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Published ahead of print on November 15, 2007, doi:10.1164/rccm.200705-659OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 309-315, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200705-659OC

Original Article

Persistent Osteopenia in Adult Cystic Fibrosis Transmembrane Conductance Regulator–deficient Mice

Christina K. Haston1, Wei Li2, Ailian Li2, Melanie Lafleur1 and Janet E. Henderson2

1 Meakins-Christie Laboratories and 2 J.T.N. Wong Laboratories for Mineralised Tissue Research, Department of Medicine, McGill University, Montreal, Quebec, Canada

Correspondence and requests for reprints should be addressed to Christina Haston, Ph.D., Meakins-Christie Laboratories, 3626 rue St. Urbain, Montreal, PQ, H2X 2P2 Canada. E-mail: christina.haston{at}mcgill.ca


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: A loss of function mutation in the cystic fibrosis transmembrane conductance regulator gene is believed to be an independent risk factor for bone disease in patients with cystic fibrosis.

Objectives: The objective of this work was to use congenic mice as a preclinical model to examine the bone phenotype of Cftr–/– mice and control littermates at 8, 12, and 28 weeks of age.

Methods: The bone phenotype of control and Cftr–/– mice was evaluated by quantitative imaging, histologic and histomorphometric analyses, and serum levels of bone biomarkers.

Measurements and Main Results: At 12 weeks of age, Cftr–/– mice were smaller, had lower bone mineral density, cortical bone thinning, and altered trabecular architecture compared with Cftr+/+ or Cftr+/– control mice. In skeletally mature 28-week-old mice, there were persistent deficits in cortical and trabecular bone structure in Cftr–/– mice despite significant, quantifiable improvements. Cftr–/– mice also had lower serum insulin-like growth factor-I levels at 12 weeks of age than did control mice, whereas parathyroid hormone and 25-hydroxyvitamin D levels were not significantly different.

Conclusions: Persistent osteopenia and structural abnormalities in adult Cftr–/– mice, in the absence of overt respiratory and gastrointestinal disease, suggest that loss of Cftr function has a direct impact on bone metabolism in Cftr–/– mice that is not sex specific or subject to haplotype insufficiency.

Key Words: bone disease • lung disease • preclinical model • genetically modified mouse



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Bone disease in patients with cystic fibrosis is multifactorial, but the contribution of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to bone disease remains undefined.

What This Study Adds to the Field
We show that adult Cftr-deficient mice have low femoral bone mineral density and compromised bone architecture in the absence of other overt disease symptoms. The results suggest that deficiency of Cftr contributes to the osteopenic phenotype of Cftr–/– mice.

 
Cystic fibrosis (CF) is a disease in which mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) in humans lead to abnormalities affecting principally the lung, intestine, and pancreas (1). Patients with CF may also suffer from bone disease, characterized by low bone mineral density (BMD) and increased fracture rates (2). In general, susceptibility to fracture is determined by a combination of factors including BMD, geometry, microarchitecture, and bone cell activity (3, 4). The diagnosis and response to treatment of osteopenia and osteoporosis, therefore, are currently monitored with tests that determine BMD and bone architecture (using peripheral quantitative micro–computed tomography) and bone cell activity (using serum biomarker assays).

In cystic fibrosis, according to the consensus statement on bone health (2), bone disease arises from a variety of factors including decreased vitamin absorption, pancreatic insufficiency, altered sex hormone production, chronic lung infection resulting in increased levels of bone-active cytokines (5, 6), physical inactivity, and steroid therapy. Studies of serum biomarkers for bone turnover in patients with CF suggest that bone resorption exceeds formation (2). It has also been suggested that low BMD in adults with CF may arise from a combination of insufficient bone accrual during puberty (7) and subsequent bone loss in young adulthood (2). King and coworkers (8) have shown that the common {Delta}F508 mutation in the chloride ion channel encoded by CFTR is an independent risk factor for bone disease in the CF population, in addition to malnutrition and lung disease and Sermet-Gaudelus and coworkers (9) report low BMD in children with CF who are younger than 6 years of age. The mechanisms through which the primary CF defect influences bone metabolism, however, remain undefined.

Mouse models that exhibit the altered electrophysiology expected to arise from mutations in Cftr have been extensively characterized (10), but there have been relatively few studies investigating their bone phenotype (11, 12). Dif and coworkers (11) evaluated 3-week-old female UNC Cftr-deficient mice, which carry a mutation that results in the absence of the Cftr protein in a mixed genetic background (13). They showed the weanling mice to have lower BMD, reduced cortical bone width, and thinner trabeculae compared with their wild-type littermates. This study suggested that bone disease in weanling Cftr-deficient mice was due primarily to the absence of Cftr, as the mice had not yet developed pancreatic insufficiency or lung disease and had not been subjected to therapeutic doses of steroids.

Under physiological conditions, skeletal maturity in mice has been determined to occur at about 16 weeks of age. Comparable to the situation seen in humans, skeletal maturity in the mouse marks the cessation of longitudinal and appositional bone growth. At this stage, net bone acquisition is replaced by remodeling to maintain bone mass. Given the changes in bone physiology with age, the current study was undertaken to determine how loss of function of the gene encoding the cystic fibrosis transmembrane conductance regulator, Cftr, influences the bone phenotype in skeletally mature, juvenile, and adult CF mice.


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Animal procedures were performed in accordance with McGill University (Montreal, PQ, Canada) guidelines set by the Canadian Council on Animal Care (Ottawa, ON, Canada). Congenic BALB Cftr UNC+/– mice were originally obtained from J. Hu of the Hospital for Sick Children (Toronto, ON, Canada) and maintained in a breeding colony at the Meakins-Christie Laboratories of McGill University. The Cftr+/– mice were intercrossed to produce Cftr+/+, Cftr+/–, and Cftr–/– mice, which were identified by genotyping as described previously (14). We used mice that are congenic in the BALB background to control for possible effects of background strain variation on the CF phenotype (14–16). To circumvent premature death of Cftr–/– mice as a result of intestinal disease, control and Cftr–/– mice were fed standard chow and received PegLyte (polyethylene glycol [17.8 mmol/L] and electrolytes; Pharmascience, Montreal, PQ, Canada) in their drinking water as described previously (17). PegLyte is used clinically to cleanse the intestinal tract while preserving electrolyte balance. To determine the effect of PegLyte on bone growth in normal mice, one group of female Cftr+/+ mice was fed regular drinking water. Mixed sex groups were used for experiments conducted at 12 weeks (Cftr–/–: 12 wk, five males and three females [5 M/3 F]; control: 12 wk, 8 M/6 F) and 28 weeks (Cftr–/–: 28 wk, 1 M/7 F; control: 28 wk, 1 M/5 F).

Radiologic Imaging
Phenotyping procedures were performed essentially as described previously (18, 19). At the indicated ages mice were weighed and killed with a lethal dose of anesthetic before capturing high-resolution X-rays of the femora and vertebrae with a Faxitron general science radiography system (model MX-20/DC2; Faxitron X-ray Corporation, Wheeling, IL), equipped with an FPX-2 imaging system (MedOptics/DALSA Life Sciences, Waterloo, ON, Canada). X-rays were used to qualitatively assess bone morphology and mineral density and to measure the lengths of femora and vertebrae. The right femur was measured from the plateau of the knee to the tip of the femoral head and the vertebral measurement represents the length from the proximal end of lumbar vertebra 3 (L3) to the distal end of L5 (L3–L5). A Lunar PIXImus 1.46 instrument (GE Healthcare Lunar, Madison, WI) was then used to measure BMD of the intact animal before dissecting bones free of soft tissue. Bones were fixed overnight in 4% paraformaldehyde, rinsed three times with phosphate-buffered saline, and scanned with a SkyScan 1072 micro–computed tomography (micro-CT) instrument (SkyScan, Antwerp, Belgium) to assess bone morphometry. Image acquisition was performed at 45 kV, 222 µA for a 2.24-second exposure at x50 for femur and x30 for lumbar vertebrae with a 0.9° rotation between frames. These two-dimensional images were used to reconstruct three-dimensional images for quantitative analysis, using 3D Creator software supplied with the instrument. The area of interest selected for quantification of trabecular bone was located immediately below the growth plate and extended for 1 mm toward the diaphysis of the femur or the midpoint of the vertebra. Reported parameters include the following: bone volume as a percentage of tissue volume (BV/TV); the structure model index (SMI), which is the ratio of rodlike structures to platelike structures, with the ideal ratio being 1.0. A high ratio is indicative of a reduction in trabecular connectivity; the thickness of individual trabeculae (TrTh); trabecular separation (TrSp), which is an indirect measure of trabecular thinning; and the number of trabeculae in a given area (TrNo).

Histology and Histochemistry
After micro-CT analysis the right femur of each of four mice from each group was embedded in polymethylmethacrylate at low temperature and 4-µm sections were cut. Sections were stained with von Kossa to identify mineralized bone and counterstained with toluidine blue to identify unmineralized tissue. Midsagittal sections, corresponding to the micro-CT images, were cut and images were captured at a magnification of x2.5, using a Zeiss Axioskop microscope (Carl Zeiss Canada Ltd, Toronto, ON, Canada) equipped with a Zeiss AxioCam MRc camera. Adjacent sections were stained for alkaline phosphatase (ALP) activity in osteoblasts or tartrate-resistant acid phosphatase (TRAP) in osteoclasts as described previously (18). Cell numbers were scored by three independent investigators, blinded to mouse age and Cftr genotype, using a single section from three different mice per group. Osteoclast numbers were determined by counting the number of TRAP-positive cells present in five individual fields of bone at a final magnification of x400, and normalizing this count to the bone volume. Osteoblast cell numbers were similarly determined by examination of von Kossa–stained sections.

Blood Analysis
Blood was collected by cardiac puncture of anesthetized animals and centrifuged, and the supernatant serum was stored at –85°C until biochemical analyses for bone biomarkers were undertaken with the following commercial assays: radioimmunoassay for 25-hydroxyvitamin D (ImmunoDiagnostic Systems, Tyne and Wear, UK), ELISA for insulin-like growth factor-I (Quantikine mouse IGF-I; R&D Systems, Minneapolis, MN), and ELISAs for parathyroid hormone and osteocalcin (Immunotopics, Inc., San Clemente, CA).

Statistical Analysis
Results are expressed as means ± SD and differences between groups of mice were determined by the Student t test and the Mann-Whitney test.


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PegLyte, Sex, and Haplotype on 12-Week-Old Control Mice
Intervention with PegLyte to minimize lethality due to the gastrointestinal phenotype enabled the survival of all the Cftr–/– mice to adulthood. To determine whether this treatment altered body weight or skeletal development of control mice we assessed the morphology, mineral density, and structure of femoral bone in 12-week-old mice receiving PegLyte in their drinking water, and compared the results with those of age- and sex-matched control mice receiving plain water. As shown in Table 1, a small but significant decrease in bone volume and altered trabecular architecture was seen in the control mice receiving PegLyte compared with untreated control mice.


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  		TABLE 1. EFFECT OF SEX AND TREATMENT WITH PEGLYTE ON BONE CHARACTERISTICS OF 12-WEEK-OLD Cftr+/+ AND Cftr+/– CONTROL MICE

 
To eliminate the possibility that haplotype insufficiency might contribute to a skeletal phenotype we analyzed the bones of each of four 3-month-old female Cftr+/+ and Cftr+/– mice. No significant differences in morphology, BMD, or architecture were observed between Cftr+/+ and Cftr+/– mice (data not shown). These data are consistent with a previous report for weanling 3-week-old littermates (11). Cftr+/+ and Cftr+/– mice were subsequently used interchangeably as control animals.

Age-related Changes in Bone Morphology and BMD in Control and Cftr–/– Mice
In a previous study it was reported that 3-week-old weanling Cftr-deficient mice had reduced BMD and osteopenia characterized by increased bone resorption relative to formation (11). Table 2 confirms and expands on the previous data by showing that the highly significant reduction in body weight, bone length, and BMD in Cftr–/– mice compared with control mice persists at 8 and 12 weeks of age. These changes were apparent in both male (n = 5) and female (n = 3) Cftr–/– mice compared with their sex-matched control mice at the age of 12 weeks and were not significantly different between 12-week-old male and female Cftr–/– mice (data not shown). Skeletally mature 28-week-old Cftr–/– mice had largely normalized with respect to skeletal morphology and BMD in the vertebra, although the femoral BMD remained significantly lower.


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  		TABLE 2. AGE-RELATED CHANGES IN MORPHOLOGY AND MINERAL DENSITY OF BONE TISSUE OF CONTROL AND Cftr–/– MICE

 
Age-related Changes in Trabecular Bone Architecture in Control and Cftr–/– Mice
To further explore the differences in the skeletal phenotypes of control and Cftr–/– mice we assessed architectural parameters by micro-CT and bone composition by classic histology. At 12 weeks of age (Figure 1), reconstruction of micro-CT images into a three-dimensional model (Figure 1a) shows little difference between the control (Figure 1, top) and Cftr–/– (Figure 1, bottom) mice, except perhaps for a thinner diaphyseal shaft in the Cftr–/– mice. Two-dimensional coronal sections adjacent to the growth plate (Figure 1b) show the reduced trabecular bone and cortical thinning in the diaphysis (Figure 1c) of Cftr–/– mice compared with control mice. These data are corroborated in the matched, midsagittal micro-CT (Figure 1d) and von Kossa– and toluidine blue–stained histologic sections (Figure 1e). Adjacent sections stained for ALP (Figure 1f) and TRAP (Figure 1g) to localize osteoblasts and osteoclasts, respectively, show there is little difference between control and Cftr–/– mice, except for a marginal increase in TRAP staining in the Cftr–/– mice. By 28 weeks of age (Figure 2) there appears to be an increase in trabecular bone (Figure 2b) and cortical thickness (Figure 2c) in the Cftr–/– mice (Figure 2, bottom) as evidenced by micro-CT and von Kossa staining (Figure 2e). ALP activity (Figure 2f) is greatly increased in Cftr–/– mice compared with control mice and TRAP activity (Figure 2g) also appears up-regulated.


Figure 1
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  		Figure 1. Architecture and composition of distal femur of 12-week-old control and Cftr–/– mice. Bones from control (top) and Cftr–/– (bottom) mice were dissected free of soft tissue, fixed, and scanned with a SkyScan 1072 micro–computed tomography (micro-CT) system equipped with 3D-Creator analytical software. Three-dimensional reconstruction (a) and two-dimensional cross-sectional scans (b–d) demonstrated fewer trabeculae and cortical thinning of Cftr–/– bones. Plastic-embedded sections of the same bones, stained with von Kossa and counterstained with toluidine blue (e) and photographed at original magnifications of x2.5 and x20 (inset), confirmed the micro-CT data. Adjacent sections stained for alkaline phosphatase (f, brown) and tartrate-resistant acid phosphatase (g, red) showed little difference between control and Cftr–/– mice in osteoblast (brown) or osteoclast (red) activity along the edges of bony trabeculae. CF = cystic fibrosis (Cftr–/– mice).

 

Figure 2
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  		Figure 2. Architecture and composition of distal femur of 28-week-old control and Cftr–/– mice. Bones from control (top) and Cftr–/– (bottom) mice were processed as described in Figure 1. Trabecular bone (b–d) and cortical width (c and d) were increased in Cftr–/– mice as compared with 12-week-old Cftr–/– mice, although the values still had not normalized relative to control mice. A significant increase in alkaline phosphatase activity (f, brown) and tartrate-resistant acid phosphatase activity (g, red) was seen along the edges of bony trabeculae in Cftr–/– mice compared with control littermates. CF = cystic fibrosis (Cftr–/– mice).

 
Quantitative micro-CT analysis of bone at 8 weeks (skeletally immature), 12 weeks (juvenile), and 28 weeks (skeletally mature adult) of age is shown in Table 3. All parameters except for trabecular thickness changed significantly between 8 and 12 weeks in the control mice and there was minimal significant change after that time. In contrast, bone architecture in the Cftr–/– mice did not change significantly between 8 and 28 weeks of age, despite the significant increase in ALP- and TRAP-positive cells. Thus, at 8 weeks of age, trabecular bone volume in Cftr–/– femora is similar to that in control littermates, despite the significant difference in BMD, but it was significantly reduced at 12 and 28 weeks of age, primarily because of reduced numbers of trabeculae. As shown in Figure 3A, there was a significant increase in TRAP-positive cells in Cftr–/– mice at both 12 and 28 weeks, whereas the increase in ALP-positive cells did not reach significance until 28 weeks. The alteration in bone cell activity in Cftr–/– mice was reflected in a decrease in cortical width of the bones of Cftr–/– mice compared with that seen in control mice. The discrepancy in cortical bone width between control and Cftr–/– mice appeared to diminish over time, as shown in Figure 3B.


Figure 3
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  		Figure 3. Trabecular bone cell numbers and cortical bone width of femur of 12- and 28-week-old control and Cftr–/– mice. Osteoblasts were counted on representative von Kossa– and toluidine blue–stained sections, and osteoclasts on representative tartrate-resistant acid phosphatase–stained sections, of plastic-embedded femoral bone from three or four female mice per group (A). Cortical width was measured at four points at the midpoint of the femoral diaphysis of three female mice per group (B). Values are expressed as means ± SD. *P < 0.05, significantly different from control data. CF = cystic fibrosis (Cftr–/– mice); OB = osteoblasts; OC = osteoclasts.

 

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  		TABLE 3. AGE-RELATED CHANGES IN FEMUR BONE ARCHITECTURE OF CONTROL AND Cftr–/– MICE

 
To determine whether alterations in the bone phenotype of Cftr–/– mice were due in part to changes in the circulating levels of bone-active factors, we examined serum biomarkers in representative mice at 12 and 28 weeks of age. As shown in Table 4, Cftr–/– mice had lower levels of IGF-I at 12 weeks and osteocalcin at 28 weeks compared with age-matched control mice. No significant differences were seen in the calciotropic hormones parathyroid hormone and 25-hydroxyvitamin D.


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  		TABLE 4. SERUM LEVELS OF BONE BIOMARKERS IN 12- AND 28-WEEK-OLD CONTROL AND Cftr–/– MICE

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study was undertaken to determine how loss of function of the Cftr gene influences bone development and metabolism. We have shown that at 12 weeks of age, Cftr–/– male and female mice are smaller, have low BMD, cortical bone thinning, and compromised trabecular architecture compared with wild-type or heterozygous control mice. At 28 weeks of age, when the mice are skeletally mature, there are persistent deficits in cortical and trabecular bone structure despite significant, quantifiable improvements. These differences were seen in the absence of overt, life-threatening lung and pancreatic disease or steroid hormone treatment and in the presence of significant alterations in bone cell numbers and activity. The data thus support the conjecture that loss of Cftr function has a direct impact on skeletal metabolism that is not sex specific or subject to haplotype insufficiency in a mouse model.

Impact of PegLyte on Survival and Bone Phenotype of Cftr–/– Mice
In this study, we used PegLyte in the drinking water to extend the life span of Cftr–/– mice as described by Gawenis and coworkers (12). The intervention had little direct effect on bone development, as evidenced by attainment of nearly normal bone lengths by 28 weeks of age in Cftr–/– mice, but did allow for examination of adult mice. It was reported by Gawenis and coworkers that, after correcting for body weight, BMD did not differ between UNC Cftr–/– mice and control littermate mice. Our own observations support their data by showing no significant decrease in femoral or vertebral BMD after correcting for differences in body weight in the Cftr–/– mice. These observations are somewhat surprising given the severe vertebral osteopenia observed in young adult patients with CF. This discrepancy might be explained by the fact that the Cftr–/– mice receiving PegLyte did not display overt gastrointestinal or respiratory complications with associated altered levels of inflammatory cytokines (6) and did not receive corticosteroids, which could lead to severe osteopenia when combined with structural abnormalities conferred by mutations in the CF gene. In contrast to the BMD data, quantitative micro-CT shows that body size is not the major determinant of the observed differences in the skeletal phenotypes of control and Cftr–/– mice. At 8, 12, and 28 weeks the Cftr–/– mice weighed 20, 44, and 19% less than control mice, respectively. However, most micro-CT parameters, which reveal changes in trabecular bone quantity and architecture that are indicative of metabolic bone disease, were significantly different at 28 weeks, but not at 8 weeks, when the differences in body weight were approximately equivalent.

Using quantitative techniques such as neutron activation and bone ash analysis, Gawenis and coworkers did not detect differences in the chemical composition of bones from Cftr–/– and control mice and concluded that Cftr does not play a direct role in bone mineralization (12). Our own studies, as well as those of Dif and coworkers (11), support this conjecture, as there was no evidence of increased osteoid in either study. We also reported normal circulating levels of parathyroid hormone and 25-hydroxyvitamin D, suggesting that mineral ion homeostasis and absorption of vitamin D via the gut were normal in Cftr–/– mice during pre- and postnatal bone development. Malabsorption would have resulted in low vitamin D and an excess of osteoid, as seen in vitamin D–deficient rickets (20). PegLyte treatment therefore had an additional indirect beneficial effect on bone by allowing for improved absorption of calcium and vitamin D, which may be compromised in patients with CF (21).

Improvement of Osteopenia over Time in BALB Cftr–/– Mice
In the current study we show that the bone phenotype of 12-week-old (juvenile) BALB Cftr–/– mice resembles that of the 3-week-old (weanling) UNC Cftr–/– mice studied by Dif and coworkers. The defects in Cftr–/– mice included low BMD; reduced cortical bone width; and fewer, thinner trabeculae compared with control littermates. The osteopenic phenotype of 3-week-old Cftr–/– mice was attributed to a decrease in bone-forming surfaces and in osteoclast numbers in trabecular bone. However, the mixed genetic background (14–16) and the immature bone age may have been confounding factors in the interpretation of data in the Dif study. In 28-week-old Cftr–/– mice, osteopenia was seen in the presence of increased numbers of trabecular osteoblasts and osteoclasts, suggesting a high turnover defect. Alternatively, the relative increase in osteoblasts and osteoclasts in the 28-week-old mice could reflect a "catch-up" phenomenon, resulting from normalization of IGF-I levels, as discussed below. Studies examining dynamic labeling of mineralization fronts and including in-depth histomorphometric analyses will be required to resolve this issue.

Cortical thinning in 3-week-old Cftr–/– mice (11) was attributed to a relative increase in cortical osteoclasts, although the cortical osteoblast surfaces were not documented. In 12-week-old Cftr–/– mice, despite the significant reduction in cortical width, we could not detect any significant differences in the number of osteoclasts or osteoblasts in cortical bone compared with the control mice. The cortical width of Cftr–/– bones in the study by Dif and coworkers was approximately 50% that of control mice at 3 weeks of age, compared with 75% at 12 weeks and 85% at 28 weeks of age in our own work. These observations, together with those documenting a gradual increase in trabecular and cortical BMD and improvement in bone architectural parameters between 12 and 28 weeks of age, suggest that attainment of peak bone mass may have been delayed in Cftr-deficient mice. However, it is also important to note that trabecular bone volume remained almost 50% lower in Cftr–/– mice at 28 weeks, with reductions in the number and thickness of individual trabeculae and an increase in the ratio of platelike to rodlike structures measured. These features, along with the continued elevation in osteoblasts and osteoclasts, support the conjecture that bone turnover is increased in Cftr–/– mice compared with age-matched control mice.

The trajectory of weight gain and bone accrual in control mice was greatest between 8 and 12 weeks of age, which would roughly correspond with childhood and adolescence in humans, and then slowed thereafter. In contrast, Cftr–/– mice gained little weight or bone mass between 8 and 12 weeks whereas significant increases in both were observed between 12 and 28 weeks. This failure to keep pace with age-matched control mice recapitulates the clinical situation, in which low bone mass in adult patients with CF has been attributed to a failure to thrive and accrue bone during childhood and adolescence (7). Supporting this, through histomorphometric analyses, Elkin and coworkers (5) revealed bone harvested from adult patients with CF with low BMD to have reduced trabecular bone volume compared with age- and sex-matched healthy control subjects, which was ascribed to low bone formation. Furthermore, a second study, in which histomorphometric analysis of bone specimens taken at autopsy from posttransplantation patients treated with corticosteroids was completed, revealed the existence of significant cortical and trabecular osteopenia in the CF specimens, in association with decreased osteoblastic and increased osteoclastic activity (22). In contrast to these clinical data, the 28-week-old Cftr–/– mice demonstrated nearly normal vertebral BMD. This discrepancy is most likely due to the absence of steroidal treatment in the mice and its presence as a confounding variable in the interpretation of the clinical findings. The increase in osteoclast numbers and the absence of excessive osteoid (osteomalacia) are common features of CF and adult Cftr–/– mouse phenotypes.

At 12 weeks of age, there was a significant reduction in circulating IGF-I in Cftr–/– mice compared with control mice. This deficiency was coincident with the largest discrepancy in body weight and bone mass between control and Cftr–/– mice. IGF-I is a major regulator of somatic cell and bone growth and has been identified as an independent predictor of low BMD in patients with CF (23) and compromised growth in Cftr–/– mice (24). Work has used genome-wide scanning of F2 mice derived from C57BL and C3H strains, with low and high BMD, respectively, to identify quantitative trait loci linked to both serum IGF-I and bone acquisition (25). Adult female congenic mice carrying a chromosome 10 locus from C3H on a C57BL background had significantly higher circulating levels of IGF-I in association with higher femoral BMD and trabecular number. In BALB Cftr–/– mice, there was a significant increase in serum IGF-I between 12 and 28 weeks and a concomitant increase in BMD and trabecular number, suggesting that the late-onset "growth spurt" in Cftr–/– mice could have been mediated by circulating IGF-I. Of additional interest in this respect are the reports of reduced peroxisome proliferator–activated receptor-{gamma} in Cftr–/– mice (26) and the negative regulation of IGF-I by activation of this receptor (27). It is conceivable that the rise in IGF-I at 28 weeks was related to an age-dependent downregulation of peroxisome proliferator–activated receptor-{gamma} in Cftr–/– mice.

The precise molecular mechanisms by which mutations in CFTR can alter bone cell function remain to be elucidated, although insight may be gained from studies of other chloride channels implicated in defective bone metabolism. For example, loss of function of the chloride channel-7 (ClC-7) results in a defect in chloride conductance required for efficient proton pumping by the H+-ATPase of the osteoclasts (28). The catabolic activity of osteoclasts depends on the active transport of hydrogen ions into the extracellular space adjacent to bone to solubilize bone mineral. Loss of chloride channel-7 activity therefore leads to accumulation of bone that would otherwise be resorbed, or to osteopetrosis (28). Similarly, Kajiya and coworkers (29) reported that the K+/Cl– cotransporter-1 expressed in mouse osteoclasts also participates in H+ extrusion during bone resorption, thus providing a second example of a chloride channel involved in the catabolic activity of osteoclasts. In our current work, bone acquisition continued in mice up to 28 weeks of age in the presence of a significant increase in osteoclast and osteoblast activity. It is therefore unlikely that there was a significant defect in either cell type, although CFTR protein has been shown to be present in both (30), but rather that a change in the bone microenvironment influenced their activity. Additional in vitro and molecular studies are required to resolve this issue.


   FOOTNOTES
 
Supported by the CCFF and Canadian Institutes of Health Research, the Basic Research and Therapy (BREATHE) Program, Valorisation Recherche Québec, and Fonds de la Recherche en Santé Québec.

Originally Published in Press as DOI: 10.1164/rccm.200705-659OC on November 15, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form May 3, 2007; accepted in final form November 9, 2007


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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