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Endocrinology, doi:10.1210/en.2002-221071
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Endocrinology Vol. 144, No. 8 3677-3682
Copyright © 2003 by The Endocrine Society

The Cell Surface Form of Colony-Stimulating Factor-1 Is Biologically Active in Bone in Vivo

Gang-Qing Yao, Jain-Jun Wu, Ben-Hua Sun, Nancy Troiano, Mary Ann Mitnick and Karl Insogna

Section of Comparative Medicine (G.-Q.Y.), Department of Internal Medicine (J.-J.W., B.-H.S., M.A.M., K.I.), and Department of Orthopedics (N.T.), Yale University School of Medicine, New Haven, Connecticut 06520-8016

Address all correspondence and requests for reprints to: Dr. Gang-Qing Yao, Section of Comparative Medicine, Yale University School of Medicine, P.O. Box 208016, New Haven, Connecticut 06520-8016. E-mail: gang-qing.yao{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The specific biological function of the cell surface or membrane-bound isoform of colony-stimulating factor-1 (mCSF-1) is not well understood. To help define the role of this isoform in bone, we developed a transgenic mouse in which targeted expression of human mCSF-1 in osteoblasts was achieved under the control of the 2.4-kb rat collagen type I {alpha} promoter. Bone density, determined by peripheral quantitative computed tomography, was reduced 7% in mCSF-1 transgenic compared with that in wild-type mice. Histomorphometric analyses indicated that the number of osteoclasts in bone (NOc/BPm, NOc/TAR, OcS/BS) was significantly increased in transgenic mice (1.7- to 1.8-fold; P < 0.05 to P < 0.01) compared with that in wild-type animals. Interestingly, the osteoblast-restricted isoform transgene corrected the osteopetrosis seen in CSF-1-deficient op/op mice. Skeletal growth and bone density in op/op mice expressing mCSF-1 in osteoblasts were similar to those in wild-type mice and were dramatically different from those in the unmanipulated op/op animals. The op/op mice expressing mCSF-1 in bone had normal incisor and molar tooth eruption, whereas the op/op mice evidenced the expected failure of tooth eruption. These findings directly support the conclusion that mCSF-1 is functionally active in bone in vivo and is probably an important local source of CSF-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MOST SECRETED CYTOKINES are synthesized as precursors that undergo intracellular posttranslational processing to the mature soluble form of the protein. These soluble molecules can act as autocrine, paracrine, and even endocrine signals, binding to cognate high affinity receptors in target cells and initiating diverse intracellular events. As an exception to this general model, a few cytokines, as a consequence of alternative gene splicing, are synthesized as transmembrane proteins (1). The specific biological function of these membrane-bound isoforms is not well understood, as most studies use the soluble isoforms because of greater ease of preparation and quantification. The available evidence suggests that soluble and membrane-bound isoforms of certain cytokines have different biological activities. Thus, the soluble and membrane-bound isoforms of stem cell factor (SCF) evidence differences in intracellular signaling kinetics and in the activation of effector cells (2, 3, 4).

Colony-stimulating factor-1 (CSF-1), another hemopoietic growth factor with significant structural homology to SCF, also has both soluble (sCSF-1) and membrane-bound (mCSF-1) isoforms (1, 5, 6). Like the SCF receptor, c-Kit, the CSF-1 receptor, c-Fms, is a receptor tyrosine kinase (1, 6). CSF-1 is required for osteoclastogenesis and is therefore a factor necessary for normal bone remodeling (7). A major unresolved question regarding the role of CSF-1 in bone is whether the two molecular forms are both biologically active and have distinct biological actions.

op/op mice are devoid of serum and tissue CSF-1 activity and, as a consequence, lack osteoclasts (8, 9, 10). This deficiency results from a single base pair insertion in the coding region of the gene for CSF-1, resulting in the production of defective CSF-1 (11, 12). Treatment of mutant mice with sCSF-1 partially corrects the defect in bone remodeling (9, 11). However, metaphaseal sclerosis persists in these treated mice despite normalization of bone structure and mass at other skeletal sites (13). Takahashi et al. (14) observed that when osteoblasts from op/op mice are cocultured with splenocytes, pharmacological amounts of sCSF-1 are required to support osteoclast formation. Antonioli-Corboz et al. (15) and Morohashi et al. (16) reported that higher concentrations of sCSF-1 are required to induce osteoclastogenesis in metatarsals from op/op mice than in bone from normal mice. These studies suggest that sCSF-1, at physiological concentrations, may not be able to completely reverse the osteopetrotic phenotype in op/op mice. One posited explanation for this finding is that mCSF-1 expression, which is also disrupted in op/op mice, plays an important role in osteoclastogenesis that cannot be compensated by sCSF-1.

Transgenic mice overexpressing full-length murine CSF-1 under the control of a 3.13-kb mouse CSF-1 promoter have been developed (17). The expression of this transgene rescued the osteopetrotic phenotype in op/op mice and corrected the neurological, hemopoietic, reproductive, and growth defects in these animals (17). The construct used in this experiment did not include the splice site for mCSF-1, and therefore in these animals only sCSF-1 was expressed. Recently, Abboud et al. (18) reported the skeletal phenotype in animals with targeted expression of sCSF-1 in osteoblasts, achieved using the osteocalcin promoter. Their results indicate that sCSF-1 overexpression in osteoblasts can rescue osteopetrosis in op/op mice. Taken together these findings demonstrate that the expression of sCSF-1 in bone is sufficient to correct the skeletal phenotype of op/op mice and provide compelling evidence that sCSF-1 is bioactive in vivo. We have used a similar model system and employed a transgenic mouse in which mCSF-1 is selectively expressed in osteoblasts to begin to explore the role of this isoform in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of the transgene construct
The mCSF-1 transgene construct was created by subcloning a 2.4-kb rat collagen type I {alpha} promoter (provided by Dr. Alex Lichtler, University of Connecticut, Farmington, CT) into Bluescript SH+ (Stratagene, La Jolla, CA). The expression of this promoter has previously been shown to be restricted principally to osteoblasts. The cDNA for human mCSF-1, purchased from American Type Culture Collection (Manassas, VA), was truncated at a point 50 bp 3' to the stop codon using a unique NdeI site to remove 3'-AU-rich instability sequences and was then cloned 3' of the collagen promoter. A 2.2-kb segment of the human GH gene containing exons 1–5 and the intervening introns were added downstream of the cDNA to provide termination/polyadenylation signals and to increase expression efficiency. The GH-coding sequences are not translated (19). This 6.0-kb minigene was excised at KpnI and XbaI restriction sites.

Generation and identification of transgenic mice
The assembled transgene was microinjected into fertilized C57BL/6xSJLF2 oocytes, and the resultant transgenic mice were identified by PCR amplification of a 171-bp sequence within exon 5 of the human GH portion of the transgene (19). The integrity of genomic DNA was assessed by coamplification of a 259-bp segment of the endogenous murine glyceraldehyde-3-phosphate dehydrogenase gene (19). The integrity of the transgene was further confirmed using PCR with human mCSF-1 primers p1 and p2, described previously (20). Transgenic lines were generated by mating founder animals to CD-1 wild-type mice, and the transgenic animals in subsequent generations were identified by PCR. The use of animals in this study was approved by the Yale animal care and use committee.

Expression of human mCSF-1 transgene in transgenic mice
mRNA expression.
Transgene expression was assessed by Northern analysis. To prepare total cellular RNA, bone and other tissues were rapidly dissected and snap-frozen in liquid nitrogen, pulverized with a mortar and pestle in the presence of powdered dry ice to keep the tissue frozen, and then solubilized in TRIzol reagent (Life Technologies, Gaithersburg, MD). Twenty micrograms of total RNA were electrophoresed on an agarose/formaldehyde gel and transferred to a nylon membrane (Hybond N, Amersham Pharmacia Biotech, Arlington Heights, IL). Northern hybridization was performed as we have previously described (21). The human CSF-1 probe was a 1.6-kb 5' fragment of the human CSF-1 cDNA (21).

mCSF-1 protein expression.
Primary murine osteoblasts were prepared from calvariae of 4- to 7-d-old mice by collagenase-dispase digestion as described previously (22). Cells were grown in MEM supplemented with 10% fetal bovine serum, penicillin, streptomycin, L-glutamine, and 20 mM HEPES. Osteoblast plasma membranes were prepared as described previously (20). Briefly, cells in the exponential phase of growth were washed with cold PBS, incubated with lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 1.5 mM MgCl2 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, 0.5 µg/ml pepstatin A, and 0.5 µg/ml leupeptin], and allowed to swell for 15 min on ice. The cells were disrupted using a ground glass homogenizer in an ice bath, and the homogenate was centrifuged at 1000 x g for 10 min to remove nuclear and cellular debris. The supernatant was overlaid on a 35% sucrose solution and centrifuged for 60 min at 20,000 x g. The plasma membranes, which concentrate in a single band at the interface of the supernatant and sucrose, were collected and centrifuged for 60 min at 100,000 x g. Membrane pellets were resuspended and stored at 70 C until used. The concentration of plasma membrane protein was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA).

To measure circulating levels of human CSF-1, blood was collected by cardiac puncture from 20 transgenic mice and 20 wild-type mice. Human mCSF-1 protein expression on plasma membranes of osteoblasts isolated from transgenic mice and in serum was quantitated using a human CSF-1 ELISA kit (R&D Systems, Minneapolis, MN).

Generation and identification of op/op-mCSF-1 mice
The op/op-mCSF-1 transgenic mice were produced by initially breeding mCSF-1 transgenic mice (mCSF-1+/-) with op/+ mice, yielding mice with an op/+ mCSF-1+ genotype. These mice were bred to mice with an op/+ genotype to yield mice with an op/op-mCSF-1+ genotype.

PCR analysis of op allelic status
The identification of op/op mice was accomplished using PCR according to previously described methods (23). The primers designed for this purpose are: P1, 5'-TGTGTCCCTTCCTCA GATTACA-3'; and P2, 5'-GGTCTCATCTATTATGTCTT GTACCAGCCAAAA-3'. The PCR generates radiolabeled DNA fragments of 195 or 196 bp depending on whether the thymidine base insertion of the op mutation is present in the template DNA. The 2-bp mismatch in the 3'-antisense primer (underlined) introduces a second BglI site spanning the extra base of the op mutation that is not present in the PCR product generated from the wild-type template. The 195/196-bp PCR products from wild-type or op/op CSF-1 tail DNA were digested with BglI. The digestion products for the two genotypes are as follows: op/+, 99, 96, 70, and 30 bp; homozygous op/op, 96, 70, and 30 bp; and wild type, 99 and 96 bp. DNA from mice identified as having the op/op genotype was then also analyzed for the mCSF-1 transgene (i.e. an op/op mCSF-1 genotype) by PCR as described above.

Bone density measurements
Peripheral quantitative computed tomography (pQCT) was used to make volumetric measurements of bone density in the proximal tibial site as described by Beamer et al. (24). A Stratec XCT-960A pQCT machine (Norland, Fort Atkinson, WI) was used to acquire scans at 3 mm below the tibial plateau. Total bone density measurements were made at a threshold of 1300 mg/cm3.

In vivo bone density measurements in op/op and op/op mCSF-1 mice and their wild-type littermates were performed by dual energy x-ray absorptiometry, using a PIXImus densitometer (Lunar Corp., Madison, WI). Measurements were made at 4, 5, and 6 wk of age. Anesthetized mice (30 mg ketamine/kg body wt and 3 mg xylazine/kg body wt, ip) were placed in the prone position, and scans were performed with a 1.270-mm diameter collimator, 0.762-mm line spacing, 0.380-mm point resolution, and an acquisition time of 5 min. The spine window is a rectangle spanning a length of the spine from T1 to the beginning of the sacrum. The femur window encompasses the entire right femur of each mouse. Bone mineral density (BMD) is expressed as milligrams per centimeter squared. The coefficient of variation for total body BMD is approximately 1.5%.

Bone histomorphometry
Static and dynamic parameters of histomorphometry were measured by methods previously reported (25, 26). All animals were injected ip with 30 mg calcein/kg body weight (Merck & Co., Rahway, NJ) 7 and 1 d before they were used. At the time of death, the tibiae were removed, stripped of soft tissue, and fixed in 70% ethanol. Tibiae were then dehydrated through graded ethanol, cleared in toluene, infiltrated with increasing concentrations of methylmethacrylate, and embedded in methylmethacrylate according to the method described preciously (25, 26). Analyses of static parameters were performed on 5-µm-thick sections stained with toluidine blue (pH 3.7). Measurement of dynamic parameters was performed on 8-µm-thick unstained sections using fluorescent microscopy. Longitudinal sections (5 µm thick) taken in the frontal plane through the cancellous bone of the proximal tibia were prepared with an RM2165 microtome (Leica, Deerfield, MI), mounted on chrom-alum-coated glass slides, and stained with toluidine blue (pH 3.7). Microscopic analysis of static and dynamic parameters was performed using a Nikon microscope (Melville, NY) interfaced with Osteomeasure system software and hardware (Osteometrics, Atlanta, GA). Measurements were obtained in an area of cancellous bone that measured approximately 2.5 mm2, containing only secondary spongiosa, and located 0.5–2.5 mm distal to the epiphyseal growth cartilage. Osteoclast perimeter, a standardized index of bone resorption; osteoblast surface, a static index of bone formation; and bone formation rate, a dynamic (tetracycline-based) index of bone formation, were measured at x250. Osteoclast surface was calculated as a percentage. The number of osteoclasts was determined per unit area (square millimeters). All indexes were defined according to the American Society of Bone and Mineral Research histomorphometry nomenclature (27).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of transgenic mice
Five of 39 mice screened were transgenic for mCSF-1. In two of the five transgenic lines, the levels of human mCSF-1 protein expression in osteoblasts, determined as described in Materials and Methods, were 53 and 100 pg/mg protein, respectively. The level of expression of mCSF-1 in osteoblast membranes isolated from wild-type animals was below the detection limit for the ELISA. The mCSF-1 transgenic line with the higher level of mCSF-1 expression was chosen for further studies.

Expression of human mCSF-1 in mCSF-1 transgenic mice
A tissue survey, by Northern analysis, demonstrated that the transgene was highly expressed in bone, whereas no expression was detected in lung, heart, skin, spleen, liver, or kidney (Fig. 1Go). The mean serum level of human CSF-1 in the transgenic animals was 89 ± 45 pg/ml. No human CSF-1 was detected in the blood of wild-type mice.



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  		FIG. 1. Tissue-specific expression of mCSF-1 RNA in mCSF-1 transgenic mice. Northern analysis of total RNA was performed using a human CSF-1 cDNA probe. The transgene was highly expressed in bone, whereas no expression was detected in lung, heart, skin, spleen, liver, or kidney.

 
Bone density analyses
To examine the effect of mCSF-1 expression in osteoblasts on bone mass, we initially measured bone density in transgenic mice and their wild-type littermates by pQCT. At 12 wk of age, the mean total tibial bone density was significantly lower in transgenic mice compared with their wild-type littermates (418 ± 60 vs. 451 ± 60 g/cm3, transgenic vs. wild-type; n = 57 and 54, respectively; P < 0.01).

Histomorphometric analyses
Histomorphometric parameters were assessed in 15 femurs each from 12-wk-old female transgenic and wild-type mice. As summarized in Table 1Go, the number of osteoclasts in bone, whether expressed as osteoclast perimeter, number of osteoclasts per unit area, or osteoclast surface, was significantly increased in transgenic mice (1.7- to 1.8-fold; P < 0.05, P < 0.01, and P < 0.05, respectively) compared with that in wild-type mice (Table 1Go). In contrast, osteoblast number and bone formation rate were equivalent in the two groups (Table 1Go).


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  		TABLE 1. Bone histomorphometry analysis

 
Osteoblast-derived mCSF-1 rescues the osteopetrotic phenotype in op/op mice
To determine whether restricted expression of mCSF-1 in bone can rescue the osteopetrotic phenotype of op/op mice, we studied whole body radiographs of 4-wk-old op/op and op/op mCSF-1 mice and their wild-type littermates (Fig. 2Go). The op/op mice exhibited severe osteopetrosis, characterized by radiopaque long bones and vertebral bodies that were dense and short. These abnormalities were fully corrected by mCSF-1 transgene expression (Fig. 2Go). As expected, there was no incisor or molar tooth eruption observed in op/op mice (Fig. 2Go, bottom, and Fig. 3Go). In contrast, the expression of the mCSF-1 transgene in op/op mCSF-1 mice resulted in normal incisor and molar tooth eruption indistinguishable from that seen in wild-type animals (Fig. 2Go, bottom, and Fig. 3Go). Op/op mice showed normal rates of growth until weaning at 3 wk. After weaning, the rate of growth in the op/op mice was severely impaired despite their being fed a nutrient-rich milk formula diet. In contrast, the growth rate of op/op mCSF-1 mice was not significantly different from that of their wild-type littermates (Fig. 4Go).



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  		FIG. 2. Osteoblast-derived mCSF-1 corrects osteopetrosis in vivo. Total skeleton radiographs in 4-wk-old op/op, op/op-mCSF-1, and wild-type littermates demonstrate that the skeletal growth and density of op/op-mCSF-1 mice are similar to those of wild-type mice and dramatically different from those of op/op mice. The severe osteopetrosis exhibited by op/op mice, as evidenced by radiopaque long bones (arrow, upper panel, right column) and vertebral bodies (lower arrow, lower panel, right column), was corrected in op/op-mCSF-1 mice (arrow, upper panel, middle column, and lower arrow, lower panel, middle column). In addition, op/op-mCSF-1 mice had normal incisor and molar tooth eruption (upper two arrows, lower panel, middle column), whereas the op/op animals had the expected failure of tooth eruption (upper two arrows, lower panel, right column). Radiographs in op/op-mCSF-1 were indistinguishable from those in wild-type littermates (left column).

 


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  		FIG. 3. The mCSF-1 transgene corrects the tooth eruption deficiency of op/op mice. The failure of tooth eruption is a typical finding in op/op mice, as bone resorption is required to open an avenue through the bone of the jaw for the eruption of teeth. This defect is completely corrected by the mCSF-1 transgene.

 


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  		FIG. 4. The growth rate and BMD of mCSF-1 op/op mice at different ages. Twenty-two op/op-mCSF-1 mice, 6 op/op mice, and 12 wild-type littermates were weighed 4, 5, and 6 wk after birth. At all three time points, the growth rate of op/op mCSF-1 mice was not significantly different from that of their wild-type littermates (upper panel). The BMD in these mice was measured at each time point by dual energy x-ray absorptiometry. The op/op mice exhibited significantly greater bone density than either of the other two groups at all time points. The op/op mCSF-1 mice had BMD values not different from those of the wild-type animals. The results shown are the mean ± SE.

 
Serial bone density measurements using PIXImus were made at 4, 5, and 6 wk in op/op mice, op/op mice rescued with the transgene, and wild-type controls. At all three time points, op/op mice exhibited significantly greater bone density than either of the other two groups (Fig. 4Go). The op/op mCSF-1 mice had bone densities not different from those of the wild-type littermates (Fig. 4Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability of sCSF-1 to promote osteoclastogenesis has been well documented in vitro and in vivo (11, 14, 17, 18, 28, 29, 30, 31). In contrast, the roles of mCSF-1 in bone remodeling have been studied only recently. We have shown that osteoblasts express mCSF-1, and that PTH and TNF regulate the expression of this isoform (20). In addition, mCSF-1 expression by mCSF-1-transfected NIH-3T3 cells or induced stromal ST2 cells is capable of supporting osteoclast formation in vitro (20, 32). Recently, we have found that the level of mCSF-1 endogenously expressed by primary murine osteoblasts supports the formation of osteoclast-like cells in a dose-dependent manner in vitro (22). Further, endogenously expressed mCSF-1 strongly enhances the effect of sCSF-1 on osteoclastogenesis (22). Our recent finding that in vitro osteoclast formation was inhibited when primary murine osteoblasts were treated with an antisense oligonucleotide further supports the idea that mCSF-1 is important for normal bone remodeling (22). This inhibitory effect was dose dependent and was not seen with a sense oligonucleotide construct.

Consistent with these in vitro results, the data presented here demonstrate that transgenic mice expressing mCSF-1 restricted to osteoblasts have significant bone loss. Bone density, as determined by pQCT, was reduced by 7.3% in the tibia of adult animals. This degree of bone loss is comparable to that seen with ovariectomy in mice (33). Histomorphometric analyses indicated that the number of osteoclasts in bone was significantly increased in transgenic mice (1.7- to 1.8-fold; P < 0.05) compared with that in wild-type animals. We could not adequately quantify marrow macrophages, but the proportion of circulating monocytes in transgenic mice (2%) was in the normal range.

Direct evidence that mCSF-1 is bioactive in vivo is provided by the finding that selective expression of mCSF-1 in osteoblasts can rescue the osteopetrotic phenotype of the op/op mouse. The normal growth rate, bone density, skeletal radiographs, and normal tooth eruption indicate that localized expression of this isoform in bone is sufficient to support normal bone modeling and remodeling. In contrast to the findings in the transgenic wild-type animal, transgene expression in the op/op mice was not associated with a reduced BMD compared with wild-type controls. The reason for this is unclear, but may reflect the lack endogenous CSF-1 in op/op mice.

It has been reported that mCSF-1 can be shed from the cell membrane (34). The bioactivity of the shed form of CSF-1 is similar to that of sCSF-1 in its ability to support osteoclast formation in vitro (35). We detected low levels of human CSF-1 in the blood of our transgenic animals, suggesting that shedding of mCSF-1 was occurring in vivo. These levels are far lower than endogenous levels in normal mice that have circulating levels of CSF-1 between 9.9–17 ng/ml (9, 17). It therefore seems unlikely that circulating mCSF-1 makes a major contribution to the biological actions of this isoform.

Our data make a compelling argument for an in vivo role for mCSF-1 in bone and compliment the recent findings of Abboud et al. (18). Taken together these results indicate that either isoform, when selectively expressed in bone, is fully bioactive. What these studies leave unanswered is whether either isoform subserves a unique nonredundant role. Isoform-specific knockouts will help to address this issue.

In summary, the findings of the present study directly support the conclusion that mCSF-1 is bioactive and plays an important role in osteoclast development in vivo. Taken together with our earlier findings, they provide evidence that osteoblasts are an important source of mCSF-1 in the bone microenvironment.


   Footnotes
 
This work was supported by NIH Grants DK-45228 and DE-12459 (to K.L.I.) and in part by the Yale Core Center for Musculoskeletal Disorders (P30-AR-46032).

Abbreviations: BMD, Bone mineral density; mCSF-1, membrane-bound isoform of colony-stimulating factor-1; pQCT, peripheral quantitative computed tomography; SCF, stem cell factor; sCSF-1, soluble isoform of colony-stimulating factor-1.

Received October 16, 2002.

Accepted for publication April 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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