bone of increased density, compared with op/opT and wt/wt mice. These abnormalities were completely reversed in 5-wk-old op/pT mice and persisted at 14 wk, with the metaphysis showing features of normal cancellous bone and the diaphysis showing bone of normal thickness with an enlarged marrow cavity comparable with wt/wt mice at each time point. Quantitative analysis of BMD at each site confirms these findings, with equivalent measurements observed in 5- and 14-wk op/opT and wt/wt mice. As shown in Fig. 3BGo, the increased BMD in the femoral diaphysis of op/op mice declined to close-to-normal values of 572 mg/cm3 in op/opT mice by 5 wk. With age, the BMD in the femoral diaphysis increased in op/opT mice and was comparable with wt littermates. The BMD in the femoral metaphysis of op/opT mice was also similar to wt/wt mice, averaging 423 mg/cm3 at 5 wk and 545 mg/cm3 at 14 wk. Although the BMD in the femoral metaphysis of op/op mice at 5 and 14 wk (data not shown at 14 wk) seems similar to that observed in op/opT and wt/wt mice, BMD cannot be accurately determined in this site in op/op femurs because of the limitations of the procedure to detect the abnormal calcified cartilage. At 14 wk, BMD in the femoral diaphysis of op/op mice (data not shown) was also similar to that observed in op/opT and wt/wt mice. However, the relatively higher BMD in op/op mice was not reflected in this measurement because of the threshold value assigned for cortical bone by the analysis system.



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  		Figure 3. Comparative BMD measurements in wt/wt, op/opT, and op/op mice at 5 and 14 wk. A, pQCT images of femurs were obtained at the metaphysis (left side of each panel) and diaphysis (right side of each panel). The color bars indicate increasing BMD as the color intensity declines. In 5- and 14-wk op/op mutants, the metaphysis shows an osteopetrotic pattern with lack of a clear cortical envelope and calcified cartilage in four quadrants (asterisk). The diaphysis shows increased unresorbed bone with marked narrowing of the marrow space at 5 wk and early expansion of the marrow cavity, visualized as an enlarged area of intermediate bone density, by 14 wk (open arrows). In contrast, BMD in op/opT mice was similar to that in wt/wt mice at each time point, with the metaphysis showing normal cancellous bone (arrowheads) and the diaphysis showing bone of normal thickness with a patent marrow cavity (arrows). B, Quantitative analysis of BMD at each site in 5- and 14-wk-old wt/wt, op/opT, and op/op mice (n = 5 mice/group). Data are expressed as mean ± SE.

 
Histologic and histomorphometric analysis of femurs in op/opT mice
Correction of the BMD in op/opT mice was visualized at the histologic level by resolution of osteopetrosis and restoration of the osteoclast population. Figure 4Go shows histologic preparations of the distal femoral metaphysis from wt/wt, op/opT, and op/op mice. In 5- and 14-wk-old op/op mutants, there are thick irregular bars of calcified cartilage below the epiphyseal plate that extend into the metaphysis and replace the marrow cavity. Few TRAP-positive cells are identified in the metaphysis or along the thick cartilaginous bars. At 5 wk, op/opT mice are completely rescued, and this effect persisted at 14 wk, with histologic findings comparable with wt/wt mice at each time point. In these femurs, metaphyseal sclerosis is absent, numerous TRAP-positive osteoclasts are identified in the metaphysis and along thin bony trabeculae, and the marrow cavity is expanded with normal hematopoietic elements.



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  		Figure 4. Comparative histological analysis of the distal femoral metaphysis from wt/wt, op/opT, and op/op mice. Decalcified, plastic-embedded tissue was sectioned in the midsagittal plane and stained for TRAP activity, followed by thionin green counterstaining. The epiphyseal plate (E) and metaphysis are shown at the top of each field. In 5- and 14-wk-old op/op mutants, thick, irregular bars of calcified cartilage extend from the epiphyseal plate into the marrow cavity (M), with few TRAP-positive cells identified. By 5 wk, op/opT mice are completely rescued, and this effect persisted at 14 wk (400x magnification).

 
The distal femoral metaphysis of op/opT, compared with wt/wt and op/op mice, was studied in more detail using histomorphometric analysis as shown in Table 1Go. The abnormal BV/TV observed in op/op mice was completely normalized in op/opT mice by 5 wk and remained comparable with wt/wt mice at 14 wk, with values of 17% and 23%, respectively. Similarly, the increased width of the growth plate in 5-wk-old op/op mice normalized in op/opT mice to 179 mm by 5 wk, an effect that persisted for 14 wk. Recovery of BV/TV and growth plate width in op/opT mice at 5 wk correlated with a marked increase in osteoclast numbers, compared with op/op mutants. At 5 wk, osteoclasts were barely detectable in op/op mice, whereas approximately 50–55 osteoclasts were measured in both op/opT and wt/wt mice. Osteoclasts remained elevated at 14 wk and mimicked the level observed in wt/wt mice. Moreover, the number of osteoclasts per millimeter of bone surface in op/opT mice was comparable with wt/wt mice. Osteoclasts in op/opT mice were multinucleated, stained strongly with TRAP, and morphologically resembled wt/wt osteoclasts, whereas those in op/op mice tended to be mononucleated and weakly positive for TRAP staining. Other histomorphometric parameters were also corrected in op/opT mice, including trabecular thickness, number and separation, total length of osteoclast surface, and percent bone surface covered by osteoclasts (osteoclast surface/BS); and results were not significantly different between op/opT and wt/wt groups.


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  		Table 1. Histomorphometric measurements

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results from this study indicate that osteoblast-targeted sCSF-1 gene expression is sufficient to drive normal osteoclast development. In op/opT mice, the osteocalcin promoter showed efficient transcriptional activity, with high levels of sCSF-1 detected in bone for at least 14 wk. This prevented the development of osteopetrosis and maintained a remarkably normal skeletal phenotype in adult op/op mice. These findings suggest that restoration of tissue and circulating levels of CSF-1 is an efficient method for regulating osteoclastogenesis. Moreover, the osteocalcin promoter provides a useful tool for delivery of CSF-1 and other cytokines to osteoblasts that, in turn, release these factors in a physiologically appropriate manner.

Analysis of CSF-1T mice before cross-breeding confirmed selective targeting of the transgene to the bone, with little or no expression of human sCSF-1 in other tissues. Circulating levels of human sCSF-1 protein were also detectable. CSF-1T mice were established on the same genetic background as op/op mice, to minimize any effect of strain variation on transgene expression. Similar levels of human sCSF-1 in bone of CSF-1T mice used for cross-breeding and in op/opT mice indicated that the transgene was efficiently transferred to op/op mice. Because op/wtT mice were interbred to generate op/opT mice, some mice would be expected to be homozygous or heterozygous for the transgene. The levels of transgene expression and resolution of the osteopetrotic defect in op/opT mice were remarkably similar among the animals examined. Successful targeting of sCSF-1 to bone in op/op T mice was evident by 21/2 wk, when eruption of upper and lower incisors was comparable with wt/wt littermates. At 5 wk, high levels of sCSF were detected in bone extracts of op/opT but not wt/wt controls. Human sCSF-1 levels in bone remained elevated at 14 wk, whereas plasma levels declined. This decrease may have resulted from enhanced CSF-1 receptor-mediated internalization and intracellular metabolism (30). Adequate transgene expression in op/op mice was manifested by normalization of growth and body weight. Tooth eruption in op/opT mice occurred at 21/2 wk, earlier than would be expected if it were attributable to spontaneous resolution (5 wk).

The radiographic changes in op/opT mice were dramatic, compared with op/op mice at 5 wk, with complete loss of metaphyseal sclerosis and radiolucent marrow spaces throughout the axial skeleton. pQCT images of femurs from 5- and 14-wk-old op/opT mice confirmed the presence of normal cancellous bone in the metaphysis and bone of normal thickness in the diaphysis, with an expanded marrow cavity comparable with wt/wt mice. These results correlated with histologic analysis of op/opT femurs that showed normalization of the growth plate width, numerous TRAP-positive osteoclasts along thin bony trabeculae in the metaphysis, and a well-developed marrow cavity. Histomorphometric analysis confirmed that the abnormal BV/TV and growth plate width observed in op/op mice was completely reversed in op/opT mice by 5 wk and persisted at 14 wk. These findings, to our knowledge, provide the most detailed analysis of the effect of sCSF-1 on op/op skeletal tissues. The lack of excessive osteoclastogenesis and osteoporosis, despite the high levels of sCSF-1, is intriguing and suggests that sCSF-1 is required for restoration of normal osteoclastogenesis but may not be a critical factor that results in excessive osteoclastogenesis and bone resorption.

Under normal physiological conditions, local production of CSF-1 by osteoblasts and their cell-to-cell interaction with osteoclast progenitors are critical for osteoclast development (21, 22, 31). Systemically administered sCSF-1 may have numerous effects on multiple organs, and it is unclear from previous studies whether the effect of sCSF-1 on bone is direct or mediated indirectly via the release of other growth factors. Our findings support a direct effect of sCSF-1 on osteoclastogenesis, with a sustained cure achieved by restoring sCSF-1 levels in the osteoblasts of op/op mice throughout murine development. To date, relapse of osteopetrosis has not been observed in op/opT mice up to 1 yr. The remarkable concordance between the op/opT and wild-type animals is intriguing. Our previous in vitro data predicted that sCSF-1 would only partially cure osteopetrosis. In coculture experiments, retrovirally transduced op/op stromal cells producing normal levels of sCSF-1 protein supported fewer numbers of osteoclasts, compared with normal stroma (32). However, in vitro studies may not mimic in vivo physiological conditions. It is likely that selective expression of sCSF-1 in op/op osteoblasts, rather than in whole-bone marrow stroma, during development provided a more physiologic milieu and restored osteoclastogenesis and corrected the skeletal abnormalities.

Similar to that in rodents, osteopetrosis in humans is a heterogenous group of skeletal disorders characterized by reduced bone resorption caused by inactive osteoclasts (33). Although a few studies have identified mutations in the gene encoding the vacuolar proton pump as a cause of certain forms of osteopetrosis, the genetic defect(s) underlying most cases of osteopetrosis remains to be determined (34, 35). Rescue of op/op mice with sCSF-1 raises the possibility that a similar CSF-1-deficiency disease may also exist among the heterogeneous forms of human osteopetrosis. The use of promoters with tissue specificity has potential therapeutic application in bone disorders (36, 37). The rat osteocalcin promoter has been shown to direct osteoblast-specific expression of GH and TGF-ß2 in transgenic mice (23, 24). It has also been used for in vivo targeting to metastatic pulmonary osteosarcoma (38). Administration iv of an adenoviral vector containing an osteocalcin-thymidine kinase construct was shown to localize to tumor cells and inhibit their growth when mice were treated with ancyclovir. More recently, mice transplanted with bone marrow-derived adherent cells containing the osteocalcin promoter showed reporter gene expression in the engrafted osteoblast cells (39). This suggests that exogenous genes could be delivered to the bone using ex vivo techniques, whereby autologous bone marrow adherent cells are expanded in culture, transduced with an osteocalcin-containing vector, and infused into the recipient. Alternatively, mature osteoblasts in the bone could be directly targeted in vivo using adenoviral-based gene therapy. Thus, targeting sCSF-1 or other exogenous genes to osteoblasts may provide a useful therapeutic approach for regulating osteoclast development and function in a variety of bone disorders, including osteopetrosis, osteoporosis, and bone metastasis.


   Acknowledgments
 
We thank Dr. Christi A. Walter for help with generating transgenic mice and for valuable discussions.


   Footnotes
 
Portions of this work were published, in abstract form, at the annual meeting of The American Society of Bone and Mineral Research, Toronto, Canada, September 22–26, 2000.

This work was supported, in part, by funding from the NIH (AR-42306, to S.L.A.), Veteran’s Administration Merit Award (to S.L.A.), and Department of Defense (DAMD17-99-1-9400, to N.G.-C).

Abbreviations: BMD, Bone mineral density; BV/TV, cancellous bone volume; pQCT, peripheral quantitative computed tomography; TRAP, tartrate resistant acid phosphatase; sCSF-1, soluble colony-stimulating factor-1.

Received August 30, 2001.

Accepted for publication January 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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